Prevention and treatment of amyloidogenic disease

专利摘要:
The present invention discloses pharmaceutical compositions and methods for preventing or treating a number of amyloid diseases, including Alzheimer's disease, prion disease, familial amyloid neuropathy and the like. The pharmaceutical composition comprises an amyloid fibril component, particularly a fibrillar forming peptide or protein, in an immunologically reactive amount. The present invention also discloses therapeutic compositions and methods using immunological agents that react with such fibril components.
公开号:KR20020025884A
申请号:KR1020017015508
申请日:2000-06-01
公开日:2002-04-04
发明作者:쉥크데일비
申请人:인슬레이, 케빈；뉴랄랩 리미티드；
IPC主号:

专利说明:

Prevention and treatment of amyloidosis disease {PREVENTION AND TREATMENT OF AMYLOIDOGENIC DISEASE}
[1] This application claims priority to US Provisional Patent Application No. 60 / 137,010, filed June 1, 1999, which is incorporated herein by reference.
[3] Amyloidosis (amyloidogenesis) is a generic term describing a number of diseases characterized by extracellular deposition of protein fibrils, which form many "amyloid deposits", which can occur locally or systemically. The fibril composition of such deposits is a feature that distinguishes various forms of amyloid disease. For example, deposits in the brain and cerebrovascular vessels, consisting primarily of fibrillar of beta amyloid peptide (β-AP), are characteristic of Alzheimer's disease (both in family and sporadic forms), and islet amyloid protein peptide (IAPP; amylin) It is characterized by the fibrils of pancreatic islet cell amyloid deposits associated with type II diabetes, and β2-microglobulin is a major component of amyloid deposits formed as a result of long-term dialysis treatment. More recently, prion-related diseases such as Creutzfeldt-Jakob disease are also recognized as amyloid diseases.
[4] Various forms of disease were classified into several classes based primarily on whether amyloidosis was associated with systemic primary disease. As such, certain diseases are considered primary amyloidosis, in which there is no evidence of existing or coexisting diseases. In general, primary amyloidosis disease is characterized by the presence of "amyloid light chain (AL)" protein fibrils, and thus the homology of the N-terminal regions of the AL fibrils to various fragments of immunoglobulin light chains (kappa or lambda). Is named for.
[5] Secondary or "reactive" amyloidosis is characterized by the deposition of type AA fibrils derived from serum amyloid A protein (ApoSSA). This form of amyloidosis is characterized by underlying chronic inflammatory or infectious disease states (eg, rheumatoid arthritis, osteomyelitis, tuberculosis, leprosy).
[6] Genetic family amyloidosis may be associated with neuropathy, kidney or cardiovascular deposits of the ATTR transthyretin type. Other hereditary family amyloidosis may include other syndromes and may include different amyloid components (eg, familial Mediterranean fever characterized by AA fibrils). Other forms of amyloidosis include topical forms characterized by pathological, sometimes tumor-like, deposits that occur in independent organs. Other amyloidosis is associated with aging and is generally characterized by plaque formation in the heart or brain. Amyloid deposits associated with prolonged hemodialysis are also common. Various forms of amyloid disease are summarized in Table 1 (Tan, SY and Pepys, Histopathology 25: 403-414, 1994; Harrison's Handbook of Internal Medicine, 13th edition, Isselbacher, KJ et al., McGraw-Hill, San Francisco, 1995). .
[7] Classification of Amyloid Disease Amyloid Protein / Peptide Protein precursor Protein variants Clinical symptoms AA Serum Amyloid A Protein (ApoSSA) Reactive (secondary) amyloidosis: familial Mediterranean fever; familial amyloid nephropathy associated with tapping and deafness (Mule-Wells syndrome) AA Serum Amyloid A Protein (ApoSSA) Reactive Systemic Amyloidosis Associated with Systemic Inflammatory Disease AL Monoclonal immunoglobulin light chains (kappa, lambda) Ak, A (e.g. AkIII) Idiopathic (primary) amyloidosis: related to myeloma or macroglobulinemia; Systemic amyloidosis associated with immune cell disorders; Monoclonal gamma globulin disorder; Latent disorders; Local nodular amyloidosis associated with chronic inflammatory diseases AH IgG (1 (γ1)) Aγ1 Heavy chain amyloidosis associated with several immune cell disorders ATTR Transthyretin (TTR) 30 or more known point mutations Familial amyloid polyneuropathy (eg Met 30, Portuguese) ATTR Transthyretin (TTR) Yes, Met 111 Familial Amyloid Myocardial Disorder (Danish Inn) ATTR Transthyretin (TTR) Wild TTR or Ile 122 Systemic aging amyloidosis AapoAI ApoAI Arg 26 Familial Amyloid Multiple Neuropathy Agel Gelsolin Asn 187 Familial Amyloidosis (Finnish) Acys Cystatin C Gln 68 Genetic cerebral hemorrhage related to amyloidosis (Icelandic) Aβ Amyloid β protein precursors (eg β-APP ₆₉₅ ) Variants: Gln 618 Alzheimer's disease Down syndrome Genetic cerebral hemorrhage Amyloidosis (Dutch) Sporadic brain amyloid angioplasty inclusion myositis AB ₂ M Beta ₂ microglobulin Chronic Hemodialysis Related Acal (Pro) calcitonin (Pro) calcitonin Thyroid medulla carcinoma AANF Atrial Sodium Excretory Factor Focal aging amyloidosis: isolated atrial amyloid
[8] AβSVEP ^a AB ₂ M β-amyloid precursor protein-beta ₂ microglobulin Cerebral sperm vesicle prostate keratin Primary topical skin amyloid (spots, papules) PrP Prion Precursor Protein (33-35 kDa Cytoplasmic Form) Scrapie protein 27-30 kDa Sporadic Creutzfeldt-Jakob Disease Cure (deliverable spongy brain disorder, Prion disease) AIAPP Islet Amyloid Polypeptide (IAPP) Island of Langerhans, Diabetes Type II, Insulin Species Peptide hormones, fragments Yes, precalcinin Exocrine amyloidosis associated with APUD tumor^a sperm vesicle exocrine protein
[9] Often, the fibrils that form the bulk of amyloid deposits are derived from one or more major precursor proteins or peptides and are usually associated with sulfated glycosaminoglycans. In addition, amyloid deposits may include a small number of proteins and peptides of various kinds, along with other components such as prothioglycans, gangliosides, and other sugars, which are described in more detail in the following paragraphs.
[10] There is currently no treatment for amyloid that is specific for amyloid disease. In the case of a primary disease or related disease state, therapies aim to reduce the production of amyloidogenic proteins by treating the original disease. An example of this is the treatment of tuberculosis with antibiotics to reduce mycobacterial accumulation, thereby reducing inflammation and the associated SSA protein. In the case of AL amyloid due to multiple myeloma, chemotherapy is applied to patients to reduce plasma cells and to reduce myeloma immunoglobulin levels. As this level decreases, AL amyloid can be removed. Co-owned US patent applications USSN09 / 201,430, filed November 30, 1998, and USSN 09 / 322,289, filed May 28, 1999, show that amyloid plaque accumulation associated with Alzheimer's disease is β-amyloid peptide (Aβ) and It has been shown that it can be significantly reduced (and prevented) by administering an agent that produces or confers an immune response against fragments thereof. The present invention has found that the induction of an immune response to various amyloid plaque components is effective in treating a wide range of amyloid diseases.
[11] Summary of the Invention
[12] The present invention relates to pharmaceutical compositions and methods for treating a number of amyloid diseases. According to one aspect, the present invention includes a pharmaceutical composition containing as an active ingredient an agent effective for inducing an immune response against an amyloid component in a patient. Such compositions generally also include excipients and, in preferred embodiments, may include adjuvants. In another preferred embodiment, the adjuvant comprises, for example, aluminum hydroxide, aluminum phosphate, MPL ^™ , QS-21 (Stimulon ^™ ) or incomplete Freund's adjuvant. According to a related embodiment, such pharmaceutical compositions may comprise a number of agents effective for inducing an immune response against one or more amyloid components in a patient.
[13] In related embodiments, the agent is effective to generate an immune response against the fibrillar peptide or protein amyloid component. Preferably such fibril peptides or proteins are derived from fibrillar precursor proteins known to be associated with certain forms of amyloid disease, as described herein. Examples of such precursor proteins include serum amyloid A protein (ApoSSA), immunoglobulin light chain, immunoglobulin heavy chain, ApoAI, transthyretin, lysozyme, fibrogen α chain, gelsolin, cystatin C, amyloid β protein precursor (β- APP), beta ₂ microglobulin, prion precursor protein (PrP), atrial sodium excretory factor, keratin, islet amyloid polypeptide, peptide hormones and synuclein, but are not limited to these. Such precursors also include mutant proteins, protein fragments and proteolytic peptides of such precursors. In a preferred embodiment, this agent is effective for inducing an immune response against neoepitopes formed by fibrillar proteins or peptides against fibrillar precursor proteins. That is, as described in more detail herein, many fibrillar forming peptides or proteins are fragments of such precursor proteins as described above. When such fragments are formed, for example by proteolytic cleavage, epitopes that are not present on the precursor can be revealed, which makes them immunologically unavailable to the immune system when the fragments are part of the precursor protein. Formulations for such epitopes may be the preferred therapeutic agents because they are less likely to induce an autoimmune response in a patient.
[14] According to a related embodiment, the pharmaceutical composition of the present invention comprises, but is not limited to, amyloid components such as AA, AL, ATTR, AApoA1, Alys, Agel, Acys, Aβ, AB ₂ M, AScr, Acal, AIAPP and Synuclin-NAC fragments Or a component selected from the group of fibril peptides or proteins. The full name and composition of such peptides is described herein. Such peptides can be prepared according to methods well known in the art, as described herein.
[15] According to another related embodiment, the agents included in such pharmaceutical compositions may also comprise certain sulfate prothioglycans. In a related embodiment, the prothioglycans are heparin sulfate glycosaminoglycans, preferably perrecan, dermatan sulfate, chondroitin-4-sulfate or pentosan polysulfate.
[16] According to another related embodiment, the invention includes a method for preventing or treating a disease characterized by amyloid deposition in a mammalian subject. According to this aspect of the invention, the subject is administered an amount of an agent effective to produce an immune response against the amyloid component that is characteristic of the amyloid disease in which the subject suffers. Essentially, such methods include administering a pharmaceutical composition comprising an immunogenic amyloid component specific for this disease, as described above. This method is also characterized by its effectiveness in inducing an immunogenic response in a subject. According to a preferred embodiment, the method is effective to generate an immunogenic response characterized by a serum titer of at least 1: 1000 for the amyloid component to which the immunogenic agent acts. In another preferred embodiment, the serum titer is at least 1: 5000 for the fibril component. According to a related embodiment, the immune response is characterized by an immunogenic amount of serum corresponding to about four times higher than the immunoreactive level of serum measured in pretreated control serum samples. This latter feature is particularly relevant when serum immunoreactivity is measured by ELISA techniques, but may be applied to any relative or absolute measurement of serum immunoreactivity. According to a preferred embodiment, the immunoreactivity is measured by diluting the serum to about 1: 100.
[17] According to another related embodiment, the present invention includes a method of measuring the prognosis of a patient who is being treated for an amyloid disease. In this case, the amount of immunoreactivity of the patient's serum to the amyloid component characteristic of the selected disease is measured, and the amount of the immunoreactivity of the patient's serum is more than four times greater than the baseline level of control serum immunoreactivity is improved for certain amyloid diseases. It shows the prognosis of the state. According to a preferred embodiment, the amount of immunoreactivity to the 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, to the amyloid component.
[18] According to another related embodiment, the invention also includes so-called "passive immunization" methods and pharmaceutical compositions for the prevention or treatment of amyloid diseases. According to this aspect of the invention, the patient is administered an effective amount of an antibody that specifically binds to the selected amyloid component, preferably the fibrillar component present in the amyloid deposit characteristic of the disease to be treated. In general, such antibodies are screened for their ability to specifically bind to various proteins, peptides, components are described for pharmaceutical compositions, and methods are described in the preceding paragraphs of this paragraph. According to related embodiments, such methods and compositions may comprise a combination of antibodies that bind to two or more amyloid fibril components. Generally, the pharmaceutical composition is administered to provide an immunoreactive amount of serum for the target amyloid component at least about four times higher than the serum level of the immunoreactivity for the component measured in the control serum sample. The antibody may also be administered with a carrier as described herein. In general, according to this aspect of the invention, such antibodies may be administered intraperitoneally, orally, nasal, subcutaneously, intramuscularly, topically or intravenously (or may be formulated for administration), but a pharmaceutically effective route (ie , Or a route effective for producing a therapeutic level indicated as described herein).
[19] According to a related embodiment, 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 to produce antibody chains in a pharmaceutically effective amount. Such polynucleotides produce heavy and light chains in a patient by encoding the heavy and chain of the antibody.
[20] According to a preferred embodiment, the aforementioned immunization method comprises administering multiple doses of an agent comprising an antibody, such as according to methods known in the art or upon the needs of a patient when evaluating an immune response. Inoculate over 6 months by booster at intervals of 6 weeks after vaccination. Optionally, such methods may also include using "delayed release" formulations as known in the art.
[21] Various objects and features of the present invention will be more fully understood from the following detailed description when read in conjunction with the accompanying drawings.
[2] The present invention relates to compositions and methods for treating amyloid related conditions in humans and other mammalian vertebrates.
[22] 1 shows antibody titers after injection of Aβ1-42 into transgenic mice.
[23] 2 shows amyloid accumulation in the hippocampus. The area ratio of hippocampal regions occupied by amyloid plaques, defined as reactivity with Aβ-specific monoclonal antibody 3D6, was determined by computerized quantitative image analysis of immune-reacted brain regions. Values for each mouse are shown, divided into treatment groups. The horizontal line for each group represents the median of the distribution.
[24] 3 shows neuritis dystrophy in the hippocampus. The area ratio of hippocampal sites occupied by dystrophic neuritis, defined as reactivity with human APP specific monoclonal 8E5, was determined by computerized quantitative image analysis of immune-reacted brain areas. Values for each mouse are shown for AN1792 treated group and PBS treated control group. The horizontal line for each group represents the median of the distribution.
[25] 4 shows astrocytosis in the plaque cortex. The area ratio of the cortical area occupied by GFAP-positive astrocytic cells was measured by computer quantitative image analysis of the immune-reacted brain area. The values for each mouse are shown broken down into treatment groups and the middle group values are indicated by horizontal lines.
[26] 5 shows geometric mean antibody titers for Aβ1-42 (“AN1792”) after immunization with eight dose ranges of AN1792 containing 0.14, 0.4, 1.2, 3.7, 11, 33, 100 or 300 μg. .
[27] 6 shows the kinetics of antibody response to AN1792 immunization. Titers are expressed as geometric mean values of the values for six animals in each group.
[28] 7 shows quantitative image analysis of cortical amyloid accumulation in PBS and AN1792 treated mice.
[29] 8 shows quantitative image analysis of neuritis plaques in PBS and AN1792 treated mice.
[30] 9 shows quantitative image analysis of the ratio of inverted cortex to astrocytic cells in PBS and AN1792 treated mice.
[31] 10 shows lymphocyte proliferation assays in spleen cells from AN1792 treated (top panel) or PBS treated (bottom panel).
[32] 11 shows the total Aβ concentration in the cortex. Scatter plot of each Aβ profile in mice immunized with Aβ or APP derivative mixed with Freund's adjuvant.
[33] FIG. 12 shows that amyloid accumulation in the cortex was determined by quantitative image analysis of the brain regions immunized against mice immunized with Aβ peptide conjugates Aβ1-5, Aβ1-12 and Aβ13-28, and full-length Aβ was determined by Aβ1-42 (" AN1792 ") and Aβ1-40 (" AN1528 ") and PBS treated controls.
[34] Figure 13 shows the geometric mean titers of Αβ-specific antibodies against a group of mice immunized with Αβ or APP derivatives mixed with Freund's adjuvant.
[35] FIG. 14 shows the geometric mean titers of Αβ-specific antibodies against a group of guinea pigs immunized with AN1792 or palmitoylated derivatives thereof mixed with various adjuvants.
[36] Figure 15 (A-E) shows the concentration of Aβ in the cortex of 12 month-old PDAPP mice treated with AN1792 or AN1528 using various adjuvants.
[37] 16 shows average titers of mice treated with polyclonal antibodies against Aβ.
[38] 17 shows the average titers of mice treated with monoclonal antibody 10D5 against Aβ.
[39] 18 shows average titers of mice treated with monoclonal antibody 2F12 against Aβ.
[40] A. Definition of terms
[41] Unless otherwise stated, all terms used herein have the same meaning as used by those skilled in the art. Those skilled in the art will be familiar with the definitions, technical terms and standard methods known in the field of biochemistry and molecular biology, particularly in Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual (2nd Edition), Cold Spring Harbor Press, Plainview, New York, and Ausubel. , FM (1998), Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY. The present invention is not limited to the specific methods, protocols and reagents described, as these changes can yield the same results.
[42] The term "adjuvant" means a compound that enhances an 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 immune responses by several mechanisms, including lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages.
[43] "Amyloid disease" or "amyloidosis" means any of a number of diseases that have an accumulation or formation of amyloid plaques as part of their symptoms or pathology.
[44] An "amyloid plaque" is an extracellular deposit consisting primarily of protein fibrils. In general, fibrils consist primarily of proteins or peptides. However, this plaque may also include additional components that are peptide or non-peptide molecules as described herein.
[45] An "amyloid component" is any molecular portion present in amyloid plaques that include the antigenic portion of such molecules. Amyloid components include, but are not limited to, proteins, peptides, prothioglycans and carbohydrates. By "specific amyloid component" is meant a molecular moiety found primarily or exclusively in the desired amyloid plaque.
[46] "Formulation" is a chemical molecule of synthetic or biological origin. In the context of the present invention, agents are generally molecules that can be used in pharmaceutical compositions.
[47] An “anti-amyloid agent” is an agent capable of generating an immune response against amyloid plaque components in vertebrate subjects when administered by active or passive immunization techniques.
[48] As used herein interchangeably, "polynucleotide" and "nucleic acid" are polymeric molecules having a backbone that supports a base capable of hydrogen bonding to a conventional polynucleotide, wherein the polymer backbone is a polymeric molecule and a conventional poly Bases are presented in a manner that allows for sequence specific hydrogen bonding between nucleotides (eg, single stranded DNA). Such bases are generally inosine, adenosine, guanosine, cytosine, uracil and thymidine. Polymeric molecules include two and one strand of RNA and DNA, and skeletal variants thereof, such as methylphosphonate bonds.
[49] As used herein, the term "polypeptide" refers to a compound consisting of a single chain of amino acid residues linked by peptide bonds. The term "protein" may have the same meaning as the term "polypeptide" or may mean a complex of two or more polypeptides.
[50] The term "peptide" refers to a compound consisting of amino acid residues linked by peptide bonds. Generally, a peptide consists of up to 100 amino acids and a polypeptide or protein consists of 100 or more amino acids. The term "protein fragment" as used herein may be interpreted to mean one peptide.
[51] By "fibrillar peptide" or "fibrillar protein" is meant a monomer or aggregate form of a protein or peptide that forms the fibrils present in amyloid plaques. Examples of such peptides and proteins are provided herein.
[52] "Pharmaceutical composition" means a chemical or biological composition suitable for administration to a mammalian subject. Such compositions are specially formulated for administration via one or more of several routes, including but not limited to oral, parenteral, intravenous, intraarterial, subcutaneous, nasal, sublingual, spinal, intraventricular, and the like. Can be.
[53] "Pharmaceutical excipient" or "pharmaceutically acceptable excipient" refers to a carrier, typically liquid, formulated with an active therapeutic agent. Excipients may generally provide chemical and / or biological stability, release properties, etc., but do not provide any pharmacological activity to the formulation. Examples of such agents can be found, for example, in Grennaro, A., Remington's Pharmaceutical Sciences (19th edition), 1995.
[54] A "glycoprotein" is a protein in which one or more carbohydrate chains (oligosaccharides) are covalently linked.
[55] A "prothioglycan" is a glycoprotein wherein at least one of the carbohydrate chains is a glycosaminoglycan, wherein glycosaminoglycans are repeats in which one member of the pair is typically a sugar acid (uronic acid) and the other is an amino sugar It is a long chain linear polymer of disaccharides.
[56] An "immunological" or "immune" or "immunogenic" response produces a humoral (antibody mediated) and / or cellular (antigen specific T cell or its secretion product) response to an antigen in vertebrate individuals. I mean. Such a response may be an active response induced by the administration of an immunogen or a passive response induced by the administration of an antibody or first antigen-stimulated T cells. The cellular immune response is characterized by the presentation of polypeptide epitopes associated 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 include the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils or other components of innate immunity. The presence of cell mediated immunological responses can be measured by standard proliferation assays (CD4 ⁺ T cells) or CTL (cytotoxic T lymphocytes) assays known in the art. The relative contribution of humoral and cellular responses to the prophylactic or therapeutic effect of the immunogen separates the immunoglobulin (IgG) and T cell fractions from the immunized genetic syngeneic animal separately and protects or treats in the second subject. It can be identified by measuring its potency.
[57] The term “immunogenic agent” or “immunogen” or “antigen” is a molecule capable of inducing an immune response to itself when administered to a patient with or without an adjuvant. Examples of such molecules include amyloid fibril peptides or fragments thereof conjugated to carrier proteins such as keyhole limpet hemocyanin, Cd3 or tetanus toxin.
[58] "Epitope" or "antigenic determinant" refers to the portion of an antigen that binds to the antigen binding region of an antibody.
[59] "Aβ", "Aβ peptide" and "amyloid β" peptides have the same meaning and refer to one or more peptide compositions consisting of about 38-43 amino acids derived from beta amyloid precursor protein (β-APP) as described herein. do. "Aβxx" means amyloid β-peptide 1-xx, where xx is a number representing the number of amino acids in the peptide, eg, Aβ42 is the same as Aβ1-42, which is also referred to herein as "AN1792" and Aβ40 is Aβ1 Same as -40, also referred to herein as "AN1578".
[60] By non-aggregated or monomeric Aβ is meant a soluble monomeric peptide unit of Aβ. One way to produce monomeric Aβ is to dissolve the lyophilized peptide in neat DMSO using ultrasound. The resulting solution is then centrifuged to remove any insoluble particulates. Aggregated Aβ is a mixture of oligomers in which monomeric units are held together by non-covalent bonds.
[61] The term "extracted polynucleotide" means a polynucleotide that does not complex with a colloidal material. Naked polynucleotides are often cloned in plasmid vectors.
[62] The term “patient” includes persons and other mammals undergoing prophylactic or therapeutic treatment.
[63] The phrases "significantly different from", "statistically significant", "significantly higher (lower)", and similar phrases indicate a comparison between data or other measurements, where two compared individuals or groups The differences between are apparently or reasonably different, or statistically significant, to the trained observer (the phrase includes the phrase "statistically", indicates some indication of a statistical test, such as a p-value, or When analyzed, the data exhibit statistical differences by standard statistical tests known in the art).
[64] Compositions or methods that “comprise” one or more of the listed elements may include other elements that are not specifically listed. For example, compositions comprising fibrillar component peptides include both isolated peptides and peptides as components of large polypeptide sequences. As a further example, a composition comprising components A and B also encompasses a composition consisting of A, B and C.
[65] B. Amyloid Disease
[66] Introduction and etiology
[67] Amyloid disease or amyloidosis includes a number of disease states with a wide variety of external symptoms. These diseases include abnormal extracellular deposits of protein fibrils, commonly known as "amyloid deposits" or "amyloid plaques," which are usually 10-100 μm in diameter and located in specific organ or tissue areas. . Such plaques consist primarily of naturally occurring soluble proteins or peptides. Such insoluble deposits generally consist of lateral aggregates of fibrils with a diameter of about 10-15 nm. Amyloid fibrils form characteristic appley green birefringence in polarization when dyed with Congo red pigment. The disease is classified based on the major fibrillar component, which forms plaque deposits, as described below.
[68] Peptides or proteins that form plaque deposits are often produced from larger precursor proteins. More specifically, the etiology of amyloid fibril deposits is generally associated with proteolytic cleavage into fragments of "abnormal" precursor proteins. These fragments generally aggregate into anti-parallel β-pleat sheets, but some non-degraded precursor protein forms are found in familial amyloid polyneuropathy (variant transthyretin fibrils) and dialysis related amyloidosis (β ₂ microglobulin fibrils). It has been reported to aggregate and form fibrils (Tan et al., 1994, homologous).
[69] 2. Clinical Syndrome
[70] This section describes the main types of amyloidosis, including the plaque fibril composition characteristic of amyloidosis. The overall finding of the present invention is that amyloid diseases can be treated by administering agents that act to stimulate the immune response to the component (s) of the various disease specific amyloid deposits. As described in more detail in paragraph C below, these components are preferably components of the fibrils forming the plaque. This paragraph is intended to illustrate the main form of amyloidosis and does not limit the invention.
[71] a. AA (reactive) amyloidosis
[72] In general, AA amyloidosis is a manifestation of several diseases that cause a sustained acute phase reaction. These diseases include chronic inflammatory diseases, chronic local or systemic microbial infections, and malignant tumors.
[73] AA fibrils are generally present in HDL particles and by proteolytic cleavage of serum amyloid A protein (apoSSA), a circulating apolipoprotein synthesized in hepatocytes in response to cytokines such as IL-1, IL-6 and TNF Consisting of 8000 Dalton fragments (AA peptide or protein) formed. Deposition can occur throughout the body and preferentially occurs in parenchymal organs. The spleen is usually the site of deposition, and the kidneys can also be affected. Deposition often occurs in the heart and gastrointestinal tract.
[74] AA amyloid diseases include, but are not limited to, inflammatory diseases such as rheumatoid arthritis, childhood chronic arthritis, ankylosing spondylitis, psoriasis, arthrosis psoriasis, lighter syndrome, adult Still's disease, Beset's syndrome, Crohn's disease. AA deposits are also produced as a result of chronic microbial infections such as leprosy, tuberculosis, bronchiectasis, pressure sores, chronic pyelonephritis, osteomyelitis and whiffles. Certain malignancies can also cause AA fibrillar amyloid deposition. These include Hodgkin's lymphoma, renal carcinoma, intestine, lung and urogenital carcinoma, basal cell carcinoma and hairy cell leukemia.
[75] b. AL amyloidosis
[76] AL amyloid deposition is generally associated with almost all disorders of the B lymphocyte lineage, from malignant tumors of plasma cells (multiple myeloma) to benign monoclonal gamma globulin disorders. Occasionally, the presence of amyloid deposits can be a major indicator of primary disease.
[77] The fibrils of AL amyloid deposits consist of monoclonal immunoglobulin light chains or fragments thereof. More specifically, these fragments are derived from the N-terminal region of the light chain (kappa or lambda) and comprise all or part of their variable (V _L ) domains. Deposits generally occur in mesenchymal tissue and cause peripheral and autologous neuropathy, carpal tunnel syndrome, blistering, limited cardiomyopathy, macroarthropathy, immune disorders, myeloma, as well as latent disorders. However, it should be noted that almost all tissues, especially visceral organs such as the heart, can be involved.
[78] c. Genetic systemic amyloidosis
[79] Many forms of genetic systemic amyloidosis exist. Although they are relatively rare, the adult onset of symptoms and their genetic patterns (typically autosomal superiority) allow these diseases to persist in the general population. In general, this syndrome is due to point mutations occurring in precursor proteins that induce the production of variant amyloidogenic peptides or proteins. Table 2 summarizes the fibril composition of representative forms of this disease.
[80] Hereditary amyloidosis ^a Fibril Peptides / Proteins Genetic variants Clinical syndrome Transthyretin and Fragments (ATTRs) Met30, many others Familial Amyloid Polyneuropathy (FAP) (primarily peripheral nerves) Transthyretin and Fragments (ATTRs) Thr45, Ala60, Ser84, Met111, Ile122 Heart Relevance Prevails Without Neuropathy N-terminal fragment of Apolipoprotein A1 (apoAI) Arg26 Familial Amyloid Polyneuropathy (FAP) (primarily peripheral nerves)
[81] N-terminal fragment of Apolipoprotein A1 (AapoAI) Arg26, Arg50, Arg60, Other Ostertag-type, non-neuropathy (mainly related to visceral) Lysozyme (Alys) Thr56, His67 Ostertag-type, non-neuropathy (mainly related to visceral) Fibrinogen α Chain Fragment Leu554, Val526 Ostertag type, non-neuropathy (mainly related to visceral) Gelsolin Fragment (Agel) Asn187, Tyr187 Cranial Neuropathy with Lattice Corneal Dystrophy Cystatin C fragment Glu68 Genetic cerebral hemorrhage (brain amyloid angiopathy)-Icelandic type Β-amyloid protein (Aβ) from amyloid precursor protein (APP) Glu693 Hereditary cerebral hemorrhage (brain amyloid angiopathy)-Danish type Β-amyloid protein (Aβ) from amyloid precursor protein (APP) Ile717, Phe717, Gly717 Familial Alzheimer's Disease Β-amyloid protein (Aβ) from amyloid precursor protein (APP) Asn670, Leu671 Familial Dementia-Presumed Alzheimer's Disease Prion protein (PrP) from PrP precursor protein 51-91 insert Leu102, Val167, Asn178, Lys200 Familial Creutzfeldt-Jakob disease; Gerstmann-Stroisler-Schainker syndrome (genetic spongy brain disorder, prion disease) AA from serum amyloid A protein (ApoSSA) Familial Mediterranean fever, kidney predominance (autosomal recessive) AA from serum amyloid A protein (ApoSSA) Muckle-Wells Syndrome, Nephropathy, Deafness, Hives, Amputee Pain Unknown Cardiomyopathy with persistent atrial arrest Unknown Skin deposits (vesicles, papules, pustules) data obtained from ^a Tan & Pepys (1994) (homologous)
[82] The data presented in Table 2 is for illustration only and is not intended to limit the scope of the invention. For example, over 40 independent point mutations in the transthyretin gene have been described, all of which cause clinically similar forms of familial amyloid multineuropathy.
[83] Transthyretin (TTR) is a 14 kD protein, sometimes called prialbumin. This protein is produced in the liver and choroid networks and serves to transport thyroid hormones and vitamin A. More than 50 protein variants, each characterized by a single amino acid change, contribute to various forms of amyloid polyneuropathy. For example, the substitution of leucine with proline at position 55 caused particularly progressive neuropathy, and the replacement of leucine with methionine at position 11 caused severe heart disease in Danish patients. It has been found that amyloid deposits isolated from cardiac tissue of patients with systemic amyloidosis consist of a heterogeneous mixture of TTR and fragments thereof (collectively called ATTR), and their full length sequences have already been identified. ATTR fibril components 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. Comm. 125: 622-628, 1984; Pras, M. et al., PNAS 80: 539-42, 1983.
