AU2013206069A1 - Prevention and treatment of amyloidogenic disease - Google Patents

Abstract

PREVENTION AND TREATMENT OF AMYLOIDOGENIC Abstract The invention provides compositions and methods for treatment of amyloidogenic diseases. Such methods entail administering an agent that induces a beneficial immune response against an amyloid deposit in the patient. The methods are particularly useful for prophylactic and therapeutic treatment of Alzheimer's disease. In such methods, a suitable agent is Apl peptide or an antibody thereto.

Description

1 PREVENTION AND TREATMENT OF AMYLOIDOGENIC DISEASE TECHNICAL FIELD The invention resides in the technical fields of immunology and medicine. 5 BACKGROUND Alzheimer's disease (AD) is a progressive disease resulting in senile dementia. See generally Selkoe, TINS 16, 403-409 (1993); Hardy et al., WO 92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53, 438-447 (1994); Duff et al., Nature 373, 476-477 (1995); Games et al., Nature 373, 523 (1995). Broadly speaking the disease falls into two 10 categories: late onset, which occurs in old age (65 + years) and early onset, which develops well before the senile period, i.e, between 35 and 60 years. In both types of disease, the pathology is the same but the abnormalities tend to be more severe and widespread in cases beginning at an earlier age. The disease is characterized by two types of lesions in the brain, senile plaques and neurofibrillary tangles. Senile plaques are areas 15 of disorganized neuropil up to 150 [tm across with extracellular amyloid deposits at the center visible by microscopic analysis of sections of brain tissue. Neurofibrillary tangles are intracellular deposits of tau protein consisting of two filaments twisted about each other in pairs .

2 The principal constituent of the plaques is a peptide termed AO or -amyloid peptide. AO peptide is an internal fragment of 39-43 amino acids of a precursor protein termed amyloid precursor protein (APP). Several mutations within the 5 APP protein have been correlated with the presence of Alzheimer's disease. See, e.g., Goate et al., Nature 349, 704) (1991) (valine 717 to isoleucine); Chartier Harlan et al. Nature 353, 844 (1991)) (valine 717 to glycine); Murrell et al., Science 254, 97 (1991) (valine 717 to phenylalanine); LO Mullan et al., Nature Genet. 1, 345 (1992) (a double mutation changing lysine 595 methionine 596 to asparagine59- leucine 5 9 6 ) Such mutations are thought to cause Alzheimer's disease by increased or altered processing of APP to AO, particularly processing of APP to increased amounts of the long form of AO L5 (i.e., A1l-42 and A01-43). Mutations in other genes, such as the presenilin genes, PS1 and PS2, are thought indirectly to affect processing of APP to generate increased amounts of long form AO (see Hardy, TINS 20, 154 (1997)). These observations indicate that AO, and particularly its long form, is a 20 causative element in Alzheimer's disease. McMichael, EP 526,511, proposes administration of homeopathic dosages (less than or equal to 10-2 mg/day) of AO to patients with preestablished AD. In a typical human with about 5 liters of plasma, even the upper limit of this dosage 25 would be expected to generate a concentration of no more than 2 pg/ml. The normal concentration of AO in human plasma is typically in the range of 50-200 pg/ml (Seubert et al., Nature 359, 325-327 (1992)). Because EP 526,511's proposed dosage would barely alter the level of endogenous circulating AO and 30 because EP 526,511 does not recommend use of an adjuvant, it seems implausible that any therapeutic benefit would result. By contrast, the present invention is directed inter alia to treatment of Alzheimer's and other amyloidogenic diseases by administration of AO or other immunogen to a patient under 35 conditions that generate a beneficial immune response in the patient. The invention thus fulfills a longstanding need for therapeutic regimes for preventing or ameliorating the neuropathology of Alzheimer's disease.

3 SUMMARY OF THE CLAIMED INVENTION In one aspect, the invention provides methods of preventing or treating a disease characterized by amyloid deposition in a patient. Such methods entail inducing an immune response 5 against a peptide component of an amyloid deposit in the patient. Such induction can be active by administration of an immunogen or passive by administration of an antibody or an active fragment or derivative of the antibody. In some patients, the amyloid deposit is aggregated A0 peptide and LO the disease is Alzheimer's disease. In some methods, the patient is asymptomatic. In some methods, the patient is under 50 years of age. In some methods, the patient has inherited risk factors indicating susceptibility to Alzheimer's disease. Such risk factors include variant L5 alleles in presenilin gene PS1 or PS2 and variant forms of APP. In other methods, the patient has no known risk factors for Alzheimer's disease. For treatment of patients suffering from Alzheimer's disease, one treatment regime entails administering a dose of 20 AO peptide to the patient to induce the immune response. In some methods, the AO peptide is administered with an adjuvant that enhances the immune response to the A# peptide. In some methods, the adjuvant is alum. In some methods, the adjuvant is MPL. The dose of A0 peptide administered to the patient is 25 typically at least 1 or 10 pg, if administered with adjuvant, and at least 50 pg if administered without adjuvant. In some methods, the dose is at least 100 pg. In some methods, the A0 peptide is A01-42. In some methods, the A0 peptide is administered in aggregated form. 30 In other methods, the A0 peptide is administered in dissociated form. In some methods, the therapeutic agent is an effective dose of a nucleic acid encoding A0 or an active fragment or derivative thereof. The nucleic acid encoding AO or fragment thereof is expressed in the patient to produce AO 35 or the active fragment thereof, which induces the immune response. In some such methods, the nucleic acid is administered through the skin, optionally via a patch. In some methods, a therapeutic agent is identified by screening a 4 library of compounds to identify a compound reactive with antibodies to AO, and administering the compound to the patient to induce the immune response. In some methods, the immune response is directed to 5 aggregated AO peptide without being directed to dissociated AO peptide. For example, the immune response can comprise antibodies that bind to aggregated AO peptide without binding to dissociated AO peptide. In some methods, the immune response comprises T-cells that bind to AO complexed with MCHI .0 or MHCII on CD8 or CD4 cells. In other methods, the immune response is induced by administering an antibody to AO to the patient. In some methods, the immune response is induced by removing T-cells from the patient, contacting the T-cells with AO peptide under conditions in which the T-cells are primed, .5 and replacing the T-cells in the patient. The therapeutic agent is typically administered orally, intranasally, intradermally, subcutaneously, intramuscularly, topically or intravenously. In some methods, the patient is monitored followed administration to assess the immune 20 response. If the monitoring indicates a reduction of the immune response over time, the patient can be given one or more further doses of the agent. In another aspect, the invention provides pharmaceutical compositions comprising AO and an excipient suitable for oral 25 and other routes of administration. The invention also provides pharmaceutical compositions comprising an agent effective to induce an immunogenic response against AO in a patient, and a pharmaceutically acceptable adjuvant. In some such compositions, the agent is AO or an active fragment 30 thereof. In some compositions, the adjuvant comprises alum. In some compositions, the adjuvant comprises an oil-in-water emulsion. In some compositions, the AO or active fragment is a component of a polylactide polyglycolide copolymer (PLPG) or other particle. The invention further provides compositions 35 comprising AO or an active fragment linked to a conjugate molecule that promotes delivery of AO to the bloodstream of a patient and/or promotes an immune response against A0. For example, the conjugate can serve to promote an immune response 5 against A0. In some compositions, the conjugate is cholera toxin. In some compositions, the conjugate is an immunoglobulin. In some compositions, the conjugate is attenuated diphtheria toxin CRM 197 (Gupta, Vaccine 15, 1341-3 5 (1997). The invention also provides pharmaceutical compositions comprising an agent effect to induce an immunogenic response against AO in a patient with the proviso that the composition is free of Complete Freund's adjuvant. The invention also .0 provides compositions comprising a viral vector encoding AO or a an active fragment thereof effective to induce an immune response against A0. Suitable viral vectors include herpes, adenovirus, adenoassociated virus, a retrovirus, sindbis, semiliki forest virus, vaccinia or avian pox. .5 The invention further provides methods of preventing or treating Alzheimer's disease. In such methods, an effective dose of AO peptide is administered to a patient. The invention further provides for the use of AO, or an antibody thereto, in the manufacture of a medicament for prevention or 0O treatment of Alzheimer's disease. In another aspect, the invention provides methods of assessing efficacy of an Alzheimer's treatment method in a patient. In these methods, a baseline amount of antibody specific for AO peptide is determined in a tissue sample from 25 the patient before treatment with an agent. An amount of antibody specific for AO peptide in the tissue sample from the patient after treatment with the agent is compared to the baseline amount of AO peptide-specific antibody. An amount of AO peptide-specific antibody measured after the treatment that 30 is significantly greater than the baseline amount of AO peptide-specific antibody indicates a positive treatment outcome. In others methods of assessing efficacy of an Alzheimer's treatment method in a patient, a baseline amount of antibody 35 specific for AO peptide in a tissue sample from a patient before treatment with an agent is determined. An amount of antibody specific for A3 peptide in the tissue sample from the subject after treatment with the agent is compared to the 6 baseline amount of AO peptide-specific antibody. A reduction or lack of significant difference between the amount of AO peptide-specific antibody measured after the treatment compared to the baseline amount of AO peptide-specific 5 antibody indicates a negative treatment outcome. In other methods of assessing efficacy of an Alzheimer's disease treatment method in a patient a control amount of antibody specific for AO peptide is determined in tissue samples from a control population. An amount of antibody LO specific for AO peptide in a tissue sample from the patient after administering an agent is compared to the control amount of AO peptide-specific antibody. An amount of AO peptide-specific antibody measured after the treatment that is significantly greater than the control amount of AO L5 peptide-specific antibody indicates a positive treatment outcome. In other methods of assessing efficacy of an Alzheimer's treatment method in a patient, a control amount of antibody specific for AO peptide in tissues samples from a control 20 population is determined. An amount of antibody specific for AO peptide in a tissue sample from the patient after administering an agent is compared to the control amount of AO peptide-specific antibody. A lack of significant difference between the amount of AO peptide-specific antibody measured 25 after beginning said treatment compared to the control amount of.AO peptide-specific antibody indicates a negative treatment outcome. Other methods of monitoring Alzheimer's disease or susceptibility thereto in a patient, comprise detecting an 30 immune response against AO peptide in a sample from the patient. In some such methods, the patient is being administered an agent effective to treat or prevent Alzheimer's disease, and the level of the response determines the future treatment regime of the patient. 35 In other methods of assessing efficacy of an Alzheimer's treatment method in a patient a value for an amount of antibody specific for AO peptide in tissue sample from a patient who has been treated with an agent is determined. The 7 value is compared with a control value determined from a population of patient experiencing amelioriation of, or freedom from, symptoms of Alzheimer's disease due to treatment with the agent. A value in the patient at least equal to the 5 control value indicates a positive response to treatment. The invention further provides diagnostic kits for performing the above methods. Such kits typically include a reagent that specifically binds to antibodies to A0 or which stimulates proliferation of T-cells reactive with A0. LO BRIEF DESCRIPTION OF THE FIGURES Fig. 1: Antibody titer after injection of transgenic mice with A01-42. Fig. 2: Amyloid burden in the hippocampus. The percentage of the area of the hippocampal region occupied by amyloid 15 plaques, defined by reactivity with the Ao-specific mA0 3D6, was determined by computer-assisted quantitative image analysis of immunoreacted brain sections. The values for individual mice are shown sorted by treatment group. The horizontal line for each grouping indicates the median value 20 of the distribution. Fig 3: Neuritic dystrophy in the hippocampus. The percentage of the area of the hippocampal region occupied by dystrophic neurites, defined by their reactivity with the human APP-specific mA0 8E5, was determined by quantitative 25 computer-assisted image analysis of immunoreacted brain sections. The values for individual mice are shown for the AN1792-treated group and the PBS-treated control group. The horizontal line for each grouping indicates the median value of the distribution. 30 Fig. 4: Astrocytosis in the retrosplenial cortex. The percentage of the area of the cortical region occupied by glial fibrillary acidic protein (GFAP)-positive astrocytes was determined by quantitative computer-assisted image analysis of immunoreacted brain sections. The values for individual mice 35 are shown sorted by treatment group and median group values are indicated by horizontal lines.

8 Fig. 5: Geometric mean antibody titers to Aol-42 following immunization with a range of eight doses of AN1792 containing 0.14, 0.4, 1.2, 3.7, 11, 33, 100, or 300 pg. Fig. 6: Kinetics of antibody response to AN1792 5 immunization. Titers are expressed as geometric means of values for the 6 animals in each group. Fig. 7: Quantitative image analysis of the cortical amyloid burden in PBS- and AN1792-treated mice. Fig. 8: Quantitative image analysis of the neuritic plaque LO burden in PBS- and AN1792-treated mice. Fig. 9: Quantitative image analysis of the percent of the retrosplenial cortex occupied by astrocytosis in PBS- and AN1792-treated mice. Fig. 10: Lymphocyte Proliferation Assay on spleen cells L5 from AN1792-treated (upper panel) or PBS-treated (lower panel). Fig. 11: Total A0 levels in the cortex. A scatterplot of individual A0 profiles in mice immunized with A0 or APP derivatives combined with Freund's adjuvant. 20 Fig. 12: Amyloid burden in the cortex was determined by quantitative image analysis of immunoreacted brain sections for mice immunized with the A0 peptide conjugates A01-5, A01 12, and A013-28; the full length A0 aggregates AN1792 (AO1-42) and AN1528 (A01-40) and the PBS-treated control group. 25 Fig. 13: Geometric mean titers of A1-specific antibody for groups of mice immunized with A0 or APP derivatives combined with Freund's adjuvant. Fig. 14: Geometric mean titers of Af-specific antibody for groups of guinea pigs immunized with AN1792, or a 30 palmitoylated derivative thereof, combined with various adjuvants. Fig: 15: A0 levels in the cortex of 12-month old PDAPP mice treated with AN1792 or AN1528 with different adjuvants. DEFINITIONS 35 The term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 65 9 percent sequence identity, preferably at least 80 or 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity or higher). Preferably, residue positions which are not 5 identical differ by conservative amino acid substitutions. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates 10 are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. 15 Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Apple. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson 20 & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Ausubel et al., 25 supra). One example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National 30 Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Typically, default program parameters can be used to perform the sequence comparison, although customized parameters can also be used. For amino acid sequences, the BLASTP program uses as defaults a 35 wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89, 10915 (1989)) 10 For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic sidechains): norleucine, met, ala, val, leu, ile; Group II (neutral hydrophilic side 5 chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino LO acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another. Therapeutic agents of the invention are typically substantially pure. This means that an agent is typically at L5 least about 50% w/w (weight/weight) purity, as well as being substantially free from interfering proteins and contaminants. Sometimes the agents are at least about 80% w/w and, more preferably at least 90 or about 95% w/w purity. However, .using conventional protein purification techniques, 20 homogeneous peptides of at least 99% w/w can be obtained. Specific binding between two entities means an affinity of at least 106, 107, 108, 109 M- 1 , or 1010 M- 1 . Affinities greater than 108 M- 1 are preferred. The term "antibody" is used to include intact antibodies 25 and binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to an antigen. Optionally, antibodies or binding fragments thereof, can be chemically conjugated to, or expressed as, fusion proteins with other proteins. 30 APP 6 9 5 , APP 7 5 1 , and APP 7 7 0 refer, respectively, to the 695, 751, and 770 amino acid residue long polypeptides encoded by the human APP gene. See Kang et al., Nature 325, 773 (1987); Ponte et al., Nature 331, 525 (1988); and Kitaguchi et al., Nature 331, 530 (1988). Amino acids within the human amyloid 35 precursor protein (APP) are assigned numbers according to the sequence of the APP770 isoform. Terms such as A039, A040, A041, A042 and A343 refer to an A0 peptide containing amino acid residues 1-39, 1-40, 1-41, 1-42 and 1-43.