[84] Patients with point mutations (eg, Gly → Arg26; Trp → Arg50; Leu → Arg60) in molecular apolipoprotein AI may be in the form of amyloidosis (“Ostertag type”) characterized by the deposition of protein apolipoprotein AI (AApoAI) or fragments thereof. ). These patients have low HDL levels and are accompanied by peripheral neuropathy or renal dysfunction.
[85] Mutations that occur in the alpha chain of the enzyme lysozyme (eg, Ile → Thr56 or Asp → His57) underlie another form of Ostertag type non-neuropathic genetic amyloid reported in English families. In this disease, fibrils of mutant lysozyme protein (Alys) are deposited and patients generally exhibit renal function impairment. Unlike most fibrillar forming proteins described herein, these proteins are typically present in their entire form (nonfragmented form) (Benson, MD et al., CIBA Fdn. Symp. 199: 104-131, 1996].
[86] β-amyloid peptide (Aβ) is a 39-43 amino acid peptide produced by the proteolysis of a large protein known as beta amyloid precursor protein (βAPP). Mutations in βAPP cause Alzheimer's disease, Down's syndrome and / or senile dementia, which are characterized by brain deposits of plaques composed of Aβ fibrils and other components (described in further detail below). Known mutations in APP associated with Alzheimer's disease occur near the cleavage site of β or γ secretory enzymes or within Aβ. For example, the 717 position is near the γ-secretase cleavage site of APP in processing with Aβ, and the 670/671 position is close to the site of β-secretase cleavage. Mutations in any of these residues may cause Alzheimer's disease, possibly by increasing the amount of the 42/43 amino acid form of Aβ produced in APP. The structures and sequences of Aβ peptides of various lengths are well known in the art. Such peptides can be prepared according to methods known in the art (e.g., Glenner and Wong, Biochem Biophys. Res. Comm. 129: 885-890, 1984); and Glenner and Wong, Biochem Biophys. Res. Comm. 122: 1131-1135, 1984). In addition, various forms of peptides are commercially available.
[87] Synuclein is a synaptic related protein similar to apoprotein and is abundant in neuronal cytoplasm and presynaptic ends. Peptide fragments derived from α-synuclein called NAC are also components of amyloid plaques of Alzheimer's disease (Clayton et al., 1998). This component also serves as a target for the immune-based therapies of the present invention, as described below.
[88] Gelsolin is a calcium binding protein that binds to fragments of actin filaments. Mutations in position 187 of this protein (eg, Asp → Asn; Asp → Tyr) result in the genetic systemic amyloidosis typically observed in Finnish patients as well as Dutch or Japanese patients. In these patients, fibrils formed from geloline fragments (Agel), which typically consist of amino acids 173-243 (68 kDa carboxy-terminal fragments), are deposited on the vessel wall and basal membrane, resulting in corneal dystrophy and cranial neuropathy. And it progresses to peripheral neuropathy, dystrophy skin changes and deposition in other organs (Kangas, H., et al., Human Mol. Genet. 5 (9): 1237-1243 (1996)).
[89] Other mutated proteins such as mutant alpha chains (AfibA) and mutant cystatin C (Acys) of fibrinogen also form fibrils and cause characteristic genetic diseases. AfibA fibrillar deposits are characteristic of non-neuropathic genetic amyloid associated with kidney disease, and Acys deposits are characteristic of genetic brain 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, including non-mutant forms of cystatin C associated with beta proteins (Nagai, A. et al., Molec. Chem. Neuropathol. 33: 63-78,1998].
[90] Certain forms of free-in disease are currently considered to be hereditary, with less than 15% of the incidence occurring, and were previously thought to be predominantly infectious (Baldwin et al., Research Advances in Alzheimer's Disease and Related Disorders, John Willy and Suns, New York, 1995]. Patients with these prion diseases develop plaques that consist of abnormal isotypes of normal prion protein (PrP ^c ). The dominant mutant isoform, PrP ^sc (also called AScr), is resistant to protease degradation, insoluble after detergent extraction, deposited in secondary lysosomes, synthesized after translation, and has a high β-pleat sheet content. It is different from normal cellular proteins. Genetic associations have been established for five or more mutations that cause Creutzfeldt-Jakob disease (CJD), Gerstmann-Stroysler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI) (Baldwin). Methods for extracting, determining the sequence, and preparing such fibrous peptides from scrapie fibrils are known in the art (see, eg, Beekes, M. et al., J. Gen. Virol. 76: 2567). -76, 1995).
[91] For example, one form of GSS is linked to a PrP mutation in codon 102, while the telelenephalic GSS is isolated with a mutation in codon 117. Mutations occurring in codons 198 and 217 cause the neuritis plaques that characterize Alzheimer's disease to cause GSS forms that contain PrP instead of Aβ peptides. Certain forms of familial CJD appear to be related to mutations occurring in codons 200 and 210, and mutations occurring in codons 129 and 178 are observed in both familial CJDs and FFIs (Baldwin, supra).
[92] d. Senile systemic amyloidosis
[93] Systemic or pathological amyloid deposition increases with age. For example, fibrils of wild-type transthyretin (TTR) are commonly found in elderly heart tissue. It may be clinically silent and asymptomatic or may cause heart attacks. Asymptomatic fibrillar local deposition can also occur in the brain (Aβ), the progesterone of the prostate (Aβ ₂ microglobulin), joints and seminal vesicles.
[94] e. Brain amyloidosis
[95] Local deposition of amyloid is common in the brain, particularly in the brain of the elderly. The most frequent type of amyloid in the brain consists mainly of Aβ peptide fibrils, leading to impaired or sporadic (non-genetic) Alzheimer's disease. In fact, the incidence of sporadic Alzheimer's disease has been shown to be much higher than that of genetic disease. The fibril peptides that form these plaques are very similar to those described above for genotype Alzheimer's disease (AD).
[96] f. Dialysis-Related Amyloidosis
[97] Plaques consisting of β ₂ microglobulin (Aβ ₂ M) fibrils are common in patients undergoing prolonged hemodialysis or peritoneal dialysis. β ₂ microglobulin is a 11.8 kDa polypeptide and is the light chain of class I MHC antigens and is present on all nucleated cells. Under normal circumstances, it is released continuously from the cell membrane and is generally filtered by the kidneys. Eliminating dysfunction, such as in the case of renal impairment, causes it to deposit in the kidneys and other areas (mainly collagen-rich tissue of the joints). Unlike other fibrillar proteins, Aβ ₂ M molecules are generally present in unfragmented form in fibrils (Benson, supra).
[98] g. Hormone-derived amyloidosis
[99] Amyloid deposition may occur in the endocrine organs, especially in older people. Hormone-secreting tumors may also include hormone-derived amyloid plaques, the fibrils of which are calcitonin (medium carcinoma of the thyroid gland), islet amyloid polypeptide (amylin; occurs in most of type II diabetics) and atrial sodium secretion peptides (Such as isolated atrial amyloidosis). The sequence and structure of these proteins are well known in the art.
[100] h. Other amyloidosis
[101] There are a variety of other forms of amyloid diseases that generally appear as local amyloid deposits. In general, such diseases may be the result of local production and / or lack of degradation of specific fibrillar precursors, or may be a result of predeposition of certain tissues (eg, joints) for fibrillar deposition. Examples of such idiopathic depositions are nodular AL amyloid, cutaneous amyloid, endocrine amyloid and tumor associated amyloid.
[102] C. Pharmaceutical Composition
[103] The present invention has found that a composition capable of inducing or providing an immune response against certain components of amyloid plaques is effective in treating or preventing the development of amyloid disease. In particular, according to the present invention described herein, when the immunostimulatory amount of an anti-amyloid agent or the corresponding anti-amyloid immune agent is administered to a patient, it prevents the progression of the accumulation of amyloid plaque in the patient and alleviates the symptoms, and It is possible to reduce the accumulation of amyloid plaques. This section describes representative anti-amyloid agents that produce active and passive immune responses of amyloid plaques and provides representative data showing the effect of treatment with such compositions on amyloid plaque accumulation.
[104] In general, the anti-amyloid preparations of the invention consist of a special plaque component, preferably a fibrillar forming component, which are generally characteristic proteins, peptides or fragments thereof, as described in the previous paragraphs and in the following paragraphs. Illustrate. More generally, therapeutic agents for use in the present invention produce or elicit an immune response against plaque, or more specifically its fibrillar component. Thus, such agents include antibodies that specifically react with amyloid components, as well as analogues and mimics of the fibrillar component itself and its variants, components which induce and / or cross-react with antibodies to the component. Or T-cells, but are not limited to such. According to an important feature, the pharmaceutical composition is not selected from nonspecific ingredients-that is, components that are generally circulating or present throughout the body. For example, serum amyloid protein (SAP) is a circulating plasma glycoprotein that is produced in the liver and binds to the best known form of amyloid deposits. Therapeutic compositions are preferred for this component.
[105] Induction of an immune response can be active when administering an immunogen to induce antibodies or T-cells that are reactive with the component in the patient, or passive if the antibody itself binds to an amyloid component in the patient by administering the antibody. Can be. Representative agents that elicit or produce an immune response against amyloid plaques are described in the sections below.
[106] The pharmaceutical compositions of the present invention may comprise, in addition to the immunogenic agent (s), an effective amount of adjuvants and / or excipients. Pharmaceutically effective useful adjuvants and excipients are well known in the art and are described in more detail in the paragraphs below.
[107] 1. Immunostimulants (active immune responses)
[108] a. Anti-fiber composition
[109] One general class of preferred anti-amyloid preparations consists of preparations derived from amyloid fibrillar proteins. As noted above, an indication of amyloid disease is the intratracheal deposition of amyloid plaques, which consist primarily of fibrils, which consist of characteristic fibrillar proteins or peptides. According to the invention, such fibrillar protein or peptide components are useful agents for inducing an anti-amyloid immune response.
[110] Tables 1 and 2 summarize representative fibrillar forming proteins that characterize various amyloid diseases. According to this aspect of the invention, administration of an immunostimulatory composition comprising an appropriate fibrillar protein or peptide, as well as analogs or fragments thereof, to a patient or susceptible person can treat or prevent amyloid disease.
[111] For example, Aβ, known as β-amyloid peptide or A4 peptide (US Pat. No. 4,666,829; Glenner & Wong, Biochem. Biophys. Res. Commun. 120, 1131 (1984)), is a peptide of 39-43 amino acids. This is a major component of plaques characteristic of Alzheimer's disease Aβ is produced by the treatment of the macroprotein APP by two enzymes called β and γ secretory enzymes (Hardy, TINS 20 , 154 (1997)). .
[112] Example 1 demonstrates the results of an experiment in support of the present invention, in which Aβ42 peptide was administered to heterozygous transgenic mice overexpressing a human APP mutated at position 717. Such mice, known as "PDAPP" mice, exhibit Alzheimer's-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 showed detectable Aβ plaque neuropathy in these brains as plaque deposition progressed over time from about 6 months of age. In the experiments described herein, aggregated Aβ 42 (AN1792) was administered to mice. Most of the treated mice (7/9) had no amyloid detected in the brain at 13 months of age, whereas severe brain amyloid accumulation was seen at this age in both control mice (saline injection or untreated) (FIG. 2). This difference was more pronounced than in the hippocampus (FIG. 3). Treated mice also showed significant serum titers against Aβ (all at 1: 1000 or higher, 8/9 at 1 / 10,000 or higher; FIG. 1, Table 3A). In general, saline treated mice showed less than 4-5 times the background antibody level against Aβ when diluted 1: 100 at all test timepoints and were therefore considered to have no significant response compared to controls (Table 3B). ). These experiments demonstrated that injection of special fibrils that form peptide Aβ provides protection against the deposition of Aβ amyloid plaques.
[113] Serum amyloid protein (SAP) is a circulating plasma glycoprotein that is produced in the liver and binds to all forms of amyloid fibrils, including those of brain amyloid plaques in Alzheimer's disease. As part of the preliminary experiment, a group of mice was injected with SAP, which produced significant serum titers for SAP (1: 1000-1: 30000) but did not show detectable serum titers for Aβ peptides. , Developed brain plaque neuropathy (FIG. 2).
[114] Another experiment detailed in Example II shows the dose dependence of the immunogenic effect of Aβ injection in mice treated between 5 and about 8 months of age. In these mice, the mean serum titer of the anti-Aβ peptide antibody increased with increasing inoculation frequency and inoculation dose, but serum titers measured after the fourth inoculation, 5 days after inoculation were more at a level of about 1: 10000. Decreased for high doses (1 to 300 μg) (FIG. 5).
[115] Another experiment supporting the present invention is described in Example III, where Aβ42 was treated in PDAPP model mice beginning after the time point when amyloid plaques were already present in the brain (about 11 months of age). In this experiment, animals were inoculated with Aβ 42 or saline and sacrificed for amyloid accumulation test at 15 or 18 months of age. As shown in FIG. 7, at 18 months of age, Aβ42 treated mice had significantly lower mean amyloid plaque accumulation rates (plaques) than control (plaque accumulation rate, 4.7%) or 12 months old untreated animals (0.28%) at 18 months of age with PBS. Accumulation rate, 0.01%), and plaque accumulation was measured by image analysis as detailed in Example XIII, Part 8. These experiments demonstrate that the treatment of the present invention is effective in reducing existing plaque accumulation and preventing the progression of plaque accumulation in patients.
[116] According to this aspect of the invention, the therapeutic agent is derived from a fibrillar peptide or protein comprising plaques characteristic of the desired disease. Optionally, such agents are antigenically sufficiently similar to components that elicit an immune response that also cross reacts with the fibril component. Tables 1 and 2 show examples of such fibril peptides and proteins, their compositions and sequences known in the art, which can be readily determined according to methods known in the art (of various fibril peptide components See the following references and references in paragraph B2 for specific teachings on extraction methods and / or compositions; examples of further fibrillar components are described below).
[117] Thus, according to the present invention, when diagnosing amyloid disease based on clinical and / or biopsy measurements, specialists can be sure of the fibril composition of the amyloid deposits and induce an immune response against fibrillar peptides or proteins. Formulations may be provided.
[118] For example, as described above, the therapeutic agent used to treat Alzheimer's disease or other amyloid disorders characterized by Aβ fibril deposition may be a naturally occurring form of Aβ peptides, especially humans ( Ie Aβ39, Aβ40, Aβ41, Aβ42 or Aβ43). The relationship between such peptide sequences and APP precursors is well known in the art (eg, Hardy et al., TINS 20, 155-158 (1997)). For example, Aβ42 has the sequence H2N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu -Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala-OH (SEQ ID NO: 1).
[119] A [beta] 41, A [beta] 40 and A [beta] 39 differ from A [beta] 42 due to missing Ala, Ala-Ile and Ala-Ile-Val, respectively, from the C-terminus of the peptide. Aβ43 differed from Aβ42 due to the presence of threonine residues at the C-terminus. According to this aspect of the invention, the therapeutic agent induces an immune response against all or part of the fibrillar component of the desired disease. For example, preferred Aβ immunogenic compositions are agents that induce antibodies specific for the free N-terminus of Aβ. Such compositions have the advantage that they are less likely to cause autoimmunity by not recognizing the precursor protein, β-APP.
[120] In another example, patients suffering from diseases characterized by the deposition of AA fibrils, such as chronic inflammatory diseases as described above, chronic local or systemic microbial infections, and malignant tumors, are known in the art of serum amyloid A protein (ApoSSA). Can be treated with AA peptide, which is an 8 kDa fragment. AA amyloid diseases include inflammatory diseases such as rheumatoid arthritis, childhood chronic arthritis, ankylosing spondylitis, psoriasis, arthrosis psoriasis, Reiter syndrome, adult Still's disease, Beset's syndrome, Crohn's disease; Chronic microbial infections such as leprosy, tuberculosis, bronchiectasis, pressure sores, chronic pyelonephritis, osteomyelitis, and whiffles, as well as malignant tumors such as Hodgkin's lymphoma, renal carcinoma, intestine, lung and genitourinary carcinoma, basal cell carcinoma and maternal There are, but are not limited to, cellular leukemia.
[121] AA peptide refers to one or more of the heterologous peptide groups derived from the N-terminus of precursor protein serum amyloid A (ApoSSA), starting at residues 1, 2 or 3 of the precursor protein and ending at any point between residues 58 and 84 AA fibrils are usually composed of residues 1-76 of ApoSSA. The exact structure and composition can be determined and suitable peptides can be synthesized according to methods well known in the art (Liepnieks, J.J. et al. Biochem. Biophys Acta 1270: 81-86,1995).
[122] For another example, an N-terminal region comprising all or a portion of the variable (V _L ) domains of an immunoglobulin light chain (kappa or lambda chain) generally comprises amyloid deposition in mesenchymal tissue, peripheral and autologous neuropathy Cartilage tunnel syndrome, heavy snow syndrome, limited cardiomyopathy, macroarthritis, immune disorders, myeloma, as well as latent disorders. The composition of the present invention preferably induces an immune response to a portion of the light chain, preferably a "neo epitope (an epitope formed as a result of fragmentation of the parent molecule)", thereby reducing possible autoimmune effects.
[123] Various genetic amyloid diseases can be treated with the treatment of the present invention. Such diseases are described in paragraph B.2. For example, familial amyloid polyneuropathy is the result of more than 50 mutant forms of transthyretin (TTR), a 14 kD protein produced in the liver, each of which is characterized by a single amino acid change. While many of these forms of disease can be distinguished based on specific pathology and / or demographic origin, therapeutic compositions may respond to one or more forms of TTR, such as a mixture of two or more forms of ATTR, including wild type TTR. It may be composed of an inducing agent to provide a therapeutic composition that is generally useful.
[124] AapoAI-containing amyloid deposits are observed in people with point mutations in the molecular apolipoprotein AI. Patients suffering from this type of disease generally exhibit peripheral neuropathy or kidney dysfunction. According to the present invention, the therapeutic composition consists of one or more of the various forms of ApoAI described herein and known in the art.
[125] Certain familial forms of Downzheimer's disease and Down's syndrome are the result of mutations in the beta amyloid precursor protein resulting in the deposition of plaques with fibrils consisting predominantly of β-amyloid peptide (Aβ). The use of Aβ peptides in the therapeutic compositions of the invention is described herein.
[126] As noted above, other agents for treating genetic forms of amyloidosis include gelsolin fragments for the treatment of genetic systemic amyloidosis, mutant lysozyme protein (Alys) for the treatment of genetic neuropathy, and non-neural neurons with apparent kidney disease. Fibrinogen mutant alpha chains (AfibA) for the symptomatic form of amyloidosis, and compositions for generating an immune response to mutant cystatin C (Acys) for the treatment of genetic cerebral hemorrhages reported in Icelandic people. In addition, some genetic forms of prion disease (eg, Creutzfeldt-Jakob disease (CJD), Gerstmann-Steuisler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI)) are mutant isotypes of prion proteins. It is characterized by PrP ^SC . Such proteins can be used in therapeutic compositions to treat and prevent the deposition of PrP plaques according to the present invention.
[127] As mentioned above, systemic or pathological amyloid deposition is also associated with aging. Another aspect of the invention is that such deposition can be prevented or treated by administering to a susceptible subject a composition consisting of one or more proteins associated with aging. Thus, plaques composed of ATTRs derived from wild type TTR are often observed in elderly heart tissue. Similarly, some older adults produce non-symptomatic fibril lesions of Αβ in the brain. As described in detail herein, Aβ peptide therapy is efficacious in such individuals. Since β ₂ microglobulin is a common component in the progesterone of the prostate, it is an additional candidate agent according to the present invention.
[128] For example, there are many other non-genetic forms of amyloid disease that are candidates for the treatment methods of the present invention. β ₂ microglobuli fibril plaques commonly occur in patients undergoing prolonged hemodialysis or peritoneal dialysis. According to the present invention such patients can be treated using a therapeutic composition for β ₂ microglobulin, more preferably its immunogenic epitope.
[129] Hormone-secreting tumors may also contain hormone-derived amyloid plaques, which composition is a general feature of certain endocrine organs. Thus, such fibrils may be composed of polypeptide hormones such as calcitonin (thyroid medulla carcinoma), islet amyloid peptide (occurring in most Type II diabetic patients), and atrial sodium excretion peptide (isolated atrial amyloidosis). Compositions for amyloid deposits formed in the aortic endothelium of atherosclerosis are also contemplated in the present invention. For example, Westermark et al. Describe 69 amino acid N-terminal fragments of apolipoprotein A that form such plaques (Westermark et al. Am. J. Path. 147: 1186-92, 1995); Therapeutic compositions of the present invention include such fragments, as well as immunological agents for the fragments themselves.
[130] The description thus far has focused on amyloid fibrillar components, which can be used as therapeutic agents to treat or prevent various forms of amyloid disease. This therapeutic agent may also be an active fragment or analog of a naturally occurring or mutant fibrillar peptide or protein comprising an epitope that induces a similar protective or therapeutic immune response when administered to a human. Immunogenic fragments have a sequence of at least 2, 3, 5, 6, 10 or 20 contiguous amino acids from natural peptides. Examples of Aβ peptide immunogenic fragments include 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 that delete one or more, sometimes five or ten, C-terminal amino acids present in naturally occurring forms of the fibrillar component are used in certain methods. For example, a fragment missing five amino acids from the C-terminal end of Aβ 43 comprises the first 38 amino acids from the N-terminal end of Aβ. Fragments of the N-terminal half of Aβ are preferred in some methods. Analogs include alleles, species and derived variants. Analogs are usually different from naturally occurring peptides at one or several positions, mainly by conservative substitutions. Analogs typically have at least 80% or 90% sequence identity with the native peptide. Certain analogs also include modifications of non-natural amino acids or N or C-terminal amino acids. Examples of non-natural amino acids include α, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline, γ-carboxyglutamate, γ-N, N, N-trimethyllysine, γ-N-acetyllysine , O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine.
[131] In general, those skilled in the art will screen for fragments and analogs designed according to this aspect of the invention for cross reactivity and / or prophylactic or therapeutic efficacy against naturally occurring fibril components in a transgenic animal model, as described below. You will know that you can. Such fragments or analogs can be used in the therapeutic compositions of the present invention as long as their immunoreactivity and animal model efficacy are approximately equal to or greater than the corresponding parameters measured for amyloid fibril components.
[132] Such peptides, proteins or fragments, analogs and other amyloidogenic peptides may be synthesized by solid phase peptide synthesis or recombinant expression according to standard methods well known in the art, or may be obtained from natural sources. Representative fibrillar compositions, fibrillar extraction methods, sequences of fibrillar peptides or protein components are provided by the many references in which particular fibrillar components provided herein are cited with description. In addition, other compositions, extraction methods, and sequencing methods are useful to those of skill in the art for preparing and using such compositions. Automated peptide synthesizers can be used to prepare such compositions and can be purchased from a variety of manufacturers, such as, for example, Appleride Biosystems, Foster City, CA, and methods for preparing synthetic peptides are known in the art. Recombinant expression is bacterial, such as E. coli. It can be carried out in coli, yeast, insect cells or mammalian cells. Alternatively, the protein can be produced using a cell-free in vitro translation system known in the art. Procedures for recombinant expression are described in Sambrook et al., Molecular Cloning: A Laboratory Manual (CSHP Press, New York 2nd Edition, 1989. In addition, certain forms of peptides and proteins, such as Aβ peptides, are described, for example, in the United States. American Peptides Company, Sunnyvale, California, Inc. and California Peptide Research, Inc., Napa, Calif., Are commonly available.
[133] The therapeutic agent may also consist of a long chain polypeptide comprising an active peptide fragment or analog with other amino acids. For example, the Αβ peptide may be present as an intact APP protein or a segment thereof, and the C-100 fragment continues from the N-terminus of Αβ and continues to the end of the APP. Such polypeptides can be screened for their prophylactic or therapeutic efficacy in animal models, as described below. Aβ peptides, analogs, active fragments or other polypeptides can be administered in bound form (ie, as amyloid peptide) or in isolated form. In addition, therapeutic agents may include multimeric or conjugated or carrier proteins of monomeric immunogenic agents, and / or may be added to other fibrillar components as described above to provide a broader range of anti-amyloid plaque activity.
[134] In further variations, immunogenic peptides, such as fragments of Αβ, may be represented by a virus or bacteria as part of an immunogenic composition. Nucleic acid encoding an immunogenic peptide is added to the episome or genome of a virus or bacterium. Optionally, nucleic acids are added such that the immunogenic peptide is expressed as a fusion protein with a bacterial dura protein or a viral outer surface protein or as a secreted protein so that the peptide appears. Viruses or bacteria used in these methods must be nonpathogenic or attenuated. Suitable viruses include adenoviruses, HSV, Venezuelan encephalitis virus and other alpha viruses, bullous stomatitis and other rhabdoviruses, scoliosis and chickenpox. Suitable bacteria include Salmonella and Shigella. Fusion of immunogenic peptides to HBsAg of HBV is particularly appropriate. Therapeutic agents also include peptides and other compounds that do not necessarily have high amino acid sequence similarity with Aβ, but nonetheless act as mimics of Aβ and elicit a similar immune response. For example, any peptides and proteins that form β-pleat sheets can be screened for suitability. Anti-idiotypic antibodies or other amyloidosis peptides against monoclonal antibodies against Αβ may be used. Such anti-Id antibodies mimic antigens and produce immune responses to them ( Essential Immunology , Roit Edit, Blackwell Scientific Publishing, Palo Alto, 6th Edition), p, 181. Agents other than Aβ peptides should elicit an immunogenic response to one or more of the preferred segments of Aβ described above (eg, 1-10, 1-7, 1-3, and 3-7). Such agents preferably induce an immunogenic response that is not directed against other segments of Aβ and specifically directed to one of these segments.
[135] In addition, random libraries of peptides or other compounds can be screened for suitability. Combinatorial libraries can be generated for various types of compounds that can be synthesized in a stepwise manner. Such compounds include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligo N-substituted glycines and oligocarbamates. Large combinatorial libraries of compounds are encoded in Affymax WO95 / 12608, Affymax WO93 / 06121, Columbia University WO94 / 08051, Pharmacopiia WO95 / 35503 and Scripps WO95 / 30642, which are incorporated herein by reference. It can be constructed by synthetic library (ESL) method. Peptide libraries can also be generated by phage display. See, for example, Devlin's WO91 / 18980.
[136] Combination libraries and other compounds were initially screened for compatibility by measuring their ability to bind to antibodies or lymphocytes (B or T) known to have specificity for Aβ or other amyloidosis peptides (eg, ATTR). For example, initial screening can be performed using any polyclonal serum or monoclonal antibody against Aβ or any corresponding amyloidogenic peptide. The compounds identified by this screening were then further analyzed for their 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 previously coated with fibril peptides, or standard ELISAs can be performed for reactive antibodies to Aβ. The compounds can then be tested for the prophylactic and therapeutic efficacy in transgenic animals with amyloidogenic disease predisposition as described below in the Examples. Examples of such animals include mice with a 717 variation of APP as described in References (Games et al., Homology) and US Pat. No. 5,612,486 to McConlogue et al., Hsiao et al. ( Science 274, 99 (1996)); Staufenbiel et al ., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997); Sturchler-Pierrat et al ., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997); Mice with a 670/671 Swedish variation of APP as described in Borchelt et al., Neuron 19, 939-945 (1997). The same screening approach can also be used for other potential agents, such as fragments of Aβ, analogs of Aβ, and longer peptides comprising Aβ.
[137] b. Other Plaque Ingredients
[138] It will be appreciated that the immune response to other amyloid plaque components is also effective in preventing, delaying or reducing plaque deposition in amyloid disease. Such components may be a minor component of the fibrils, or may be associated with fibrillar or fibrillar formation in plaques, provided that they are present throughout the body or that are relatively nonspecific to amyloid deposits. Generally less suitable for use as.
[139] Thus, another finding of the present invention is that agents that induce an immune response against specific plaque components are useful for treating or preventing the progression of amyloid disease. This section provides the basis for some examples of amyloid plaque related molecules. In accordance with the present invention, an immune response to the molecule is administered by administering any of these molecules, alone or in combination with the immunogenic therapeutic composition to the fibrillar component or to any of the other non-fibrils forming components described below. Induction provides additional anti-amyloid therapies. Another part of the present invention is passive immunization based on these plaque components described herein.
[140] For example, synuclein is a protein that is structurally similar to the apolipoprotein found in the neuronal cytoplasm, especially at the end of warnas. There are three or more forms of these proteins, called α, β and γ synuclein. Recently, α and β synuclein have been found to be involved in the nucleation of amyloid deposition in certain amyloid diseases, especially Alzheimer's disease (Clayton, D.F. et al. TINS 21 (6): 249-255,1998). More specifically, fragments of the NAC domains of α and β synuclein (residues 61-95) were isolated from amyloid plaques in Alzheimer's patients, and in fact these fragments remained insoluble after solubilization with sodium dodecyl sulfate (SDS). About 10% of plaques (George, JM et al., Neurosci. News 1: 12-17, 1995). In addition, full-length αsynuclein and its NAC fragments have been reported to accelerate the aggregation of β-amyloid peptides into insoluble amyloids in vitro (Clayton, homolog).
[141] Another component associated with amyloid plaques includes nonpeptide components. For example, Perrecan and Perrecan-derived glycosaminoglycans are giant heparin sulfate prothioglycans present in Aβ-containing amyloid plaques, including amyloid plaques associated with diabetes, and Alzheimer's disease and other CNS and systemic amyloidosis . These compounds have been shown to enhance Aβ fibril formation. Both the core protein and glycosaminoglycan chain of Perrecan have been shown to be involved in binding to Aβ. Other glycosaminoglycans, specifically dermatan sulfate, chondroitin-4-sulfate and pentosan polysulfate, are frequently observed in various types of amyloid plaques and have been shown to enhance fibril formation. Dextran sulfate also has this property. This strengthening is significantly reduced when the molecule is desulfated. Immunogenic therapeutics for the sulfate form of glycosaminoglycans, including the particular glycosaminoglycans themselves, form another aspect of the invention as primary or secondary therapeutics. Preparation of such molecules, as well as suitable therapeutic compositions comprising such molecules, are within the skill of the art.
[142] 2. Agents That Induce a Passive Immune Response
[143] Therapeutic agents of the present invention also include immune agents such as antibodies that specifically bind to fibril peptides or other components of amyloid plaques. Such antibodies may be monoclonal or polyclonal and have binding specificities that match the type of amyloid disease being treated. Therapeutic compositions and therapies may comprise antibodies against a single binding domain or epitope on a particular fibrillar or non-fibrils component of the plaque, or antibodies against two or more epitopes on the same component or antibodies to epitopes on several components of the plaque It may include.
[144] For example, polyclonal anti-Aβ42 or monoclonal anti-Aβ42 prepared for specific epitopes of Αβ peptides in PDAPP mice of 8 1/2 to 10 1/2 months of age in experiments conducted to support the present invention. Antibodies, or saline, were intraperitoneally injected (as described in Example XI). In this experiment, circulating antibody concentrations were monitored and boosted when necessary to ensure that the antibody had a circulating antibody concentration of at least 1: 1000 for the particular antigen produced. Compared with the control group, a decrease in total Aβ levels was observed in the cortex, hippocampus, and cerebellum of the brain regions of antibody-treated mice, with the largest decrease in mice treated with polyclonal antibodies.
[145] In another experiment conducted to support the present invention, a prospective ex vivo assay (Example XIV) was used to test the removal of antibodies to synuclein fragments called NAC. Synuclin is known to be an amyloid plaque related protein. Antibodies to NAC were contacted with brain tissue samples including amyloid plaques and microglia. Rabbit serum was used as a control. Subsequent monitoring showed a significant reduction in the number and size of plaques indicating the clearance activity of the antibody.
[146] From these data, it can be seen that amyloid plaque accumulation associated with Alzheimer's disease and other amyloid diseases can be significantly reduced by administration of an immune agent to epitopes of Αβ peptides or NAC fragments of synuclein. Various antibodies can also be used in such compositions. Antibodies that do not bind to the isolated form and specifically bind to aggregated Aβ are suitable for use in the present invention, and antibodies that bind specifically to the isolated form without binding to the aggregated form are also suitable. Antibodies that bind to both aggregated and isolated forms are also suitable. Some of these antibodies do not bind to naturally occurring long chains of Aβ (ie Aβ42 and Aβ43) but bind to naturally short chains of Aβ (ie Aβ39, 40 or 41). Some antibodies do not bind to short chains, but to long chains. Some antibodies do not bind to the full length amyloid precursor protein, but rather to Aβ. Some antibodies bind to Aβ with a binding affinity of at least about 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ or 10 ¹⁰ M ⁻¹ .
[147] Polyclonal sera typically comprise a mixed population of antibodies that bind to several epitopes along the length of Aβ. Monoclonal antibodies bind specific epitopes within Aβ, which can be conformational or nonstereoscopic epitopes. Some monoclonal antibodies bind to epitopes within residues 1-28 of Αβ (including the first N terminal residues of native Αβ indicated by 1). Other monoclonal antibodies bind to epitopes having residues 1-10 of Aβ. There are also monoclonal antibodies that bind to epitopes having residues 1-16 of Aβ. Other monoclonal antibodies bind to epitopes having residues 1-25 of Aβ. Some monoclonal antibodies bind to epitopes having 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 the antibodies can be tested using the transgenic animal model procedures described in the Examples.
[148] More generally, in accordance with the teachings of the present invention, those skilled in the art will use antibodies to the compositions described herein and other amyloid components to identify fibrous proteins such as those described in paragraph 2 or peptides characteristic of other amyloid diseases. Antibodies can be designed, manufactured and tested.
[149] a. General features of immunoglobulins
[150] Basic antibody structural units are known to contain tetramers of subunits. Each tetramer consists 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 comprises variable regions of at least about 100-110 amino acids that are primarily involved in antigen recognition. The carboxy terminal portion of each chain defines constant regions that are primarily involved in effector function.
[151] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, and the isotypes of the antibodies are defined as IgG, IgM, IgA, IgD and IgE, respectively. In the light and heavy chains, the variable and constant regions are linked by the "J" sites of about 12 or more amino acids, and the heavy chain also includes the "D" sites of about 10 or more amino acids. Fundamental Immunology, edited by Paul W., Raven Press, New York, 1989, Ch. 7, which is incorporated herein by reference.
[152] The variable region of each light / heavy chain pair forms the antibody binding site. Thus, an intact antibody has two binding sites. The two binding sites are identical except for bifunctional or bispecific antibodies. The chains represent the same general structure of relative conserved backbone sites (FRs) linked by three hypervariable sites called complementarity determining sites or CDRs. CDRs from each pair of two chains are aligned by framework regions, which are capable of binding specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chains comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The arrangement of amino acids into each domain is described by Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health Bethesda, MD, 1987 and 1991, or Chothia & Lesk, J. Mol. Biol. 196: 901-917 (1987) Chothia et al ., Nature 342: 878-883 (1989).
[153] b. Generation of non-human antibodies
[154] The production of non-human monoclonal antibodies such as rats, guinea pigs, primates, rabbits or rats can be performed, for example, by immunizing animals with plaque components such as Aβ or other fibrillar components. Longer polypeptides comprising A [beta] or immunogenic fragments of A [beta] or anti-idiotypic antibodies directed against antibodies to A [beta] can also be used. Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988), incorporated herein by reference. Such immunogens can be obtained from natural sources by peptide synthesis or by recombinant expression. Optionally, the immunogen can be administered to fusion or complex with the carrier protein as described below. Optionally, the immunogen can be administered with an adjuvant. Several auxiliaries can be used as described below. Use of complete Freund's adjuvant followed by incomplete adjuvant is preferred for immunization of laboratory animals. Rabbit or guinea pigs are used to generate polyclonal antibodies. Mice are commonly used to generate monoclonal antibodies. Antibodies are screened for specific binding to the immunogen. Optionally, antibodies are screened for binding to specific sites of the immunogen. For example, if the immunogen is an Aβ peptide, this screening can be done by measuring the binding of the antibody to the collection of deletion mutations of the Aβ peptide and determining whether the deletion mutation binds to the antibody. Binding can be assessed by, for example, Western blot or ELISA. Minimal fragments showing specific binding to the antibody form the epitope of the antibody. Alternatively, epitope specificity can be determined by competition assays in which test and reference antibodies compete for binding to a component. When the test and reference antibodies are complete, they bind to the same epitope (s) that are close enough that binding of one antibody interferes with binding of the other.
[155] c. Chimeric and Humanized Antibodies
[156] Chimeric and humanized antibodies have the same or similar binding specificities and affinity as mouse or other non-human antibodies that provide starting materials for the construction of chimeric or humanized antibodies. Chimeric antibodies were constructed by processing light and heavy chains by genetic engineering techniques, typically from immunoglobulin gene segments belonging to different species. For example, variable (V) segments of genes from mouse monoclonal antibodies can be bound to human constant (C) segments such as IgG1 and IgG4. Common chimeric antibodies are hybrid proteins consisting of a V or antigen binding domain from a mouse antibody or a C or effector domain from a human antibody.
[157] Humanized antibodies have substantially variable region backbone residues from human antibodies (called receptor antibodies) and complementarity (called donor immunoglobulins) that substantially determines sites from mouse antibodies. Queen et al . , Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989) and WO90 / 07861, US Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101 and Winter, US 5,225,539 (all incorporated herein by reference)] . If the constant site (s) is present, it is also derived from substantially or almost human immunoglobulins. Human variable domains generally appear to have high sequence identity with the murine variable region domains from which CDRs are derived. Heavy and light chain variable region backbone residues can be derived from the same or different human antibody sequences. The human antibody sequence can be the sequence of a natural human antibody or can be the matched sequence of several human antibodies. Carter et al., WO 92/22653. Certain amino acids from human variable region framework residues are selected for substitution based on possible effects on the CDR conformation and / or binding to the antigen. Investigation of such possible effects is made by investigating the characterization of amino acids at specific positions or by modeling experimental observation of the effect of substitution or mutation of specific amino acids.
[158] For example, if an amino acid differs between a murine variable region backbone residue and a variable region backbone residue of the selected person, the amino acid
[159] (1) form a non-covalent bond directly to the antigen,
[160] (2) neighboring the CDR sites,
[161] (3) interact with the CDR site (ie within about 6 A of the CDR site), or
[162] (4) When participating at the VL-VH interface, it must be substituted by an equivalent backbone amino acid from the mouse antibody.
[163] Others for substitution include receptor human backbone amino acids that are abnormal for human immunoglobulins at that position. Such amino acids may be substituted with amino acids from the equivalent positions of the mouse donor antibody or from the equivalent positions of the more common human immunoglobulin. Other examples of substitutions include receptor human skeletal amino acids that are abnormal for human immunoglobulins at that location. Humanized immunoglobulin variable region backbones typically exhibit at least 85% sequence identity to a human variable region backbone sequence or to a match of such sequence.
[164] d. Human antibodies
[165] Human antibodies against Αβ are provided by various techniques described below. Some human antibodies are selected by competitive binding experiments or selected to have the same epitope specificity as certain mouse antibodies, such as one of the mouse monoclonals described in Example XI. Human antibodies can also be screened for specific epitope specificities by using only segments of Aβ as an immunogen and / or by screening antibodies for collection of deletion mutations of Aβ.
[166] (1) trioma methodology
[167] Basic approaches and exemplary cell fusion partners SPAZ-4 for use in this approach are described in Oestberg et al., Hybridoma 2: 361-367 (1983); Oestberg, US Pat. No. 4,634,664 and Engleman et al., US Pat. No. 4,634,666; All of which are incorporated herein by reference. The antibody producing cell line obtained by this method is called trioma because it is derived from three cells, ie two humans and one mouse. Initially, mouse myeloma strains are fused with human B-lymph cells to obtain non-antibody producing xenogeneic hybrid cells, such as the SPAZ-4 cell line described in Oestberg, homology. Triomas have been found to produce more stable antibodies than conventional hybridomas produced from human cells.
[168] Immunized B-lymphocytes are obtained from the blood, spleen, lymph nodes or bone marrow of human donors. If an antibody against a specific antigen or epitope is desired, it is preferred to use the antigen or epitope thereof for immunization. Immunization can be performed in vivo or in vitro. For in vivo immunization, B cells are typically isolated from humans immunized with Aβ, fragments thereof, or large polypeptides containing Aβ or fragments, or anti-specific antibodies to antibodies against Aβ. In some methods, B cells are isolated from the same patient who is ultimately going to administer the antibody treatment. For in vitro immunization, B-lymphocytes are typically exposed to antigen for 7-14 days in medium such as RPMI-1640 (Engleman, homolog) supplemented with 10% human plasma.
[169] Immunized B-lymphocytes are fused to xenogeneic hybrid cells such as SPAZ-4 by known methods. For example, these cells are treated with 40-50% polyethylene glycol at a molecular weight of 1000-4000, about 37 ° C., for about 5-10 minutes. The cells are separated from the fusion mixture and propagated in a medium having selectivity for a given hybrid (eg HAT or AH). Clonal secreting antibodies with the essential binding specificities are identified by analyzing trioma culture media for their ability to bind Aβ or fragments thereof. Trioma producing human antibodies with certain specificities are subcloned by restriction dilution techniques and grown in vitro in culture medium. The resulting trioma cell line is tested for the ability to bind Aβ or a fragment thereof.
[170] Although trioma is genetically stable, it does not produce antibodies at high concentrations. Expression can also be increased by cloning the antibody gene from trioma into one or more expression vectors and by modifying the vector to standard mammalian, bacterial or yeast cell lines according to methods well known in the art.
[171] (2) transgenic non-human mammals
[172] In addition, human antibodies against Αβ may be generated from non-human transgenic mammals having mutations encoding at least one segment of human immunoglobulin positions. In general, endogenous immunoglobulin sites in such transgenic mammals are inactive. The segment of human immunoglobulin position preferably comprises the unaligned sequences of the heavy and light chain components. Inactivation of endogenous immunoglobulin genes and introduction of exogenous immunoglobulin genes can be by targeted homologous recombination or by YAC chromosome introduction. Transgenic mammals generated from this method can operatively align immunoglobulin component sequences and express repeats of various isotype antibodies encoded by human immunoglobulin genes without expressing endogenous immunoglobulin genes. have. The production and properties of mammals having these characteristics are described in Lonberg et al., WO93 / 12227 (1993), US Pat. Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991), all of which are incorporated herein by reference. Cited) in detail. Transgenic mice are particularly suitable. Anti-Aβ antibodies are obtained by immunizing a transgenic non-human mammal with Aβ or a fragment thereof as described in kucherlapati, homology. Monoclonal antibodies are produced, for example, by fusion of B-cells from mammals into an appropriate myeloma cell line using conventional Kohler-Milstein techniques. In addition, human polyclonal antibodies can be provided in the form of serum from a person immunized with an immunizing agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using Aβ or other amyloid peptides as affinity agents.
[173] (3) phage display method
[174] Additional methods for obtaining human anti-Aβ antibodies are intended to screen DNA libraries from human B cells by the general protocol outlined in Huse et al., Science 246: 1275-1281 (1989). As detailed above in the trioma technique, such B cells can be obtained from humans immunized with longer polypeptides or anti-idiotype antibodies comprising Aβ, fragment, Aβ or fragment. Optionally such B cells are obtained from a patient who finally wants to receive antibody treatment. Antibodies bound to epitopes of the desired amyloid component such as Aβ or fragments thereof are selected. The sequences encoding these antibodies (or binding fragments) are cloned and amplified. The protocol described in Huse's literature is more effective in combination with phage display techniques. Dower et al., WO91 / 17271 and McCafferty et al./01047, US Pat. Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743 and 5,565,332, all of which are incorporated herein by reference. . In this method, a phage library is generated in which the component displays various antibodies on its outer surface. Antibodies are generally displayed as Fv or Fab fragments. Phage display antibodies with certain specificities are selected by affinity enrichment for Aβ peptides or fragments thereof.
[175] In a variation of the phage display method, human antibodies can be generated having the binding specificity of the selected murine antibody. Winter, WO 92/20791. In this method, the light or heavy chain variable region of the selected murine antibody is used as starting material. For example, when light chain variable regions are used as starting materials, a phage library is constructed that displays the same light chain variable regions (ie, mouse starting materials) and various heavy chain variable regions with the same components. The heavy chain variable region is obtained from a library of aligned human heavy chain variable regions. Phage showing strong specificity binding to the desired component (ie, at least 10 ⁸ M ⁻¹ , preferably at least 10 ⁹ M ⁻¹ ) are selected. Human heavy chain variable regions from these phages serve as starting material for constructing additional phage libraries. In such libraries, each phage displays the same heavy chain variable region (ie, the region identified from the first display library) and different light chain variable regions. Light chain variable regions are obtained from a library of aligned human variable light chain regions. Again, phages showing strong specific binding to amyloid peptide components are selected. Such phage fully display the variable region of the human anti-amyloid peptide antibody. Such antibodies generally have the same or similar epitope specificity as the murine starting material.
[176] e. Invariant site selection
[177] The heavy and light chain variable regions of the chimeric, humanized or human antibody can be bound to at least a portion of the human constant region. The choice of constant regions is determined in part by whether antibody dependent complement and / or cell mediated toxicity is desired. For example, isotype IgG1 and IgG3 have complementary activities, while isotype IgG2 and IgG4 do not have this activity. The choice of isotypes also affects the passage of antibodies into the brain. The light chain constant region can be lambda or kappa. Antibodies are tetramers comprising two light chains and two heavy chains, as separate heavy chains, light chains, as Fab, Fab 'F (ab') 2 and Fv, with a single chain in which the heavy and light chain variable domains are bound through a spacer It can be expressed as an antibody.
[178] f. Expression of Recombinant Antibodies
[179] Chimeric, humanized and human antibodies are commonly produced by recombinant expression. Recombinant polynucleotide constructs typically include expression control sequences that are operably linked to the coding sequence of the antibody chain, including natural binding or heterologous promoter sites. Expression control sequences are preferably eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector is incorporated into the appropriate host, the host is maintained under conditions suitable for high expression of the nucleotide sequence, collection and purification of the antibody to be cross reacted.
[180] These expression vectors are typically replicable in the host organism as episomes or as an endogenous part of the host chromosomal DNA. In general, expression vectors can detect these cells transformed with a given DNA sequence, including selection markers such as ampicillin resistance or hygromycin resistance.
[181] 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 are preferred yeast hosts, and suitable vectors include, as necessary, expression control sequences, origins of replication, termination sequences, and the like. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include alcohol dehydrogenase, isocytochrome C and enzymes involved in the use of maltose and galactose.
[182] Mammalian cells are preferred hosts that express nucleotide segments or fragments thereof that encode immunoglobulins. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Many suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, such as CHO cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. These expression vectors for cells include expression control sequences such as origins of replication, promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), necessary processing information sites such as ribosomal binding sites, RNA splice sites, Polyadenylation sites and transcription termination sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus and the like. Co et al., J. Immunol. See 148: 1149 (1992).
[183] Alternatively, the sequence encoding the antibody can be integrated into a transgene for introduction into the genome of the transgenic animal and then expressed in the milk of the transgenic animal (eg US Pat. No. 5,741,957, US). See method described in patent 5,304,489 and US Pat. No. 5,849,992, incorporated herein by reference). Suitable transgenes include light and / or heavy chain coding sequences operably linked with promoters and enhancers derived from mammary gland specific genes such as casein or beta galactoglobulin.
[184] The vector containing the DNA segment can be delivered into the host cell by a known method depending on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics or viral based transfection can be used for other host cells. 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 generation of transgenic animals, the transgene can be microinjected into fertilized oocytes, or integrated into the genome of embryonic hepatocytes, the nucleus of which is delivered to the nucleated oocytes.
[185] Once expressed, antibodies 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-Verlag, NY, 1982)).
[186] 4. Other Treatments
[187] Therapeutic agents for use in the present invention also include T-cells that bind to plaque components such as Aβ peptides. For example, by expressing human MHC class I and human β-2 microglobulin genes from insect cell lines, T-cells can be activated against Aβ peptides, resulting in the formation of empty complexes on the cell surface that will bind to Aβ peptides. Can be. T-cells contacted with the cell line are specifically activated for the peptide. See US Pat. No. 5,314,813 for this. Insect cell lines expressing MHC class II antigens can similarly be used to activate CD4 T cells.
[188] 5. Carrier Protein
[189] Some agents for inducing an immune response include a suitable epitope that induces an immune response to amyloid deposition, but is too small to show immunogenicity. Under these circumstances, peptide immunogens can be linked to appropriate carriers to aid in induction of immune responses. Suitable carriers include toxoids from serum albumin, keyhole limpet hemocyanin, immunoglobulin molecule, tyroglobulin, egg albumin, tetanus toxoid or other pathogenic bacteria (e.g. diphtheria, E. coli, cholera or H. pylori), or Directed toxin derivatives; Other carriers include T-cell epitopes that bind to a plurality of MHC alleles, such as at least 75% of all human MHC alleles. Such carriers are often known in the art as "universal T-cell epitopes." Examples of universal T-cell epitopes are as follows.
[190] Influenza erythrocyte aggregates: (SEQ ID NO 1)
[191] PADRE (common residues in bold): (SEQ ID NO: 2)
[192] Malaria CS: T3 epitope EKKIAKMEKASSVFNV (SEQ ID NO: 3)
[193] Hepatitis B surface antigen: (SEQ ID NO 4)
[194] Heat shock protein 65: (SEQ ID NO: 5)
[195] Bacille Calmette-Guerin (BCG): (SEQ ID NO: 6)
[196] Tetanus Toxoid: (SEQ ID NO 7)
[197] Tetanus Toxoid: (SEQ ID NO 8)
[198] HIV gp120 T1: (SEQ ID NO: 9)
[199] Other carriers that stimulate or enhance the immune response include cytokines such as IL-1, IL-1 α and β peptides, IL-2, γINF, IL-10 and GM-CSF, and chemotin such as MIP1α and β and RANTES. It includes. Immunogenic agents can be linked to peptides that increase transport through tissue, as disclosed in WO 97/17613 and WO97 / 17614 to O'Mahony et al.
[200] Immunogenic agents may be linked to the carrier by chemical crosslinking. Techniques for linking immunogens to carriers include N-succinimidyl-3- (2-pyridyl-thio) propionate (SPDP) and succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate ( Formation of disulfide bonds (SMCC) (if the peptide lacks sulfhydryl groups, cysteine residues can be added to provide this). These reagents themselves form disulfide bonds between peptide cysteine residues on one protein and amide bonds through ε-amino or other free amino groups on other amino acids on lysine. Various disulfide / amide forming reagents are described in Immun. 62, 185 (1982). Other bifunctional coupling agents form thioethers rather than disulfide bonds. Many of these thio-ether-forming agents are commercially available, examples of which include 6-maleimidocaproic acid, 2-bromoacetic acid and 2-iodoacetic acid, 4- (N-maleimido-methyl) cyclohexane-1 -Reactive esters of carboxylic acids. A carboxyl group can be activated by combining these with succinimide, 1-hydroxyl 2-nitro-4-sulfonic acid, and a sodium salt.
[201] The immunogenic peptide can be expressed as a fusion protein with a carrier (ie, a heterologous peptide). The immunogenic peptide may bind to the carrier at the amino terminus, at the carboxy terminus, or at both termini. If desired, several repeats of an immunogenic peptide may be present in the fusion protein. If desired, immunogenic peptides may bind to multiple copies of the heterologous peptide, eg, at both ends of the N and C of the peptide. Some carrier peptides act to induce a helper T-cell response to the carrier peptide. Induced helper T-cells induce a B-cell response against an immunogenic peptide that in turn is bound to a carrier peptide.
[202] Some formulations of the present invention comprise a fusion protein wherein the N-terminal fragment of Aβ is linked at the C-terminus to a carrier peptide. In such formulations, the N-terminal residue of the fragment of Aβ constitutes the N-terminal residue of the fusion protein. Thus, such fusion proteins are effective in inducing antibodies that bind the N-terminal residues of Aβ to epitopes that require free form. Some formulations of the invention comprise multiple repeats of the N-terminal segment of Aβ linked to one or more copies of the carrier peptide at the C-terminus. N-terminal fragments of Aβ integrated into such fusion proteins often start at Aβ1-3 and end at 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 Aβ in series. For example, the fusion protein may comprise Aβ1-7, Aβ1-3, heterologous peptides in sequence.
[203] In some fusion proteins, the N-terminal segment of Aβ is fused to a heterologous carrier peptide at the N-terminus. As in C-terminal fusion, the same kind of N-terminal segment of Aβ can be used. Some fusion proteins include peptides of heterostructures linked to the N-terminus of the N-terminal segment of Aβ, which are connected in series to one or more additional N-terminal segments of Aβ.
[204] Some examples of fusion proteins suitable for use in the present invention are set forth below. Some of these fusion proteins include segments of Aβ linked to tetanus toxoid epitopes as disclosed in US Pat. No. 5,196,512, EP 378,881 and EP 427,347. Some fusion proteins comprise segments of Aβ linked to a carrier peptide as disclosed in US Pat. No. 5,736,142. Some heterologous peptides are universal T-cell epitopes. In some methods, the formulation for administration is simply a single fusion protein having an Aβ segment linked to a segment of heterostructure in a linear structure. In some methods, the agent is a multimer of fusion proteins represented by the formula 2 ^× where x is an integer from 1 to 5. It is preferable that x is 1, 2 or 3, and it is most preferable that it is 2. When x is 2, such multimers carry four fusion proteins linked to the desired structure referred to as MAP4 (see US 5,229,490). Epitopes of Αβ are underlined.
[205] The MAP4 structure is shown below. Branched structures are prepared by initiating peptide synthesis at both the branched amine and N-terminus of lysine. Depending on the number of times lysine has been incorporated into the sequence and branched, the resulting structure provides a plurality of N-terminus. In this example, four identical N-terminus were generated in the branched lysine containing core. Such multiplicity significantly increases the responsiveness of cognate B cells.
[206]
[207] AN90549 (Aβ1-7 / Tetanus Toxoid 830-844 with MAP4 structure):
[208] DAEFRHD QYIKANSKFIGITEL (SEQ ID NO: 10)
[209] AN90550 (Aβ1-7 / Tetanus Toxoid 947-967 with MAP4 structure):
[210] DAEFRHD FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 11)
[211] AN90542 (Linear Aβ1-7 / Tetus Toxoid 830-844 + 947-967)
[212] DAEFRHD QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 12)
[213] AN90576 (Aβ3-9 / Tetanus Toxoid 830-844 with MAP4 Structure)
[214] EFRHDSG QYIKANSKFIGITEL (SEQ ID NO: 13)
[215] Peptides disclosed in US Pat. No. 5,736,142, all of which are linear structures
[216] AN90562 (Aβ1-7 / peptide) AKXVAAWTLKAAA DAEFRHD (SEQ ID NO: 14)
[217] AN90543 (Aβ1-7 x 3 / peptide): DAEFRHDDAEFRHDDAEFRHD AKXVAAWTLKAAA (SEQ ID NO: 15)
[218] Another example of a fusion protein (Aβ immunogenic epitopes are shown in bold) is as follows.
[219] AKXVAAWTLKAAA -DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO: 16)
[220] DAEFRHD -AKXVAAWTLKAAA (SEQ ID NO: 17)
[221] DAEFRHD -ISQAVHAAHAEINEAGR (SEQ ID NO: 18)
[222] FRHDSGY -ISQAVHAAHAEINEAGR (SEQ ID NO: 19)
[223] EFRHDSG -ISQAVHAAHAEINEAGR (SEQ ID NO: 20)
[224] PKYVKQNTLKLAT- DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO: 21)
[225] DAEFRHD -PKYVKQNTLKLAT- DAEFRHD (SEQ ID NO: 22)
[226] DAEFRHD-DAEFRHD-DAEFRHD -PKYVKQNTLKLAT (SEQ ID NO: 23)
[227] DAEFRHD-DAEFRHD- PKYVKQNTLKLAT (SEQ ID NO: 24)
[228] DAEFRHD- PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE- DAEFRHD-DAEFRHD-DAEFRHD-DAEFRHD- QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE
[229] DAEFRHD -QYIKANSKFIGITEL C FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 26)
[230] DAEFRHD -QYIKANSKFIGITEL C FNNFTVSFWLRVPKVSASHLE- DAEFRHD (SEQ ID NO: 27)
[231] DAEFRHD -QYIKANSKFIGITEL (SEQ ID NO: 28) on 2 branch resins
[232]
[233] EQVTNVGGA ISQAVHAAHAEINEAGR (synuclein fusion protein of MAP-4 structure, SEQ ID NO: 29)
[234] The same or similar carrier proteins and linking methods can be used to form immunogens used in the formation of antibodies against Aβ for use in passive immunization. For example, Aβ or fragment linked to a carrier can be administered to a laboratory animal in the preparation of a monoclonal antibody against Aβ.
[235] 6. Nucleic Acids Encoding Therapeutics
[236] Immune responses to amyloid deposition can be induced by administering nucleic acids, or antibodies, and components thereof that encode selected peptide immunogens. Such nucleic acid may be DNA or RNA. Nucleic acid segments encoding immunogens are typically linked to regulatory sequences such as promoters and enhancers that express DNA segments in the patient's intended target cells. For expression in blood cells preferred for induction of an immune response, promoter and enhancer components derived from the light or heavy chain immunoglobulin genes or CMV major immediate primitive promoters and enhancers are suitable for direct expression. Linked regulatory components and coding sequences are often cloned into the vector. For administration of double stranded antibodies, the two chains can be cloned in the same or separate vectors.
[237] Many viral vector systems, such as retroviral systems (see, eg, Lawrie and Tumin, Cur. Opin. Genet. Development. 3, 102-109 (1993)); Adenovirus 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 Fox family, including vaccinia virus and avian fox virus, Sindbis and SFV (Semliki). Alpha virus species, such as those derived from Forest Virus (see, eg, Dubensky et al., J. Virol. 70, 508-519 (1996)), Venezuelan equine encephalitis virus (see US Pat. No. 5,643,576) and rhabdovirus ) Such as bullous stomatitis virus (WO 96/34625) and papillomavirus (Ohe et al., Human Gene Therapy 6, 325-333 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma, Nucleic Acids. 24, 2630-2622 (1996)), and the like can be used.
[238] DNA encoding an immunogen, or a vector containing the same, can be packaged into liposomes. Suitable lipids and related analogs are disclosed in US Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and 5,283,185. DNA and vectors encoding immunogens can be adsorbed to or associated with particulate carriers, examples of which include polymethyl methacrylate polymers and polylactides and poly (lactide-co-glycolides). . See, eg, McGee et al., J. Micro Encap. (1996).