11 The term "epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a 5 protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 LO amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. L5 (1996). Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. T-cells recognize continuous epitopes of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 20 cells. T cells that recognize the epitope can be identified by in vitro assays that measure antigen-dependent proliferation, as determined by 3 H-thymidine incorporation by primed T cells in response to an epitope (Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), by antigen-dependent killing 25 (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. 156, 3901-3910) or by cytokine secretion. The term "immunological" or "immune" response is the development of a beneficial humoral (antibody mediated) and/or a cellular (mediated by antigen-specific T cells or their 30 secretion products) response directed against an amyloid peptide in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T-cells. A cellular immune response is elicited by the 35 presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4+ T helper cells and/or CD8* cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK 12 cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4+ T cells) or CTL 5 (cytotoxic T lymphocyte) assays (see Burke, supra; Tigges, supra). The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating IgG and T-cells from an immunized syngeneic animal and measuring 10 protective or therapeutic effect in a second subject. An "immunogenic agent" or "immunogen" is capable of inducing an immunological response against itself on administration to a patient, optionally in conjunction with an adjuvant. 15 The term "naked polynucleotide" refers to a polynucleotide not complexed with colloidal materials. Naked polynucleotides are sometimes cloned in a plasmid vector. The term "adjuvant" refers to a compound that when administered in conjunction with an antigen augments the 20 immune response to the antigen, but when administered alone does not generate an immune response to the antigen. Adjuvants can augment an immune response by several mechanisms including lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of macrophages. 25 The term "patient" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. Disaggregated or monomeric A0 means soluble, monomeric peptide units of A,6. One method to prepare monomeric A0 is to 30 dissolve lyophilized peptide in neat DMSO with sonication. The resulting solution is centrifuged to remove any nonsoluble particulates. Aggregated A0 is a mixture of oligomers in which the monomeric units are held together by noncovalent bonds. 35 Compositions or methods "comprising" one or more recited elements may include other elements not specifically recited. For example, a composition that comprises A0 peptide 13 encompasses both an isolated A0 peptide and AO peptide as a component of a larger polypeptide sequence. DETAILED DESCRIPTION I. General 5 The invention provides pharmaceutical compositions and methods for prophylactic and therapeutic treatment of diseases characterized by accumulation of amyloid deposits. Amyloid deposits comprise a peptide aggregated to an insoluble mass. The nature of the peptide varies in different diseases but in 10 most cases, the aggregate has a f-pleated sheet structure and stains with Congo Red dye. Diseases characterized by amyloid deposits include Alzheimer's disease (AD), both late and early onset. In both diseases, the amyloid deposit comprises a peptide termed A0, which accumulates in the brain of affected 15 individuals. Examples of some other diseases characterized by amyloid deposits are SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, and spongiform encephalopathies, including mad cow disease, Creutzfeldt Jakob disease, sheep scrapie, and mink spongiform encephalopathy (see Weissmann et 20 al., Curr. Opin. Neurobiol. 7, 695-700 (1997); Smits et al., Veterinary Quarterly 19, 101-105 (1997); Nathanson et al., Am. J. Epidemiol. 145, 959-969 (1997)). The peptides forming the aggregates in these diseases are serum amyloid A, cystantin C, IgG kappa light chain respectively for the first three, and 25 prion protein for the others. II. Therapeutic Agents 1. Alzheimer's Disease Therapeutic agents for use in the present invention induce an immune response against A0 peptide. These agents include 30 A0 peptide itself and variants thereof, analogs and mimetics of AO peptide that induce and/or crossreact with antibodies to AO peptide, and antibodies or T-cells reactive with A0 peptide. Induction of an immune response can be active as when an immunogen is administered to induce antibodies or T 35 cells reactive with A0 in a patient, or passive, as when an antibody is administered that itself binds to A0 in patient.

14 AO, also known as -amyloid peptide, or A4 peptide (see US 4,666,829; Glenner & Wong, Biochem. Biophys. Res. Commun. 120, 1131 (1984)), is a peptide of 39-43 amino acids, which is the principal component of characteristic plaques of Alzheimer's 5 disease. AO is generated by processing of a larger protein APP by two enzymes, termed f and y secretases (see Hardy, TINS 20, 154 (1997)). Known mutations in APP associated with Alzheimer's disease occur proximate to the site of # or y secretase, or within A0. For example, position 717 is LO proximate to the site of T-secretase cleavage of APP in its processing to AO, and positions 670/671 are proximate to the site of -secretase cleavage. It is believed that the mutations cause AD disease by interacting with the cleavage reactions by which AO is formed so as to increase the amount L5 of the 42/43 amino acid form of AO generated. AO has the unusual property that it can fix and activate both classical and alternate complement cascades. In particular, it binds to Clq and ultimately to C3bi. This association facilitates binding to macrophages leading to 20 activation of B cells. In addition, C3bi breaks down further and then binds to CR2 on B cells in a T cell dependent manner leading to a 10,000 increase in activation of these cells. This mechanism causes AO to generate an immune response in excess of that of other antigens. 25 The therapeutic agent used in the claimed methods can be any of the naturally occurring forms of AO peptide, and particularly the human forms (i.e., A339, A040, A041, A342 or A043). The sequences of these peptides and their relationship to the APP precursor are illustrated by Fig. 1 of Hardy et 30 al., TINS 20, 155-158 (1997). For example, A042 has the sequence:

H

2 N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-IIe-Ala-OH. 35 A041, A40 and A39 differ from A42 by the omission of Ala, Ala-Ile, and Ala-Ile-Val respectively from the C-terminal end. A43 differs from A42 by the presence of a threonine residue 15 at the C-terminus. The therapeutic agent can also be an active fragment or analog of a natural AO peptide that contains an epitope that induces a similar protective or therapeutic immune response on administration to a human. 5 Immunogenic fragments typically have a sequence of at least 3, 5, 6, 10 or 20 contiguous amino acids from a natural peptide. Immunogenic fragments include A01-5, 1-6, 1-12, 13-28, 17-28, 25-25, 35-40 and 35-42. Fragments from the N-terminal half of AO are preferred in some methods. Analogs include allelic, LO species and induced variants. Analogs typically differ from naturally occurring peptides at one or a few positions, often by virtue of conservative substitutions. Analogs typically exhibit at least 80 or 90* sequence identity with natural peptides. Some analogs also include unnatural amino acids or L5 modifications of N or C terminal amino acids. Examples of unnatural amino acids are a,a-disubstituted amino acids, N alkyl amino acids, lactic acid, 4-hydroxyproline, 7 carboxyglutamate, e-N,N,N-trimethyllysine, e-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3 20 methylhistidine, 5-hydroxylysine, w-N-methylarginine. Fragments and analogs can be screened for prophylactic or therapeutic efficacy in transgenic animal models as described below. AO, its fragments, analogs and other amyloidogenic peptides 25 can be synthesized by solid phase peptide synthesis or recombinant expression, or can be obtained from natural sources. Automatic peptide synthesizers are commercially available from numerous suppliers, such as Applied Biosystems, Foster City, California. Recombinant expression can be in 30 bacteria, such as E. coli, yeast, insect cells or mammalian cells. Procedures for recombinant expression are described by Sambrook et al., Molecular Cloning: A Laboratory Manual (C.S.H.P. Press, NY 2d ed., 1989). Some forms of AO peptide are also available commercially (e.g., American Peptides 35 Company, Inc., Sunnyvale, CA and California Peptide Research, Inc. Napa, CA). Therapeutic agents also include longer polypeptides that include, for example, an AO peptide, active fragment or analog 16 together with other amino acids. For example, AO peptide can be present as intact APP protein or a segment thereof, such as the C-100 fragment that begins at the N-terminus of AO and continues to the end of APP. Such polypeptides can be 5 screened for prophylactic or therapeutic efficacy in animal models as described below. The AO peptide, analog, active fragment or other polypeptide can be administered in associated form (i.e., as an amyloid peptide) or in dissociated form. Therapeutic agents also include multimers 10 of monomeric immunogenic agents. In a further variation, an immunogenic peptide, such as AO, can be presented as a viral or bacterial vaccine. A nucleic acid encoding the immunogenic peptide is incorporated into a genome or episome of the virus or bacteria. Optionally, the 15 nucleic acid is incorporated in such a manner that the immunogenic peptide is expressed as a secreted protein or as a fusion protein with an outersurface protein of a virus or a transmembrane protein of a bacteria so that the peptide is displayed. Viruses or bacteria used in such methods should 20 be nonpathogenic or attenuated. Suitable viruses include adenovirus, HSV, vaccinia and fowl pox. Fusion of an immunogenic peptide to HBsAg of HBV is particularly suitable. Therapeutic agents also include peptides and other compounds that do not necessarily have a significant amino acid sequence 25 similarity with AO but nevertheless serve as mimetics of AO an~d induce a similar immune response. For example, any peptides and proteins forming -pleated sheets can be screened for suitability. Anti-idiotypic antibodies against monoclonal antibodies to AO or other amyloidogenic peptides can also be 30 used. Such anti-Id antibodies mimic the antigen and generate an immune response to it (see Essential Immunology (Roit ed., Blackwell Scientific Publications, Palo Alto, 6th ed.), p. 181). Random libraries of peptides or other compounds can also be 35 screened for suitability. Combinatorial libraries can be produced for many types of compounds that can be synthesized in a step-by-step fashion. Such compounds include polypeptides, beta-turn mimetics, polysaccharides, 17 phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates. Large combinatorial libraries of the compounds can be constructed by 5 the encoded synthetic libraries (ESL) method described in Affymax, WO 95/12608, Affymax, WO 93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 (each of which is incorporated by reference for all purposes). Peptide libraries can also be 10 generated by phage display methods. See, e.g., Devlin, WO 91/18980. Combinatorial libraries and other compounds are initially screened for suitability by determining their capacity to bind to antibodies or lymphocytes (B or T) known to be specific for 15 AO or other amyloidogenic peptides. For example, initial screens can be performed with any polyclonal sera or monoclonal antibody to AO or other amyloidogenic peptide. Compounds identified by such screens are then further analyzed for capacity to induce antibodies or reactive lymphocytes to 20 AO or other amyloidogenic peptide. For example, multiple dilutions of sera can be tested on microtiter plates that have been precoated with AO peptide and a standard ELISA can be performed to test for reactive antibodies to A0. Compounds can then be tested for prophylactic and therapeutic efficacy 25 in transgenic animals predisposed to an amyloidogenic disease, as.described in the Examples. Such animals include, for example, mice bearing a 717 mutation of APP described by Games et al., supra, and mice bearing a Swedish mutation of APP such as described by McConlogue et al., US 5,612,486 and Hsiao et 30 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); Borchelt et al., Neuron 19, 939-945 (1997)). The same screening approach can be used on other potential agents such 35 as fragments of AO, analogs of AO and longer peptides including AO, described above. Therapeutic agents of the invention also include antibodies that specifically bind to A0. Such antibodies can be 18 monoclonal or polyclonal. Some such antibodies bind specifically to the aggregated form of AO without binding to the dissociated form. Some bind specifically to the dissociated form without binding to the aggregated form. Some 5 bind to both aggregated and dissociated forms. The production of non-human monoclonal antibodies, e.g., murine or rat, can be accomplished by, for example, immunizing the animal with A0. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Such 10 an immunogen can be obtained from a natural source, by peptides synthesis or by recombinant expression. Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et 15 al., Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989) and WO 90/07861 (incorporated by reference for all purposes). Human antibodies can be obtained using phage-display methods. See, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047. In these methods, libraries of phage are 20 produced in which members display different antibodies on their outersurfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity are selected by affinity enrichment to AO, or fragments thereof. Human antibodies against AO can also be 25 produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus. See, e.g., Lonberg et al., W093/12227 (1993); Kucherlapati, WO 91/10741 (1991) (each of which is incorporated by reference in 30 its entirety for all purposes). Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Such antibodies are particularly likely to share the useful functional properties of the mouse antibodies. 35 Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by 19 affinity purification using AO or other amyloid peptide as an affinity reagent. Human or humanized antibodies can be designed to have IgG, IgD, IgA and IgE constant region, and any isotype, including 5 IgG1, IgG2, IgG3 and IgG4. Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab' F(ab') 2 , and Fv, or as single chain antibodies in which heavy and light chain variable domains are linked through a spacer. 10 Therapeutic agents for use in the present methods also include T-cells that bind to AO peptide. For example, T-cells can be activated against AO peptide by expressing a human MHC class I gene and a human 0-2-microglobulin gene from an insect cell line, whereby an empty complex is formed on the surface 15 of the cells and can bind to AO peptide. T-cells contacted with the cell line become specifically activated against the peptide. See Peterson et al., US 5,314,813. Insect cell lines expressing an MHC class II antigen can similarly be used to activate CD4 T cells. 20 2. Other Diseases The same or analogous principles determine production of therapeutic agents for treatment of other amyloidogenic diseases. In general, the agents noted above for use in treatment of Alzheimer's disease can also be used for 25 treatment early onset Alzheimer's disease associated with Down's syndrome. In mad cow disease, prion peptide, active fragments, and analogs, and antibodies to prion peptide are used in place of AO peptide, active fragments, analogs and antibodies to AO peptide in treatment of Alzheimer's disease. 30 In treatment of multiple myeloma, IgG light chain and analogs and antibodies thereto are used, and so forth in other diseases. 3. Carrier Proteins Some agents for inducing an immune response contain the 35 appropriate epitope for inducing an immune response against amyloid deposits but are too small to be immunogenic. In this 20 situation, a peptide immunogen can be linked to a suitable carrier to help elicit an immune response. Suitable carriers include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus 5 toxoid, or a toxoid from other pathogenic bacteria, such as diphtheria, E. coli, cholera, or H. pylori, or an attenuated toxin derivative. Other carriers for stimulating or enhancing an immune response include cytokines such as IL-1, IL-1 a and 3 peptides, IL-2, TINF, IL-10, GM-CSF, and chemokines, such as 10 MlPla and 0 and RANTES. Immunogenic agents can also be linked to peptides that enhance transport across tissues, as described in O'Mahony, WO 97/17613 and WO 97/17614. Immunogenic agents can be linked to carriers by chemical crosslinking. Techniques for linking an immunogen to a 15 carrier include the formation of disulfide linkages using N succinimidyl-3-(2-pyridyl-thio) propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-i-carboxylate (SMCC) (if the peptide lacks a sulfhydryl group, this can be provided by addition of a cysteine residue). These reagents 20 create a disulfide linkage between themselves and peptide cysteine resides on one protein and an amide linkage through the E-amino on a lysine, or other free amino group in other amino acids. A variety of such disulfide/amide-forming agents are described by Immun. Rev. 62, 185 (1982). Other 25 bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thio-ether-forming agents are commercially available and include reactive esters of 6 maleimidocaproic acid, 2-bromoacetic acid, and 2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-i-carboxylic acid. 30 The carboxyl groups can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. Immunogenic peptides can also be expressed as fusion proteins with carriers. The immunogenic peptide can be linked 35 at the amino terminus, the carboxyl terminus, or internally to the carrier. Optionally, multiple repeats of the immunogenic peptide can be present in the fusion protein.