[239] Gene therapy vectors or naked DNA are typically individually administered by systemic administration (eg, intravenous, intraperitoneal, nasal, stomach, intradermal, intramuscular, subcutaneous or intracranial injection) or topical application (see, eg, US Pat. No. 5,399,346). It can be administered to a patient and delivered in vivo. Such vectors further include promoters such as bupivacine (US Pat. No. 5,593,970). Gene guns can also be used to administer DNA. See Xiao & Brandsma, supra. DNA encoding the immunogen is deposited on the surface of the fine metal beads. Microprojectiles are accelerated with shock waves or expanded helium gas to infiltrate tissue into the core of some cell layers. For example, the Accel ^™ gene delivery device manufactured by Agacetus Incorporated, Middleleton, Wisconsin is suitable. Alternatively, naked DNA can be passed through the skin into the bloodstream by simply spotting the DNA on the skin with chemical or mechanical stimuli (see WO 95/05853).
[240] In another variation, a vector encoding an immunogen is delivered from an individual patient to cells cultured in vitro (eg, lymphocytes, bone marrow inhalation, tissue biopsy) or universal donor hematopoietic stem cells in vitro and then generally integrated into the vector. After selection, the cells can be transplanted to the patient.
[241] 7. Antibody Screening for Removal Activity
[242] Example XIV provides a method for screening antibodies for activity that eliminates amyloid deposition. To screen for activity against amyloid deposition, tissue samples from brains of amyloidosis patients, such as brain tissues from Alzheimer's disease, or animal models with characteristic Alzheimer's disease pathologies, are obtained from phagocytic cells (eg bovine cells) carrying Fc receptors in in vitro medium. Glial cells) and the antibody to be tested. The phagocytes can be cell lines or primary cultures such as BV-2, C8-B4 or THP-1. These components can be coupled to microscope slides to facilitate microscopy monitoring or to perform several reactions simultaneously in the wells of a microtiterplate. In this format, separate small microscope slides can be installed in separate wells, or non-microscopic detection formats such as ELISA detection of Aβ can be used. Before proceeding with the reaction, it is advisable to make a series of measurements of amyloid deposition in the in vitro reaction mixture and one or more test values of the reaction, starting from the reference value. Antigens can be detected, for example, by staining with fluorescently labeled antibodies against Aβ or other components of amyloid plaques. The antibody used for staining may or may not be the same as the antibody under test for clearance activity. The reduction shown compared to baseline in the course of the amyloid deposition reaction suggests that the antibody under test retains elimination activity. Such antibodies are likely to be useful for preventing or treating Alzheimer's and other amyloidogenic diseases. As mentioned above, experiments conducted in support of the present invention showed that antibodies to NAC fragments of synuclein were effective in removing amyloid plaques characteristic of Alzheimer's disease using this assay.
[243] D. Treatable Patients
[244] Treatable patients include individuals who are at risk of disease but do not exhibit symptoms and patients who are currently presenting symptoms. In the case of Alzheimer's disease, if a man and a woman live long enough, there is a risk that anyone will eventually develop Alzheimer's disease. Thus, the methods of the present invention can be performed prophylactically in a general population without having to assess the risk of the subject patient. The methods of the invention are particularly useful for individuals who carry the genetic risk of any of known Alzheimer's disease or other genetic amyloid disease. Such individuals include those with relatives who have experienced the disease and those whose risk has been determined by analysis of genetic or biochemical markers. Genetic markers of risk for Alzheimer's disease include mutations in the APP gene, in particular mutations at positions 717 and positions 670 and 671, called Hardy and Swedish mutations, respectively (Hardy, TINS, supra). Other risk markers include the presenilin gene, PS1 and PS2 and mutations in ApoE, which are a family history of AD, hypercholesterolemia or atherosclerosis. Individuals currently suffering from Alzheimer's disease can be identified from the presence of characteristic dementia and the aforementioned risk factors. In addition, several diagnostic tests are available to identify individuals with AD. These include measuring CSF tau and Aβ42 levels. Elevated tau and decreased levels of Aβ 42 indicate the presence of AD. Individuals suffering from Alzheimer's disease can be diagnosed on the ADRDA criteria as discussed in the Examples section.
[245] In asymptomatic patients, treatment may commence at any age (eg, 10, 20, 30). Generally, however, when a patient reaches 40, 50, 60 or 70 years old, there is no need to initiate treatment. Treatment typically involves several doses over a period of time. Treatment can be monitored by analyzing antibodies, or activated T-cells or B-cells that respond to a therapeutic agent (eg, Aβ peptide) for a period of time, as described in Examples I and II herein. If the response is lowered, an additional antigen is administered. In patients with potential Down syndrome, treatment may be initiated before birth by administering the treatment to the mother or immediately after birth.
[246] If certain signs of the disease are not suspected, other forms of amyloidosis are often not diagnosed. One major symptom is the presence of heart or kidney disease in middle to old patients who also show signs of involvement with other organs. Low voltage or extreme off-axis and thickened ventricular tissue of the ECG can be an indication of cardiac relevance. Proteinuria is a sign of kidney relatedness. If hepatomegaly is detected on the patient's physical examination, liver relevance may also be suspected. Peripheral neuropathy can also occur in certain amyloidosis forms, and autonomic neuropathy characterized by postural hypotension can also be observed. Amyloidosis should be suspected in all persons with progressive neuropathy of uncertain origin. Accurate diagnosis of this disease can be performed using tissue biopsies that can utilize the affected organ (s). For systemic amyloidosis, fat pad inhalation or rectal biopsy samples may be used. When the biopsy material is stained with Congo red, the positive sample shows apple green birefringence under polarized light microscope.
[247] E. Treatment
[248] For prophylaxis, a pharmaceutical composition and medicament is administered to a patient susceptible to or at risk for a particular disease in an amount sufficient to eliminate or reduce the risk or onset of the disease. For treatment, a pharmaceutical composition and medicament is administered to a patient suspected of having or already suffering from a disease, in an amount effective to treat, or at least partially stop the progression of, the symptoms of the disease and its complications. Amounts suitable to achieve a therapeutic or prophylactic treatment are defined as therapeutically or prophylactically effective amounts. In prophylaxis and treatment, the formulations are generally administered in several doses until a sufficient immune response is obtained. Typically, the immune response is monitored and repeated doses are given as the immune response begins to weaken.
[249] An effective amount of a composition of the present invention for treating the above-mentioned symptoms may be determined by several different factors such as means of administration, target site, physiological state of the patient, whether the patient is human or animal, other drugs administered, and therapies for preventing It depends on whether it is cognitive or therapeutic. Generally, the patient is a human, but some diseases, such as prion protein related mad cow disease, can also treat non-human mammals such as cattle. The therapeutic dose should be titrated to optimize safety and efficacy. The amount of immunogen depends on the administration of the adjuvant and the dose in the absence of the adjuvant should be higher. The amount of immunogen for administration is sometimes from 1 to 500 μg per patient, more generally from 5 to 500 μg per injection for human administration. In some cases, a high dose of 0.5 to 5 mg per injection is used. Typically in humans at least about 10, 20, 50 or 100 μg is used per injection. The timing of injection can vary considerably from once a day to once a year, or even once a decade, with the "up-dose" of continuous immunogens being somewhat preferred. In general, according to the teachings provided herein, a bodily fluid, generally a serum sample, is obtained for an immunogen from a patient using a method well known in the art and readily applicable to the specific antigen to be measured. The effective amount can be monitored by measuring the titer of the antigen thus obtained. Ideally, samples should be taken before the first dose, followed by subsequent samples, and titered after each inoculation. Generally, a dose or dose schedule that provides a detectable titer of at least 4 times higher than the control or "background" level when the serum is diluted 1: 100 is preferred, where the background is relative to the control serum or plate background in the ELISA assay. Is defined. According to the invention a titer of 1: 1000 or 1: 5000 or higher is preferred.
[250] On the day of administration of the immunogen, the dose is generally at least 1 μg, preferably at least 10 μg per patient in the presence of an adjuvant and at least 10 μg, generally at least 100 μg, in the absence of the adjuvant. Dosages of individual immunogens selected in accordance with the present invention are measured in accordance with standard dosing and potency assays in combination with the methods taught herein. Conventional methods consist of performing additional antigen injections at time intervals (eg, 6 week intervals) after immunization. Another method consists of injection of additional antigen 1, 2 and 12 months after immunization. Another way is to inject once every two months of life. Alternatively, additional antigen injections can be performed irregularly as indicated by monitoring the immune response.
[251] For passive immunization with antibodies, doses range from 0.0001 to 100 mg, more usually 0.01 to 5 mg per kg body weight of the host. For example, the dose may be 1 mg or 10 mg per kg of body weight. Exemplary therapies are administered once every two weeks or once a month or once every three to six months. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered falls within the specified range. Antibodies are generally administered several times. The interval between single doses can be weekly, monthly or yearly. The interval may be irregular as shown by measuring blood levels of the antibody against the patient's Aβ. Alternatively, the antibody can be administered as a sustained release formulation, in which case the frequency of administration is reduced. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies have the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. Dosage and frequency may vary depending on whether the treatment is prophylactic or therapeutic. For prophylaxis, relatively low doses are administered over relatively long periods of time. Some patients continue to be treated for the rest of their lives. For therapeutic use, relatively high doses are required at relatively short intervals until the progression of the disease is reduced or terminated, preferably until the patient exhibits partial or complete improvement of the disease symptoms. The patient may then be prevented.
[252] Doses for nucleic acids encoding immunogens range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30 to 300 μg DNA per patient. Doses for infectious viral vectors vary from 10 to 100 or more virions per dose.
[253] Agents that induce an immune response can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, 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) injections, but other routes are equally effective. Intramuscular injection is most commonly given to the arm or leg muscles. In some methods, the agent is injected directly, eg intracranially, into a particular tissue where deposits accumulate. Intramuscular injection upon intravenous infusion is preferred for antibody administration. In some methods, certain therapeutic antibodies are injected directly into the skull. In some methods, the antibody is administered as a sustained release composition or device (eg, a Medipad ^™ device).
[254] The formulations of the present invention may optionally be administered with other agents that are at least partially effective for the treatment of amyloidogenic diseases. In the case of Alzheimer's and Down's syndrome, where amyloid deposition occurs in the brain, the formulations of the invention may be administered in combination with other formulations that increase the passage of the formulations of the invention across the blood brain barrier. In addition, therapeutic cocktails comprising immunogens designed to elicit an immune response against one or more amyloid components are also contemplated as belonging to the present invention, and combinations of antibodies against one plaque component and immunogens for different plaque components are also present invention. Included in
[255] Immunogenic agents of the invention, such as peptides, are sometimes administered with adjuvant. Various adjuvant may be used with peptides such as Aβ to elicit an immune response. Preferred adjuvants augment the intrinsic response to the immunogen without causing structural changes in the immunogen that affect the nature of the response. Examples of preferred auxiliaries include aluminum hydroxide and aluminum phosphate, 3-de-O-acylated monophosphoryl lipid A (MPL ^™ ) (GB 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now part of Corixa), and the like. Stimulon ^™ QS-21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (Vaccine Design; The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY). (1995); see U.S. Patent 5,057,540) (Aquila BioPharmaceuticals, Framingham, Mass.) Other adjuvants are oil-in-water emulsions (e.g. squalene or peanut oil) together with an immunostimulant such as monophosphoryl lipid A, if appropriate (Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)) Another adjuvant is CpG (WO 98/40100) Alternatively, Aβ can be coupled to the adjuvant. Such coupling affects the nature of the immune response Should not substantially alter the structure of A [beta] .The adjuvant may be administered as an ingredient of the therapeutic composition with the active agent, or may be administered separately from the administration of the therapeutic agent, or may be administered before, concurrently or after the administration of the therapeutic agent. .
[256] Preferred classes of auxiliaries are aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvants may be used with or without other specific immunostimulants such as MPL or 3-DMP, QS-21 or polymer or monomeric amino acids such as polyglutamic acid or polylysine. Another class of adjuvants is oil-in-water emulsion formulations. Such adjuvants include other specific immunostimulating agents, such as Muramyl peptides (eg, N-acetylmuramil-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normurayl-L-alanine-D- Isoglutamine (nor-MDP), N-acetylmurayl-L-alanyl-D-isoglutamyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycerol-3-hydroxy Phosphoryloxy) -ethylamine (MTP-PE), N-acetylglucosaminyl-N-acetylmuramil-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) terra Mead TM), or other bacterial cell wall components. Oil-in-water emulsions are (a) 5% squalene, 0.5% twin formulated into particles of sub-micron size using microfluidizing agents such as Model 110Y microfluidizing agent (Microfluidics, Newton, Mass.). MF59 (WO 90/14837) containing 80 and 0.5% Span 85 (optionally containing varying amounts of MTP-PE); (b) SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic block polymer L121 and thr-MDP, which are microfluidized or vortexed into emulsions of submicron size to form larger particle size emulsions, And (c) 2% squalene, 0.2% Tween 80, and monophosphoryl lipid A (MPL), trehalose dicholate (TDM) and cell wall backbone (CWS), preferably MPL + CSW (Detox ^™ ) Ribi ^™ reinforcement system (RAS) containing a selected one or more bacterial cell wall components (Livi Immunchem, Hamilton, Montana, USA). Another class of preferred adjuvants are saponin aids such as particles formed from Stimulon ^™ (QS-21, Aquila, Framingham, Mass.) Or ISCOM (immunostimulatory complex) and ISCOMATRIX. Other adjuvants include complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA). Other adjuvants include cytokines such as interleukins (IL-1, IL-2 and IL-12), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), and the like. Such adjuvants can be purchased generally.
[257] Adjuvants may be administered with the immunogen as a single composition. Alternatively, the adjuvant may be administered before, concurrently or after administration of the immunogen. Immunogens and adjuvants may be packaged and supplied in the same bottle or packaged in separate bottles and mixed before use. Immunogens and adjuvants are usually packaged with a label indicating the desired therapeutic use. If the immunogen and adjuvant are packaged separately, the package usually includes instructions for mixing prior to use. The choice of adjuvant and / or carrier depends on the stability of the immunogenic agent containing the adjuvant, the route of administration, the dosing regimen, and the efficacy of the adjuvant for the species to be vaccinated. Licensed or may be licensed for human administration. In humans, examples of preferred auxiliaries are alum, MPL and QS-21. Optionally, two or more different adjuvants may be used simultaneously. Preferred combinations include alum and MPL, alum and QS-21, MPL and QS-21, alum, QS-21 and MPL together. Incomplete Freund's adjuvant may also be used, optionally with alum, QS-21 and MPL and all combinations thereof (Chang et al., Advanced Drug Delivery Reviews 32, 173-186 (1998)).
[258] Formulations of the present invention are often administered as pharmaceutical compositions comprising the active therapeutic agent and various other pharmaceutically acceptable ingredients. Remington's Pharmaceutical Science (19th ed., Mack Publishing Company, Easton, Pennsylvania, 1995). Preferred forms depend on the mode of administration desired and the therapeutic use. The composition may comprise, depending on the desired formulation, a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as an excipient commonly used in formulating pharmaceutical compositions for animal or human administration. Diluents are chosen so as not to affect biological activity in combination. Examples of this diluent include distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution and Hanks' solution. The pharmaceutical compositions or combinations may also include other carriers, adjuvants, or nontoxic, non-therapeutic, non-immunogenic stabilizers, and the like.
[259] Pharmaceutical compositions are large and slowly metabolized macromolecules such as polysaccharides such as proteins, chitosan, polylactic acid, polyglycolic acid and copolymers (e.g. latex functionalized sepharose, agarose, cellulose, etc.), amino acid polymers, amino acid copolymers And lipid aggregates (eg, oil drops or liposomes). In addition, these carriers can act as immunostimulants (ie, adjuvants).
[260] For parenteral administration, the formulations of the present invention may be administered in injectable doses of solutions or suspensions of substances in pharmaceutical carriers and physiologically acceptable diluents, which may be sterile solutions such as water, oil, saline, glycerol or ethanol. In addition, auxiliary materials such as wetting or emulsifying agents, surfactants, pH buffer materials and the like may be present in the composition. Other components of the pharmaceutical composition include those of petroleum, animal, plant or synthetic origin oils such as peanut oil, soybean oil and mineral oil. In general, glycols such as polypropylene glycol or polyethylene glycol are particularly preferred liquid carriers for injectable solutions. The antibody can be administered in the form of a depot injection or implantable formulation that can be formulated in a sustained release of the active ingredient. Exemplary compositions comprise 5 mg / ml of monoclonal antibody composed of 50 mM L-histidine, 150 mM NaCl and formulated in aqueous buffer adjusted to pH 6.0 with HCl.
[261] Typically, the composition is prepared injectable as a liquid solution or suspension. Solutions or suspensions in liquid excipients may be prepared in suitable solid form prior to injection. The formulations may be emulsified or encapsulated in microparticles, such as liposomes or polylactides, polyglycolide or adjuvant copolymers as described above (Langer, Science 249, 1527 (1990) and Hanes). , Advanced Drug Delivery Reviews 28, 97-119 (1997). The formulations of the present invention may be administered in the form of depot injection or implantable formulations which may be formulated in a manner that allows for sustained or pulsatile release of the active ingredient.
[262] Additional formulations suitable for other modes of administration include oral, intranasal, pulmonary formulations, suppositories, and transdermal formulations.
[263] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5-10%, preferably 1-2%. Oral formulations include excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose and magnesium carbonate. These compositions may take the form of solvents, suspensions, tablets, pills, capsules, delayed release formulations or powders and comprise 10 to 95%, preferably 25 to 70%, of the active ingredient.
[264] Topical administration may result in transdermal or intradermal delivery. The cholera toxin or detoxified derivative or subunit or other similar bacterial toxin can be easily administered by co-administration (Glenn et al., Nature 391, 851 (1998)). Co-administration may be carried out using the components as a mixture or binding molecule obtained by chemical crosslinking or expression as a fusion protein.
[265] Alternatively, transdermal delivery can be accomplished using skin patches or transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998).
[266] F. Diagnostic Method
[267] The present invention provides a method for detecting an immune response to an Αβ peptide in a patient suffering from or susceptible to Alzheimer's disease. The method is particularly useful for monitoring the course of treatment administered to a patient. This method can be used to monitor both treatment treatment in asymptomatic patients and preventive treatment in asymptomatic patients. This method is useful for monitoring active immunization (eg, antibodies produced in response to administration of an immunogen) and passive immunization (eg, measuring the level of antibody administered).
[268] 1. Active Immunization
[269] Some methods include measuring a baseline of the patient's immune response prior to administering a dose of the agent, and comparing the immune response value to the baseline after treatment. Significant increases in the immune response (ie greater than the usual limit of experimental error in repeated measurements of the same sample, expressed as one standard deviation from the mean of these measurements) resulted in a positive treatment result (i.e. Achieve or augment an immune response). If the value for the immune response does not change or decrease significantly, it is a negative therapeutic control. In general, patients undergoing an initial course of treatment with immunogenic agents are expected to show an increase in immune response to successive administrations, which eventually reach a flat phase. Administration of the agent generally continues while the immune response increases. Squamous arrival is an indicator that the treatment performed can be stopped or reduced in terms of dose and frequency.
[270] In another method, the control values (ie mean and standard deviation) of the immune response are measured against the control. Typically, individuals in the control population did not receive prior treatment. After administration of the therapeutic agent, the measurement of the patient's immune response is compared with the control. Significant increases relative to the control (eg, an increase over the standard deviation obtained from the mean) indicate a positive treatment result. Lack of or a significant increase in negative treatment results. Administration of the agent generally continues while the immune response increases relative to the control. As noted above, squamous arrival to the control is an indicator that treatment practice may be stopped or reduced in terms of dose and frequency.
[271] In another method, the control values (eg, mean and mean deviation) of the immune response are determined from a control group of individuals treated with the therapeutic agent and whose immune response has reached a flat phase in response to the treatment. The value of the immune response measured in the patient is compared with the control. If the patient's readings do not differ significantly from the control (eg, beyond one standard deviation), the treatment is discontinued. If the patient's level is significantly lower than the control, continuous administration of the formulation is allowed. If the patient's value persists below the control, the therapy can be altered, such as with the use of other adjuvants.
[272] In another method, patients who are not currently undergoing treatment but have previously undergone a course of treatment are monitored for immune responses to determine if resumption of treatment is necessary. The measured value of the patient's immune response can be compared with the value of the previously obtained immune response from the patient after the previous course of treatment. A significant reduction compared to previous measurements (ie, the amount of reduction beyond the usual limits of error in repeated measurements of the same sample) is an indication that treatment can be resumed. Alternatively, the value measured in the patient can be compared with the control value (mean + standard deviation) measured in the patient's population after experiencing the course of treatment. Alternatively, the measured value of the patient can be compared with a control in a population of prophylactically treated patients with no symptoms of the disease, or with a control of a population of therapeutically treated patients indicating an improvement in disease characteristics. Can be compared. In all these cases, a significant reduction (ie, above the standard deviation) compared to the control value is an indication that the treatment of the patient should be resumed.
[273] Analytical tissue samples are typically blood, plasma, serum, mucosal or cerebrospinal fluid from a patient. This sample is analyzed for any form of Aβ peptide, typically an indicator of an immune response to Aβ 42. Immune responses can be determined from the presence of antibodies or T cells that specifically bind to a desired component, such as an Aβ peptide. ELISA methods for detecting antibodies specific for Αβ are disclosed in the Examples section and may be applied to other peptide antigens. Methods for detecting reactive T-cells are well known in the art.
[274] 2. Passive Immunization
[275] In general, the procedure for monitoring passive immunization is similar to that selected for monitoring active immunization described above. However, antibody profiles after passive immunization typically show an immediate peak of antibody concentration followed by exponential attenuation. Without further dosage, the attenuation approaches the pretreatment level within days to months, depending on the half-life of the antibody administered. For example, the half-life of some human antibodies is in the 20-day range.
[276] In some methods, a baseline measurement of the antibody against the patient's Aβ is performed prior to administration, a second measurement is made immediately after measuring the peak antibody level, and one or more additional measurements are made at intervals monitoring the attenuation of the antibody level. Conduct. Additional levels of antibody are administered when the level of the antibody is lowered to a predetermined rate (eg 50%, 25% or 10%) of the baseline or below baseline. In some methods, levels below the peak or subsequent measured background are compared to reference levels already measured to constitute an advantageous prophylactic or therapeutic treatment for other patients. If the measured antibody level is significantly lower than the reference level (e.g., when the standard deviation is lower than the limit value in the mean of the reference value of the population of patients for whom treatment was beneficial), further administration of the antibody is indicated.
[277] 3. Diagnostic Kit
[278] The present invention further provides a diagnostic kit for performing the aforementioned diagnostic method. Typically, such kits contain an agent that specifically binds to an antibody against an amyloid plaque component such as Aβ and reacts with T-cells specific to that component. The kit may comprise a label. For detection of antibodies to Αβ, the labels are usually in the form of labeled anti-iodotype antibodies. For detection of the antibody, the agent may be supplied prebound with a solid phase, such as a well of a microtiter plate. For detection of reactive T-cells, labels for measuring proliferative responses can be supplied in 3H-thymidine form. Kits also typically include a label that provides instructions for use of the kit. The label may comprise a chart or other corresponding method that correlates the measured label level with the level of T-cells reactive with Aβ or antibodies against Aβ. By label is meant any written or recorded material attached to or otherwise included in a kit at any point in the process of manufacture, transport, sale or use. For example, covers include advertising leaflets and pamphlets, packaging materials, instructions, audio or video cassettes, computer disks, as well as documents imprinted directly on the kit.
[279] I. Preventive Effect of Aβ on Alzheimer's Disease (AD)
[280] These examples _disclose the administration of Aβ42 peptides to transgenic mice overexpressing APP (APP _{717V → F} ) with a mutation at position 717 that facilitates the formation of Alzheimer's-like neuropathology. The production and properties of these mice (PDAPP mice) are disclosed in Games et al., Natrue, supra. These animals of the heterozygous type begin to deposit Aβ at 6 months of age. By 15 months of age, they exhibit the same level of Aβ deposition as observed in Alzheimer's disease. PDAPP mice were injected with aggregated Aβ ₄₂ (aggregated Aβ ₄₂ ) or phosphate buffered saline. Aggregated Aβ ₄₂ was chosen because of its ability to induce antibodies against multiple epitopes of Aβ.
[281] A. Method
[282] 1. Mouse Source
[283] Thirty PDAPP heterologous female mice were randomly divided into the following groups: 10 mice to be injected with aggregated Aβ42 (one died in transit), 5 mice to be injected with PBS / adjuvant or PBS, And 10 non-injected controls. Five mice were injected with peptides derived from the sequence of serum amyloid protein (SAP).
[284] 2. Immunogen Preparation
[285] Preparation of Aggregated Aβ 42: 2 mg of Aβ 42 (US Peptide, Lot K42-12) was dissolved in 0.9 mL of water and made 1 mL by adding 0.1 mL 10 × PBS. It was vortexed and incubated overnight at 37 ° C. Peptides aggregated under these conditions. Any Aβ not used was stored as lyophilized powder at −20 ° C. until the next injection.
[286] When using such commercially available peptides, it should be noted that the dry weight can include the salt weight (weight recorded in all examples) unless otherwise noted. The exact mass of the peptide can be measured using standard assays of the formulation, such as nitrogen determination, with known compositions.
[287] 3. Injection Preparation
[288] For each injection, 100 μg of aggregated Aβ42 per mouse was emulsified 1: 1 with complete Freund's adjuvant (CFA) in a final volume of 400 μl emulsion for first immunization, followed by incomplete Freund's adjuvant (2 weeks). IFA) provided additional antigens with the same immunogen. Two additional doses of IFA were given at monthly intervals. Subsequent immunizations were performed at 500 μl PBS at 1 month intervals. Injections were delivered intraperitoneally (i.p.).
[289] PBS injection followed the same scheme and mice were dosed with 400 μl of PBS / adjuvant 1: 1 mixture, or 500 μl of PBS. Similarly, SAP injections were performed according to the same scheme using a dose of 100 μl per injection.
[290] 4. Determination of Mouse Blood, Tissue Formulation, and Immunochemical Titer
[291] The method is disclosed in the following general materials and methods.
[292] B. Results
[293] PDAPP mice were injected with aggregated Aβ42 (aggregated Aβ42), SAP peptide or phosphate buffered saline. One group of PDAPP mice was left as a positive control without injection. Titers of mice against aggregated Aβ42 were monitored every other month after the fourth additional antigen administration until the mice were 1 year old. Mice were killed at 13 months of age. At all time points examined, 8 out of 9 aggregated Aβ42 mice showed high antibody titers, which remained high through a series of injections (titers over 1/10000). The titer of the ninth mouse was low, but was a measurable titer of approximately 1/1000 (FIG. 1, Table 3). Titers for immunogens of mice injected with SAPP ranged from 1: 1,000 to 1: 30,000, with only one mouse exceeding 1: 10,000.
[294] Titer at 50% maximal O.D. of mice injected with aggregated Aβ Age of PDAPP Mouse# 100# 101# 102# 103# 104# 105# 106# 107# 108 470000150000150001200001000150005000060000100000 61500065000300005500030015000150005000060000 82000055000500005000040015000180005000060000 104000020000600005000090015000500002000040000 1225000300006000040000270020000700002500020000
[295] Titer at 50% maximum O.D. for two immunogens diluted 1/100 in mice injected with PBS. Age of PDAPP Mouse# 113# 114# 115# 116# 117 6<4 x bkg<4 x bkg<4 x bkg<4 x bkg<4 x bkg 105 x bkg<4 x bkg<4 x bkg<4 x bkg<4 x bkg 12<4 x bkg<4 x bkg<4 x bkg<4 x bkg<4 x bkg