21 4. Nucleic Acid Encoding Immunogens Immune responses against amyloid deposits can also be induced by administration of nucleic acids encoding AO peptide or other peptide immunogens. Such nucleic acids can be DNA or 5 RNA. A nucleic acid segment encoding the immunogen is typically linked to regulatory elements, such as a promoter and enhancer, that allow expression of the DNA segment in the intended target cells of a patient. For expression in blood cells, as is desirable for induction of an immune response, 10 promoter and enhancer elements from light or heavy chain immunoglobulin genes or the CMV major intermediate early promoter and enhancer are suitable to direct expression. The linked regulatory elements and coding sequences are often cloned into a vector. 15 A number of viral vector systems are available including retroviral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol. 67, 5911 (1993)); adeno associated virus vectors (see, e.g., Zhou et al., J. Exp. Med. 20 179, 1867 (1994)), viral vectors from the pox family including vaccinia virus and the avian pox viruses, viral vectors from the alpha virus genus such as those derived from Sindbis and Semliki Forest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519 (1996)), and papillomaviruses (Ohe et al., Human 25 Gene Therapy 6, 325-333 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma, Nucleic Acids. Res. 24, 2630-2622 (1996)). DNA encoding an immunogen, or a vector containing the same, can be packaged into liposomes. Suitable lipids and related analogs are described by US 5,208,036, 5,264,618, 5,279,833 30 and 5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed to or associated with particulate carriers, examples of which include polymethyl methacrylate polymers and polylactides and poly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap. (1996). 35 Gene therapy vectors or naked DNA can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, nasal, gastric, intradermal, intramuscular, subdermal, or 22 intracranial infusion) or topical application (see e.g., US 5,399,346). DNA can also be administered using a gene gun. See Xiao & Brandsma, supra. The DNA encoding an immunogen is precipitated onto the surface of microscopic 5 metal beads. The microprojectiles are accelerated with a shock wave or expanding helium gas, and penetrate tissues to a depth of several cell layers. For example, The AccelTM Gene Delivery Device manufactured by Agacetus, Inc. Middleton WI is suitable. Alternatively, naked DNA can pass through skin into .0 the blood stream simply by spotting the DNA onto skin with chemical or mechanical irritation (see WO 95/05853). In a further variation, vectors encoding immunogens can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, L5 tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector. III. Patients Amenable to Treatment 20 Patients amenable to treatment include individuals at risk of disease but not showing symptoms, as well as patients presently showing symptoms. In the case of Alzheimer's disease, virtually anyone is at risk of suffering from Alzheimer's disease if he or she lives long enough. 25 Therefore, the present methods can be administered prophylactically to the general population without any assessment of the risk of the subject patient. The present methods are especially useful for individuals who do have a known genetic risk of Alzheimer's disease. Such individuals 30 include those having relatives who have experienced this disease, and those whose risk is determined by analysis of genetic or biochemical markers. Genetic markers of risk toward Alzheimer's disease include mutations in the APP gene, particularly mutations at position 717 and positions 670 and 35 671 referred to as the Hardy and Swedish mutations respectively (see Hardy, TINS, supra). Other markers of risk are mutations in the presenilin genes, PS1 and PS2, and ApoE4, 23 family history of AD, hypercholesterolemia or atherosclerosis. Individuals presently suffering from Alzheimer's disease can be recognized from characteristic dementia, as well as the presence of risk factors described above. In addition, a 5 number of diagnostic tests are available for identifying individuals who have AD. These include measurement of CSF tau and A342 levels. Elevated tau and decreased A042 levels signify the presence of AD. Individuals suffering from Alzheimer's disease can also be diagnosed by MMSE or ADRDA 10 criteria as discussed in the Examples section. In asymptomatic patients, treatment can begin at any age (e.g., 10, 20, 30). Usually, however, it is not necessary to begin treatment until a patient reaches 40, 50, 60 or 70. Treatment typically entails multiple dosages over a period of 15 time. Treatment can be monitored by assaying antibody, or activated T-cell or B-cell responses to the therapeutic agent (e.g., AO peptide) over time. If the response falls, a booster dosage is indicated. In the case of potential Down's syndrome patients, treatment can begin antenatally by 20 administering therapeutic agent to the mother or shortly after birth. IV. Treatment Regimes In prophylactic applications, pharmaceutical compositions or medicants are administered to a patient susceptible to, or 25 otherwise at risk of, a particular disease in an amount sufficient to eliminate or reduce the risk or delay the outset of the disease. In therapeutic applications, compositions or medicants are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient 30 to cure, or at least partially arrest, the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a therapeutically- or pharmaceutically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in 35 several dosages until a sufficient immune response has been achieved. Typically, the immune response is monitored and 24 repeated dosages are given if the immune response starts to fade. Effective doses of the compositions of the present invention, for the treatment of the above described conditions 5 vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human, LO but in some diseases, such as mad cow disease, the patient can be a nonhuman mammal, such as a bovine. Treatment dosages need to be titrated to optimize safety and efficacy. The amount of immunogen depends on whether adjuvant is also administered, with higher dosages being required in the 15 absence of adjuvant. The amount of an immunogen for administration sometimes varies from 1 pg-500 pg per patient and more usually from 5-500 pg per injection for human administration. Occasionally, a higher dose of 1-2 mg per injection is used. Typically about 10, 20, 50 or 100 pg is 20 used for each human injection. The timing of injections can vary significantly from once a day, to once a year, to once a decade. On any given day that a dosage of immunogen is given, the dosage is greater than 1 pg/patient and usually greater than 10 pg/ patient if adjuvant is also administered, and 25 greater than 10 pg/patient and usually greater than 100 pg/patient in the absence of adjuvant. A typical regimen consists of an immunization followed by booster injections at 6 weekly intervals. Another regimen consists of an immunization followed by booster injections 1, 2 and 12 months 30 later. Another regimen entails an injection every two months for life. Alternatively, booster injections can be on an irregular basis as indicated by monitoring of immune response. For passive immunization with an antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 35 mg/kg of the host body weight. Doses for nucleic acids encoding immunogens range from about 10 ng to 1 g, 100 ng to 100 mg, 1 gg to 10 mg, or 30-300 pg DNA per patient. Doses 25 for infectious viral vectors vary from 10-109, or more, virions per dose. Agents for inducing an immune response can be administered by parenteral, topical, intravenous, oral, subcutaneous, 5 intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment. The most typical route of administration is subcutaneous although others can be equally effective. The next most common is intramuscular injection. This type of injection is most typically performed LO in the arm or leg muscles. Intravenous injections as well as intraperitoneal injections, intraarterial, intracranial, or intradermal injections are also effective in generating an immune response. In some methods, agents are injected directly into a particular tissue where deposits have L5 accumulated. Agents of the invention can optionally be administered in combination with other agents that are at least partly effective in treatment of amyloidogenic disease. In the case of Alzheimer's and Down's syndrome, in which amyloid deposits 20 occur in the brain, agents of the invention can also be administered in conjunction with other agents that increase passage of the agents of the invention across the blood-brain barrier. Immunogenic agents of the invention, such as peptides, are 25 sometimes administered in combination with an adjuvant. A variety of adjuvants can be used in combination with a peptide, such as A0, to elicit an immune response. Preferred adjuvants augment the intrinsic response to an immunogen without causing conformational changes in the immunogen that 30 affect the qualitative form of the response. Preferred adjuvants include alum, 3 De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211). QS21 is a triterpene glycoside or saponin isolated from the bark of the Quillaja Saponaria Molina tree found in South America (see Kensil et al., in 35 Vaccine Design: The Subunit and Ajuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); US Patent No. 5,057,540). Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune 26 stimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (Bioworld Today, Nov. 15, 1998). Alternatively, AO can be coupled to an adjuvant. For example, a lipopeptide version of 5 AO can be prepared by coupling palmitic acid or other lipids directly to the N-terminus of AO as described for hepatitis B antigen vaccination (Livingston, J. Immunrol. 159, 1383-1392 (1997)). However, such coupling should not substantially change the conformation of AO so as to affect the nature of LO the immune response thereto. Adjuvants can be administered as a component of a therapeutic composition with an active agent or can be administered separately, before, concurrently with, or after administration of the therapeutic agent. A preferred class of adjuvants is aluminum salts (alum), L5 such as aluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvants can be used with or without other specific immunostimulating agents such as MPL or 3-DMP, QS21, polymeric or monomeric amino acids such as polyglutamic acid or polylysine. Another class of adjuvants is oil-in-water 20 emulsion formulations. Such adjuvants can be used with or without other specific immunostimulating agents such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D 25 isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3 hydroxyphosphoryloxy)-ethylamine (MTP-PE), N acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala dipalmitoxy propylamide (DTP-DPP) theramideTM), or other bacterial cell wall components. Oil-in-water emulsions 30 include (a) MF59 (WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton MA), (b) SAF, containing 10% Squalane, 35 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi" adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) 27 containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox m ). 5 Another class of preferred adjuvants is saponin adjuvants, such as Stimulon" (QS21, Aquila, Worcester, MA) or particles generated therefrom such as ISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvants include Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant 10 (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). An adjuvant can be administered with an immunogen as a single composition, or can be administered before, concurrent 15 with or after administration of the immunogen. Immunogen and adjuvant can be packaged and supplied in the same vial or can be packaged in separate vials and mixed before use. Immunogen and adjuvant are typically packaged with a label indicating the intended therapeutic application. If immunogen and 20 adjuvant are packaged separately, the packaging typically includes instructions for mixing before use. The choice of an adjuvant and/or carrier depends on the stability of the vaccine containing the adjuvant, the route of administration, the dosing schedule, the efficacy of the adjuvant for the 25 species being vaccinated, and, in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies. For example, Complete Freund's adjuvant is not suitable for human administration. Alum, MPL and QS21 are 30 preferred. Optionally, two or more different adjuvants can be used simultaneously. Preferred combinations include alum with MPL, alum with QS21, MPL with QS21, and alum, QS21 and MPL together. Also, Incomplete Freund's ajuvant can be used (Chang et al., Advanced Drug Delivery Reviews 32, 173-186 35 (1998)), optionally in combination with any of alum, QS21, and MPL and all combinations thereof. Agents of the invention are often administered as pharmaceutical compositions comprising an active therapeutic 28 agent, i.e., and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pennsylvania, 1980). The preferred form depends on the intended mode of 5 administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human LO administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or L5 formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. However, some reagents suitable for administration to animals, such as Complete Freund's adjuvant are not typically included in compositions for human use. 20 Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid 25 aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants). For parenteral administration, agents of the invention can be administered as injectable dosages of a solution or 30 suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as water oils, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and 35 the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as 29 propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for 5 solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (see Langer, Science 249, 10 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97 119 (1997). The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. 15 Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications. For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such 20 suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% 2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium 25 carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%. Topical application can result in transdermal or 30 intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins (See Glenn et al., Nature 391, 851 (1998)). Co-administration can be achieved by using the 35 components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein. Alternatively, transdermal delivery can be achieved using a skin path or using transferosomes (Paul et al., Eur. J.

30 Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)). V. Methods of Diagnosis The invention provides methods of detecting an immune 5 response against AO peptide in a patient suffering from or susceptible to Alzheimer's disease. The methods are particularly useful for monitoring a course of treatment being administered to a patient. The methods can be used to monitor both therapeutic treatment on symptomatic patients and 10 prophylactic treatment on asymptomatic patients. Some methods entail determining a baseline value of an immune response in a patient before administering a dosage of agent, and comparing this with a value for the immune response after treatment. A significant increase (i.e., greater than 15 the typical margin of experimental error in repeat measurements of the same sample, expressed as one standard deviation from the mean of such measurments) in value of the immune response signals a positive treatment outcome (i.e., that administration of the agent has achieved or augmented an 20 immune response). If the value for immune response does not change significantly, or decreases, a negative treatment outcome is indicated. In general, patients undergoing an initial course of treatment with an agent are expected to show an increase in immune response with successive dosages, which 25 eventually reaches a plateau. Administration of agent is generally continued while the immune response is increasing. Attainment of the plateau is an indicator that the administered of treatment can be discontinued or reduced in dosage or frequency. 30 In other methods, a control value (i.e., a mean and standard deviation) of immune response is determined for a control population. Typically the individuals in the control population have not received prior treatment. Measured values of immune response in a patient after administering a 35 therapeutic agent are then compared with the control value. A significant increase relative to the control value (e.g., greater than one standard deviation from the mean) signals a 31 positive treatment outcome. A lack of significant increase or a decrease signals a negative treatment outcome. Administration of agent is generally continued while the immune response is increasing relative to the control value. 5 As before, attainment of a plateau relative to control values in an indicator that the administration of treatment can be discontinued or reduced in dosage or frequency. In other methods, a control value of immune response (e.g., a mean and standard deviation) is determined from a control 10 population of individuals who have undergone treatment with a therapeutic agent and whose immune responses have plateaued in response to treatment. Measured values of immune response in a patient are compared with the control value. If the measured level in a patient is not significantly different 15 (e.g., more than one standard deviation) from the control value, treatment can be discontinued. If the level in a patient is significantly below the control value, continued administration of agent is warranted. If the level in the patient persists below the control value, then a change in 20 treatment regime, for example, use of a different adjuvant may be indicated. In other methods, a patient who is not presently receiving treatment but has undergone a previous course of treatment is monitored for immune response to determine whether a 25 resumption of treatment is required. The measured value of immune response in the patient can be compared with a value of immune response previously achieved in the patient after a previous course of treatment. A significant decrease relative to the previous measurement (i.e., greater than a typical 30 margin of error in repeat measurements of the same sample) is an indication that treatment can be resumed. Alternatively, the value measured in patient can be compared with a control value (mean plus standard deviation) determined in population of patients after undergoing a course of treatment. 35 Alternatively, the measured value in a patient can be compared with a control value in populations of prophylactically treated patients who remain free of symptoms of disease, or populations of therapeutically treated patients who show 32 amelioration of disease characteristics. In all of these cases, a significant decrease relative to the control level (i.e., more than a standard deviation) is an indicator that treatment should be resumed in a patient. 5 The tissue sample for analysis is typically blood, plasma, serum, mucus or cerebral spinal fluid from the patient. The sample is analyzed for indicia of an immune response to any form of AO peptide, typically A042. The immune response can be determined from the presence of, e.g., antibodies or T 10 cells that specifically bind to AO peptide. ELISA methods of detecting antibodies specific to AO are described in the Examples section. Methods of detecting reactive T-cells have been described above (see Definitions). The invention further provides diagnostic kits for 15 performing the diagnostic methods described above. Typically, such kits contain an agent that specifically binds to antibodies to AO or reacts with T-cells specific for A0. The kit can also include a label. For detection of antibodies to AO, the label is typically in the form of labelled anti 20 idiotypic antibodies. For detection of antibodies, the agent can be supplied prebound to a solid phase, such as to the wells of a microtiter dish. For detection of reactive T cells, the label can be supplied as 3 H-thymidine to measure a proliferative response. Kits also typically contain labelling 25 providing directions for use of the kit. The labelling may also include a chart or other correspondence regime correlating levels of measured label with levels of antibodies to AO or T-cells reactive with A0. The term labelling refers to any written or recorded material that is attached to, or 30 otherwise accompanies a kit at any time during its manufacture, transport, sale or use. For example, the term labelling encompasses advertising leaflets and brochures, packaging materials, instructions, audio or video cassettes, computer discs, as well as writing imprinted directly on kits. 35 EXAMPLES I. Prophylactic Efficacy of A8 Against AD 33 These examples describe administration of A042 peptide to transgenic mice overexpressing APP with a mutation at position 717 (APP717V4F) that predisposes them to develop Alzheimer's like neuropathology. Production and characteristics of these 5 mice (PDAPP mice) is described in Games et al., Nature, supra. These animals, in their heterozygote form, begin to deposit AO at six months of age forward. By fifteen months of age they exhibit levels of AO deposition equivalent to that seen in Alzheimer's disease. PDAPP mice were injected with aggregated 10 A0 4 2 (aggregated A0 4 2 ) or phosphate buffered saline. Aggregated A0 42 was chosen because of its ability to induce antibodies to multiple epitopes of A0.

34 A. Methods 1. Source of Mice Thirty PDAPP heterogenic female mice were randomly divided into the following groups: 10 mice to be injected with 5 aggregated A3 42 (one died in transit), 5 mice to be injected with PBS/adjuvant or PBS, and 10 uninjected controls. Five mice were injected with serum amyloid protein (SAP). 2. Preparation of Immunogens Preparation of aggregated A3 42 : two milligrams of A3 42 (US .0 Peptides Inc, lot K-42-12) was dissolved in 0.9 ml water and made up to 1 ml by adding 0.1 ml 10 x PBS. This was vortexed and allowed to incubate overnight 370 C, under which conditions the peptide aggregated. Any unused AO was stored as a dry lyophilized powder at -200 C until the next .5 injection. 3. Preparation of Iniections 100 pg of aggregated A0 42 in PBS per mouse was emulsified 1:1 with Complete Freund's adjuvant (CFA) in a final volume of 400 pl emulsion for the first immunization, followed by a .0 boost of the same amount of immunogen in Incomplete Freund's adjuvant (IFA) at 2 weeks. Two additional doses in IFA were given at monthly intervals. The subsequent immunizations were done at monthly intervals in 500 pl of PBS. Injections were delivered intraperitoneally (i.p.). 25 PBS injections followed the same schedule and mice were injected with a 1:1 mix of PBS/ Adjuvant at 400 pl per mouse, or 500 Al of PBS per mouse. SAP injections likewise followed the same schedule using a dose of 100 pg per injection. 4. Titration of Mouse Bleeds, Tissue Preparation 30 and Immunohistochemistry The above methods are described infra in General Materials and Methods. B. Results PDAPP mice were injected with either aggregated A0 42 35 (aggregated A0 4 2 ), SAP peptides, or phosphate buffered saline.