[296] Serum of PBS-treated mice was measured for aggregated Aβ42 at 6, 10 and 12 months. At 1/100 dilution, titers for aggregated Aβ42 were only four times greater than background at one data point and four times less than background at all data points (Table 3). SAP specific responses were negligible at these points where all titers were less than 300.
[297] Seven of the nine mice in the Aβ1-42 treated group were free of detectable amyloid in the brain. In contrast, brain tissue from mice of the SAP and PBS groups contained a large number of amyloid deposits in the hippocampus and frontal and cingulate cortex. The deposition pattern characteristically of fragile subregions, such as the outer molecular layer of the hippocampal dentate gyrus, was similar to that of the untreated control. One mouse from the group injected with Aβ1-42 had significantly reduced amyloid loading, limited to the hippocampus. Isolated plaques were identified in another Aβ1-42 treated mouse.
[298] Quantitative image analysis of amyloid load in the hippocampus demonstrated the rapid reduction achieved in Aβ42 (AN1792) -treated animals (FIG. 2). The median amyloid loading for the PBS group (2.22%) and untreated control (2.65%) was significantly greater than that immunized with AN1792 (0.00%, p = 0.0005). In contrast, the median for the group immunized with SAP peptide (SAPP) was 5.74%. Brain tissue from untreated control mice contained a number of Aβ amyloid deposits visualized with Aβ specific monoclonal antibody (mAb) 3D6 in the hippocampus as well as in the retrosplenial cortex. Similar patterns of amyloid deposition were observed in mice immunized with SAPP or PBS (FIG. 2). In addition, for these later three groups, there was a feature in all three of these groups that involved fragile subregions of the brain that were classically observed in AD, such as the outer molecular layers of hippocampal cerebral gyrus.
[299] In addition, brains that do not contain Aβ deposits do not have neural plaques commonly visualized in PDAPP mice using human APP antibody 8E5. All brains from the remaining group (SAP injection mice, PBS mice and untreated mice) had a large number of neural plaques common in untreated PDAPP mice. A few neural plaques were present in one mouse treated with AN1792, and a single cluster of dystrophy neurites was found in a second mouse treated with AN1792. Image analysis of the hippocampus and the figure of FIG. 3 demonstrated parenchymal removal of dystrophy neurites in AN1792 treated mice (median 0.00%) compared to PBS receptor (median 0.28%, p = 0.0005).
[300] The astrocytic character of plaque-associated inflammation was not present in the brains of the Aβ1-42 injection group. Brains from other groups of mice contained GFAP positive stellate cells that were abundant and clustered typical of Aβ plaque associated gliosis. Subsets of GFAP-related slides were control stained with thioflavin S to place Aβ deposits. GFAP positive stellate cells were associated with SAP, PBS and untreated controls. This association was not observed in plaque negative Aβ1-42 treated mice, but minimal plaque-associated neurogliosis was identified in one mouse treated with AN1792.
[301] The image analysis shown in FIG. 4 for bulging posterior cortex showed that the reduction in astrocytosis was significant, when treated with AN1792 for median value of 6% or more in the group immunized with SAS peptide, PBS or untreated group. A median of 1.56% was shown (p = 0.0017).
[302] Evidence from a subset of mice injected with Aβ1-42 and a mouse injected with PBS suggests that plaque-related MHC II immunoreactivity was absent in Aβ1-42 injected mice, consistent with a deficiency of Aβ-related inflammatory response. Is the result.
[303] Sections of the mouse brain were reacted with monoclonal antibodies (mAb) specific for MAC-1 (cell surface protein). MAC-1 (CD11b) is a member of the integrin family and exists as a dimer with CD18. CD11b / CD18 complexes are present in protein, macrophages, neutrophils and natural killer cells (Mak and Simard). The brain's intrinsic MAC-1 reactive cell type is likely to be small neuroglial cells based on similar phenotypic behavior in MAC-1 immunoreacted sections. Plaque-related MAC-1 labeling was lower in the brain of mice treated with AN1792 compared to the PBS control, a finding consistent with the lack of Aβ-induced inflammatory response.
[304] C. Conclusion
[305] Deficiency of Aβ plaques and reactive neurons and glial changes in the brain of Aβ1-42 injected mice suggests no or little deposition of amyloid in the brain and the absence of pathological consequences such as gliosis and neurite pathology. PDAPP mice treated with Aβ1-42 exhibit substantially the same pathology deficiency as control mice not transgenic. Thus, Aβ1-42 injections are very effective in preventing the deposition or removal of human Aβ from brain tissue and in eliminating subsequent neurons and inflammatory degenerative changes. Thus, administration of Αβ peptides may have prophylactic and therapeutic benefits in preventing AD.
[306] II. Dose response experiment
[307] A group of 5 week old female Swiss Webster mice (6 per group) were immunized by intraperitoneal injection of Aβ300, 100, 33, 11, 3.7, 1.2, 0.4 or 0.13 μg formulated in CFA / IFA. Three doses were inoculated at two week intervals, followed by a fourth dose one month later. The first dose was emulsified with CFA and the remaining doses were emulsified with IFA. To determine antibody titers, blood was collected 4-7 days after each inoculation, starting after the second inoculation, and antibody titers were measured after the second inoculation. Attenuation of the antibody response over the dose range of the immunogenic agent was monitored by additional blood collection at nearly monthly intervals for four months after the fourth inoculation in a subset of groups of animals inoculated with antigen 11, 33 or 300 μg. . These animals were given the final fifth inoculation seven months after the start of the experiment. After one week these animals were killed and antibody responses to AN1792 were measured and toxicity assayed.
[308] Attenuation dose response was observed at 300-3.7 μg with no response at the two lowest doses. The average antibody titer was about 1: 1000 after three inoculations with antigens 11 to 300 μg and 1: 10,000 after four inoculations (see FIG. 5).
[309] Antibody titers increased rapidly after the 3rd inoculation except for the lowest dose group, and the range of increase of GMT was 5-25 fold. Thereafter, a low antibody response could be detected even at 0.4 µg receptor. The 1.2 and 3.7 μg groups showed similar titers with a GMT of about 1000, with the highest dose of four dose groups having a GMT of about 25,000, with the lower 33 μg group having a lower GMT 3000. After the fourth inoculation, titers increased more slowly in most groups. A clear dose response was seen across the lesser doses of the 0.14-11 μg group, with no antibody detected for the 0.14 μg receptor and 36,000 GMT for the 11 μg receptor. In addition, titers for the four highest dose groups of 11 to 300 μg were concentrated. Thus, after two inoculations, antibody titers were dependent on antigen dose over a wide range of 0.4-300 μg. By tertiary inoculation, the titers of the highest four dose groups were all similar and this titer remained constant after booster vaccination.
[310] One month after the fourth inoculation, titers were 2-3 times higher in the 300 μg group than measured in blood drawn 5 days after inoculation (FIG. 6). This observation suggests that the peak history antibody response occurs 5 days after inoculation. A gentler (50%) increase was observed at this time in the 33 μg group. In the 300 μg group, GMT decreased rapidly by about 70% two months after the last dose. After another month, the reduction was less abrupt, 45% (100 μg), about 14% for 33 μg and 11 μg administration. Thus, the rate of decrease in circulating antibody titers after stopping the inoculation appears to be bimodal, which decreases rapidly in the first month after the peak reaction and then at a slower rate.
[311] The antibody titers and reaction kinetics of these Swiss Webster mice are similar to those of young heterozygous PDAPP transgenic mice inoculated in the same manner. The dose effective for inducing an immune response in humans is generally similar to the dose effective in mice.
[312] III. Screening of Therapeutic Efficacy on Established AD
[313] This assay is designed to test immunogenic agents for activity that delays or reverses the neuropathological properties of AD in older animals. At any point where amyloid plaques were already present in the brains of PDAPP mice, inoculation of 42 amino acids long β (AN1792) was started.
[314] Over time used in this experiment, untreated PDPAA mice showed many neurodegenerative changes similar to those observed in AD (Games et al., Homology and Johnson-Wood et al., Proc. Natl. Acad. Sci. USA 94, 1550-1555 (1997)]. Deposits of amyloid into amyloid plaques are associated with a degenerative nervous response called dystrophy neurites, consisting of abnormal axon and dendrites. Amyloid deposits surrounded by and comprising dystrophic neurites are called neurogenic plaques. Dystrophic neurites in both AD and PDAPP mice have a unique spherical structure, are immunoreactive with a panel of antibodies that recognize APP and cytoskeleton components, and exhibit complex cytostatic degenerative changes at the ultrastructure level. This property allows for disease-related, selective scaffolding and reproducible measurements of neuritis plaque formation in the PDAPP brain. The dystrophic neuronal components of PDAPP neuritis plaques can be easily visualized with antibodies specific to human APP (monoclonal antibody 8E5) and easily measured by computer aided 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.
[315] Astrocytes and microglia are non-neuronal cells that respond to and reflect the degree of nerve damage. CFAP-positive astrocytes and MHC II-positive microglia are commonly observed in AD, and their activation increases with disease severity. Therefore, we also monitored the development of reactive astrocytosis and microglia in AN1792-treated mice.
[316] A. Materials and Methods
[317] 48 heterozygous female PDAPP mice, aged 11 to 11.5 months old, obtained from the Charles River were randomly divided into two groups: 24 were inoculated with 100 μg of AN1792 in combination with Freund's adjuvant, and the remaining 24 were Freund's adjuvant. PBS in combination with was inoculated. When mice reached ˜15 months of age, AN1792 group and PBS group were subdivided. At 15 months of age, approximately half of each group of AN1792 and PBS treated animals were euthanized (n = 10 and 9, respectively) and the remainder was continued inoculation until the end of ˜18 weeks (n = 9 and 12, respectively). A total of eight animals (5 AN1792, 3 PBS) died during the experiment. In addition to immunized animals, untreated PDPAA mice aged 1 (n = 10), 15 months (n = 10) and 18 months (n = 10) were included in the comparison using ELISA to measure Aβ and APP levels in the brain. In addition, 1-year-old animals were included in immunohistochemical analysis.
[318] The method was the same as in Example 1 unless otherwise noted. UA peptide lot 12 of AN1792 and California peptide lot ME0339 were used to prepare antigens for 6 inoculations before the 15 month time point. The California peptide lots ME0339 and ME0439 were used for three boosters between 15 and 18 weeks.
[319] For immunization, only 100 μg of AN1792 or PBS in 200 μl PBS was emulsified in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) or PBS 1: 1 (volume: volume) to a final volume of 400 μl. It was. In the first inoculation, CFA was used as an adjuvant, followed by 4 inoculations using IFA, and the last 4 inoculations using only PBS without adding adjuvant. A total of nine inoculations were performed over seven months, with the first three inoculations being two weeks apart and the remaining inoculations being four weeks apart. The four-month treatment group, which received the first six doses only, was euthanized at 15 months of age.
[320] B. Results
[321] 1. Effect of AN1792 Treatment on Amyloid Deposits
[322] The results of the effect of AN1792 treatment on cortical amyloid deposits as determined by quantitative image analysis are shown in FIG. 7. The median cortical amyloid deposition was 0.28% in the group of untreated 12-month-old PDAPP mice, which was indicative of plaque deposition in mice at the start of the experiment. At 18 months, amyloid deposition increased more than 17-fold to 4.87% in PBS treated mice, whereas AN1792 treated mice exhibited significantly reduced amyloid deposition of only 0.01%, significantly less than 12-month-old and 15- and 18-month-old PBS-treated groups. It was. Amyloid deposition was significantly reduced at AN1792 receptor at both 15 months (96% decrease; p = 0.003) and 18 months (> 99% decrease; p = 0.0002).
[323] In general, cortical amyloid deposition in PDAPP mice begins in the frontal and retrosplenial cortex (RSC) and progresses in the ventral-outward direction, affecting the temple enterorhinal cortex (EC). Little or no amyloid was found in EC of 12-month-old mice, about the same age as when AN1792 was first administered. After 4 months of AN1792 treatment, amyloid deposition in RSC was markedly reduced, and the progressive involvement of EC was also completely eliminated by AN1792 treatment. Subsequent observations showed that AN1792 can normally stop the progression of amyloid penetrating the temples and ventral cortex, as well as delay or reverse deposition in RSC.
[324] The profound effect of AN1792 on ongoing cortical amyloid deposition in PDAPP mice was further demonstrated in the 18-month-old group treated for 7 months. Almost no cortical amyloid was found in AN1792 treated mice, as well as the complete disappearance of diffuse plaques, as well as a dense deposition.
[325] 2. Cellular Changes and Morphological Changes Associated with AN1792 Treatment
[326] Populations of Aβ-positive cells have generally been observed in areas of the brain that include amyloid deposition. Surprisingly, in some brains from AN1792 receptor, little or no extracellular cortical amyloid plaques were found. Most of the Αβ immunoreactivity was shown to be contained in cells with large lobules or aggregated cell bodies. The phenotype showed that these cells were similar to activated microglia or monocytes. They were immunoreactive with antibodies recognizing ligands expressed by activated monocytes and microglia (MHC II and CD11b) and sometimes bound to the walls or lumens of blood vessels. Comparison of adjacent fragments labeled with Aβ and MHC II specific antibodies showed that a similar pattern of these cells was recognized by both antibodies. Detailed observations of the AN1792 treated brain revealed that MHC II-positive cells were confined to the vicinity of the restricted amyloid remaining in these animals. Under the fixed conditions employed, the cells did not show immunoreactivity with antibodies recognizing T cell (CD3, CD3e) or B cell (CD45RA, CD45RB) ligands or leukocyte common antigen (CD45), but did not react with leukoalin that cross-reacts with monocytes. It was reactive with an antibody that recognizes (CD43). These cells were not found in PBS treated mice.
[327] PDAPP mice always generate many amyloid deposits in the outer molecular layer of the hippocampus dentate gyrus. Deposition forms a distinctive stripe in the passageway, i.e., a subregion comprising amyloid plaques generally in AD. Characteristic aspects of this deposition in PBS treated mice were similar to those previously identified in untreated PDAPP mice. Amyloid deposition consisted of both diffuse and dense plaques in the form of continuous bands. In contrast, in many brains derived from AN1792 treated mice, this pattern changed dramatically. Hippocampal amyloid deposition no longer contained diffuse amyloidol and the band pattern completely collapsed. Instead, there are a number of strange puncture structures that are reactive with anti-Aβ antibodies, some of which have been shown to be amyloid containing hepotes.
[328] MHC-II positive cells were often observed in the vicinity of extracellular amyloid in AN1792 treated animals. The pattern of association of Αβ-positive cells with amyloid was very similar in some brains from AN1792 treated mice. This monocyte distribution was confined to the vicinity of the deposited amyloid and was not present at all in other brain regions without Aβ plaques. Confocal microscopy of MHCII- and Aβ-labeled sections showed that plaque material was contained in many monocytes.
[329] Quantitative image analysis of MHC II and MAC I labeled sections showed a trend towards increased immunoreactivity in RSC and hippocampus of AN1792 treated mice compared to the PBS group, with no significant difference in the magnitude of MAC I reactivity in the hippocampus. there was.
[330] These results indicate that there is an active, cell-mediated clearance of amyloid in plaque-bearing brain regions.
[331] 3. Effect of AN1792 on Aβ Level: ELISA Measurement
[332] (a) cortical levels
[333] In untreated PDAPP mice, the median level of total Aβ in the cortex at 12 months was 1,600 ng / g, which increased to 8,700 ng / g by 15 months (Table 4). At 18 months, this value was 22,000 ng / g, an increase of more than 10-fold over the course of the experiment. PBS treated animals had a total Aβ of 8,600 ng / g at 15 months and this value increased to 19,000 ng / g at 18 months. In contrast, AN1792 treated animals had 81% less total Aβ (1,600 ng / g) than the PBS inoculated group at 15 months. The total Aβ (5,200 ng / g) was significantly less (p = 0.0001) at 18 months when comparing AN1792 and PBS groups (Table 4), indicating a 72% reduction in Aβ present without treatment. When comparing cortical levels of Aβ 42, similar results were obtained: the AN1792 treatment group contained much less Aβ 42, but in this case the difference between AN1792 and PBS groups was 15 months (p = 0.04) and 18 months (p = 0.0001). , Table 4) were remarkable in both.
[334] Intermediate Aβ levels in the cortex (ng / g) UntreatedPBSAN1792 AgegunAβ42(n)gunAβ42(n)gunAβ42(n) 121,6001,30010 158,7008,300108,6007,200(9)1,6001,300 ^* 10 1822,20018,5001019,00015,900(12)5,200 ^** 4,000 ^** (9) * p = 0.0412 ** P = 0.0001
[335] (b) hippocampus levels
[336] In untreated PDAPP mice, the median hippocampus level of total Aβ at 12 months of age was 15,000 ng / g, which increased to 51,000 ng / g at 15 months and 81,000 ng / g at 18 months (Table 5). Similarly, mice inoculated with PBS were 40,000 ng / g at 15 months and 65,000 ng / g at 18 months. Animals inoculated with AN1792 showed less total Aβ42, specifically 25,000 ng / g at 15 months and 51,000 ng / g at 18 months. The value of the 18 months AN1792 treatment group was much less than the PBS treatment group value (p = 0.0105; Table 5). The measurement of Aβ42 at 18 months of evaluation showed the same pattern of results: the levels of the AN1792 treated group were much less than in the PBS group (39,000 ng / g vs 57,000 ng / g, respectively; p = 0.002) (Table 5).
[337] Medium Aβ Level in the Hippocampus (ng / g) UntreatedPBSAN1792 AgegunAβ42(n)gunAβ42(n)gunAβ42(n) 1215,50011,10010 1551,50044,4001040,10035,700(9)24,50022,10010 1880,80064,2001065,400057,100(12)50,90038,900 ^** (9) * p = 0.0105 ** P = 0.0022
[338] (c) cerebellar level
[339] In 12-month-old untreated PDAPP mice, the median brain level of total Aβ was 15 ng / g (Table 6). At 15 months, this median increased to 28 ng / g and to 18 ng by 35 ng / g. PBS treated animals had a median total Aβ level of 21 ng / g at 15 months and 43 ng / g at 18 months. AN1792 treated animals were found to have a total Aβ level of 22 ng / g at 15 months, with significantly lower total Aβ at 18 months than the corresponding PBS group (25 ng / g) (p = 0.002) (Table 6). .
[340] Medium Aβ Level in Cerebellum (ng / g) UntreatedPBSAN1792 AgeTotal Aβ(n)Total Aβ(n)Total Aβ(n) 1215.610 1527.71020.8(9)21.710 1835.01043.1(12)24.8 ^* (9) * p = 0.0018
[341] 4. AN1792 Effect of Processing on APP Level
[342] Since both APP-α and full-length APP molecules contain all or part of the Aβ sequence, they may be potentially affected by the formation of the AN1792 commanded immune response. In previous studies, a slight increase in APP levels has been shown to increase neuropathology in PDAPP mice. In the cortex, APP-α / FL (full length) or APP-α levels were substantially decreased by treatment, except that APP-α was 19% reduced at 18 months for AN1792 treated group compared to PBS treated group. Unchanged. The 18 month AN1792 treated APP values were not significantly different from those of the 12 and 15 month untreated, and 15 month PBS groups. In all cases, APP values did not change within the range generally observed in PDAPP mice.
[343] 5. Effect of AN1792 Treatment on Neurodegeneration and Glial Pathology
[344] Neuritis plaque deposition was significantly reduced at 15 months of age (84%; p = 0.03) and 18 months of age (55%; p = 0.01) for the frontal cortex of AN1792 treated mice compared to the PBS group (FIG. 8). Median neuritis plaque deposition increased from 0.32% to 0.49% for the PBS group between 15 and 18 months of age. This is in contrast to the markedly reduced development of neuritis plaques in the AN1792 group, with median neuritis plaque deposition values of 0.05% and 0.02% in the 15- and 18-month-old groups, respectively.
[345] Immunization with AN1792 was shown to be resistant and reactive astrocytosis was also seen in both RSC in AN1792 treated mice at 15 months of age (56%; p = 0.011) and 18 months of age (39%; p = 0.028) compared to the PBS group. It was significantly reduced (FIG. 9). The median rate of astrocytosis in the PBS group increased from 4.26% to 5.21% between 15 and 18 months of age. AN1792 treatment inhibited astrocytosis at 1.89% and 3.2%, respectively. This suggests that the neural network was not damaged by the elimination process.
[346] 6. Antibody Reactions
[347] As described above, heterozygous PDAPP mice (N = 24) at 11 months of age were intraperitoneally injected with 100 μg of AN1792 emulsified with Freund's adjuvant at 0, 2, 4, 8 and 12 weeks and inoculated 6 times. Was performed at 16 weeks with PBS only (no Freund's adjuvant). As a negative control, the same set of transgenic mice with the same age of 24 months were inoculated with PBS emulsified with the same adjuvant according to the same schedule. Blood was collected within 3 to 7 days after each inoculation, beginning after the second inoculation. Antibodies to AN1792 were measured by ELISA. For animals inoculated with AN1792, the geometric mean titers (GMT) after the second, third and final (sixth) inoculations were about 1,900, 7,600 and 45,000, respectively. No Αβ-specific antibodies were detected in control animals after the sixth inoculation.
[348] About half of the animals were treated for three more months, at about 10, 24 and 27 weeks of inoculation. Each of these doses was administered with only PBS excipients without the addition of Freund's adjuvant. Average antibody titers did not change over this period. In fact, antibody titers have been shown to remain stable from 4 th to 8 th blood collection, corresponding to the period from 5 th to 9 th inoculations.
[349] To determine if Aβ-specific antibodies produced by immunization detected in the serum of AN1792 treated mice were also associated with deposited brain amyloid, one subset of sections from AN1792- and PBS treated mice was directed to mouse IgG. React with specific antibodies. In contrast to the PBS group, Aβ plaques of AN1792 treated brain were coated with endogenous IgG. This difference between the two groups was observed in both 15 and 18 month-old groups. What was particularly surprising was that despite the presence of many amyloid deposits in these mice, they were not labeled in the PBS group. These results show that immunization with synthetic Αβ protein produces antibodies that recognize and bind Αβ in amyloid plaques in vivo.
[350] 7. Cell Mediated Immune Response
[351] Spleens were removed after the ninth inoculation from nine PDAPP mice inoculated with AN1792 of 18 months of age and 12 PDAPP mice inoculated with PBS. Splenocytes were isolated and incubated for 72 hours in the presence of Aβ40, Aβ42, or Aβ40-1 (reverse sequence protein). Mitogen Con A was used as a positive control. Optimal response was obtained at> 1.7 μM protein. Cells obtained from all nine AN1792 treated mice were grown by inserting Aβ1-40 or Aβ1-42 protein at the same level to react (FIG. 10, top panel). It did not respond to Aβ40-1 retrograde protein. Cells from control animals did not respond to any of the Aβ protein (FIG. 10, bottom panel).
[352] C. Conclusion
[353] The results of this experiment show that AN1792 immunization of PDAPP mice processing existing amyloid deposits mitigates and prevents progressive amyloid deposition and delays the resulting neuropathological changes in aged PDAPP mouse brains. Immunization with AN1792 substantially stopped amyloid development in structures succumbing to amyloidosis. Thus, inoculation of Aβ peptides provides a therapeutic advantage in the treatment of AD.
[354] IV. Screening of Aβ Fragments
[355] To determine the epitope conferring an effective response, 100 PDAPP mice aged 9-11 months were inoculated with nine different APP and Aβ regions. Nine different immunogens and one control group were intraperitoneally injected as described above. Immunogens include four human Aβ peptide conjugates 1-12, 13-28, 32-42, 1-5 (all of which are bound to both anti-mouse IgG via a cysteine link), APP polypeptide amino acids 592-695, Aggregated human Aβ1-40 and aggregated human Aβ25-35 and aggregated rodent Aβ42. Aggregated Aβ 42 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 of inoculation. Histochemical analysis, Aβ level analysis and toxicity analysis were determined post hoc.
[356] A. Materials and Methods
[357] 1. Preparation of Immunogen
[358] Preparation of Coupled Aβ Peptides: Four human Aβ peptide conjugates (amino acid residues 1-5, 1-12, 13-28 and 33-42, respectively conjugated to both anti-mouse IgGs) using crosslinker sulfo-EMCS By coupling through artificial cysteine added to the Aβ peptide. Aβ peptide derivatives were synthesized with the following final amino acid sequences. In each case, the position of the inserted cysteine residues is underlined. As indicated for the Aβ 13-28 peptide derivative, two glycine residues were added before the carboxyl terminal cysteine.
[359] Aβ1-12 Peptide NH2-DAEFRHDSGYEV C- COOH (SEQ ID NO: 30)
[360] Aβ1-5 Peptide NH2-DAEFR C- COOH (SEQ ID NO: 31)
[361] Aβ 33-42 Peptide NH2-C-amino-heptanoic acid-GLMVGGVVIA-COOH (SEQ ID NO: 32)
[362] Aβ13-28 Peptide Ac-NH-HHQKLVFFAEDVGSNKGG C -COOH (SEQ ID NO: 33)
[363] To prepare the coupling reaction, 10 mg of both anti-mouse IgG (Jackson Immunore Laboratories) were dialyzed overnight against 10 mM sodium borate buffer (pH 8.5). The dialyzed antibody was then concentrated to a volume of 2 ml using Amicon Centripepep tube. 10 mg of sulfo-EMCS [N (ε-maleimidocuproyloxy) succinimide] (Molecular Sciences Company) was dissolved in 1 ml of deionized water. A 40-fold molar excess of sulfo-EMCS was added dropwise to both anti-mouse IgGs with stirring, then the solution was further stirred for 10 minutes. Both activated anti-mouse IgGs were purified and passed through a 10 ml gel filtration column (Pierce Presto column, Pierce Chemicals) equilibrated with 0.1 M NaPO ₄ , 5 mM EDTA, pH 6.5. Fractions containing the antibody identified by absorbance at 280 nm were collected and diluted to a concentration of about 1 mg / ml using 1.4 mg per OD as an absorption coefficient. A 40-fold molar excess of Αβ peptide was dissolved in 20 mL of 10 mM NaPO ₄ (pH 8.0), except that for Aβ33-42 peptide, 10 mg was dissolved in 0.5 mL of DMSO first, followed by 20 with 10 mM NaPO ₄ buffer. Dilute to mL. Peptide solutions were each added to 10 ml of both activated anti-mouse IgG and shaken for 4 hours at room temperature. The conjugate thus obtained was concentrated to a final volume of less than 10 ml using Amicon Centriprep tube and then dialyzed against PBS to exchange buffer and remove free peptide. The conjugate was passed through a 0.22 μm diameter filter for sterilization, then partitioned into 1 mg fractions and stored frozen at −20 ° C. Concentrations of the conjugates were measured using BCA protein analysis (Pierce Chemicals) and equine IgG for standard curves. Conjugation was recorded as the molecular weight increase of the conjugated peptide relative to the molecular weight of both activated anti-mouse IgG. Aβ1-5 both anti-mouse conjugates were two pools of conjugates, the rest from a single agent.
[364] 2. Preparation of Aggregated Aβ Peptides
[365] Human 1-40 (AN1528; California Peptides Incorporated, Lot ME0541), Human 1-42 (AN1792; California Peptides Incorporated) to prepare each injection set from lyophilized powder stored moistureproof at −20 ° C. Tide, Lot ME0339 and ME0439), Human 25-35 and Rodent 1-42 (California Peptides, Lot ME0218) peptides were newly solubilized. For this purpose, 2 mg of peptide was added to 0.9 ml of deionized water and the mixture was shaken to form a relatively uniform solution or suspension. Of the four, AN1528 was the only soluble peptide at this stage. Next, 100 μl of 10 × PBS (1 × PBS: 0.15 N NaCl, 0.01 M sodium phosphate, pH 7.5) was added at the time when AN1528 began to precipitate. This suspension was shaken again and incubated overnight at 37 ° C. for next day use.
[366] Preparation of pBx6 Protein: Expression plasmids encoding the fusion protein pBx6 consisting of the 100 amino acid bacteriophage MS-2 polymerase N-terminal leader sequence followed by amino acids 592-695 (βAPP) of APP are described in Oltersdorf et al. J. Biol. Chem. 265, 4492-4497 (1990). This plasmid was transfected with E. coli, the promoter was induced and the protein expressed. The bacteria were dissolved in 8M urea and pBx6 was partially purified by preparative SDS PAGE. Fractions containing pBx6 were identified by Western blot using rabbit anti-pBx polyclonal antibodies, then collected and concentrated using Amicon centriprep tubes and dialyzed against PBS. The purity of the formulation evaluated by SDS PAGE stained with Coomassie blue was about 5-10%.
[367] B. Results and Discussion
[368] 1. Experimental design
[369] Male and female heterozygous PDAPP transgenic mice of 9-11 months of age were obtained from Charles River Laboratories and Taconic Laboratories. Mice were divided into 10 groups to inoculate different regions of Aβ or APP combined with Freund's adjuvant. Animals in the group were distributed so that sex, age, lineage, and source match as closely as possible. Immunogens were four Αβ peptides from human sequences 1-5, 1-12, 13-28 and 33-42 conjugated to both anti-mouse IgGs, respectively; Four aggregated Αβ peptides, human 1-40 (AN1528), human 1-42 (AN1792), human 25-35 and rodent 1-42; And a fusion polypeptide represented by pBx6 comprising an APP amino acid residues 592-695. The tenth group was inoculated with PBS in combination with adjuvant as a control.
[370] At each inoculation, 100 μg of each Aβ peptide in 200 μl PBS or 200 μg of APP derivative pBx6 in the same volume of PBS alone or 400 μl of PBS alone was used to complete 1: 1 Freund's adjuvant (CFA) and 1: 1 (volume: volume). First inoculation by emulsification, followed by four additional inoculations with the same amount of immunogen in incomplete Freund's adjuvant (IFA), and finally inoculated with PBS. Intraperitoneal injections were made every two weeks for the first three times thereafter. Beginning after the second inoculation, blood collection was performed 4 to 7 days after each inoculation to measure antibody titers. Animals were euthanized about one week after the last inoculation.
[371] 2. Aβ and APP Levels in the Brain
[372] After about four months of immunization with various Aβ peptides or APP derivatives, brains were removed from saline-perfused animals. One hemisphere was prepared for immunohistologic analysis and the other half was used for quantification of Aβ and APP levels. To measure the concentration of various forms of beta amyloid peptide and amyloid precursor protein, hemispheres were cut and homogenates of hippocampus, cortex and cerebellar regions were prepared in 5 M guanidine. These were diluted and quantified amyloid or APP levels compared to serial dilutions of known concentrations of Aβ peptide or APP in ELISA format.
[373] The median concentration of total Aβ was 5.8 times higher in the hippocampus than in the control group inoculated with PBS (median 24,318 ng / g of hippocampal tissue, median of cortex 4,221 ng / g). The median level of cerebellum in the control group (23.4 ng / g) was about 1,000 times lower than in the hippocampus. This level is similar to what we previously reported for heterozygous PDAPP transgenic mice of this age (see Johnson-Woods et al., 1997, supra).
[374] For the cortex, one subset of the treatment groups showed significantly different mean total Aβ and Aβ1-42 levels than those of the control group (p <0.05). For treatment animals, AN1792, rodent Aβ1-42, as shown in FIG. Or Aβ1-peptide conjugates. For these treatment groups the median levels of total Aβ were reduced by 75%, 79% and 61%, respectively, compared to the control. There was no characteristic association between Aβ-specific antibody titers for any group and Aβ levels in cortical regions of the brain.
[375] In the hippocampus, the median decrease in total Aβ associated with AN1792 treatment (46%, p = 0.0543) was not as great as that observed in the cortex (75%, p = 0.0021). However, the degree of reduction was much greater in the hippocampus than in the cortex, with a net decrease in the hippocampus of 11,186 ng per g tissue (3) and 3,171 ng per g tissue (g) in the cortex. For animals receiving rodent Aβ1-42 or Aβ1-5, median total Aβ levels were reduced by 36% and 26%, respectively. However, given the small group size and high variability of amyloid peptide levels between the animals in the two groups, this reduction was not significant. When measuring the levels of Aβ1-42 in the hippocampus, none of the decreases caused by treatment was significant. Thus, due to less Aβ deposition in the cortex, changes in this area become a more sensitive measure of therapeutic effect. Changes in Aβ levels in the cortex measured by ELISA were similar but not identical to the results of immunohistologic analysis (see below).
[376] Total Αβ was also measured in the cerebellum, the region generally least affected by AD pathology. None of the median Aβ concentrations of any group inoculated with the various Aβ peptides or APP derivatives differed from the concentrations of the control in this region of the brain. These results suggest that non-pathological levels of Αβ are not affected by treatment.
[377] APP concentrations were also measured using ELISA in the cortex and cerebellum of treated and control mice. Two different APP assays were used. The first assay, called APP-α / FL, recognizes both APP-alpha (α, the secreted form of APP cleaved within the Aβ sequence) and the full-length form of APP (FL), while the second assay only identifies APP-α. Recognize. In contrast to Aβ reduction associated with treatment in a subset of treatment groups, APP levels did not change in all treatment groups as compared to control animals. These results indicate that immunization with Αβ peptide does not deplete APP, but rather that the therapeutic effect is specific for Αβ.
[378] In summary, total Aβ and Aβ1-42 levels were significantly reduced by treatment with AN1792, rodent Aβ1-42 or Aβ1-5 conjugates. In the hippocampus, total Aβ was reduced only by AN1792 treatment. No other treatment related changes in Aβ or APP levels were significant in the hippocampus, cortex or cerebellum area.
[379] 2. Immunohistologic Analysis
[380] Brains were prepared for immunohistologic analysis from one subset of six groups, three groups inoculated with Aβ peptide conjugates Aβ1-5, Aβ1-12 and Aβ13-28; Two groups inoculated with full length Aβ aggregates AN1792 and AN1528 and PBS treated controls. The image analysis results of amyloid deposition of brain sections obtained in these groups are shown in FIG. 12. Reduction of amyloid deposition was significant in the cortical areas of the three treated animals compared to the control animals. The greatest reduction in amyloid deposition was observed in the AN1792 group, with a median of 97% reduction (p = 0.001). Significant decrease was also seen in animals treated with AN1528 (95%, p = 0.005) and Aβ1-5 peptide conjugate (67%, p = 0.02).
[381] The results obtained by quantifying total Aβ or Aβ1-42 by ELISA and amyloid deposition by image analysis were somewhat different. Treatment of AN1528 had a significant effect on cortical amyloid deposition levels as determined by quantitative image analysis, but did not affect total Aβ concentrations in the same area as measured by ELISA. The difference between these two results is thought to be due to the specificity of the analysis. Image analysis measures only insoluble Aβ aggregated into plaques. In contrast, ELISA measures all forms of Aβ, both insoluble and soluble, monomers and aggregates. Because disease pathology is thought to be associated with an insoluble plaque associated type of Αβ, image analysis techniques may have greater sensitivity to exhibit therapeutic effects. ELISA is faster and easier to analyze, which is very useful for screening purposes. In addition, the treatment related reduction of Aβ is greater for plaque related types than for total Aβ.
[382] To determine if Αβ-specific antibodies produced by immunization in treated animals react with deposited brain amyloid, react one antibody of mouse IgG with a subset of sections from treated animals and control mice. I was. In contrast to the PBS group, A [beta] -containing plaques include A [beta] peptide conjugates A [beta] 1-5, A [beta] 1-12 and A [beta] 13-28; And animals inoculated with full length Aβ aggregates AN1792 and AN1528 were coated with endogenous IgG. This assay was not performed with brains from animals immunized with other Aβ peptides or APP peptide pBx6.
[383] 3. Determination of Antibody Titers
[384] Starting from the second inoculation, a total of five blood samples were collected from the mice 4 to 7 days after each inoculation. Antibody titers were measured as Aβ1-42 binding antibodies using a sandwich ELISA using a plastic multiwell plate coated with Aβ1-42. As shown in FIG. 13, peak antibody titers were generated after the fourth inoculation for the four immunogenic compositions that produced the highest titers of AN1792 specific antibodies: 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). In these groups titers decreased slightly after 5 and 6 inoculations. For the remaining five doses of immunogen, peak titers were reached after the fifth or sixth inoculation, which were much less than those of the four highest titer groups: Aβ1-5 conjugate (peak GMT: 2356), pBx6 (Peak GMT: 1,986), Aβ 13-28 conjugate (peak GMT: 1,183), Aβ 33-42 conjugate (peak GMT: 658), Aβ 25-35 (peak GMT: 125). Antibody titers were also determined for homologous peptides using the same ELISA sandwich format for one subset of immunogens, these groups inoculated with Aβ1-5, Aβ13-28, Aβ25-35, Aβ33-42 or rodent Aβ1-42 It was. This titer was the same as that measured for Aβ1-42, except that for rodent Aβ1-42 immunogens, antibody titers to homologous immunogens were at least about two-fold. The size of the AN1792 specific antibody titers or the mean value of the treatment groups in the individual animals was not related to the efficacy measured as a decrease in Aβ in the cortex.
[385] 4. Lymphocytic reaction
[386] Αβ-dependent lymphocyte proliferative responses were measured using splenocytes recovered about one week after the last sixth inoculation. Freshly recovered cells (105 per well) were incubated for 5 days in the presence of Aβ1-40 at a concentration of 5 μM for stimulation. Cells obtained from a subset of 7 of 10 groups were also cultured in the presence of retrograde peptide Aβ40-1. As a positive control, another cell was incubated with PHA, a T cell mitogen, and as a negative control, cells without addition of peptides were cultured.
[387] Lymph from most animals proliferated in response to PHA. There was no significant response to the Aβ40-1 retrograde peptide. Cells from animals inoculated with the larger aggregated Aβ peptide, AN1792, rodent Aβ1-42 and AN1528, proliferated strongly when stimulated with Aβ1-40 with the largest cpm at the receptor of AN1792. One of each group inoculated with Aβ1-12 conjugate, Aβ13-28 conjugate, and Aβ25-35 grew in response to Aβ1-40. The remaining groups receiving Aβ1-5 conjugates, Aβ33-42 conjugates, pBx6 or PBS had no animals exhibiting Aβ stimulatory responses. These results are summarized in Table 7 below.
[388] ImmunogenConjugateAβ amino acidReacting animals Aβ1-5U5-mer0/7 Aβ1-12U12-mer1/8 Aβ13-28U16-mer1/9 Aβ25-35 11-mer1/9 Aβ33-42U10-mer0/10 Aβ1-40 40-mer5/8 Aβ1-42 42-mer9/9 rAβ1-42 42-mer8/8 pBx6 0/8 PBS 0-mer0/8
[389] These results indicate that AN1792 and AN1528 stimulate potent T cell responses most similar to the CD4 + phenotype. The absence of Aβ-specific T cells in animals inoculated with Aβ1-5 is surprising because peptide epitopes recognized by CD4 + T cells are typically about 15 amino acids long, although shorter peptides sometimes function with less efficiency. no. Thus, for the four conjugate peptides, most of the helper T cell epitopes appear to be located in the IgG conjugate partner, not the Aβ region. This assumption is supported by the very low incidence of proliferative responses for animals in each of these treatment groups. Since A [beta] 1-5 conjugates are effective while significantly reducing A [beta] levels in the brain even in the absence of A [beta] -specific T cells, the main effector immune response induced by immunization with these peptides appears to be antibodies.
[390] The lack of T-cells and the low antibody response from the fusion protein pBx6 comprising the APP amino acids 592-695, which includes all parts of the Aβ residues, may be due to the low immunogenicity of this particular agent. The low immunogenicity of Aβ25-35 aggregates is believed to be due to the peptide being so small that it cannot contain good T cell epitopes to help induce the antibody response. If this peptide is conjugated to a carrier protein, its immunogenicity may be greater.
[391] V. Preparation of Polyclonal Antibodies for Passive Protection
[392] 125 non-transformed mice were inoculated with Aβ and adjuvant and euthanized 4-5 months later. Blood was collected from the immunized mice. IgG was isolated from other blood components. Antibodies specific for immunogens were partially purified via affinity chromatography. An average of about 0.5-1 mg of immunogen specific antibody was obtained per mouse, totaling 60-120 mg.
[393] VI. Passive Immunization with Antibodies to Aβ
[394] Each group of PDAPP mice at 7 to 9 months of age was injected with 0.5 mg of polyclonal anti-Aβ or specific anti-Aβ monoclonal in PBS as described below. All antibody preparations were purified to have low endotoxin levels. Certain fragments by injecting mice with fragments or longer forms of Aβ, preparing hybridomas, and screening hybridomas for antibodies that specifically bind to the desired fragment of Aβ without binding to other overlapping fragments of Aβ. Monoclonal antibodies against can be prepared.
[395] AntibodiesEpitope 2H3Aβ1-12 10D5Aβ1-12 266Aβ13-28 21F12Aβ33-42 Mouse Polyclonal Anti-human Aβ42Anti-aggregated Aβ42
[396] Mice were intraperitoneally injected over 4 months as needed to ensure that circulating antibody concentrations measured by ELISA maintained titers greater than 1/1000 defined by ELISA for Aβ42 or other immunogens. Titers were monitored as described above and euthanized at the end of 6 months of inoculation. Immunohistologic analysis, Aβ level analysis and toxicity test were performed post mortem. Ten per group were used. Further experiments of passive immunization are described in Examples XI and XII below.
[397] VII. Comparison of Different Supplements
[398] This example compares the ability of CFA, alum, oil-in-water emulsions, and MPL to stimulate an immune response.
[399] A. Materials and Methods
[400] 1. Experimental design
[401] One hundred six-week-old Hartley species guinea pig females from Elm Hill Breeding Laboratories (Chelmsford, Mass.) Were divided into 10 groups and immunized with AN1792 or palmitoylated derivatives thereof combined with various adjuvants. Seven groups included a) PBS, b) Freund's adjuvant, c) MPL, d) squalene, e) MPL / squalene, f) low-dose alum, or g) high-dose alum (unless otherwise noted 33 Μg) was injected. Two groups were injected with a) PBS or b) palmitoylated derivatives of AN1792 (33 μg) combined with squalene. The last ten groups were injected with only PBS without antigen or additional adjuvant. For the group injected with Freund's adjuvant, the first dose was emulsified with CFA and the remaining four doses were emulsified with IFA. Antigen was administered at a dose of 33 μg for all groups, except for 300 μg of AN1792 in the high dose alum group. The injections were intraperitoneally injected for CFA / IFA and intramuscularly injected to the lateral quadriceps of the left and right sides for all other groups. The first three doses were given at two week intervals, followed by two doses at one month intervals. Beginning after the second inoculation, blood collection was performed 6 to 7 days after each inoculation to measure antibody titers.
[402] 2. Preparation of Immunogen
[403] 2 mg of Aβ 42 (California peptide, lot ME0339) was added to 0.9 mL of deionized water and shaken to form a relatively uniform suspension. To this was added 100 μl of an aliquot of 10 × PBS (1 × PBS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5). The suspension was shaken again and incubated overnight at 37 ° C. for use the next day. Unused Aβ1-42 was stored in the form of lyophilized powder with a desiccant at -20 ° C.
[404] Palmitoylated derivatives of AN1792 were prepared by binding palmitic anhydride dissolved in dimethyl formamide to the amino terminal residues of AN1792, followed by hydrofluoric acid to remove the initial peptide from the resin.
[405] To prepare a dosage formulation (group 2) comprising complete Freund's adjuvant (CFA), 33 μg of AN1792 in 200 μl of PBS for the first inoculation and 1: 1 (volume: volume) with CFA to a final volume of 400 μl. Emulsified. For subsequent inoculations, they were similarly emulsified with incomplete Freund's adjuvant (IFA).
[406] For groups 5 and 8, to prepare a dosage formulation comprising MPL, lyophilized powder (Livi Immunochem Research, Incorporated, Hamilton, MT) was added to 0.2% aqueous triethylamine to give a final concentration. 1 mg / ml and shaken. The mixture was heated at 65-70 ° C. for 30 seconds to give a slightly opaque uniform micelle suspension. This solution was prepared fresh for each set of injections. For each injection in Group 5, 33 μg AN1792, 50 μg MPL (50 μl) and 162 μl PBS in 16.5 μl PBS were mixed in a borosilicate tube immediately before use.
[407] To prepare a dosage formulation comprising a small oil-in-water suspension, AN1792 in PBS was added to 5% squalene, 0.5% Tween 80, 0.5% Span 85 in PBS to give a final single dose concentration of 33 μg AN1792 in 250 μl. (Group 6). This mixture was passed through a two-chamber hand-held fixture to emulsify 15-20 times until the diameter of the emulsion droplets looked almost the same as a standard latex bead of 1.0 μm diameter when viewed under a microscope. The suspension thus obtained was milky white, milky white. Emulsions were freshly prepared with each series of injections. For group 8, MPL in 0.2% triethylamine was added at a concentration of 50 μg per dose to the squalene and detergent mixture for emulsification as described above. For palmitoyl derivatives (group 7), 33 μg per palmitoyl-NH-Aβ1-42 dose was added to squalene and shaken. Tween 80 and Span 85 were then added with shaking. This mixture was added to PBS to a final concentration of 5% squalene, 0.5% Tween 80, 0.5% Span 85, and the mixture was emulsified as described above.
[408] To prepare a dosage formulation (groups 9 and 10) comprising alum, AN1792 in PBS was added to an alhydrogel (aluminum hydroxide gel, Accurate, Westberry, NY) to alum 5 in a 250 μl final dose volume. The concentration of AN1792 per mg was set to 33 μg (low dose, group 9) or 300 μg (high dose, group 10). This suspension was gently mixed for 4 hours at room temperature.
[409] 3. Determination of Antibody Titers
[410] A total of four blood samples were collected from guinea pigs about 6 to 7 days after the second inoculation. Antibody titers against Aβ 42 were measured by ELISA as described in Overall Materials and Methods.
[411] 4. Prepare the organization
[412] After about 14 weeks, all guinea pigs were euthanized by administering CO ₂ . Cerebrospinal fluid was collected and brains were removed to dissect three brain regions (the hippocampus, cortex and cerebellum) and used to measure the concentration of total Aβ protein using ELISA.
[413] B. Results
[414] 1. Antibody Reactions
[415] The range of efficacy of various adjuvants varied as measured as antibody response to AN1792 after inoculation. As shown in FIG. 14, when AN1792 was inoculated in PBS, no antibody was detected after the second or third inoculation, and the geometric mean titer (GMT) at the fourth and fifth inoculations was only about 45. A degree of response was detected. Oil-in-water emulsions induced moderate titers after the 3rd inoculation (GMT 255), which remained after the 4th inoculation (GMT 301) and decreased at the 5th inoculation (GMT 54). There was a clear antigenic response to 300 μg of AN1792 bound to alum, which was more immunogenic than 33 μg at all time points. At the peak of the antibody response after the fourth inoculation, the difference in the two doses was 43% with GMT of about 1940 (33 μg) and 3400 (300 μg). The antibody response to 33 μg AN1792 + MPL was similar to the extent produced by the nearly 10-fold higher dose of antigen (300 μg) bound to alum. Adding MPL to an oil-in-water emulsion reduced formulation efficacy by 75% compared to using MPL as a sole adjuvant. Palmitoylated derivatives of AN1792 were completely non-immunogenic when administered in PBS and exhibited moderate titers of GMT 340 and 105 for tertiary and quaternary blood collection when administered in oil-in-water emulsions. The highest antibody titer was seen when administered with Freund's adjuvant with a peak GMT of about 87,000, almost 30 times higher than the next two most potent agents, MPL and high dose AN1792 / alum GMT. It was.
[416] The most potent aids identified in this experiment were MPL and alum. Of these two, MPL is more preferred because, in the case of MPL, the dose of antibody required to produce the same antibody response obtained by alum is 10 times less. Response may be increased by increasing the dose of antigen and / or adjuvant and optimizing the inoculation schedule. The oil-in-water emulsion was a very weak adjuvant for AN1792, and adding the oil-in-water emulsion to the MPL adjuvant reduced the inherent adjuvant activity of MPL alone.
[417] 2. Aβ levels in the brain
[418] At about 14 weeks, guinea pigs were deeply anesthetized, cerebrospinal fluid (CSF) was taken out, and brains dissected from a subset of the groups, which were grouped with Freund's vaccination group (group 2) and MPL inoculation group (group 5). ), High dose alum inoculation group, AN1792 300 μg inoculation group (group 10) and PBS inoculation control group (group 3). To measure Αβ peptide levels, one hemisphere was dissected and homogenates of hippocampus, cortex and cerebellar regions in 5 M guanidine were prepared. They were diluted and quantified in comparison to serial dilutions of Aβ standard proteins of known concentration in ELISA format. The antibody response to Aβ induced by these agents was extensive, but Aβ protein levels in the hippocampus, cortex and cerebellum were very similar in all four groups. Mean Aβ levels were about 25 ng per g tissue in the hippocampus, 21 ng / g in the cortex and 12 ng / g in the cerebellum. Thus, the presence of high circulating antibody titers against Αβ for almost three months in some of these animals did not change their total Αβ levels in their brains. The levels of Αβ in CSF were also very similar among the groups. The lack of significant effect of AN1792 immunization on endogenous Αβ indicates that the immune response is concentrated on the pathogenesis of Αβ.
[419] VIII. Immune Responses to Different Adjuvant in Mice
[420] In this experiment, 6-week-old female Swiss Webster mice were used with 10 to 13 mice per group. At 0, 14, 28, 60, 90 and 20 days, the injection volume was subcutaneously injected at 200 μl. Seven days after each inoculation, starting after the second inoculation, blood was collected from these animals and antibody titers were analyzed using ELISA. The treatment of each group is summarized in Table 9.
[421]
[422] week:
[423] ^a The number of mice in each group at the start of the experiment
[424] ^b supplements. The buffer used for all these formulations was PBS. In group 8, no adjuvant and antigen were used.
[425] The ELISA titers of antibodies to Aβ 42 in each group are shown in Table 10 below.
[426]
[427] The table showed the highest titers were obtained for groups 4, 5 and 18, in which the adjuvant was 125 μg MPL, 50 μg MPL and QS-21 + MPL.
[428] IX. Therapeutic efficacy of different adjuvants
[429] Therapeutic efficacy experiments were performed in PDAPP transgenic mice using a set of adjuvants suitable for use in humans to determine whether they intensify the immune response to Aβ and induce immune mediated elimination of amyloid deposition in the brain. .
[430] The male and female heterozygous PDAPP transgenic mice of 7.5 to 8.5 months of age were obtained from Charles River Laboratories. Mice were divided into 9 groups with 15-23 mice per group, inoculated with AN1792 or AN1528 combined with various adjuvants. Animals were grouped to be as close as possible to gender, age, and ancestry within the group. Adjuvants included alum, MPL and QS-21, each combined with two antigens, and AN1792 only with Freund's adjuvant (FA). Another group was inoculated with no adjuvant AN1792 + preservative thimerosal prepared in PBS buffer. The ninth group was inoculated with only PBS as a negative control.
[431] Preparation of Aggregated Aβ Peptides: Human Aβ1-40 (AN1528; California Peptides, Napa, CA; Lot ME0541) and Human Aβ1-42 (AN1792; California Peptides, Lot ME0439) peptides Freshly solubilized for preparation of each injection set from lyophilized powder and stored moistureproof at -20 ° C. For this purpose, 2 mg of peptide was added to 0.9 ml of deionized water and the mixture was shaken to form a relatively uniform solution or suspension. Unlike AN1728, AN1528 was soluble at this stage. Then 100 μl aliquots of 10 × PBS (1 × PBS: 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5) were added, at which time AN1528 began to precipitate. The suspension was shaken again and incubated overnight at 37 ° C. for use the next day.
[432] To prepare a dosage formulation comprising groups of alum (groups 1 and 5), A peptide in PBS was added to an alhydrogel (2% aluminum hydroxide gel, positive incorporated, Clifton, NJ) to increase the concentration of alum. 100 μg of A peptide per mg. 10 × PBS was added so that the final dose volume was 200 μl in 1 × PBS. 10 × PBS was added to bring the final dose volume to 200 μl in 1 × PBS. This suspension was then mixed gently for about 4 hours at room temperature before injection.
[433] To prepare a dosage formulation (Groups 2 and 6) comprising MPL, lyophilized powder (Livi Immunochem Research, Incorporated, Hamilton, MT; Lot 67039-E0896B) was added to 0.2% aqueous triethylamine. Added to a final concentration of 1 mg / ml and shaken. The mixture was heated at 65-70 ° C. for 30 seconds to give a slightly opaque uniform micelle suspension. This solution was stored at 4 ° C. For each injection set, 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 before use.
[434] To prepare dosage formulations (groups 3 and 7) comprising QS-21, lyophilized powders (Aquilia, Framingham, MA; Lot A7018R) were added to PBS (pH 6.6-6.7) to give a final concentration of 1 MG / mL and shaken. This solution was stored at -20 ° C. For each set of injections, 100 μg of peptide in 50 μl of PBS per dose, 25 μg of QS-21 per dose in 25 μl of PBS and 125 μl of PBS per dose were mixed in the borosilicate tube immediately before use.
[435] To prepare a dosage formulation comprising Freund's adjuvant (group 4), 100 μg of AN1792 in 200 μl PBS was emulsified with complete Freund's adjuvant (CFA) and 1: 1 (volume: volume) for final inoculation. The volume was brought to 400 μl. For subsequent inoculations, they were similarly emulsified with incomplete Freund's adjuvant (IFA). For preparations with adjuvant alum, MPL or QS-21, 100 μg per dose of AN1792 or AN1528 is administered in a final volume of 200 μl PBS alum (1 mg per dose), MPL (50 μg per dose) or QS-21 ( 25 μg per dose) and subcutaneously injected between the shoulder line and the back. For the FA-administered group, 100 μg of AN1792 was emulsified with complete Freund's adjuvant (CFA) and 1: 1 (volume: volume) to a final volume of 400 μl followed by intraperitoneal injection for the first inoculation followed by five subsequent doses. Inoculation was boosted with the same amount of immunogen in incomplete Freund's adjuvant (IFA). For groups inoculated with AN1792 without adjuvant, 10 μg of AN1792 was combined subcutaneously with 5 μg thimerosal in a final volume of 50 μl PBS. In the ninth control group, only 200 μl of PBS was injected subcutaneously. The first three doses were given at 2 week intervals and then at 0, 16, 28, 56, 85 and 112 days at monthly intervals. Beginning after the second inoculation, blood collection was performed 6 to 7 days after each inoculation to measure antibody titers. Animals were euthanized about one week after the last inoculation. Results were determined by ELISA analysis of Aβ and APP levels in the brain and histological immunoassay of the presence of amyloid plaques in brain sections. In addition, Αβ specific antibody titers and Αβ dependent proliferative and cytokine responses were measured.
[436] Table 10 shows that the highest antibody titers against Aβ1-42 were induced by FA and AN1792, which titers peaked after the fourth inoculation (peak GMT: 75,386) and then decreased 59% after the last six inoculations. Shows. Peak averages induced by AN1792 and MPL were 62% lower than those produced with FA (peak GMT: 28,867), again reaching early in the inoculation plan, ie after three inoculations, and then peak values after the sixth inoculation Reduced to 28%. The peak mean titer produced by QS-21 in combination with AN1792 was about 5 times lower than that obtained in MPL (GMT: 1,511). In addition, the rate of reaction was slower, since further inoculation was required to reach the peak response. The titer formed by alum bound AN1792 was slightly higher than the titer obtained by QS-21 and the reaction rate was faster. For AN1792 delivered in PBS with thimerosal, the frequency and titer size were slightly larger than with PBS alone. The peak titers generated for MPL and AN1528 were about 9 times lower than those produced by AN1792. Alum-coupled AN1528 showed very low immunogenicity, showing low titers produced only in some of the animals. No antibody response was observed in control rounds inoculated with PBS only.
[437]
[438] week:
[439] geometric mean antibody titers measured against ^a Αβ1-42
[440] ^b Number of animals responding per group
[441] The results measured by ELISA of treatment of AN1792 or AN1528 with various adjuvants, or thimerosal, on cortical amyloid deposition in 12-month-old mice are shown in FIG. 15. In PBS control PDAPP mice, the median level of total Aβ in the cortex at 12 months was 1,817 ng / g. Significantly decreased levels of Aβ were observed in mice treated with AN1792 + CFA / IFA, AN1792 + alum, AN1792 + MPL, and QS-21 + AN1792. Only in the case of AN1792 + CFA / IFA, the reduction reached a statistically significant level (p <0.05). However, as shown in Examples I and III, the effect of immunization in reducing Aβ levels was substantially further increased in mice at 15 months and 18 months of age. Thus, at least AN1792 + alum, AN1792 + MPL and AN1792 + QS-21 compositions are expected to reach statistically significant levels in the treatment of older mice. In contrast, AN1792 + preservative thimerosal showed almost the same levels of Aβ mean as in PBS treated mice. Similar results were obtained when comparing the cortical levels of Aβ 42. The median level of Aβ 42 in the PBS control group was 1624 ng / g. Significantly reduced intermediate levels 403, 1149, 620 and 714 were observed in mice treated with AN1792 + CFA / IFA, AN1792 + alum, AN1792 + MPL and AN1792 + QS-21, respectively, for the AN1792 CFA / IFA treated group The reduction reached a statistically significant level (p = 0.05). The median level in AN1792 thimerosal treated mice was 1619 ng / g Aβ42.
[442] X. Toxicity Assay
[443] Tissues were harvested for histopathology at the end of the experiments described in Examples II, III and VII. In addition, hematological and clinical chemistry analyzes were performed on the final blood samples of Examples III and VI. Most of the major organs were evaluated, including the brain, lungs, lymphocytes, gastrointestinal tract, liver, kidneys, adrenal glands and gonads. Sporadic lesions were observed in the experimental animals, but there were no obvious differences in the damaged tissue or lesion severity between AN1792 treated and untreated animals. No specific histopathological lesions were observed in AN-1528 immunized animals compared to PBS treated or untreated animals. There was no difference in the clinical chemistry profile between the adjuvant group and the PBS treated animals in Example VI. Although there was a significant increase in some of the hematology parameters between AN1792 and Freund's adjuvant compared to PBS treated animals in Example VI, this kind of effect is predictable from Freund's adjuvant and associated peritonitis. It does not show any adverse effects resulting from AN1792 treatment. Although not part of the toxicity assessment, PDAPP mouse brain pathology has been widely observed as part of the efficacy endpoint. No signs of adverse effects on treatment-related brain morphology emerged during the experiment. These results indicate that AN1792 treatment is well tolerated and at least substantially free of side effects.
[444] XI. Therapeutic Treatment with Anti-Aβ Antibodies
[445] The experiments described in this section were performed to test the ability of various monoclonal and polyclonal antibodies to inhibit A [beta] accumulation in the brain of heterozygous transgenic mice.
[446] A. Experiment 1
[447] 1. Experimental design
[448] Sixty male and female heterozygous PDAPP transgenic mice, aged 8.5-10.5 months, were obtained from the Charles River Laboratories. Mice were divided into 6 groups and treated with various antibodies to Aβ. Animals were distributed in groups as close as possible to sex, age, household and source. As shown in Table 12, the antibodies are four murine Aβ-specific monoclonal antibodies, 2H3 (for Aβ residues 1-12), 10D5 (for Aβ residues 1-16), 266 (Aβ residues 13- 28, binding to monomer AN1792, not aggregated AN1792), 21F12 (for Aβ residues 33-42). The fifth group was treated with Aβ-specific polyclonal antibody fractions (obtained by inoculation with aggregated AN1792). Negative controls were inoculated only with diluent PBS without antibody.
[449] Monoclonal antibodies were injected at a dose of about 10 mg / kg (assume 50 g of mouse weight). Inoculation by intraperitoneal injection every 7 days averaged to maintain anti-Aβ titers of 1000 or more. Lower titers were measured for mAb266 because they did not bind well to aggregated AN1792 used as capture antigen in the assay, but the same inoculation schedule was maintained in this group. The group receiving monoclonal antibody 2H3 was discontinued within the first three weeks because the antibody was removed too quickly in vivo. Blood was drawn from animals before each inoculation for determination of antibody titers. Treatment continued over six months for a total of 196 days. Animals were euthanized one week after the last inoculation.
[450] Experiment design Treatment groupNaTreatment antibodyAntibody SpecificityAntibody isotype One9No (PBS only)NA ^b NA 210PolyclonalAβ1-42mix 30mAb ^c 2H3A1β-12IgG1 48mAb10D5A1β-16IgG1 56mAb266Aβ13-28IgG1 68mAb21F12Aβ33-42IgG2 ^a
[451] week
[452] a. Number of mice in group at end of experiment. All groups started with 10 per group.
[453] b. NA: Not applicable.
[454] c. mAb: monoclonal antibody
[455] 2. Materials and Methods
[456] a. Preparation of Antibodies
[457] Anti Aβ polyclonal antibodies were prepared from blood taken from two groups of animals. The first group consisted of 100 female Swiss Webster mice, 6-8 weeks old. These animals were inoculated with 100 μg of AN1792 in combination with CFA / IFA on days 0, 15 and 29. The fourth inoculation was inoculated with 1/2 dose of AN1792 at 36 days. Animals were sacrificed at 42 days and then bled to obtain serum, which was collected to a total of 64 ml. The second group consisted of PDAPP mice and 24 female mice of the same genotype, with the exception of nontransgenic mice directed to human APP genes of 6 to 9 weeks of age. These animals were inoculated with 100 μg of AN1792 combined with CFA / IFA on days 0, 14, 28 and 56. These animals were also sacrificed and bled on day 63 to prepare serum and collected to a total of 14 ml. Two lots of serum were collected. The antibody fraction was purified by two successive precipitations with 50% saturated ammonium sulfate. The final precipitate was dialyzed against PBS and tested for endotoxin. The level of endotoxin was less than 1 EU / mg.
[458] Anti-Aβ monoclonal antibodies were prepared from ascites fluid. To the ice-cold aqueous solution, concentrated sodium dextran sulfate was first added and stirred on ice to remove lipids to a final concentration of 0.238%. The concentrated CaCl ₂ was centrifuged at 10,000 × g and the pellet was discarded. This solution was centrifuged at 10,000 xg and the pellet removed. The supernatant was stirred on ice with the addition of saturated ammonium sulfate in the eastern blood. This solution was again centrifuged at 10,000 xg and the supernatant was removed. 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 to elute with a reverse concentration gradient of 0.275 M NaCl at 0.4 M in 20 mM Tris-HCl, pH 7.5.
[459] Absorbance at 280 nm confirmed antibody peaks and the appropriate fractions were collected. Purified antibody preparations were characterized by measuring protein concentration using the BCA method and purity using SDS-PAGE. This pool was also tested for endotoxin. The level of endotoxin was less than 1 EU / mg. Titers below 100 randomly assigned a titer value of 25.
[460] 3. Aβ and APP levels in the brain
[461] After about 6 months of treatment with various anti-Αβ antibody preparations, saline perfusion was performed and brains were removed from the animals. One hemisphere was prepared for immunohistologic analysis and the other was used for quantification of Aβ and APP levels. To measure the concentration of various forms of beta amyloid peptide and amyloid precursor protein (APP), hemispheres were developed and homogenates of hippocampus, cortex and cerebellar regions were prepared in 5 M guanidine. These were serially diluted and quantified amyloid peptide or APP levels compared to serial dilutions of known concentrations of Aβ peptides or APP standards in ELISA format.
[462] The levels of total Aβ and Aβ1-12 and total Aβ in the cerebellum, as measured by ELISA, are shown in Tables 11, 12 and 13, respectively. The median concentration of total Aβ was 3.6-fold higher in the hippocampus than in the hippocampal control group (median 63,389 ng / g of hippocampal tissue, median of cortex 17,818 ng / g). The median level of cerebellum in the control group (30.6 ng / tissue g) was about 2,000 times lower than in the hippocampus. This level is similar to what we previously reported for heterozygous PDAPP transgenic mice of this age (see Johnson-Woods et al., 1997, supra).
[463] For the cortex, one treatment group showed significantly different (p <0.05) median Aβ levels from those of the control group, as measured by Aβ1-42. These animals showed polyclonal anti-Aβ antibodies as shown in FIG. Was administered. The median level of Aβ1-42 was reduced by 65% compared to the control for this treatment group. The median level of Aβ1-42 was also significantly reduced by 55% compared to the control group in another treatment group (animals receiving mAb 10D5) (p = 0.0433).
[464]
[465] week:
[466] a. Number of animals per group at the end of the experiment
[467] b. ng / tissue (g)
[468] c. Man Whitney Analysis
[469] d. NA: Not applicable
[470] e. Standard Deviation