35 A group of PDAPP mice were also left as uninjected, positive controls. The titers of the mice to aggregated A0 4 2 were monitored every other month from the fourth boost until the mice were one year of age. Mice were sacrificed at 13 months. 5 At all time points examined, eight of the nine aggregated A0 42 mice developed a high antibody titer, which remained high throughout the series of injections (titers greater than 1/10000). The ninth mouse had a low, but measurable titer of approximately 1/1000 (Figure 1, Table 1). SAPP-injected mice .0 had titers of 1:1,000 to 1:30,000 for this immunogen with only a single mice exceeding 1:10,0000. The PBS-treated mice were titered against aggregated A0 42 at six, ten and twelve months. At a 1/100 dilution the PBS mice when titered against aggregated A0 4 2 only exceeded 4 .5 times background at one data point, otherwise, they were less than 4 times background at all time points (Table 1). The SAP-specific response was negligible at these time points with all titers less than 300. Seven out of the nine mice in the aggregated Ao1-42 group 0 had no detectable amyloid in their brains. In contrast, brain tissue from mice in the SAP and PBS groups contained numerous 3D6-positive amyloid deposits in the hippocampus, as well as in the frontal and cingulate cortices. The pattern of deposition was similar to that of untreated controls, with 25 characteristic involvement of vulnerable subregions, such as the outer molecular layer of the hippocampal dentate gyrus. One mouse from the AO 1-42-injected group had a greatly reduced amyloid burden, confined to the hippocampus. An isolated plaque was identified in another A0 1-42-treated 30 mouse. Quantitative image analyses of the amyloid burden in the hippocampus verified the dramatic reduction achieved in the AN1792-treated animals (Fig. 2). The median values of the amyloid burden for the PBS group (2.22%), and for the 35 untreated control group (2.65%) were significantly greater than for those immunized with AN1792 (0.00%, p=0.0005). In contrast, the median value for the group immunized with SAP peptides (SAPP) was 5.74%. Brain tissue from the untreated, 36 control mice contained numerous AO amyloid deposits visualized with the A-specific monoclonal antibody (mAb) 3D6 in the hippocampus, as well as in the retrosplenial cortex. A similar pattern of amyloid deposition was also seen in mice 5 immunized with SAPP or PBS (Fig. 2). In addition, in these latter three groups there was a characteristic involvement of vulnerable subregions of the brain classically seen in AD, such as the outer molecular layer of the hippocampal dentate gyrus, in all three of these groups. 10 The brains that contained no AO deposits were also devoid of neuritic plaques that are typically visualized in PDAPP mice with the human APP antibody 8E5. All of brains from the remaining groups (SAP-injected, PBS and uninjected mice) had numerous neuritic plaques typical of untreated PDAPP mice. A 15 small number of neuritic plaques were present in one mouse treated with AN1792, and a single cluster of dystrophic neurites was found in a second mouse treated with AN1792. Image analyses of the hippocampus, and shown in Fig. 3, demonstrated the virtual elimination of dystrophic neurites in 20 AN1792-treated mice (median 0.00%) compared to the PBS recipients (median 0.28%, p = 0.0005). Astrocytosis characteristic of plaque-associated inflammation was also absent in the brains of the Al-42 injected group. The brains from the mice in the other groups 25 contained abundant and clustered GFAP-positive astrocytes typical of AO plaque-associated gliosis. A subset of the GFAP-reacted slides were counter-stained with Thioflavin S to localize the AO deposits. The GFAP-positive astrocytes were associated with AO plaques in the SAP, PBS and untreated 30 controls. No such association was found in the plaque-negative Aol-42 treated mice, while minimal plaque-associated gliosis was identified in one mouse treated with AN1792. Image analyses, shown in Fig. 4 for the retrosplenial 35 cortex, verified that the reduction in astrocytosis was significant with a median value of 1.56% for those treated with AN1792 versus median values greater than 6% for groups immunized with SAP peptides, PBS or untreated (p=0.001 7

)

37 Evidence from a subset of the AO 1-42- and PBS-injected mice indicated plaque-associated MHC II immunoreactivity was absent in the A31-42 injected mice, consistent with lack of an A-related inflammatory response. 5 Sections of the mouse brains were also reacted with a mAO specific for MAC-1, a cell surface protein. MAC-1 (CD11b) is an integrin family member and exists as a heterodimer with CD18. The CD11b/CD18 complex is present on monocytes, macrophages, neutrophils and natural killer cells (Mak and LO Simard). The resident MAC-1-reactive cell type in the brain is likely to be microglia based on similar phenotypic morphology in MAC-1 immunoreacted sections. Plaque-associated MAC-1 labeling was lower in the brains of mice treated with AN1792 compared to the PBS control group, a finding consistent L5 with the lack of an A-induced inflammatory response. C. Conclusion The lack of AO plaques and reactive neuronal and gliotic changes in the brains of the A01-42-injected mice indicate that no or extremely little amyloid was deposited in their 20 brains, and pathological consequences, such as gliosis and neuritic pathology, were absent. PDAPP mice treated with A31-42 show essentially the same lack of pathology as control nontransgenic mice. Therefore, Ao1-42 injections are highly effective in the prevention of deposition or clearance of 25 human AO from brain tissue, and elimination of subsequent neuronal and inflammatory degenerative changes. Thus, administration of AO peptide has therapeutic benefit in prevention of AD. II. Dose Response Study 30 Groups of five-week old, female Swiss Webster mice (N=6 per group) were immunized with 300, 100, 33, 11, 3.7, 1.2, 0.4, or 0.13 ug of AO formulated in CFA/IFA administered intraperitoneally. Three doses were given at biweekly intervals followed by a fourth dose one month later. The 35 first dose was emulsified with CFA and the remaining doses were emulsified with IFA. Animals were bled 4-7 days 38 following each immunization starting after the second dose for measurement of antibody titers. Animals in a subset of three groups, those immunized with 11, 33, or 300 pg of antigen, were additionally bled at approximately monthly 5 intervals for four months following the fourth immunization to monitor the decay of the antibody response across a range of vaccine doses. These animals received a final fifth immunization at seven months after study initiation. They were sacrificed one week later to measure antibody responses .0 to AN1792 and to perform toxicological analyses. A declining dose response was observed from 300 to 3.7 pg with no response at the two lowest doses. Mean antibody titers are about 1:1000 after 3 doses and about 1:10,000 after 4 doses of 11-300 pg of antigen (see Fig. 5). .5 Antibody titers rose dramatically for all but the lowest dose group following the third immunization with increases in GMTs ranging from 5- to 25-fold. Low antibody responses were then detectable for even the 0.4 pg recipients. The 1.2 and 3.7 pg groups had comparable titers with GMTs of about 1000 0O and the highest four doses clustered together with GMTs of about 25,000, with the exception of the 33 pg dose group with a lower GMT of 3000. Following the fourth immunization, the titer increase was more modest for most groups. There was a clear dose response across the lower antigen dose groups from 15 0.14 pug to 11 pg ranging from no detectable antibody for recipients of 0.14 pg to a GMT of 36,000 for recipients of 11 pg. Again, titers for the four highest dose groups of 11 to 300 pg clustered together. Thus following two immunizations, the antibody titer was dependent on the antigen 30 dose across the broad range from 0.4 to 300 pg. By the third immunization, titers of the highest four doses were all comparable and they remained at a plateau after an additional immunization. One month following the fourth immunization, titers were 2 35 to 3-fold higher in the 300 pg group than those measured from blood drawn five days following the immunization (Fig. 6). This observation suggests that the peak anamnestic antibody response occurred later than 5 days post-immunization. A more 39 modest (50%) increase was seen at this time in the 33 pg group. In the 300 pg dose group at two months following the last dose, GMTs declined steeply by about 70%. After another month, the decline was less steep at 45% (100 pg) and about 5 14% for the 33 and 11 pg doses. Thus, the rate of decline in circulating antibody titers following cessation of immunization appears to be biphasic with a steep decline the first month following peak response followed by a more modest rate of decrease thereafter. o The antibody titers and the kinetics of the response of these Swiss Webster mice are similar to those of young heterozygous PDAPP transgenic mice immunized in a parallel manner. Dosages effective to induce an immune response in humans are typically similar to dosages effective in mice. .5 III. Screen For Therapeutic Efficacy Against Established AD This assay is designed to test immunogenic agents for activity in arresting or reversing neuropathological characteristics of AD in aged animals. Immunizations with 42 amino acid long A0 (AN1792) were begun at a timepoint when 0 amyloid plaques are already present in the brains of the PDAPP mice. Over the timecourse used in this study, untreated PDAPP mice develop a number of neurodegenerative changes that resemble those found in AD (Games et al., supra and Johnson 25 Wood et al., Proc. Natl. Acad. Sci. USA 94, 1550-1555 (1997)). The deposition of AO into amyloid plaques is associated with a degenerative neuronal response consisting of aberrant axonal and dendritic elements, called dystrophic neurites. Amyloid deposits that are surrounded by and contain dystrophic 30 neurites called neuritic plaques. In both AD and the PDAPP mouse, dystrophic neurites have a distinctive globular structure, are immunoreactive with a panel of antibodies recognizing APP and cytoskeletal components, and display complex subcellular degenerative changes at the 35 ultrastructural level. These characteristics allow for disease-relevant, selective and reproducible measurements of neuritic plaque formation in the PDAPP brains. The dystrophic 40 neuronal component of PDAPP neuritic plaques is easily visualized with an antibody specific for human APP (mA 8E5), and is readily measurable by computer-assisted image analysis. Therefore, in addition to measuring the effects of AN1792 on 5 amyloid plaque formation, we monitored the effects of this treatment on the development of neuritic dystrophy. Astrocytes and microglia are non-neuronal cells that respond to and reflect the degree of neuronal injury. GFAP positive astrocytes and MHC II-positive microglia are commonly 10 observed in AD, and their activation increases with the severity of the disease. Therefore, we also monitored the development of reactive astrocytosis and microgliosis in the AN1792-treated mice. A. Materials and Methods 15 Forty-eight, heterozygous female PDAPP mice, 11 to 11.5 months of age, obtained from Charles River, were randomly divided into two groups: 24 mice to be immunized with 100 pg of AN1792 and 24 mice to be immunized with PBS, each combined with Freund's adjuvant. The AN1792 and PBS groups were again 20 divided when they reached ~15 months of age. At 15 months of age approximately half of each group of the AN1792- and PBS treated animals were euthanized (n=10 and 9, respectively), the remainder continued to receive immunizations until termination at ~18 months (n=9 and 12, respectively). A total 25 of.8 animals (5 AN1792, 3 PBS) died during the study. In addition to the immunized animals, one-year old (n=10), 15 month old (n=10) and 18-month old (n=10) untreated PDAPP mice were included for comparison in the ELISAs to measure aO and APP levels in the brain; the one-year old animals were also 30 included in the immunohistochemical analyses. Methodology was as in Example 1 unless otherwise indicated. US Peptides lot 12 and California Peptides lot ME0339 of AN1792 were used to prepare the antigen for the six immunizations administered prior to the 15-month timepoint. 35 California Peptides lots ME0339 and ME0439 were used for the three additional immunizations administered between 15 and 18 months.

41 For immunizations, 100 pg of AN1792 in 200 pl PBS or PBS alone was emulsified 1:1 (vol:vol) with Complete Freund's adjuvant (CFA) or Incomplete Freund's adjuvant (IFA) or PBS in a final volume of 400 pl. The first immunization was 5 delivered with CFA as adjuvant, the next four doses were given with IFA and the final four doses with PBS alone without added adjuvant. A total of nine immunizations were given over the seven-month period on a two-week schedule for the first three doses followed by a four-week interval for the remaining 10 injections. The four-month treatment group, euthanized at 15 months of age, received only the first 6 immunizations. B. Results 1. Effects of AN1792 Treatment on Amyloid Burden The results of AN1792 treatment on cortical amyloid burden 15 determined by quantitative image analysis are shown in Fig. 7. The median value of cortical amyloid burden was 0.28% in a group of untreated 12-month old PDAPP mice, a value representative of the plaque load in mice at the study's initiation. At 18 months, the amyloid burden increased over 20 17-fold to 4.87% in PBS-treated mice, while AN1792-treated mice had a greatly reduced amyloid burden of only 0.01%, notably less than the 12-month untreated and both the 15- and 18-month PBS-treated groups. The amyloid burden was significantly reduced in the AN1792 recipients at both 15 (96% 25 reduction; p=0.003) and 18 (>99% reduction; p=0.0002) months. Typically, cortical amyloid deposition in PDAPP mice initiates in the frontal and retrosplenial cortices (RSC) and progresses in a ventral-lateral direction to involve the temporal and entorhinal cortices (EC). Little or no amyloid 30 was found in the EC of 12 month-old mice, the approximate age at which AN1792 was first administered. After 4 months of AN1792 treatment, amyloid deposition was greatly diminished in the RSC, and the progressive involvement of the EC was entirely eliminated by AN1792 treatment. The latter 35 observation showed that AN1792 completely halted the progression of amyloid that would normally invade the temporal 42 and ventral cortices, as well as arrested or possibly reversed deposition in the RSC. The profound effects of AN1792 treatment on developing cortical amyloid burden in the PDAPP mice are further 5 demonstrated by the 18-month group, which had been treated for seven months. A near complete absence of cortical amyloid was found in the AN1792-treated mouse, with a total lack of diffuse plaques, as well as a reduction in compacted deposits. 2. AN1792 Treatment-associated Cellular and Morphological .0 Changes A population of A-positive cells was found in brain regions that typically contain amyloid deposits. Remarkably, in several brains from AN1792 recipients, very few or no extracellular cortical amyloid plaques were found. Most of .5 the AO immunoreactivity appeared to be contained within cells with large lobular or clumped soma. Phenotypically, these cells resembled activated microglia or monocytes. They were immunoreactive with antibodies recognizing ligands expressed by activated monocytes and microglia (MHC II and CD11b) and 0O were occasionally associated with the wall or lumen of blood vessels. Comparison of near-adjacent sections labeled with AO and MHC II-specific antibodies revealed that similar patterns of these cells were recognized by both classes of antibodies. Detailed examination of the AN1792-treated brains revealed 25 that the MHC II-positive cells were restricted to the vicinity of the limited amyloid remaining in these animals. Under the fixation conditions employed, the cells were not immunoreactive with antibodies that recognize T cell (CD3, CD3e) or B cell (CD45RA, CD45RB) ligands or leukocyte common 30 antigen (CD45), but were reactive with an antibody recognizing leukosialin (CD43) which cross-reacts with monocytes. No such cells were found in any of the PBS-treated mice. PDAPP mice invariably develop heavy amyloid deposition in the outer molecular layer of the hippocampal dentate gyrus. 35 The deposition forms a distinct streak within the perforant pathway, a subregion that classically contains amyloid plaques in AD. The characteristic appearance of these deposits in PBS-treated mice resembled that previously characterized in 43 untreated PDAPP mice. The amyloid deposition consisted of both diffuse and compacted plaques in a continuous band. In contrast, in a number of brains from AN1792-treated mice this pattern was drastically altered. The hippocampal amyloid 5 deposition no longer contained diffuse amyloid, and the banded pattern was completely disrupted. Instead, a number of unusual punctate structures were present that are reactive with anti-A3 antibodies, several of which appeared to be amyloid-containing cells. 10 MHC II-positive cells were frequently observed in the vicinity of extracellular amyloid in AN1792-treated animals. The pattern of association of A-positive cells with amyloid was very similar in several brains from AN1792-treated mice. The distribution of these monocytic cells was restricted to 15 the proximity of the deposited amyloid and was entirely absent from other brain regions devoid of AO plaques. Quantitative image analysis of MHC II and MAC I-labeled sections revealed a trend towards increased immunoreactivity in the RSC and hippocampus of AN1792-treated mice compared to 20 the PBS group which reached significance with the measure of MAC 1 reactivity in hippocampus. These results are indicative of active, cell-mediated removal of amyloid in plaque-bearing brain regions. 3. AN1792 Effects on AU Levels: ELISA Determinations 25 (a) Cortical Levels In untreated PDAPP mice, the median level of total AO in the cortex at 12 months was 1,600 ng/g, which increased to 8,700 ng/g by 15 months (Table 2). At 18 months the value was 22,000 ng/g, an increase of over 10-fold during the time 30 course of the experiment. PBS-treated animals had 8,600 ng/g total AO at 15 months which increased to 19,000 ng/g at 18 months. In contrast, AN1792-treated animals had 81% less total A# at 15 months (1,600 ng/g) than the PBS-immunized group. Significantly less (p=0.0001) total AO (5,200 ng/g) 35 was found at 18 months when the AN1792 and PBS groups were compared (Table 2), representing a 72% reduction in the AO that would otherwise be present. Similar results were 44 obtained when cortical levels of A342 were compared, namely that the AN1792-treated group contained much less A042, but in this case the differences between the AN1792 and PBS groups were significant at both 15 months (p=0.04) and 18 months 5 (p=0.0001, Table 2). Table 2: Median AO Levels (ng/g) in Cortex UNTREATED PBS AN1792 Age Total A# A#42 (n) Total AO A#42 (n) Total A#42 (n) 12 1,600 1,300 (10) 10 15 8,700 8,300 (10) 8,600 7,200 (9) 1,600 1,300- (10) 18 22,200 18,500 (10) 19,000 15,900 (12) 5,200" 4,000" (9) * p = 0.0412 ** p = 0.0001 (b) Hippocampal Levels 15 In untreated PDAPP mice, median hippocampal levels of total AO at twelve months of age were 15,000 ng/g which increased to 51,000 ng/g at 15 months and further to 81,000 ng/g at 18 months (Table 3). Similarly, PBS immunized mice showed values of 40,000 ng/g and 65,000 ng/g at 15 months and 18 months, 20 respectively. AN1792 immunized animals exhibited less total AO, specifically 25,000 ng/g and 51,000 ng/g at the respective 15-month and 18-month timepoints. The 18-month AN1792-treated group value was significantly lower than that of the PBS treated group (p= 0.0105; Table 3). Measurement of 25 A342 gave the same pattern of results, namely that levels in the AN1792-treated group were significantly lower than in the PBS group (39,000 ng/g vs. 57,000 ng/g, respectively; p=0.0022) at the 18-month evaluation (Table 3).