[471] In the hippocampus, the median percentage reduction (50%, p = 0.0055) associated with polyclonal anti Aβ antibody treatment was not as large as that observed in the cortex (65%). However, the absolute magnitude of the reduction was almost three times greater in the hippocampus than in the cortex, with a net reduction in the hippocampus of 31,683 ng per gram of tissue and 11,658 ng per gram of tissue in the cortex. When measured as the level of Aβ, Aβ1-42, which is the larger Aβ form of amyloidosis rather than total Aβ, the reduction made by polyclonal antibodies was significant (p = 0.0025). Median levels of the groups treated with mAbs 10D5 and 266 were reduced by 33% and 21%, respectively.
[472]
[473] week:
[474] a. Number of animals per group at the end of the experiment
[475] b. ng / tissue (g)
[476] c. Man Whitney Analysis
[477] d. NA: Not applicable
[478] e. Standard Deviation
[479] Total Aβ was also measured in the cerebellum (Table 15). The group receiving polyclonal anti Aβ and 266 antibodies showed a significant decrease in the level of total Aβ (43% and 46%, respectively, p = 0.0033 and p = 0.0184), and the group treated with 10D5 had a significant level. A decrease close to (29%, p = 0.0675).
[480]
[481] week:
[482] a. Number of animals per group at the end of the experiment
[483] b. ng / tissue (g)
[484] c. Man Whitney Analysis
[485] d. NA: Not applicable
[486] e. Standard Deviation
[487] APP concentrations were also measured by ELISA in the cortex and cerebellum obtained from antibody-treated, control, and PBS-treated mice. Two different APP assays were used. The first assay, called APP-α / FL, recognizes both APP-alpha (α, the secreted form of APP cleaved within the Aβ sequence) and the full-length form of APP (FL), while the second assay recognizes APP-α. Recognize only. In contrast to Aβ reduction associated with treatment in a subset of treatment groups, APP levels did not change in all treatment groups as compared to control animals. These results indicate that immunization with Αβ antibodies did not deplete APP, but rather depleted Αβ.
[488] In summary, total Aβ was significantly reduced in the cortex, hippocampus and cerebellum in animals treated with polyclonal antibodies against AN1792. Monoclonal antibodies against the amino terminal regions of Aβ1-42, particularly amino acids 1-16 and 13-28, also showed a less significant therapeutic effect.
[489] 4. Immunohistologic Analysis
[490] The morphology of Aβ immunoreactive plaques in a subset of brains from mice in PBS, polyclonal Aβ42, 21F12, 266 and 10D5 treated groups was quantitatively compared to that of previous experiments that performed standard immunization procedures with Aβ42.
[491] The largest change in both the degree and appearance of amyloid plaques occurred in animals inoculated with polyclonal Aβ42 antibody. Amyloid deposition, corroded plaque morphology and cell mediated Αβ immunoreactivity were very similar to the effects produced by standard immune procedures. This observation supports the ELISA results that a significant reduction in both total Aβ and Aβ42 is achieved by inoculation of polyclonal A42 antibodies.
[492] In a similar quantitative evaluation, amyloid plaques of the 10D5 group also decreased in number and appearance, demonstrating some cell-mediated Aβ immunoreactivity. No significant difference was observed when the 21F12 and 266 groups were compared with the PBS group.
[493] 5. Determination of Antibody Titers
[494] A subset of three randomly selected mice from each group was bled 30 times prior to each intraperitoneal injection. Antibody titers were measured as Aβ1-42 binding antibodies using a sandwich ELISA in plastic multi-wells coated with Aβ1-42 as described in the overall materials and methods. Average titers for each blood collection are shown in FIGS. 16-18 for polyclonal antibodies and monoclonal 10D5 and 21F12, respectively. Titer averaged over 1000 over this period for polyclonal antibody preparations and slightly higher than this level for 10D15 and 21F12 treated animals.
[495] 6. Lymphocytic reaction
[496] Ap-dependent lymphoproliferative responses were measured using splenocytes recovered 8 days after the last antibody inoculation. Freshly harvested cells (10 ^{5 per} well) were incubated for 5 days in the presence of 5 μM concentration of Aβ1-40 for stimulation. As a positive control, another cell was incubated with T cell mitogen, PHA, and as a negative control it was incubated without adding peptide.
[497] Splenocytes from old PDAPP mice passively immunized with various Αβ antibodies were stimulated in vitro with AN1792 and proliferative and cytokine responses were measured. The purpose of these assays was to determine whether passive immunization facilitated antibody presentation, thereby initiating T cell responses specific for AN1792. No AN1792 specific proliferative or cytokine responses were observed in any mice passively immunized with anti-Αβ antibody.
[498] B. Experiment 2
[499] In the second experiment, the treatment of 10D5 was repeated and two other anti-Αβ antibodies, monoclonal 3D6 (Aβ _1-5 ) and 16C1 (Aβ _1-5 ), were tested. The control group was administered with PBS or an unrelated isotype pair of antibodies (TM2a). Mice were older than in the previous experiment (11.5-12 months of age heterozygosity) and the rest of the experimental design was the same. Once again, after 6 weeks of treatment, 10D5 had reduced plaque deposition by at least 80% compared to the PBS or isotype match antibody controls (p = 0.003). One of the other antibodies against Aβ, 3D6 was equally effective, reducing 86% (p = 0.003). In contrast, the third antibody against peptide, 16C11, had no effect on plaque deposition. Similar results were obtained for Aβ42 ELISA measurements. These results indicate that although the antibody response to Αβ peptide is sufficient to reduce amyloid deposition in PDAPP mice in the absence of T cell immunity, not all anti Αβ antibodies are potent. Antibodies to epitopes comprising amino acids 1-5 or 3-7 of Aβ were particularly effective.
[500] These experiments showed that the antibodies against passively administered Αβ reduced the degree of plaque deposition in mouse models of Alzheimer's disease. When maintained at an appropriate blood serum concentration (25-70 μg / ml), the antibody could access the CNS to a degree sufficient to adorn amyloid plaques. The access of the antibody to the CNS was not due to abnormal outflow of the blood flow-brain barrier, as the vascular permeability did not increase as measured by Evans Blue in PDAPP mice. In addition, the antibody concentration in the brain parenchyma of old PDAPP mice was the same as in the nontransgenic mice, and the concentration of antibody in serum was 0.1% (regardless of isotype).
[501] C. Experiment 3: Monitoring of Antibody Binding
[502] To determine whether antibodies against Αβ can act directly in the CNS, the presence of antibodies administered systemically to the brain taken from saline-perfused mice later in Example XII was observed. Unfixed cryogen brain sections were exposed to fluorescent material for mouse immunoglobulin (goat anti-mouse IgG-Cy3). Plaques in the brain of the 10D5 and 3D6 groups were heavily decorated with antibodies but were not stained in the 16C11 group. To determine the overall extent of plaque deposition, serial sections of each brain were first immunized with anti-Αβ antibody and then with secondary material. After systemic administration, 10D5 and 3D6 were able to access most plaques in the CNS. Plaque deposition was significantly reduced in these treatment groups compared to the 16C11 group. These data indicate that systemically administered antibodies can invade the CNS where they can directly initiate amyloid clearance. 16C11 also has access to plaques but appears to be unable to bind.
[503] XVII. Prevention and Treatment of Human Subjects
[504] Single dose Phase I trials are performed to determine stability in humans. The therapeutic agent is administered to different patients with increasing dose at a rate of 3 starting at the expected efficacy level of about 0.01 to a level of about 10 times reaching the mouse effective amount.
[505] Phase II trials are performed to measure the efficacy of treatment. Alzheimer's disease and related disorder combination (ADRDA) criteria for possible AD are used to select patients from early to intermediate Alzheimer's disease. Appropriate patients were scored in the 12-26 range on the Mini-Mental State Exam (MMSE). Another screening criterion is that there are no complex problems such as the use of concomitant medications that may survive and interfere with the duration of the trial. Baseline assessment of patient function was performed using classical psychometric methods such as MMSE and ADAS, which is an overall scale for assessing patients with Alzheimer's status and action. This psychometric measure is a measure of progression of Alzheimer's status. Appropriate quantitative life scale may be used to monitor the treatment. Disease progression can also be monitored by MRI. Blood profiles of patients can also be monitored, including analysis of immunogen specific antibodies and T-cell responses.
[506] After baseline measurement, the patient begins treatment. Patients are randomly divided to treat the treatment or placebo blindly. Patients are monitored at least every six weeks. Efficacy is determined to be that the progress of the treatment group is significantly reduced compared to the placebo group.
[507] The second phase II trial assesses the transition to possible Alzheimer's disease, as defined by the ADRDA criteria, in patients with non-Alzheimer's disease early memory loss, often referred to as age-related memory disorder (AAMI) or mid-term mild cognitive impairment (MCI) To be performed. Screening controls for early signs of memory loss or other difficulties associated with pre-Alzheimer's syndrome, family history of Alzheimer's disease, genetic risk factors, age, sex and other features found to predict high risk of Alzheimer's disease Patients at high risk for screening are selected. Baseline scores on appropriate matrices, including MMSE and ADAS, are collected along with other matrices designed to assess more normal populations. This patient population is divided into appropriate groups to have a placebo comparison group for alternative administrations of the formulation. Performed in this patient population at about 6 month intervals to determine if at each patient's endpoint they transition to the end of observation a possible Alzheimer's disease as defined by the ADRDA criteria.
[508] XIII. Overall materials and methods
[509] 1. Measurement of Antibody Titer
[510] A small scratch was taken on the tail vein of the mouse to collect blood and about 200 μl of blood was collected in a microfuge tube. First, the hair of the hind limb segment was shaved, and then, using an 18 gauge needle, the guinea pig blood was collected in a microfuge tube by puncturing into the metatarsal vein. The blood was left at room temperature for 1 hour to allow coagulation, shaking and centrifugation at 14,000 x g for 10 minutes to separate coagulum from serum. The serum was then transferred to a clean microfuge tube and stored at 4 ° C. until titer was measured.
[511] Antibody titers were measured by ELISA. 96-well microtiter plates (Costa EIA plates) were plated in well coating buffer (0.1 M sodium phosphate, pH 8.5, 0.1% sodium azide) with 10 μg / ml of Aβ42 or SAPP or as described in each individual report. Coated with 100 μl of solution) and left overnight at room temperature. Wells were aspirated and serum was added to the wells beginning with a 1/100 dilution in sample dilutions (0.014 M sodium phosphate, pH 7.4, 0.15 NaCl, 0.6% bovine serum albumin, 0.05% thimerosal). Seven consecutive dilution samples were diluted directly on the plate in three steps to finally dilute to 1 / 218,700. Dilutions were incubated in coated plate wells for 1 hour at room temperature. The plates were then washed four times with PBS containing 0.05% Tween 20. Goat anti-mouse Ig conjugated to a second antibody, horseradish peroxidase (by Schöllinger Mannheim), was added to the wells at 100 μl of a 1/3000 dilution in sample buffer and incubated for 1 hour at room temperature. The plate was again washed four times with PBS, Tween 20. To develop the colorant, slow TMB (3,3 ', 5,5'-tetramethyl benzidine from Pierce Chemicals) was added to each well and incubated for 15 minutes at room temperature. The reaction was stopped by adding 25 μl 2 MH ₂ SO ₄ . Thereafter, the color density was read at 450-650 nm on the molecular device Vmax.
[512] Titer was defined as the inverse of the dilution of serum representing half of the maximum OD. Maximum OD was generally taken at the first 1/100 dilution except for very high titers, where higher initial dilutions were required to establish the maximum OD. If the 50% point is located between the two dilutions, a straight line estimate is drawn to calculate the final titer. To calculate the geometric mean antibody titers, titers less than 100 were arbitrarily set to titers of 25.
[513] 2. Lymphocyte proliferation assay
[514] Mice were anesthetized with isoflurane. The spleen is removed and washed twice with 5 ml of PBS (PBS-FBS) containing 10% heat inactive fetal bovine serum, which is then 50 ° centricon unit (Dako A / S, Denmark) in medicine (Dako). Homogenized for 10 seconds at 100 rpm in 1.5 ml of PBS-FBS at and filtered through a 100 micron diameter nylon mesh. The splenocytes were washed once with 15 ml of PBS-FBS and then centrifuged at 200 × g for 5 minutes to make pellets. Erythrocytes were lysed by resuspending the pellet for 5 minutes at room temperature in 5 ml of buffer containing 0.15 M NH ₄ Cl, 1 M KHCO ₃ , 0.1 M NaEDTA, pH 7.4. Thereafter, the white blood cells were washed as described above. Freshly isolated spleen cells (10 ⁵ cells per well) were tripled in RPMI 1640 medium (JHR Biosciences, Renex, KS) supplemented with 2.05 mM L glutamine, 1% penicillin / streptomycin and 10% heat inactive FBS. 96 well U bottom tissue culture treated microtiter plates (Corning, Cambridge, MA) were incubated at 37 ° C. for 96 hours. Various A [beta] peptides, A [beta] 1-16, A [beta] 1-40, A [beta] 1-42 or A [beta] 40-1 retrograde sequence proteins were also added in dose steps ranging from 5 to 0.18 micromolar in four steps. Cells from control wells were incubated with Concanavalin A (ConA) (Sigma, Cat. No. C-5275, 1 μg / ml) without the addition of protein. Cells were pulsed for the last 24 hours with 3H-thymidine (1 μCi / well, Amersham Corporation, Arlington Heights, IL). Cells were then harvested into unifilter plates and counted on a top count microplate scintillation counter (Packard Instruments, Downers Grove, IL). The results are expressed in counts per minute (cpm) of radioactivity incorporated into insoluble macromolecules.
[515] 4. Brain Tissue Preparation
[516] After anesthesia, the brain was taken out and one hemisphere was prepared for immunohistologic analysis, and three brain regions (the hippocampus, the cortex and the cerebellum region) were cut from the other hemisphere and a special ELISA (see Johnson-Wood et al.). It was used to measure the concentration of various Aβ protein and APP form.
[517] Tissues for ELISA were homogenized in 10-fold volume of ice cold guanidine buffer (5.0 M guanidine-HCl, 50 mM Tris-HCl, pH 8.0). Homogenates were mixed with gentle shaking using Adams Neuter (Fischer) at room temperature for 3-4 hours and then stored at -20 ° C prior to quantification of Aβ and APP. Preliminary experiments showed that the analytes were stable under these storage conditions and that synthetic Aβ protein (Bachem) could be recovered quantitatively when spiked into homogenates of control brain tissue from single-row mice (Johnson-S., Supra). Wood et al.).
[518] 5. Measurement of Aβ Level
[519] 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 lupetin), and then 16,000 Centrifuged at 4 ° C. for 20 minutes at xg. 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. A "total" Aβ sandwich ELISA uses a monoclonal antibody monoclonal antibody 266 (Seubert et al.) Specific for amino acids 13-28 of Aβ as a capture antibody and a biotinylated monoclonal specific for amino acids 1-5 of Aβ Null antibody 3D6 was used as reporter antibody. The 3D6 monoclonal antibody does not recognize secreted APP or full-length APP, but detects only Aβ species bearing amino terminal aspartic acid. The lower sensitivity limit of this assay is ˜50 ng / ml (11 nM) and does not show cross-reactivity with endogenous murine Aβ protein at concentrations of 1 ng / ml or less (Johnson-Wood et al.).
[520] Aβ1-42 specific sandwich ELISA uses mAβ21F12 specific for amino acids 33-42 of Aβ as a capture antibody (Johnson-Wood et al., Supra). Biotinylated mAβ3D6 is also a reporter antibody in this assay, with a lower sensitivity limit of about 125 μg / ml (28 μM, Johnson-Wood et al.). For AβELISA, 100 μl of mAβ266 (10 μg / ml) or mAβ21F12 (5 μg / ml) was coated on 96 well immunoassay plates (Costa) and incubated overnight at room temperature. This solution was removed by inhalation and added to the wells with 200 μl of 0.25% human serum albumin in PBS buffer and left for 1 hour at room temperature for blocking. The blocking solution was removed and stored moistureproof at 4 ° C. until the plate was used. The plates were rehydrated with wash buffer [Tris buffered saline (0.15 M NaCl, 0.01 M Tris-HCl, pH 7.5) + 0.05% Tween 20). Samples and standard samples were added in triplicate aliquots of 100 μl per well and then incubated at 4 ° C. overnight. Plates were washed three more times with wash buffer at each assay step. Biotinylated mA 3D6 diluted to 0.5 μg / ml in casein assay buffer (0.25% casein, PBS, 0.05% Tween 20, pH 7.4) was added to the wells and incubated for 1 hour at room temperature. Avidin-horseradish peroxidase conjugate (Avidin-HRP, Vector, Burlinggame, CA) diluted 1: 4000 in casein assay buffer was added to the wells and left for 1 hour at room temperature. Slow TMB-ELISA (Pierce), a chromogenic substrate, was added and allowed to react at room temperature for 15 minutes, and then 25 μl of 2 NH ₂ SO ₄ was added to stop the enzyme reaction. The reaction product was quantified by measuring the difference in absorbance at 450 nm and 650 nm using Molecular Devices Vmax.
[521] 6. Measurement of APP level
[522] Two different APP assays were used. The first analysis, called APP-α / FL, recognizes both APP-alpha (α) and the full length (FL) type of APP. The second assay is specific only to APP-α. The APP-α / FL assay recognizes secreted APP including the first 12 amino acids of Aβ. Since the reporter antibody (2H3) is not specific for the α-clip-site that occurs between amino acids 612-613 of APP695 (Esch et al., Science 248, 1122-1124 (1990)), this assay is a full length APP ( APP-FL) is also recognized. Preliminary experiments with immobilized APP antibodies against the cytoplasmic tail of APP-FL to deplete brain homogenates of APP-FL indicate that about 30-40% of APP-α / FL APP is FL (data not shown) ). The capture antibody for both APP-α / FL and APP-α assays is mAb 8E5 generated against 444-592 in APP695 form (Games et al., Supra). The reporter mAb for APP-α / FL analysis is mAb 2H3 specific for amino acids 597-608 of APP695 (Johnson-Wood et al., Supra), and the reporter antibody for APP-α analysis is directed to amino acids 605-611 of APP. The resulting biotinylated mAb 16H9 derivative. The lower limit of the sensitivity of the APP-αFL assay is about 11 ng / ml (150 pM) (Johnson-Wood et al.) And the lower limit of the sensitivity of the APP-α specific assay is 22 ng / ml (0.3 nM). In both APP assays, mAb 8E5 was coated in wells of 96 well EIA plates as described above for mAb 266. Secreted APP-α of purified recombination was used as a reference for APP-α analysis and APP-α / FL analysis (Esch et al., Supra). Brain homogenate samples in 5 M guanidine were diluted 1:10 in ELISA sample dilutions (0.014 M sodium phosphate, pH 7.4, 0.6% bovine serum albumin, 0.05% thimerosal, 0.5 M NaCl, 0.1% NP40). Then they were diluted 1: 4 in sample buffer containing 0.5 M guanidine. The diluted homogenates were then centrifuged at 16,000 x g for 15 seconds at room temperature. APP standards and samples were added to the plates in duplicate aliquots and incubated for 1.5 hours at room temperature. Biotinylated reporter antibody 2H3 or 16H9 was incubated for 1 hour at room temperature. Streptavidin-alkaline phosphatase (Charlinger Mannheim) diluted 1: 1000 in sample dilutions was incubated in the wells for 1 hour at room temperature. Fluorescent substrate 4-methyl-umbeliferyl-phosphate was incubated for 30 minutes at room temperature and the plate was read with 365 nm excitation and 450 nm emission on a cytofluor tm 2350 fluorometer (Millipore).
[523] 7. Immunohistochemical Analysis
[524] Brains were fixed for 3 days at 40 ° C. in 4% paraformaldehyde in PBS, then stored at 4 ° C. for 1-7 days until cleavage in 1% paraformaldehyde, PBS. 40 μm thick parietal sections were cut in vibratom at room temperature and stored in a cryostat (30% glycerol in phosphate buffer, 30% ethylene glycol) at −20 ° C. prior to immunohistochemical analysis. For each brain, six sections at hippocampus levels, each separated at consecutive 240 μm intervals, were incubated overnight with one of the following antibodies: (1) 2 μg / ml in PBS and 1% horse serum. Biotinylated anti Aβ diluted at concentrations (mAb, 3D6, specific for human Aβ); Or (2) biotinylated mAb specific for human APP, 8E5, diluted to 3 μg / ml concentration in PBS and 1.0% horse serum; Or (3) mAb (GFAP; Sigma Chemical Company) specific for glial fibrillar protein diluted 1: 500 with 0.25% Triton X-100 and 1% horse serum in Tris buffered saline (pH7.4) (TBS). ; (4) CD11b, a mAb specific for MAC-1 antigen (Chemicon International) diluted 1: 100 with 0.25% Triton X-100 and 1% rabbit serum in TBS; Or (5) mAbs specific for MHC II antigen (Pharmingen) diluted 1: 100 in 0.25% Triton X-100 and 1% rabbit serum in TBS; Or (6) rat mAbs specific for CD43 (pharmingen) diluted 1: 100 with 1% rabbit serum in PBS; Or (7) rat mAbs specific for CD45RA diluted 1: 100 with 1% rabbit serum in PBS (Pharmingen); Or (8) rat monoclonal Αβ (pharming) specific for CD45RB diluted 1: 100 with 1% rabbit serum in PBS; Or (9) rat monoclonal Αβ (pharming) specific for CD45 diluted 1: 100 with 1% rabbit serum in PBS; Or (10) biotinylated polyclonal hamster Aβ (pharmingen) specific for CD3e diluted 1: 100 with 1% rabbit serum in PBS; Or (11) rat mAbs specific for CD3 (Serotec) diluted 1: 200 with 1% rabbit serum in PBS; Or PBS solution without primary antibody comprising 1% normal horse serum.
[525] The sections reacted with the antibody solutions described in 1,2 and 6-12 were pretreated for 20 minutes at room temperature with 1.0% Triton X-100, 0.4% hydrogen peroxide in PBS to block endogenous peroxidase. They were then incubated with primary antibody overnight at 4 ° C. Then, the components reacted with 3D6 or 8E5 or CD3e mAb were diluted to 1:75 in PBS with kit components "A" and "B" and horseradish peroxidase-avidin-biotin-complex (vector elite standard kit, vector Rabs, Burlingame, CA) were reacted at room temperature for 1 hour. Sections reacted with antibodies specific for CD 45RA, CD 45RB, CD 45, CD3 and PBS solution without primary antibody were diluted 1:75 in PBS, respectively 1:75 in anti-rat IgG (vector) or PBS Dilute with biotinylated anti-mouse IgG (vector) for 1 hour at room temperature. Next, the kit components "A" and "B" diluted sections at 1:75 in PBS and the horseradish peroxidase-avidin-biotin-complex (Vector Elite Standard Kit, Vector Labs, Burlingame, CA) and room temperature The reaction was carried out for 1 hour at.
[526] Sections were developed at room temperature in 0.01% hydrogen peroxide, 0.05% 3,3'-diaminobenzidine (DAB). Sections for incubation with GFAP-, MAC-1- and HMC II-specific antibodies were pretreated with 0.6% hydrogen peroxide at room temperature to block endogenous peroxidase and then incubated with primary antibody overnight at 4 ° C. Sections reacted with GFAP antibody were incubated for 1 hour at room temperature with biotinylated anti-mouse IgG (Vector Laboratories; Baxtastein Elite ABC Kit) produced in horses diluted 1: 200 in TBS. . The sections were then reacted with the avidin-biotin-peroxidase complex (Vector Laboratories; Vitastein Elite ABC Kit) diluted 1: 1000 in TBS for 1 hour. Sections incubated with MAC-1 or MHC II-specific monoclonal antibodies as primary antibodies reacted with biotinylated anti-rat IgG produced in rabbits diluted 1: 200 in TBS for 1 hour at room temperature It was then incubated with avidin-biotin-peroxidase complex diluted 1: 1000 with TBS for 1 hour. The incubated sections with GFAP-, MAC-1- and MHC II-specific antibodies were then treated with 0.05% DAB, 0.01% hydrogen peroxide, 0.04% nickel chloride at room temperature, respectively, and then for 11 minutes at 4 ° C. with TBS. Treated and visualized.
[527] Immunolabeled sections were mounted on glass slides (VWR, superfrost slides), dried in air overnight, and immersed in propar (anatec) to cover coverslips with permount (Fisher) as mounting medium.
[528] To reverse stain Aβ plaques, a subset of GFAP-positive sections were mounted on superfrost slides and incubated for 7 minutes in aqueous 1% thioflavin S (Sigma) followed by immunohistochemical analysis. The sections were then dehydrated and the profar removed, and then covered with coverslips mounted with permount.
[529] 8. Image Analysis
[530] A Videometric 150 image analysis system (Onco Inc., Gatorsburg, MD), connected to a Nikon MyProport-FX microscope via a CCD video camera and a Sony Trinitron monitor, was used to quantify immunoreactive slides. Images of the sections were stored in a video buffer and thresholds based on color and saturation were measured to select and calculate total pixels occupied by immunolabeled structures. For each section, the hippocampus was manually outlined to calculate the total pixel area occupied by the hippocampus. % Amyloid deposition was determined as x100 (part of the hippocampal region comprising Aβ deposits immunoreactive with mAb 3D6). Similarly, the percentage of neuritis deposits was determined as x100 (part of the hippocampal region including dystrophic neurons reactive with monoclonal antibody 8E5). C-imaging systems (Complex Incorporated, Canbury Township, PA), which run the Simple 32 Software Application System program, are connected to a Nikon Microport-FX microscope via an Optronics camera, GFAP-positive astrocytes, and It was used to quantify the proportion of inverted cortex occupied by MAC-1- and MHC II-positive microglia. Images of the immunoreacted sections were stored in a video buffer and monochromatic thresholds were determined to screen and calculate the total pixel area occupied by immunolabeled cells. For each section, the inverted plate cortex (RSC) was manually outlined and the total pixel area occupied by the RSC was calculated. The percentage of astrocytosis was defined as (part of RSC occupied by GFAP reactive stellate cells) x 100. Similarly, the percentage of microglia was defined as x) (part of RSC occupied by MAC-1- or MHC II reactive microglia). For all image analyzes, six sections at the hippocampus level, each separated at successive 240 μm intervals, were quantified for each animal. In all cases, the treatment status of the animals was not informed to the observer.
[531] XIV: In Vitro Screening Assay for the Activity of Antibodies on Amyloid Deposition
[532] To investigate the effect of antibodies on plaque removal, an in vitro assay was performed in which myoblast microglia cells were incubated with unfixed cryo sections of PDAPP mice or human AD brains. Microglia cells were obtained from the cerebral cortex of newborn DBA / 2N mice (1-3 days). The cortex HBSS ^- using (Hanks' Balanced Salt Solution, Sigma) in 50 ㎍ / ㎖ in DNaseI (Sigma) was mechanically separated. The isolated cells were filtered with a 100 μm cell strainer (Falcon) and centrifuged at 1000 rpm for 5 minutes. The pellet was resuspended in growth medium (high glucose DMEM, 10% FBS, 25 ng / ml rmGH-CSF) and the cells were plated at a density of 2 brains per T-75 plastic culture flask. After 7-9 days, the flask was shaken at 37 rpm for 2 hours at 200 rpm on an orbital shaker. The cell suspension was centrifuged at 1000 rpm and resuspended in assay medium.
[533] 10 μm cryogen sections of PDAPP mice or human AD brains (post interval <3 hours) were thawed and mounted on polylysine coated round glass coverslips and placed in wells of a 24 well tissue culture plate. Coverslips were washed twice with assay medium consisting of H-SFM (Hybridoma-serum-free medium, Gibco BRL) containing 1% FBS, glutamine, penicillin / streptomycin and 5 ng / ml rmGM-CSF (R & D) It was. Control or anti Aβ antibody was added at 2 × concentration (final 5 μg / ml) for 1 hour. Microglia were then seeded at a density of 0.8 × 10 ⁶ cells per ml of assay medium. Cultures were maintained for at least 24 hours in a humidified thermostat (37 ° C., 5% CO ₂ ). Later incubation, the cultures were fixed with 4% paraformaldehyde and infiltrated with 0.1% Triton-X 100. Sections were stained with biotinylated 3D6 and then stained with streptavidin / Cy3 conjugate (Jackson Immunoresearch). Exogenous microglia cells were visualized with nuclear stain (DAPI). Cultures were observed with a reversed-phase fluorescence microscope (Nikon, TE300) and micrographed with a SPOT digital camera using SPOT software (Diagnostic Instruments). For Western blot analysis, cultures were extracted in 8M urea and diluted 1: 1 in reducing Tricine Sample Buffer and loaded onto 16% Tricine Gel (Novex). After transfer to immobilon, the blots were exposed to 5 μg / ml pabAβ42 and then exposed to HRP-conjugated anti-mouse antibody and developed with ECL (Amersham).
[534] When analysis was performed with PDAPP brain sections in the presence of 16C11 (one of the antibodies against Aβ that was in vivo ineffective), β-amyloid plaques were preserved intact and no phagocytosis was observed. In contrast, when adjacent sections were cultured in the presence of 10D5, amyloid deposits usually disappeared and microglia cells showed many phagocytic vesicles containing Aβ. The same result was obtained with AD brain sections; 10D5 induced phagocytosis of AD plaque, whereas 16C11 was ineffective. In addition, similar results were obtained when analysis was performed with mouse or human microglia cells against Aβ and mouse, rabbit, or primate antibodies.
[535] Table 16 shows the results obtained for the various antibodies against Aβ comparing the ability to induce phagocytosis in in vitro assays and the ability to reduce in vivo plaque deposition in passive delivery experiments. Although 16C11 and 21F12 bind to aggregated synthetic Aβ peptides with great binding power, these antibodies were unable to react with β-amyloid plaques in unfixed brain sections, and did not cause phagocytosis in in vitro assays, and in vivo There was no efficacy. Polyclonal antibodies against 10D5, 3D6, and Aβ showed activity in all three measurements. The 22C8 antibody binds more strongly to the analogous form of native Αβ with aspartic acid at positions 1 and 7 replaced with iso aspartic acid. These results indicate that in vivo efficacy is due to direct antibody mediated elimination of plaques in the CNS, and that ex vivo assays can predict in vivo efficacy.
[536] The same assay was used to test the removal of antibodies to fragments of synuclein called NAC. Synuclin is known to be an amyloid plaque related protein. Antibodies against NAC were contacted with brain tissue samples, microglia, containing amyloid plaques as described above. Rabbit serum was used as a control. Subsequent monitoring showed a significant reduction in the number and size of plaques indicating antibody removal activity.
[537] In vitro assays to predict efficacy in vivo AntibodiesIsotypeBinding force to aggregated Aβ (pM)Binding to β-amyloid PlaquesEx vivo efficacyIn vivo efficacy Monoclonal3D6IgG2b470+++ 10D5IgG143+++ 16C11IgG190--- 21F12IgG2a500--- TM2aIgG1---- Polyclonal1-42mixture600+++
[538] Confocal microscopy was used to confirm that Aβ was absorbed during the ex vivo analysis. In the presence of control antibodies, exogenous microglia remained in the confocal plane of the tissue, there were no phagocytic vesicles containing Aβ and plaques were preserved intact in sections. In the presence of 10D5 almost all plaque material was contained in the vesicles within the exogenous microglia. To determine the fate of the internalized peptides, 10D5 treated cultures were extracted with 8 M urea at various time points and observed by Western blot analysis. At 1 hour, when no phagocytosis had occurred yet, the response of the polyclonal antibody to Aβ showed a dark 4 kD band (corresponding to Aβ peptide). Αβ immunoreactivity decreased on day 1 and disappeared by day 3. Thus, antibody mediated phagocytosis of Αβ causes degradation of Αβ.
[539] To determine whether phagocytosis in in vitro analysis was Fc-mediated, F (ab ′) 2 fragments of anti-Aβ antibody 3D6 were prepared. Although F (ab ') 2 fragments retained full capacity to react with plaques, they could not induce phagocytosis by microglia. In addition, phagocytosis using whole antibodies could be blocked by preparations for murine Fc receptors (anti-CD16 / 32). These data indicate that in vivo clearance of Αβ occurs via Fc-mediated phagocytosis.
[540] XV: Penetration of the Blood Brain Barrier of Antibodies
[541] This experiment was performed to provide information about the ability of antibodies to pass into the brain after intravenous injection into peripheral tissues of normal or PDAPP mice and to provide a method for measuring the concentration of antibodies delivered to the brain. These measurements are useful for predicting and determining effective amounts.
[542] PDAPP or control normal mice were perfused with 0.9% NaCl. Brain regions (hippocampus or cortex) were dissected and snap frozen. Brains were homogenized in 0.1% Triton + protease inhibitors. Immunoglobulins in the extracts were detected using ELISA. Fab'2 goat anti-mouse IgG was coated onto RIA plates as capture reagent. Serum or brain extracts were incubated for 1 hour. Isotypes were detected using anti-mouse IgG-HRP or IgG2a-HRP or IgG2b-HRP (Carlteg). Regardless of the isotype, the antibody was present in the CNS with a concentration of 1: 1000 when found in the blood. For example, when the concentration of IgG1 was three times the concentration of IgG2a in the blood, there was also three times the IgG2a in the brain, both with 0.1% of each level in the blood. This result was observed in both transgenic and nontransgenic mice, so PDAPP does not have a specific shed blood brain barrier.
[543] While the foregoing invention has been described in detail for purposes of clarity of understanding, it will be apparent that certain modifications may be made within the scope of the appended claims. All publications and patent documents cited herein are hereby incorporated by reference in their entirety for all purposes to the extent that each is individually indicated.