45 Table 3: Median Afl Levels (ng/g) in Hippocampus UNTREATED PBS AN1792 Age Total AO A,342 (n) Total AO A042 (n) Total A342 (n) 12 15,500 11,100 (10) 5 15 51,500 44,400 (10) 40,100 35,700 (9) 24,500 22,100 (10) 18 80,800 64,200 (10) 65,400 57,100 (12) 50,900' 38,900~ (9) * p = 0.0105 ** p = 0.0022 (c) Cerebellar Levels In 12-month untreated PDAPP mice, the median cerebellar level of total AO was 15 ng/g (Table 4). At 15 months, this .0 median increased to 28 ng/g and by 18 months had risen to 35 ng/g. PBS-treated animals displayed median total AO values of 21 ng/g at 15 months and 43 ng/g at 18 months. AN1792-treated animals were found to have 22 ng/g total AO at 15 months and significantly less (p=0.002) total AO at 18 months (25 ng/g) L5 than the corresponding PBS group (Table 4). Table 4: Median Af Levels (ng/g) in Cerebellum UNTREATED PBS AN1792 Age (months) Total A# (n) Total A# (n) Total A# (n) 12 15.6 (10) 15 27.7 (10) 20.8 (9) 21.7 (10) 20 18 35.0 (10) 43.1 (12) 24.8* (9) * p = 0.0018 4. Effects of AN1792 Treatment on APP Levels APP-a and the full-length APP molecule both contain all or part of the AO sequence and thus could be potentially impacted by the generation of an AN1792-directed immune response. In 25 studies to date, a slight increase in APP levels has been noted as neuropathology increases in the PDAPP mouse. In the cortex, levels of either APP-a/FL (full length) or APP-a were essentially unchanged by treatment with the exception that APP-a was reduced by 19% at the 18-month timepoint in the 30 AN1792-treated vs. the PBS-treated group. The 18-month AN1792-treated APP values were not significantly different 46 from values of the 12-month and 15-month untreated and 15 month PBS groups. In all cases the APP values remained within the ranges that are normally found in PDAPP mice. 5. Effects of AN1792 Treatment on Neurodegenerative and 5 Gliotic Pathology Neuritic plaque burden was significantly reduced in the frontal cortex of AN1792-treated mice compared to the PBS group at both 15 (84%; p=0.03) and 18 (55%; p=0.01) months of age (Fig. 8). The median value of the neuritic plaque burden 10 increased from 0.32% to 0.49% in the PBS group between 15 and 18 months of age. This contrasted with the greatly reduced development of neuritic plaques in the AN1792 group, with median neuritic plaque burden values of 0.05% and 0.22%, in the 15 and 18 month groups, respectively. 15 Immunizations with AN1792 seemed well tolerated and reactive astrocytosis was also significantly reduced in the RSC of AN1792-treated mice when compared to the PBS group at both 15 (56%; p=0.011) and 18 (39%; p=0.028) months of age (Fig. 9). Median values of the percent of astrocytosis in 20 the PBS group increased between 15 and 18 months from 4.26% to 5.21%. AN1792-treatment suppressed the development of astrocytosis at both time points to 1.89% and 3.2%, respectively. This suggests the neuropil was not being damaged by the clearance process. 25 6. Antibody Responses As described above, eleven-month old, heterozygous PDAPP mice (N=24) received a series of 5 immunizations of 100 pg of AN1792 emulsified with Freund's adjuvant and administered intraperitoneally at weeks 0, 2, 4, 8, and 12, and a sixth 30 immunization with PBS alone (no Freund's adjuvant) at week 16. As a negative control, a parallel set of 24 age-matched transgenic mice received immunizations of PBS emulsified with the same adjuvants and delivered on the same schedule. Animals were bled within three to seven days following each 35 immunization starting after the second dose. Antibody responses to AN1792 were measured by ELISA. Geometric mean titers (GMT) for the animals that were immunized with AN1792 47 were approximately 1,900, 7,600, and 45,000 following the second, third and last (sixth) doses respectively. No A3 specific antibody was measured in control animals following the sixth immunization. 5 Approximately one-half of the animals were treated for an additional three months, receiving immunizations at about 20, 24 and 27 weeks. Each of these doses was delivered in PBS vehicle alone without Freund's adjuvant. Mean antibody titers remained unchanged over this time period. In fact, antibody .0 titers appeared to remain stable from the fourth to the eighth bleed corresponding to a period covering the fifth to the ninth injections. To determine if the A3-specific antibodies elicited by immunization that were detected in the sera of AN1792-treated .5 mice were also associated with deposited brain amyloid, a subset of sections from the AN1792- and PBS-treated mice were reacted with an antibody specific for mouse IgG. In contrast to the PBS group, A0 plaques in AN1792-treated brains were coated with endogenous IgG. This difference between the two 20 groups was seen in both 15-and 18-month groups. Particularly striking was the lack of labeling in the PBS group, despite the presence of a heavy amyloid burden in these mice. These results show that immunization with a synthetic A0 protein generates antibodies that recognize and bind in vivo to the AO 25 in amyloid plaques. 7. Cellular-Mediated Immune Responses Spleens were removed from nine AN1792-immunized and 12 PBS-immunized 18-month old PDAPP mice 7 days after the ninth immunization. Splenocytes were isolated and cultured for 72 h 30 in the presence of A340, A42, or A040-1 (reverse order protein). The mitogen Con A served as a positive control. Optimum responses were obtained with >1.7 pM protein. Cells from all nine AN1792-treated animals proliferated in response to either A1-40 or A1-42 protein, with equal levels of 35 incorporation for both proteins (Fig. 10, Upper Panel). There was no response to the A40-1 reverse protein. Cells from 48 control animals did not respond to any of the AO proteins (Fig. 10, Lower Panel). C. Conclusion The results of this study show that AN1792 immunization of 5 PDAPP mice possessing existing amyloid deposits slows and prevents progressive amyloid deposition and retard consequential neuropathological changes in the aged PDAPP mouse brain. Immunizations with AN1792 essentially halted amyloid developing in structures that would normally succumb 10 to amyloidosis. Thus, administration of AO peptide has therapeutic benefit in the treatment of AD. IV. Screen of A3 Fragments 100 PDAPP mice age 9-11 months are immunized with 9 different regions of APP and AO to determine which epitopes 15 convey the response. The 9 different immunogens and one control are injected i.p. as described above. The immunogens include four human AO peptide conjugates 1-12, 13-28, 32-42, 1-5, all coupled to sheep anti-mouse IgG via a cystine link; an APP polypeptide aa 592-695, aggregated human AO 1-40, and 20 aggregated human AO 25-35, and aggregated rodent A042. Aggregated A342 and PBS are used as controls. Ten mice are used per treatment group. Titers are monitored as above and mice are euthanized at the end of 4 months of injections. Histochemistry, AO levels, and toxicology are determined post 25 mortem. A. Materials and Methods 1. Preparation of Immunogens Preparation of coupled AO peptides: four human AO peptide conjugates (amino acid residues 1-5, 1-12, 13-28, and 33-42, 30 each conjugated to sheep anti-mouse IgG) were prepared by coupling through an artificial cysteine added to the AO peptide using the crosslinking reagent sulfo-EMCS. The AO peptide derivatives were synthesized with the following final amino acid sequences. In each case, the location of the 35 inserted cysteine residue is indicated by underlining. The 49 A013-28 peptide derivative also had two glycine residues added prior to the carboxyl terminal cysteine as indicated. A01-12 peptide NH 2 -DAEFRHDSGYEVC COOH A31-5 peptide NH 2 -DAEFRC COOH 5 A33-42 peptide NH 2 -C-amino-hevtanoic acid-GLMVGGVVIA COOH A13-28 peptide Ac-NH-HHQKLVFFAEDVGSNKGGC-COOH To prepare for the coupling reaction, ten mg of sheep anti-mouse IgG (Jackson ImmunoResearch Laboratories) was dialyzed overnight against 10 mM sodium borate buffer, pH 8.5. 10 The dialyzed antibody was then concentrated to a volume of 2 mL using an Amicon Centriprep tube. Ten mg sulfo-EMCS [N (E-maleimidocuproyloxy) succinimide] (Molecular Sciences Co.) was dissolved in one mL deionized water. A 40-fold molar excess of sulfo-EMCS was added dropwise with stirring to the 15 sheep anti-mouse IgG and then the solution was stirred for an additional ten min. The activated sheep anti-mouse IgG was purified and buffer exchanged by passage over a 10 mL gel filtration column (Pierce Presto Column, obtained from Pierce Chemicals) equilibrated with 0.1 M NaPO 4 , 5 mM EDTA, pH 6.5. 20 Antibody containing fractions, identified by absorbance at 280 nm, were pooled and diluted to a concentration of approximately 1 mg/mL, using 1.4 mg per OD as the extinction coefficient. A 40-fold molar excess of AO peptide was dissolved in 20 mL of 10 mM NaPO 4 , pH 8.0, with the exception 25 of the A033-42 peptide for which 10 mg was first dissolved in 0.5 mL of DMSO and then diluted to 20 mL with the 10 mM NaPO 4 buffer. The peptide solutions were each added to 10 mL of activated sheep anti-mouse IgG and rocked at room temperature for 4 hr. The resulting conjugates were concentrated to a 30 final volume of less than 10 mL using an Amicon Centriprep tube and then dialyzed against PBS to buffer exchange the buffer and remove free peptide. The conjugates were passed through 0.22 y-pore size filters for sterilization and then aliquoted into fractions of 1 mg and stored frozen at -20 0 C. 35 The concentrations of the conjugates were determined using the BCA protein assay (Pierce Chemicals) with horse IgG for the 50 standard curve. Conjugation was documented by the molecular weight increase of the conjugated peptides relative to that of the activated sheep anti-mouse IgG. The A0 1-5 sheep anti mouse conjugate was a pool of two conjugations, the rest were 5 from a single preparation. 2. Preparation of aqqregated A3 peptides Human 1-40 (AN1528; California Peptides Inc., Lot ME0541), human 1-42 (AN1792; California Peptides Inc., Lots ME0339 and ME0439), human 25-35, and rodent 1-42 (California Peptides 0 Inc., Lot ME0218) peptides were freshly solubilized for the preparation of each set of injections from lyophilized powders that had been stored desiccated at -20 0 C. For this purpose, two mg of peptide were added to 0.9 ml of deionized water and the mixture was vortexed to generate a relatively uniform 5 solution or suspension. Of the four, AN1528 was the only peptide soluble at this step. A 100 sl aliquot of 1oX PBS (1X PBS: 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5) was then added at which point AN1528 began to precipitate. The suspension was vortexed again and incubated overnight at 37*C 0 for use the next day. Preparation of the pBx6 protein: An expression plasmid encoding pBx6, a fusion protein consisting of the 100-amino acid bacteriophage MS-2 polymerase N-terminal leader sequence followed by amino acids 592-695 of APP (3APP) was constructed 25 as described by Oltersdorf et al., J. Biol. Chem. 265, 4492 4497 (1990). The plasmid was transfected into E. coli and the protein was expressed after induction of the promoter. The bacteria were lysed in 8M urea and pBx6 was partially purified by preparative SDS PAGE. Fractions containing pBx6 were 30 identified by Western blot using a rabbit anti-pBx6 polyclonal antibody, pooled, concentrated using an Amicon Centriprep tube and dialysed against PBS. The purity of the preparation, estimated by Coomassie Blue stained SDS PAGE, was approximately 5 to 10%.

51 B. Results and Discussion 1. Study Design One hundred male and female, nine- to eleven-month old heterozygous PDAPP transgenic mice were obtained from Charles 5 River Laboratory and Taconic Laboratory. The mice were sorted into ten groups to be immunized with different regions of A0 or APP combined with Freund's adjuvant. Animals were distributed to match the gender, age, parentage and source of the animals within the groups as closely as possible. The 10 immunogens included four AO peptides derived from the human sequence, 1-5, 1-12, 13-28, and 33-42, each conjugated to sheep anti-mouse IgG; four aggregated A0 peptides, human 1-40 (AN1528), human 1-42 (AN1792), human 25-35, and rodent 1-42; and a fusion polypeptide, designated as pBx6, containing APP 15 amino acid residues 592-695. A tenth group was immunized with PBS combined with adjuvant as a control. For each immunization, 100 pg of each AO peptide in 200 pl PBS or 200 pg of the APP derivative pBx6 in the same volume of PBS or PBS alone was emulsified 1:1 (vol:vol) with Complete 20 Freund's adjuvant (CFA) in a final volume of 400 pl for the first immunization, followed by a boost of the same amount of immunogen in Incomplete Freund's adjuvant (IFA) for the subsequent four doses and with PBS for the final dose. Immunizations were delivered intraperitoneally on a biweekly 25 schedule for the first three doses, then on a monthly schedule thereafter. Animals were bled four to seven days following each immunization starting after the second dose for the measurement of antibody titers. Animals were euthanized approximately one week after the final dose. 30 2. A8 and APP Levels in the Brain Following about four months of immunization with the various A0 peptides or the APP derivative, brains were removed from saline-perfused animals. One hemisphere was prepared for immunohistochemical analysis and the second was used for the 35 quantitation of A0 and APP levels. To measure the concentrations of various forms of beta amyloid peptide and amyloid precursor protein, the hemisphere was dissected and 52 homogenates of the hippocampal, cortical, and cerebellar regions were prepared in 5 M guanidine. These were diluted and the level of amyloid or APP was quantitated by comparison to a series of dilutions of standards of AO peptide or APP of 5 known concentrations in an ELISA format. The median concentration of total AO for the control group immunized with PBS was 5.8-fold higher in the hippocampus than in the cortex (median of 24,318 ng/g hippocampal tissue compared to 4,221 ng/g for the cortex). The median level in 10 the cerebellum of the control group (23.4 ng/g tissue) was about 1,000-fold lower than in the hippocampus. These levels are similar to those that we have previously reported for heterozygous PDAPP transgenic mice of this age (Johnson-Woods et al., 1997, supra). 15 For the cortex, a subset of treatment groups had median total AO and A1l-42 levels which differed significantly from those of the control group (p < 0.05), those animals receiving AN1792, rodent Aol-42 or the A01-5 peptide conjugate as shown in Fig. 11. The median levels of total AO were reduced by 20 75%, 79% and 61%, respectively, compared to the control for these treatment groups. There were no discernable correlations between AO-specific antibody titers and AO levels in the cortical region of the brain for any of the groups. In the hippocampus, the median reduction of total AO 25 associated with AN1792 treatment (46%, p = 0.0543) was not as great as that observed in the cortex (75%, p = 0.0021). However, the magnitude of the reduction was far greater in the hippocampus than in the cortex, a net reduction of 11,186 ng/g tissue in the hippocampus versus 3,171 ng/g tissue in the 30 cortex. For groups of animals receiving rodent Ao1-42 or A01-5, the median total AO levels were reduced by 36% and 26%, respectively. However, given the small group sizes and the high variability of the amyloid peptide levels from animal to animal within both groups, these reductions were not 35 significant. When the levels of Aol-42 were measured in the hippocampus, none of the treatment-induced reductions reached significance. Thus, due to the smaller AO burden in the cortex, changes in this region are a more sensitive indicator 53 of treatment effects. The changes in AO levels measured by ELISA in the cortex are similar, but not identical, to the results from the immunohistochemical analysis (see below). Total AO was also measured in the cerebellum, a region 5 typically unaffected in the AD pathology. None of the median AO concentrations of any of the groups immunized with the various AO peptides or the APP derivative differed from that of the control group in this region of the brain. This result suggests that non-pathological levels of AO are unaffected by .0 treatment. APP concentration was also determined by ELISA in the cortex and cerebellum from treated and control mice. Two different APP assays were utilized. The first, designated APP-a/FL, recognizes both APP-alpha (a, the secreted form of .5 APP which has been cleaved within the AO sequence), and full length forms (FL) of APP, while the second recognizes only APP-a. In contrast to the treatment-associated diminution of AO in a subset of treatment groups, the levels of APP were unchanged in all of the treated compared to the control 20 animals. These results indicate that the immunizations with AO peptides are not depleting APP; rather the treatment effect is specific to A0. In summary, total AO and A1l-42 levels were significantly reduced in the cortex by treatment with AN1792, rodent Ao1-42 25 or A01-5 conjugate. In the hippocampus, total AO was significantly reduced only by AN1792 treatment. No other treatment-associated changes in AO or APP levels in the hippocampal, cortical or cerebellar regions were significant. 2. Histochemical Analyses 30 Brains from a subset of six groups were prepared for immunohistochemical analysis, three groups immunized with the AO peptide conjugates A01-5, A01-12, and A013-28; two groups immunized with the full length AO aggregates AN1792 and AN1528 and the PBS-treated control group. The results of image 35 analyses of the amyloid burden in brain sections from these groups are shown in Fig. 12. There were significant reductions of amyloid burden in the cortical regions of three 54 of the treatment groups versus control animals. The greatest reduction of amyloid burden was observed in the group receiving AN1792 where the mean value was reduced by 97% (p = 0.001). Significant reductions were also observed for those 5 animals treated with AN1528 (95%, p = 0.005) and the A01-5 peptide conjugate (67%, p = 0.02). The results obtained by quantitation of total AO or Ao1-42 by ELISA and amyloid burden by image analysis differ to some extent. Treatment with AN1528 had a significant impact on the 10 level of cortical amyloid burden when measured by quantitative image analysis but not on the concentration of total AO in the same region when measured by ELISA. The difference between these two results is likely to be due to the specificities of the assays. Image analysis measures only insoluble AO 15 aggregated into plaques. In contrast, the ELISA measures all forms of AO, both soluble and insoluble, monomeric and aggregated. Since the disease pathology is thought to be associated with the insoluble plaque-associated form of AO, the image analysis technique may have more sensitivity to 20 reveal treatment effects. However since the ELISA is a more rapid and easier assay, it is very useful for screening purposes. Moreover it may reveal that the treatment associated reduction of AO is greater for plaque-associated than total A0. 25 To determine if the AO-specific antibodies elicited by immunization in the treated animals reacted with deposited brain amyloid, a subset of the sections from the treated animals and the control mice were reacted with an antibody specific for mouse IgG. In contrast to the PBS group, A3 30 containing plaques were coated with endogenous IgG for animals immunized with the AO peptide conjugates A01-5, A01-12, and A013-28; and the full length AO aggregates AN1792 and AN1528. Brains from animals immunized with the other AO peptides or the APP peptide pBx6 were not analyzed by this assay. 35 3. Measurement of Antibody Titers Mice were bled four to seven days following each immunization starting after the second immunization, for a 55 total of five bleeds. Antibody titers were measured as A1 42-binding antibody using a sandwich ELISA with plastic multi well plates coated with A01-42. As shown in Fig. 13, peak antibody titers were elicited following the fourth dose for 5 those four vaccines which elicited the highest titers of AN1792-specific antibodies: AN1792 (peak GMT: 94,647), AN1528 (peak GMT: 88,231), Ao1-12 conjugate (peak GMT: 47,216)and rodent Ao1-42 (peak GMT: 10,766). Titers for these groups declined somewhat following the fifth and sixth doses. For 10 the remaining five immunogens, peak titers were reached following the fifth or the sixth dose and these were of much lower magnitude than those of the four highest titer groups: A01-5 conjugate (peak GMT: 2,356), pBx6 (peak GMT: 1,986), A13-28 conjugate (peak GMT: 1,183), A33-42 conjugate (peak 15 GMT: 658), A025-35 (peak GMT: 125). Antibody titers were also measured against the homologous peptides using the same ELISA sandwich format for a subset of the immunogens, those groups immunized with A01-5, A013-28, A025-35, A33-42 or rodent A01-42. These titers were about the same as those measured 20 against A1-42 except for the rodent A1-42 immunogen in which case antibody titers against the homologous immunogen were about two-fold higher. The magnitude of the AN1792-specific antibody titer of individual animals or the mean values of treatment groups did not correlate with efficacy measured as 25 the reduction of AO in the cortex. 4. Lymphoproliferative Responses A4-dependent lymphoproliferation was measured using spleen cells harvested approximately one week following the final, sixth, immunization. Freshly harvested cells, 105 per well, 30 were cultured for 5 days in the presence of A1-40 at a concentration of 5 yM for stimulation. Cells from a subset of seven of the ten groups were also cultured in the presence of the reverse peptide, A040-1. As a positive control, additional cells were cultured with the T cell mitogen, PHA, 35 and, as a negative control, cells were cultured without added peptide.

56 Lymphocytes from a majority of the animals proliferated in response to PHA. There were no significant responses to the A40-1 reverse peptide. Cells from animals immunized with the larger aggregated A0 peptides, AN1792, rodent Ao1-42 and 5 AN1528 proliferated robustly when stimulated with Ao1-40 with the highest cpm in the recipients of AN1792. One animal in each of the groups immunized with A31-12 conjugate, A013-28 conjugate and A25-35 proliferated in response to A01-40. The remaining groups receiving A01-5 conjugate, A033-42 conjugate LO pBx6 or PBS had no animals with an Ao-stimulated response. These results are summarized in Table 5 below. Table 5 Immunogen Conjugate A0 Amino Acids Responders A01-5 yes 5-mer 0/7 L5 A1l-12 yes 12-mer 1/8 A013-28 yes 16-mer 1/9 A25-35 11-mer 1/9 A033-42 yes 10-mer 0/10 A1-40 40-mer 5/8 20 Aol-42 42-mer 9/9 r Ao1-42 42-mer 8/8 pBx6 0/8 PBS 0-mer 0/8 These results show that AN1792 and AN1528 stimulate strong 25 T cell responses, most likely of the CD4+ phenotype. The absence of an Ao-specific T cell response in animals immunized with A01-5 is not surprising since peptide epitopes recognized by CD4+ T cells are usually about 15 amino acids in length, although shorter peptides can sometimes function with less 30 efficiency. Thus the majority of helper T cell epitopes for the four conjugate peptides are likely to reside in the IgG conjugate partner, not in the A0 region. This hypothesis is supported by the very low incidence of proliferative responses for animals in each of these treatment groups. Since the A01 35 5 conjugate was effective at significantly reducing the level 57 of A0 in the brain, in the apparent absence of A-specific T cells, the key effector immune response induced by immunization with this peptide appears to be antibody. Lack of T-cell and low antibody response from fusion peptide 5 pBx6, encompassing APP amino acids 592-695 including all of the A0 residues may be due to the poor immunogenicity of this particular preparation. The poor immunogenicity of the A325 35 aggregate is likely due to the peptide being too small to be likely to contain a good T cell epitope to help the 10 induction of an antibody response. If this peptide were conjugated to a carrier protein, it would probably be more immunogenic. V. Preparation of Polyclonal Antibodies for Passive Protection 15 20 non-transgenic mice are immunized with A0 or other immunogen, optionally plus adjuvant, and are euthanized at 4-5 months. Blood is collected from immunized mice. Optionally, IgG is separated from other blood components. Antibody specific for the immunogen may be partially purified by 20 affinity chromatography. An average of about 0.5-1 mg of immunogen-specific antibody is obtained per mouse, giving a total of 5-10 mg. VI. Passive Immunization with Antibodies to A3 Groups of 7-9 month old PDAPP mice each are injected with 25 0.5 mg in PBS of polyclonal anti-A3 or specific anti-A0 monoclonals as shown below. All antibody preparations are purified to have low endotoxin levels. Monoclonals can be prepared against a fragment by injecting the fragment or longer form of A into a mouse, preparing hybridomas and 30 screening the hybridomas for an antibody that specifically 58 binds to a desired fragment of AO without binding to other nonoverlapping fragments of A0. Table 6 Antibody Epitope 5 2H3 AO 1-12 10D5 AO 1-12 266 AO 13-28 21F12 AO 33-42 Mouse polyclonal Anti-Aggregated A042 LO anti-human A042 Mice are injected ip as needed over a 4 month period to maintain a circulating antibody concentration measured by ELISA titer of greater than 1/1000 defined by ELISA to A342 or other immunogen. Titers are monitored as above and mice are 15 euthanized at the end of 4 months of injections. Histochemistry, AO levels and toxicology are performed post mortem. Ten mice are used per group. VII. Comparison of Different Adiuvants This examples compares CFA, alum, an oil-in water emulsion 20 and MPL for capacity to stimulate an immune response.

59 A. Materials and Methods 1. Study Design One hundred female Hartley strain six-week old guinea pigs, obtained from Elm Hill, were sorted into ten groups to 5 be immunized with AN1792 or a palmitoylated derivative thereof combined with various adjuvants. Seven groups received injections of AN1792 (33pg unless otherwise specified) combined with a) PBS, b) Freund's adjuvant, c) MPL, d) squalene, e) MPL/squalene f) low dose alum, or g) high dose LO alum (300pg AN1792). Two groups received injections of a palmitoylated derivative of AN1792 (33 pg) combined with a) PBS or b) squalene. A final, tenth group received PBS alone without antigen or additional adjuvant. For the group receiving Freund's adjuvant, the first dose was emulsified 15 with CFA and the remaining four doses with IFA. Antigen was administered at a dose of 33 pg for all groups except the high dose alum group, which received 300 pg of AN1792. Injections were administered intraperitoneally for CFA/IFA and intramuscularly in the hind limb quadriceps alternately on the 20 right and left side for all other groups. The first three doses were given on a biweekly schedule followed by two doses at a monthly interval). Blood was drawn six to seven days following each immunization, starting after the second dose, for measurement of antibody titers. 25 2. Preparation of Immunogens Two mg A042 (California Peptide, Lot ME0339) was added to 0.9 ml of deionized water and the mixture was vortexed to generate a relatively uniform suspension. A 100 sl aliquot of 1OX PBS (1X PBS, 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5) 30 was added. The suspension was vortexed again and incubated overnight at 370C for use the next day. Unused Aol-42 was stored with desiccant as a lyophilized powder at -20 0

C.

60 A palmitoylated derivative of AN1792 was prepared by coupling palmitic anhydride, dissolved in dimethyl formamide, to the amino terminal residue of AN1792 prior to removal of the nascent peptide from the resin by treatment with 5 hydrofluoric acid. To prepare vaccine doses with Complete Freund's adjuvant (CFA) (group 2), 33 pg of AN1792 in 200 l PBS was emulsified 1:1 (vol:vol) with CFA in a final volume of 400 pl for the first immunization. For subsequent immunizations, the antigen 10 was similarly emulsified with Incomplete Freund's adjuvant (IFA). To prepare vaccine doses with MPL for groups 5 and 8, lyophilized powder (Ribi ImmunoChem Research, Inc., Hamilton, MT) was added to 0.2% aqueous triethylamine to a final 15 concentration of 1 mg/ml and vortexed. The mixture was heated to 65 to 70 0 C for 30 sec to create a slightly opaque uniform suspension of micelles. The solution was freshly prepared for each set of injections. For each injection in group 5, 33 pg of AN1792 in 16.5 pl PBS, 50 pg of MPL (50 4l) and 162 l of 20 PBS were mixed in a borosilicate tube immediately before use. To prepare vaccine doses with the low oil-in-water emulsion, AN1792 in PBS was added to 5% squalene, 0.5% Tween 80., 0.5% Span 85 in PBS to reach a final single dose concentration of 33 pg AN1792 in 250 l (group 6). The mixture 25 was emulsified by passing through a two-chambered hand-held device 15 to 20 times until the emulsion droplets appeared to be about equal in diameter to a 1.0 pm diameter standard latex bead when viewed under a microscope. The resulting suspension was opalescent, milky white. The emulsions were freshly 30 prepared for each series of injections. For group 8, MPL in 0.2% triethylamine was added at a concentration of 50 pg per dose to the squalene and detergent mixture for emulsification as noted above. For the palmitoyl derivative (group 7), 33 pg per dose of palmitoyl-NH-Aol-42 was added to squalene and 35 vortexed. Tween 80 and Span 85 were then added with 61 vortexing. This mixture was added to PBS to reach final concentrations of 5% squalene, 0.5% Tween 80, 0.5% Span 85 and the mixture was emulsified as noted above. To prepare vaccine doses with alum (groups 9 and 10), 5 AN1792 in PBS was added to Alhydrogel (aluminum hydroxide gel, Accurate, Westbury, NY) to reach concentrations of 33 pg (low dose, group 9) or 300 pg (high dose, group 10) AN1792 per 5 mg of alum in a final dose volume of 250 pl. The suspension was gently mixed for 4 hr at RT. 10 3. Measurement of Antibody Titers Guinea pigs were bled six to seven days following immunization starting after the second immunization for a total of four bleeds. Antibody titers against A042 were measured by ELISA as described in General Materials and 15 Methods. 4. Tissue Preparation . After about 14 weeks, all guinea pigs were administered

CO

2 . Cerebrospinal fluid was collected and the brains were 20 removed and three brain regions (hippocampus, cortex and cerebellum) were dissected and used to measure the concentration of total AO protein using ELISA.

62 B. Results 1. Antibody Responses There was a wide range in the potency of the various adjuvants when measured as the antibody response to AN1792 5 following immunization. As shown in Fig. 14, when AN1792 was administered in PBS, no antibody was detected following two or three immunizations and negligible responses were detected following the fourth and fifth doses with geometric mean titers (GMTs) of only about 45. The o/w emulsion induced -0 modest titers following the third dose (GMT 255) that were maintained following the fourth dose (GMT 301) and fell with the final dose (GMT 54). There was a clear antigen dose response for AN1792 bound to alum with 300 pg being more immunogenic at all time points than 33 pg. At the peak of the .5 antibody response, following the fourth immunization, the difference between the two doses was 43% with GMTs of about 1940 (33 pg) and 3400 (300 Ag). The antibody response to 33 pg AN1792 plus MPL was very similar to that generated with almost a ten-fold higher dose of antigen (300 pg) bound to 20 alum. The addition of MPL to an o/w emulsion decreased the potency of the vaccine relative to that with MPL as the sole adjuvant by as much as 75%. A palmitoylated derivative of AN1792 was completely non-immunogenic when administered in PBS and gave modest titers when presented in an o/w emulsion with 25 GMTs of 340 and 105 for the third and fourth bleeds. The highest antibody titers were generated with Freund's adjuvant with a peak GMT of about 87,000, a value almost 30-fold greater than the GMTs of the next two most potent vaccines, MPL and high dose AN1792/alum. 30 The most promising adjuvants identified in this study are MPL and alum. Of these two, MPL appears preferable because a 10-fold lower antigen dose was required to generate the same antibody response as obtained with alum. The response can be increased by increasing the dose of antigen and /or adjuvant 35 and by optimizing the immunization schedule. The o/w emulsion 63 was a very weak adjuvant for AN1792 and adding an o/w emulsion to MPL adjuvant diminished the intrinsic adjuvant activity of MPL alone. 2. A8 Levels In The Brain 5 At about 14 weeks the guinea pigs were deeply anesthetized, the cerebrospinal fluid (CSF) was drawn and brains were excised from animals in a subset of the groups, those immunized with Freund's adjuvant (group 2), MPL (group 5), alum with a high dose, 300 pg, of AN1792 (group 10) and 10 the PBS immunized control group (group 3). To measure the level of AO peptide, one hemisphere was dissected and homogenates of the hippocampal, cortical, and cerebellar regions were prepared in 5 M guanidine. These were diluted and quantitated by comparison to a series of dilutions of AO 15 standard protein of known concentrations in an ELISA format. The levels of AO protein in the hippocampus, the cortex and the cerebellum were very similar for all four groups despite the wide range of antibody responses to AO elicited by these vaccines. Mean AO levels of about 25 ng/g tissue were 20 measured in the hippocampus, 21 ng/g in the cortex, and 12 ng/g in the cerebellum. Thus, the presence of a high circulating antibody titer to AO for almost three months in some of these animals did not alter the total AO levels in their brains. The levels of AO in the CSF were also quite 25 similar between the groups. The lack of large effect of AN1792 immunization on endogenous AO indicates that the immune response is focused on pathological formations of A0. VIII. Immune Response to Different Adiuvants in Mice Six-week old female Swiss Webster mice were used for this 30 study with 10-13 animals per group. Immunizations were given 64 on days 0, 14, 28, 60, 90 and 20 administered subcutaneously in a dose volume of 200 pl. PBS was used as the buffer for all formulations. Animals were bleed seven days following each immunization starting after the second dose for analysis 5 of antibody titers by ELISA. The treatment regime of each group is summarized in Table 7.