权利要求:
Claims (57)
[1" claim-type="Currently amended] A pharmaceutical composition comprising a pharmaceutical excipient and an agent effective for inducing an immune response against an amyloid component in a patient.
[2" claim-type="Currently amended] The pharmaceutical composition of claim 1, wherein the amyloid component is a fibril peptide or protein.
[3" claim-type="Currently amended] The amyloid component of claim 2, wherein the amyloid component is serum amyloid A protein (ApoSSA), immunoglobulin light chain, immunoglobulin heavy chain, ApoAI, transthyretin, lysozyme, fibrinogen α chain, gelsolin, cystatin C, amyloid β protein precursor (β). -APP), a protein or peptide consisting of beta ₂ microglobulin, prion precursor protein (PrP), atrial sodium excretory factor, keratin, islet amyloid polypeptide, peptide hormone and synuclein, and mutant proteins, protein fragments and proteolytic peptides thereof Pharmaceutical composition, characterized in that derived from fibril precursor protein selected from the group consisting of.
[4" claim-type="Currently amended] 4. A pharmaceutical composition according to claim 3, wherein said agent induces an immune response against neoepitopes formed by fibrillar proteins or peptides against fibrillar precursor proteins.
[5" claim-type="Currently amended] The method of claim 3, wherein the amyloid component is selected from the group consisting of AA, AL, ATTR, AApoA1, Alys, Agel, Acys, Aβ, AB ₂ M, AScr, Acal, AIAPP and Synuclein-NAC fragments. Pharmaceutical composition.
[6" claim-type="Currently amended] The pharmaceutical composition of claim 5, wherein the agent is selected from the group consisting of AA, AL, ATTR, AApoA1, Agel, Acys, Aβ, AB ₂ M, AScr, Acal, AIAPP and Sinuclin-NAC fragments.
[7" claim-type="Currently amended] The pharmaceutical composition of claim 1, wherein the composition comprises an agent effective for inducing an immunogenic response to two or more different amyloid components.
[8" claim-type="Currently amended] The pharmaceutical composition of claim 1, wherein the agent is a peptide linked to a carrier protein.
[9" claim-type="Currently amended] The pharmaceutical composition according to any one of claims 1 to 8, wherein the composition comprises an adjuvant.
[10" claim-type="Currently amended] The pharmaceutical composition of claim 9, wherein the adjuvant is selected from the group consisting of QS21, monophosphoryl lipids, alum and Freund's adjuvant.
[11" claim-type="Currently amended] A method for preventing or treating a disease characterized by amyloid deposition in a mammalian subject comprising administering to the subject an effective amount of an agent effective to produce an immune response against the amyloid component characteristic of the disease.
[12" claim-type="Currently amended] 12. The method of claim 11, wherein said amyloid component is a fibrillar protein or peptide.
[13" claim-type="Currently amended] The method according to claim 12, wherein the immune response is serum amyloid A protein (ApoSSA), immunoglobulin light chain, immunoglobulin heavy chain, ApoAI, transthyretin, lysozyme, fibrinogen α chain, gelsolin, cystatin C, amyloid β protein precursor ( β-APP), beta ₂ microglobulin, prion precursor protein (PrP), atrial sodium excretory factor, keratin, islet amyloid polypeptide, peptide hormones and synuclein, and mutant proteins, protein fragments or peptides thereof Characterized in that for the fibrillar component derived from the precursor protein.
[14" claim-type="Currently amended] The method of claim 13, wherein the agent induces an immune response against neoepitopes formed by the amyloid component to the precursor protein.
[15" claim-type="Currently amended] The method according to claim 13, wherein the amyloid component is selected from the group consisting of AA, AL, ATTR, AapoA1, Alys, Agel, Acys, Aβ, AB ₂ M, AScr, Acal, AIAPP and Synuclin-NAC fragments. Way.
[16" claim-type="Currently amended] The method of claim 15, wherein said agent is selected from the group consisting of AA, AL, ATTR, AapoA1, Agel, Acys, Aβ, AB ₂ M, AScr, Acal, AIAPP and Synuclin-NAC fragments.
[17" claim-type="Currently amended] The method of claim 11, wherein said agent is effective for inducing an immune response against two or more different amyloid components.
[18" claim-type="Currently amended] 18. The method of claim 17, wherein said administering comprises administering two or more amyloid fibril components.
[19" claim-type="Currently amended] The method of claim 11, wherein said agent is a peptide linked to a carrier protein.
[20" claim-type="Currently amended] 20. The method of any one of claims 11 to 19, wherein said administration further comprises an adjuvant.
[21" claim-type="Currently amended] The method of claim 20, wherein said adjuvant is selected from the group consisting of QS21, monophosphoryl lipids, alum and Freund's adjuvant.
[22" claim-type="Currently amended] The method of claim 11, wherein said immunological response is characterized by a serum titer of at least 1: 1000 relative to the amyloid component.
[23" claim-type="Currently amended] 23. The method of claim 22, wherein said serum titer is greater than 1: 5000 relative to the fibril component.
[24" claim-type="Currently amended] The method of claim 11, wherein said immunological response is characterized by a serum immunoreactive amount corresponding to at least about four times higher than the serum immunoreactive level measured in a pretreated control serum sample.
[25" claim-type="Currently amended] The method of claim 24, wherein the serum immunoreactive amount is measured by diluting the serum about 1: 100.
[26" claim-type="Currently amended] A method of determining the prognosis of a patient being treated for an amyloid disease comprising measuring a patient serum immunoreactive amount for a selected amyloid component, wherein the patient serum immunoreactive amount is at least four times the baseline control serum immunoreactivity level. For an indicator of the prognosis of the improved state.
[27" claim-type="Currently amended] The method of claim 26, wherein the amount of patient serum immunoreactivity to the selected amyloid component is a serum titer of at least about 1: 1000.
[28" claim-type="Currently amended] The method of claim 27, wherein the patient serum immunoreactive amount for the selected amyloid component is a serum titer of at least about 1: 5000.
[29" claim-type="Currently amended] A method of preventing or treating a disease comprising administering to a patient suffering from a disease characterized by amyloid deposition an effective amount of an antibody or antibody fragment that specifically binds to an amyloid component present in the deposit.
[30" claim-type="Currently amended] 30. The method of claim 29, wherein the amyloid component is a fibrillar component.
[31" claim-type="Currently amended] 31. The method of claim 30, wherein said antibody or antibody fragment binds to an epitope of a fibril component.
[32" claim-type="Currently amended] The method of claim 31, wherein the antibody or antibody fragment specifically binds to the fibrillar component without binding to the precursor of the fibrillar component.
[33" claim-type="Currently amended] 31. The method of claim 30, wherein the antibody is a human antibody against a fibrillar component prepared from B cells obtained from a person immunized with a fibrillar component epitope.
[34" claim-type="Currently amended] The amyloid component of claim 30, wherein the amyloid component is serum amyloid A protein (ApoSSA), immunoglobulin light chain, immunoglobulin heavy chain, ApoAI, transthyretin, lysozyme, fibrinogen α chain, gelsolin, cystatin C, amyloid β protein precursor ( β-APP), beta ₂ microglobulin, prion precursor protein (PrP), atrial sodium excretory factor, keratin, islet amyloid polypeptide, peptide hormones and synuclein, and mutant proteins, protein fragments or peptides thereof Characterized in that it is derived from a precursor protein.
[35" claim-type="Currently amended] 35. The method of claim 34, wherein said amyloid fibril component is selected from the group consisting of AA, AL, ATTR, AapoA1, Alys, Agel, Acys, Aβ, AB ₂ M, AScr, Acal, AIAPP and Synuclin-NAC fragments. Characteristic method.
[36" claim-type="Currently amended] The method of claim 29, wherein said administering comprises administering an antibody that binds two or more amyloid fibril components.
[37" claim-type="Currently amended] 30. The method of claim 29, wherein said effective amount is a level of a patient of said serum immunoreactive amount for said amyloid component that is at least about four times higher than the serum immunoreactive level for said component measured in a pretreated control serum sample. .
[38" claim-type="Currently amended] The method of claim 29, wherein the antibody or antibody fragment is administered with a carrier as a pharmaceutical composition.
[39" claim-type="Currently amended] The method of claim 29, wherein the antibody or antibody fragment is administered intraperitoneally, orally, subcutaneously, intramuscularly, intranasally, topically or intravenously.
[40" claim-type="Currently amended] The method of claim 29, wherein the antibody is administered by administering to the patient a polynucleotide encoding at least one antibody chain, wherein the polynucleotide is expressed in the patient to produce an antibody chain.
[41" claim-type="Currently amended] 41. The method of claim 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.
[42" claim-type="Currently amended] The method of claim 29, wherein the antibody or antibody fragment is administered in multiple administrations over six months or more.
[43" claim-type="Currently amended] The method of claim 29, wherein the antibody is administered as a sustained release composition.
[44" claim-type="Currently amended] A pharmaceutical composition for preventing or treating a disease comprising administering to a patient suffering from a disease characterized by amyloid deposition an effective amount of an antibody or antibody fragment that specifically binds to an amyloid component present in the deposit.
[45" claim-type="Currently amended] 45. The pharmaceutical composition of claim 44, wherein the amyloid component is a fibrillar component.
[46" claim-type="Currently amended] 46. The pharmaceutical composition of claim 45, wherein the antibody binds to an epitope of a fibril component.
[47" claim-type="Currently amended] The pharmaceutical composition of claim 46, wherein the antibody specifically binds to the fibrillar component without binding to the precursor of the fibrillar component.
[48" claim-type="Currently amended] 47. The pharmaceutical composition of claim 46, wherein the antibody is a human antibody against a fibrillar component produced in B cells obtained from a person immunized with a fibrillar component epitope.
[49" claim-type="Currently amended] 46. The method of claim 45, wherein the amyloid fibril component is serum amyloid A protein (ApoSSA), immunoglobulin light chain, immunoglobulin heavy chain, ApoAI, transthyretin, lysozyme, fibrinogen α chain, gelzoline, cystatin C, amyloid β protein In the group consisting of precursor (β-APP), beta ₂ microglobulin, prion precursor protein (PrP), atrial sodium excretory factor, keratin, islet amyloid polypeptide, peptide hormone and synuclein, and mutant proteins, protein fragments or peptides thereof Pharmaceutical composition, characterized in that it is derived from the precursor protein of choice.
[50" claim-type="Currently amended] 50. The method of claim 49, wherein the amyloid fibril component is selected from the group consisting of AA, AL, ATTR, AapoA1, Alys, Agel, Acys, Aβ, AB ₂ M, AScr, Acal, AIAPP and Synuclin-NAC fragments. Characterized pharmaceutical composition.
[51" claim-type="Currently amended] 45. The pharmaceutical composition of claim 44, wherein the composition comprises an antibody or antibody fragment that binds two or more amyloid fibril components.
[52" claim-type="Currently amended] 45. The pharmaceutical according to claim 44, wherein said effective amount is a level of a patient of said serum immunoreactive amount for said amyloid component that is at least about four times higher than the serum immunoreactive level for said component measured in a pretreated control serum sample. Composition.
[53" claim-type="Currently amended] 45. The pharmaceutical composition of claim 44, wherein the pharmaceutical composition comprises a carrier.
[54" claim-type="Currently amended] 45. The pharmaceutical composition of claim 44, wherein the pharmaceutical composition is formulated for intraperitoneal, oral, subcutaneous, intramuscular, nasal, topical or intravenous administration.
[55" claim-type="Currently amended] 45. The pharmaceutical composition of claim 44, wherein the pharmaceutical composition comprises a polynucleotide encoding at least one antibody chain effective to express the antibody chain in a patient.
[56" claim-type="Currently amended] The pharmaceutical composition of claim 55, wherein the polynucleotide encodes the heavy and light chains of the antibody and the polynucleotide can be expressed in a patient to produce a heavy and light chain.
[57" claim-type="Currently amended] 45. The pharmaceutical composition of claim 44, wherein the pharmaceutical composition is formulated as a sustained release composition.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1999-06-01|Priority to US13701099P

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1999-06-01|Priority to US60/137,010

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2000-06-01|Application filed by 인슬레이, 케빈, 뉴랄랩 리미티드

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2000-06-01|Priority to PCT/US2000/015239

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2002-04-04|Publication of KR20020025884A

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2009-12-09|Application granted

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2009-12-09|Publication of KR100930559B1

优先权:

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申请号 | 申请日 | 专利标题

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US13701099P| true| 1999-06-01|1999-06-01|

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US60/137,010|1999-06-01|

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PCT/US2000/015239|WO2000072876A2|1999-06-01|2000-06-01|Prevention and treatment of amyloidogenic disease|