65 Table 7 Experimental Design of Betabloc Study 010 Group N a Adjuvant b Dose Antigen Dose (pg) 1 10 MPL 12.5 pg AN1792 33 5 2 10 MPL 25 pg AN1792 33 3 10 MPL 50 pg AN1792 33 4 13 MPL 125 pg AN1792 33 5 13 MPL 50 pg AN1792 150 6 13 MPL 50 pg AN1528 33 LO 7 10 PBS AN1792 33 8 10 PBS none 9 10 Squalene 5% AN1792 33 emulsified 10 10 Squalene 5% - AN1792 33 admixed 11 10 Alum 2 mg AN1792 33 15 12 13 MPL + Alum 50 pg/2 mg AN1792 33 13 10 QS21 5 pg AN1792 33 14 10 QS21 10 pg AN1792 33 15 10 QS21 25 pg AN1792 33 16 13 QS21 25 pg AN1792 150 20 17 13 QS21 25 pg AN1528 33 18 13 QS21 + MPL 25 pg/50 pg AN1792 33 19 13 QS21 + Alum 25 pg/2 mg AN1792 33 Footnotes: a Number of mice in each group at the initiation of the experiment. 25 h The adjuvants are noted. The buffer for all these formulations was PBS. For group 8, there was no adjuvant and no antigen. The ELISA titers of antibodies against A042 in each group are shown in Table 8 below.

66 Table 8. Geometric Mean Antibody Titers Week of Bleed Treatment 5 Group 2.9 5.0 8.7 12.9 16.7 1 248 1797 2577 6180 4177 2 598 3114 3984 5287 6878 3 1372 5000 7159 12333 12781 4 1278 20791 14368 20097 25631 -0 5 3288 26242 13229 9315 23742 6 61 2536 2301 1442 4504 7 37 395 484 972 2149 8 25 25 25 25 25 9 25 183 744 952 1823 LS 10 25 89 311 513 817 11 29 708 2618 2165 3666 12 198 1458 1079 612 797 13 38 433 566 1080 626 14 104 541 3247 1609 838 20 15 212 2630 2472 1224 1496 16 183 2616 6680 2085 1631 17 28 201 375 222 1540 18 31699 15544 23095 6412 9059 19 63 243 554 299 441 25 The table shows that the highest titers were obtained for groups 4, 5 and 18, in which the adjuvants were 125 pg MPL, 50 pg MPL and QS21 plus MPL. IX. Therapeutic Efficacy of Different Adiuvants A therapeutic efficacy study was conducted in PDAPP transgenic mice with a set of adjuvants suitable for use in 30 humans to determine their ability to potentiate immune responses to AO and to induce the immune-mediated clearance of amyloid deposits in the brain.

67 One hundred eighty male and female, 7.5- to 8.5-month old heterozygous PDAPP transgenic mice were obtained from Charles River Laboratories. The mice were sorted into nine groups containing 15 to 23 animals per group to be immunized with 5 AN1792 or AN1528 combined with various adjuvants. Animals were distributed to match the gender, age, and parentage of the animals within the groups as closely as possible. The adjuvants included alum, MPL, and QS21, each combined with both antigens, and Freund's adjuvant (FA) combined with only .0 AN1792. An additional group was immunized with AN1792 formulated in PBS buffer plus the preservative thimerosal without adjuvant. A ninth group was immunized with PBS alone as a negative control. Preparation of aggregated AO peptides: human A31-40 .5 (AN1528; California Peptides Inc., Napa, CA; Lot ME0541) and human A31-42 (AN1792; California Peptides Inc., Lot ME0439) peptides were freshly solubilized for the preparation of each set of injections from lyophilized powders that had been stored desiccated at -20 0 C. For this purpose, two mg of ?0 peptide were added to 0.9 ml of deionized water and the mixture was vortexed to generate a relatively uniform solution or suspension. AN1528 was soluble at this step, in contrast to AN1792. A 100 pl aliquot of 1OX PBS (1X PBS: 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5) was then added at which point 25 AN1528 began to precipitate. The suspensions were vortexed again and incubated overnight at 37'C for use the next day. To prepare vaccine doses with alum (Groups 1 and 5), AO peptide in PBS was added to Alhydrogel (two percent aqueous aluminum hydroxide gel, Sargeant, Inc., Clifton, NJ) to reach 30 concentrations of 100 pg A0 peptide per 1 mg of alum. 1OX PBS was added to a final dose volume of 200 pl in 1X PBS. The suspension was then gently mixed for approximately 4 hr at RT prior to injection. To prepare vaccine doses for with MPL (Groups 2 and 6), 35 lyophilized powder (Ribi ImmunoChem Research, Inc., Hamilton, 68 MT; Lot 67039-E0896B) was added to 0.2% aqueous triethylamine to a final concentration of 1 mg/ml and vortexed. The mixture was heated to 65 to 70 0 C for 30 sec to create a slightly opaque uniform suspension of micelles. The solution was 5 stored at 4 0 C. For each set of injections, 100 pg of peptide per dose in 50 pl PBS, 50 pg of MPL per dose (50 pl) and 100 Al of PBS per dose were mixed in a borosilicate tube immediately before use. To prepare vaccine doses with QS21 (Groups 3 and 7), .0 lyophilized powder (Aquila, Framingham, MA; Lot A7018R) was added to PBS, pH 6.6-6.7 to a final concentration of 1 mg/ml and vortexed. The solution was stored at -20 0 C. For each set of injections, 100 pg of peptide per dose in 50 pl PBS, 25 pg of QS21 per dose in 25 pzl PBS and 125 pl of PBS per dose were L5 mixed in a borosilicate tube immediately before use. To prepare vaccine doses with Freund's Adjuvant (Group 4), 100 pg of AN1792 in 200 pl PBS was emulsified 1:1 (vol:vol) with Complete Freund's Adjuvant (CFA) in a final volume of 400 sl for the first immunization. For subsequent immunizations, 20 the antigen was similarly emulsified with Incomplete Freund's Adjuvant (IFA). For the vaccines containing the adjuvants alum, MPL or QS21, 100 pg per dose of AN1792 or AN1528 was combined with alum (1 mg per dose) or MPL (50 pg per dose) or QS21 (25 pg per dose) in a final volume of 200 pl PBS and 25 delivered by subcutaneous inoculation on the back between the shoulder blades. For the group receiving FA, 100 pg of AN1792 was emulsified 1:1 (vol:vol) with Complete Freund's adjuvant (CFA) in a final volume of 400 pl and delivered intraperitoneally for the first immunization, followed by a 30 boost of the same amount of immunogen in Incomplete Freund's adjuvant (IFA) for the subsequent five doses. For the group receiving AN1792 without adjuvant, 10 pg AN1792 was combined with 5 pg thimerosal in a final volume of 50 pl PBS and delivered subcutaneously. The ninth, control group received 35 only 200 gl PBS delivered subcutaneously. Immunizations were given on a biweekly schedule for the first three doses, then 69 on a monthly schedule thereafter on days 0, 16, 28, 56, 85 and 112. Animals were bled six to seven days following each immunization starting after the second dose for the measurement of antibody titers. Animals were euthanized 5 approximately one week after the final dose. Outcomes were measured by ELISA assay of A0 and APP levels in brain and by immunohistochemical evaluation of the presence of amyloid plaques in brain sections. In addition, A3-specific antibody titers, and A-dependent proliferative and cytokine responses .0 were determined. Table 9 shows that the highest antibody titers to A1l-42 were elicited with FA and AN1792, titers which peaked following the fourth immunization (peak GMT: 75,386) and then declined by 59% after the final, sixth immunization. The peak L5 mean titer elicited by MPL with AN1792 was 62% lower than that generated with FA (peak GMT: 28,867) and was also reached early in the immunization scheme, after 3 doses, followed by a decline to 28% of the peak value after the sixth immunization. The peak mean titer generated with QS21 combined with AN1792 20 (GMT: 1,511) was about 5-fold lower than obtained with MPL. In addition, the kinetics of the response were slower, since an additional immunization was required to reach the peak response. Titers generated by alum-bound AN1792 were marginally greater than those obtained with QS21 and the 25 response kinetics were more rapid. For AN1792 delivered in PBS with thimerosal the frequency and size of titers were barely greater than that for PBS alone. The peak titers generated with MPL and AN1528 (peak GMT 3099) were about 9 fold lower than those with AN1792. Alum-bound AN1528 was very 30 poorly immunogenic with low titers generated in only some of the animals. No antibody responses were observed in the control animals immunized with PBS alone.

70 Table 9 Geometric Mean Antibody Titersa Week of Bleed Treatment 3.3 5.0 9.0 13.0 17.0 5 Alum/ 102 1,081 2,366 1,083 572 AN1792 ( 12

/

2 1 )b (17/20) (21/21) (19/21) (18/21) MPL/ 6241 28,867 1,1242 5,665 8,204 AN1792 (21/21) (21/21) (21/21) (20/20) (20/20) QS21/ 30 227 327 1,511 1,188 LO AN1792 (1/20) (10/19) (10/19) (17/18) (14/18) CFA/ 10,076 71,279 75,386 41,628 30,574 AN1792 (15/15) (15/15) (15/15) (15/15) (15/15) Alum/ 25 33 39 37 31 AN1528 (0/21) (1/21) (3/20) (1/20) (2/20) L5 MPL/ 184 2,591 1,63 1,156 3,099 AN1528 (15/21) (20/21) (21/21) (20/20) (20/20) QS21/ 29 221 51 820 2,994 AN1528 (1/22) (13/22) (4/22) (20/22) (21/22) PBS plus 25 33 39 37 47 20 Thimerosal (0/16) (2/16) (4/16) (3/16) (4/16) PBS 25 25 25 25 25 (0/16) (0/16) (0/15) (0/12) (0/16) Footnotes: * Geometric mean antibody titers measured against Ao1-42 h Number of responders per group 25 The results of AN1792 or AN1592 treatment with various adjuvants, or thimerosal on cortical amyloid burden in 12 month old mice determined by ELISA are shown in Fig. 15. In PBS control PDAPP mice, the median level of total AO in the 71 cortex at 12 months was 1,817 ng/g. Notably reduced levels of AO were observed in mice treated with AN1792 plus CFA/IFA, AN1792 plus alum, AN1792 plus MPL and QS21 plus AN1792. The reduction reached statistical significance (p<0.05) only for 5 AN1792 plus CFA/IFA. However, as shown in Examples I and III, the effects of immunization in reducing AO levels become substantially greater in 15 month and 18 month old mice. Thus, it is expected that at least the AN1792 plus alum, AN1792 plus MPL and AN1792 plus QS21 compositions will achieve .0 statistical significance in treatment of older mice. By contrast, the AN1792 plus the preservative thimerosal showed a median level of AO about the same as that in the PBS treated mice. Similar results were obtained when cortical levels of A342 were compared. The median level of A342 in PBS controls -5 was 1624 ng/g. Notably reduced median levels of 403, 1149, 620 and 714 were observed in the mice treated with AN1792 plus CFA/IFA, AN1792 plus alum, AN1792 plus MPL and AN1792 plus QS21 respectively, with the reduction achieving statistical significance (p=0.05) for the AN1792 CFA/IFA treatment group. 20 The median level in the AN1792 thimerosal treated mice was 1619 ng/g A#42. X. Toxicity Analysis Tissues were collected for histopathologic examination at the termination of studies described in Examples 2, 3 and 7. 25 In addition, hematology and clinical chemistry were performed on terminal blood samples from Examples 3 and 7. Most of the major organs were evaluated, including brain, pulmonary, lymphoid, gastrointestinal, liver, kidney, adrenal and gonads. Although sporadic lesions were observed in the study animals, 30 there were no obvious differences, either in tissues affected or lesion severity, between AN1792 treated and untreated animals. There were no unique histopathological lesions noted in AN-1782-immunized animals compared to PBS-treated or untreated animals. There were also no differences in the 72 clinical chemistry profile between adjuvant groups and the PBS treated animals in Example 7. Although there were significant increases in several of the hematology parameters between animals treated with AN1792 and Freund's adjuvant in Example 7 5 relative to PBS treated animals, these type of effects are expected from Freund's adjuvant treatment and the accompanying peritonitis and do not indicate any adverse effects from AN1792 treatment. Although not part of the toxicological evaluation, PDAPP mouse brain pathology was extensively 0 examined as part of the efficacy endpoints. No sign of treatment related adverse effect on brain morphology was noted in any of the studies. These results indicate that AN1792 treatment is well tolerated and at least substantially free of side effects. 5 XI. Prevention and Treatment of Subiects A single-dose phase I trial is performed to determine safety. A therapeutic agent is administered in increasing dosages to different patients starting from about 0.01 the level of presumed efficacy, and increasing by a factor of 0 three until a level of about 10 times the effective mouse dosage is reached. A phase II trial is performed to determine therapeutic efficacy. Patients with early to mid Alzheimer's Disease defined using Alzheimer's disease and Related Disorders ?5 Association (ADRDA) criteria for probable AD are selected. Suitable patients score in the 12-26 range on the Mini-Mental State Exam (MMSE). Other selection criteria are that patients are likely to survive the duration of the study and lack complicating issues such as use of concomitant medications 30 that may interfere. Baseline evaluations of patient function are made using classic psychometric measures, such as the MMSE, and the ADAS, which is a comprehensive scale for evaluating patients with Alzheimer's Disease status and 73 function. These psychometric scales provide a measure of progression of the Alzheimer's condition. Suitable qualitative life scales can also be used to monitor treatment. Disease progression can also be monitored by MRI. Blood profiles of patients can also be monitored including assays of immunogen-specific antibodies and T-cells responses. Following baseline measures, patients begin receiving treatment. They are randomized and treated with either therapeutic agent or placebo in a blinded fashion. Patients are monitored at least every six months. Efficacy is determined by a significant reduction in progression of a treatment group relative to a placebo group. A second phase II trial is performed to evaluate conversion of patients from non-Alzheimer's Disease early memory loss, sometimes referred to as age-associated memory impairment (AAMI), to probable Alzheimer's disease as defined as by ADRDA criteria. Patients with high risk for conversion to Alzheimer's Disease are selected from a non-clinical population by screening reference populations for early signs ) of memory loss or other difficulties associated with pre Alzheimer's symptomatology, a family history of Alzheimer's Disease, genetic risk factors, age, sex, and other features found to predict high-risk for Alzheimer's Disease. Baseline scores on suitable metrics including the MMSE and the ADAS 5 together with other metrics designed to evaluate a more normal population are collected. These patient populations are divided into suitable groups with placebo comparison against dosing alternatives with the agent. These patient populations are followed at intervals of about six months, and the 0 endpoint for each patient is whether or not he or she converts to probable Alzheimer's Disease as defined by ADRDA criteria at the end of the observation.

74 XII. General Materials and Methods 1. Measurement of Antibody Titers Mice were bled by making a small nick in the tail vein and collecting about 200 pl of blood into a microfuge tube. 5 Guinea pigs were bled by first shaving the back hock area and then using an 18 gauge needle to nick the metatarsal vein and collecting the blood into microfuge tubes. Blood was allowed to clot for one hr at room temperature (RT), vortexed, then centrifuged at 14,000 x g for 10 min to separate the clot from 10 the serum. Serum was then transferred to a clean microfuge tube and stored at 40 C until titered. Antibody titers were measured by ELISA. 96-well microtiter plates (Costar EIA plates) were coated with 100 pl of a solution containing either 10 pg/ml either A042 or SAPP or 15 other antigens as noted in each of the individual reports in Well Coating Buffer (0.1 M sodium phosphate, pH 8.5, 0.1% sodium azide) and held overnight at RT. The wells were aspirated and sera were added to the wells starting at a 1/100 dilution in Specimen Diluent (0.014 M sodium phosphate, pH 20 7.4, 0.15 M NaCl, 0.6% bovine serum albumin, 0.05% thimerosal). Seven serial dilutions of the samples were made directly in the plates in three-fold steps to reach a final dilution of 1/218,700. The dilutions were incubated in the coated-plate wells for one hr at RT. The plates were then 25 washed four times with PBS containing 0.05% Tween 20. The second antibody, a goat anti-mouse Ig conjugated to horseradish peroxidase (obtained from Boehringer Mannheim), was added to the wells as 100 pl of a 1/3000 dilution in Specimen Diluent and incubated for one hr at RT. Plates were 30 again washed four times in PBS, Tween 20. To develop the chromogen, 100 pl of Slow TMB (3,3',5,5'-tetramethyl benzidine obtained from Pierce Chemicals) was added to each well and incubated for 15 min at RT. The reaction was stopped by the addition of 25 pl of 2 M H 2

SO

4 . The color intensity was then 35 read on a Molecular Devices Vmax at (450 nm - 650 nm).

75 Titers were defined as the reciprocal of the dilution of serum giving one half the maximum OD. Maximal OD was generally taken from an initial 1/100 dilution, except in cases with very high titers, in which case a higher initial 5 dilution was necessary to establish the maximal OD. If the 50% point fell between two dilutions, a linear extrapolation was made to calculate the final titer. To calculate geometric mean antibody titers, titers less than 100 were arbitrarily assigned a titer value of 25. LO 2. Lymphocyte proliferation assay Mice were anesthetized with isoflurane. Spleens were removed and rinsed twice with 5 ml PBS containing 10% heat inactivated fetal bovine serum (PBS-FBS) and then homogenized in a 50 y Centricon unit (Dako A/S, Denmark) in 1.5 ml PBS-FBS L5 for 10 sec at 100 rpm in a Medimachine (Dako) followed by filtration through a 100 y pore size nylon mesh. Splenocytes were washed once with 15 ml PBS-FBS, then pelleted by centrifugation at 200 x g for 5 min. Red blood cells were lysed by resuspending the pellet in 5 mL buffer containing 20 0.15 M NH 4 Cl, 1 M KHCO 3 , 0.1 M NaEDTA, pH 7.4 for five min at RT. Leukocytes were then washed as above. Freshly isolated spleen cells (10s cells per well) were cultured in triplicate sets in 96-well U-bottomed tissue culture-treated microtiter plates (Corning, Cambridge, MA) in RPMI 1640 medium (JRH 25 Biosciences, Lenexa, KS) supplemented with 2.05 mM L glutamine, 1% Penicillin/Streptomycin, and 10% heat inactivated FBS, for 96 hr at 370C. Various AO peptides, A01 16, A01-40, Aol-42 or A$40-1 reverse sequence protein were also added at doses ranging from 5 AM to 0.18 yM in four 30 steps. Cells in control wells were cultured with Concanavalin A (Con A) (Sigma, cat. # C-5275, at 1 pg/ml) without added protein. Cells were pulsed for the final 24 hr with 3

H

thymidine (1 pCi/well obtained from Amersham Corp., Arlington Heights IL). Cells were then harvested onto UniFilter plates 76 and counted in a Top Count Microplate Scintillation Counter (Packard Instruments, Downers Grove, IL). Results are expressed as counts per minute (cpm) of radioactivity incorporated into insoluble macromolecules. 5 4. Brain Tissue Preparation After euthanasia, the brains were removed and one hemisphere was prepared for immunohistochemical analysis, while three brain regions (hippocampus, cortex and cerebellum) were dissected from the other hemisphere and used to measure LO the concentration of various AO proteins and APP forms using specific ELISAs (Johnson-Wood et al., supra). Tissues destined for ELISAs were homogenized in 10 volumes of ice-cold guanidine buffer (5.0 M guanidine-HCl, 50 mM Tris HCl, pH 8.0). The homogenates were mixed by gentle agitation L5 using an Adams Nutator (Fisher) for three to four hr at RT, then stored at -20 0 C prior to quantitation of AO and APP. Previous experiments had shown that the analytes were stable under this storage condition, and that synthetic AO protein (Bachem) could be quantitatively recovered when spiked into 20 homogenates of control brain tissue from mouse littermates (Johnson-Wood et al., supra). 5. Measurement of A8 Levels The brain homogenates were diluted 1:10 with ice cold Casein Diluent (0.25% casein, PBS, 0.05% sodium azide, 20 25 pg/ml aprotinin, 5 mM EDTA pH 8.0, 10 pg/ml leupeptin) and then centrifuged at 16,000 x g for 20 min at 40 C. The synthetic AO protein standards (1-42 amino acids) and the APP standards were prepared to include 0.5 M guanidine and 0.1% bovine serum albumin (BSA) in the final composition. The 77 "total" AO sandwich ELISA utilizes monoclonal antibody (mAO) 266, specific for amino acids 13-28 of AO (Seubert, et al.), as the capture antibody, and biotinylated mA3 3D6, specific for amino acids 1-5 of AO (Johnson-Wood, et al), as the 5 reporter antibody. The 3D6 mA3 does not recognize secreted APP or full-length APP, but detects only AO species with an amino-terminal aspartic acid. This assay has a lower limit of sensitivity of ~50 pg/ml (11 pM) and shows no cross-reactivity to the endogenous murine AO protein at concentrations up to 1 0 ng/ml (Johnson-Wood et al., supra). The Ao1-42 specific sandwich ELISA employs mA 21F12, specific for amino acids 33-42 of AO (Johnson-Wood, et al.), as the capture antibody. Biotinylated mA3 3D6 is also the reporter antibody in this assay which has a lower limit of 5 sensitivity of about 125 pg/ml (28 pM, Johnson-Wood et al.). For the AO ELISAs, 100 pl of either mA 266 (at 10 pg/ml) or mAO 21F12 at (5 pg/ml) was coated into the wells of 96-well immunoassay plates (Costar) by overnight incubation at RT. The solution was removed by aspiration and the wells were 0O blocked by the addition of 200 pl of 0.25% human serum albumin in PBS buffer for at least 1 hr at RT. Blocking solution was removed and the plates were stored desiccated at 4 0 C until used. The plates were rehydrated with Wash Buffer [Tris buffered saline (0.15 M NaCl, 0.01 M Tris-HCl, pH 7.5), plus 25 0.05% Tween 201 prior to use. The samples and standards were added in triplicate aliquots of 100 pl per well and then incubated overnight at 40 C. The plates were washed at least three times with Wash Buffer between each step of the assay. The biotinylated mAO 3D6, diluted to 0.5 pg/ml in Casein Assay 30 Buffer (0.25% casein, PBS, 0.05% Tween 20, pH 7.4), was added and incubated in the wells for 1 hr at RT. An avidin horseradish peroxidase conjugate, (Avidin-HRP obtained from Vector, Burlingame, CA), diluted 1:4000 in Casein Assay Buffer, was added to the wells for 1 hr at RT. The 35 colorimetric substrate, Slow TMB-ELISA (Pierce), was added and allowed to react for 15 minutes at RT, after which the enzymatic reaction was stopped by the addition of 25 pl 2 N 78

H

2

SO

4 . The reaction product was quantified using a Molecular Devices Vmax measuring the difference in absorbance at 450 nm and 650 nm. 6. Measurement of APP Levels 5 Two different APP assays were utilized. The first, designated APP-a/FL, recognizes both APP-alpha (a) and full length (FL) forms of APP. The second assay is specific for APP-a. The APP-u/FL assay recognizes secreted APP including the first 12 amino acids of A0. Since the reporter antibody 0 (2H3) is not specific to the a-clip-site, occurring between amino acids 612-613 of APP695 (Esch et al., Science 248, 1122 1124 (1990)); this assay also recognizes full length APP (APP FL). Preliminary experiments using immobilized APP antibodies to the cytoplasmic tail of APP-FL to deplete brain homogenates 5 of APP-FL suggest that approximately 30-40% of the APP-a/FL APP is FL (data not shown). The capture antibody for both the APP-a/FL and APP-a assays is mA3 8E5, raised against amino acids 444 to 592 of the APP695 form (Games et al., supra). The reporter mAO for the APP-u/FL assay is mAO 2H3, specific 0 for amino acids 597-608 of APP695 (Johnson-Wood et al., supra) and the reporter antibody for the APP-a assay is a bio.tinylated derivative of mA3 16H9, raised to amino acids 605 to 611 of APP. The lower limit of sensitivity of the APP-a/FL assay is about 11 ng/ml (150 pM) (Johnson-Wood et al.) and 25 that of the APP-a specific assay is 22 ng/ml (0.3 nM). For both APP assays, mAO 8E5 was coated onto the wells of 96-well EIA plates as described above for m.AO 266. Purified, recombinant secreted APP-a was used as the reference standard for the APP-a assay and the APP-a/FL assay (Esch et al., 30 supra). The brain homogenate samples in 5 M guanidine were diluted 1:10 in ELISA Specimen Diluent (0.014 M phosphate buffer, pH 7.4, 0.6% bovine serum albumin, 0.05% thimerosal, 0.5 M NaCl, 0.1% NP40). They were then diluted 1:4 in Specimen Diluent containing 0.5 M guanidine. Diluted 79 homogenates were then centrifuged at 16,000 x g for 15 seconds at RT. The APP standards and samples were added to the plate in duplicate aliquots and incubated for 1.5 hr at RT. The biotinylated reporter antibody 2H3 or 16H9 was incubated with 5 samples for 1 hr at RT. Streptavidin-alkaline phosphatase (Boehringer Mannheim), diluted 1:1000 in specimen diluent, was incubated in the wells for 1 hr at RT. The fluorescent substrate 4-methyl-umbellipheryl-phosphate was added for a 30 min RT incubation and the plates were read on a Cytofluor tm .0 2350 fluorimeter (Millipore) at 365 nm excitation and 450 nm emission. 7. Immunohistochemistry Brains were fixed for three days at 4 0 C in 4% paraformaldehyde in PBS and then stored from one to seven days .5 at 40C in 1% paraformaldehyde, PBS until sectioned. Forty micron-thick coronal sections were cut on a vibratome at RT and stored in cryoprotectant (30% glycerol, 30% ethylene glycol in phosphate buffer) at -200C prior to immunohistochemical processing. For each brain, six sections !0 at the level of the dorsal hippocampus, each separated by consecutive 240 pm intervals, were incubated overnight with one. of the following antibodies: (1) a biotinylated anti-AO (mAO, 3D6, specific for human AO) diluted to a concentration of 2 pg/ml in PBS and 1% horse serum; or (2) a biotinylated 25 mAO specific for human APP, 8E5, diluted to a concentration of 3 pg/ml in PBS and 1.0% horse serum; or (3) a mAO specific for glial fibrillary acidic protein (GFAP; Sigma Chemical Co.) diluted 1:500 with 0.25% Triton X-100 and 1% horse serum, in Tris-buffered saline, pH 7.4 (TES); or (4) a mAO specific for 30 CD11b, MAC-1 antigen, (Chemicon International) diluted 1:100 with 0.25% Triton X-100 and 1% rabbit serum in TBS; or (5) a mAO specific for MHC II antigen, (Pharmingen) diluted 1:100 with 0.25% Triton X-100 and 1% rabbit serum in TBS; or (6) a rat mAO specific for CD 43 (Pharmingen) diluted 1:100 with 1% 80 rabbit serum in PBS or (7) a rat mA3 specific for CD 45RA (Pharmingen) diluted 1:100 with 1% rabbit serum in PBS; or (8) a rat monoclonal AO specific for CD 45RB (Pharmingen) diluted 1:100 with 1% rabbit serum in PBS; or (9) a rat monoclonal AO 5 specific for CD 45 (Pharmingen) diluted 1:100 with 1% rabbit serum in PBS; or (10) a biotinylated polyclonal hamster AO specific for CD3e (Pharmingen) diluted 1:100 with 1% rabbit serum in PBS or (11) a rat mA3 specific for CD3 (Serotec) diluted 1:200 with 1% rabbit serum in PBS; or with (12) a 0 solution of PBS lacking a primary antibody containing 1% normal horse serum. Sections reacted with antibody solutions listed in 1,2 and 6-12 above were pretreated with 1.0% Triton X-100, 0.4% hydrogen peroxide in PBS for 20 min at RT to block endogenous 5 peroxidase. They were next incubated overnight at 4 0 C with primary antibody. Sections reacted with 3D6 or 8E5 or CD3e mAos were then reacted for one hr at RT with a horseradish peroxidase-avidin-biotin-complex with kit components "A" and "B" diluted 1:75 in PBS (Vector Elite Standard Kit, Vector 0 Labs, Burlingame, CA.). Sections reacted with antibodies specific for CD 45RA, CD 45RB, CD 45, CD3 and the PBS solution devoid of primary antibody were incubated for 1 hour at RT with biotinylated anti--rat IgG (Vector) diluted 1:75 in PBS or biotinylated anti-mouse IgG (Vector) diluted 1:75 in PBS, 25 respectively. Sections were then reacted for one hr at RT with a horseradish peroxidase-avidin-biotin-complex with kit components "A" and "B" diluted 1:75 in PBS (Vector Elite Standard Kit, Vector Labs, Burlingame, CA.). Sections were developed in 0.01% hydrogen peroxide, 0.05% 30 3,3'-diaminobenzidine (DAB) at RT. Sections destined for incubation with the GFAP-, MAC-1- AND MHC II-specific antibodies were pretreated with 0.6% hydrogen peroxide at RT to block endogenous peroxidase then incubated overnight with the primary antibody at 4 0 C. Sections reacted with the GFAP 35 antibody were incubated for 1 hr at RT with biotinylated anti mouse IgG made in horse (Vector Laboratories; Vectastain Elite 81 ABC Kit) diluted 1:200 with TBS. The sections were next reacted for one hr with an avidin-biotin-peroxidase complex (Vector Laboratories; Vectastain Elite ABC Kit) diluted 1:1000 with TBS. Sections incubated with the MAC-1-or MHC II 5 specific mAO as the primary antibody were subsequently reacted for 1 hr at RT with biotinylated anti-rat IgG made in rabbit diluted 1:200 with TBS, followed by incubation for one hr with avidin-biotin-peroxidase complex diluted 1:1000 with TBS. Sections incubated with GFAP-, MAC-1- and MHC II-specific 0 antibodies were then visualized by treatment at RT with 0.05% DAB, 0.01% hydrogen peroxide, 0.04% nickel chloride, TBS for 4 and 11 min, respectively. Immunolabeled sections were mounted on glass slides (VWR, Superfrost slides), air dried overnight, dipped in Propar 5 (Anatech) and overlaid with coverslips using Permount (Fisher) as the mounting medium. To counterstain AO plaques, a subset of the GFAP-positive sections were mounted on Superfrost slides and incubated in aqueous 1% Thioflavin S (Sigma) for 7 min following 0 immunohistochemical processing. Sections were then dehydrated and cleared in Propar, then overlaid with coverslips mounted with Permount. 8. Image Analysis A Videometric 150 Image Analysis System (Oncor, Inc., 25 Gaithersburg, MD) linked to a Nikon Microphot-FX microscope through a CCD video camera and a Sony Trinitron monitor was used for quantification of the immunoreactive slides. The image of the section was stored in a video buffer and a color and saturation-based threshold was determined to select and 30 calculate the total pixel area occupied by the immunolabeled structures. For each section, the hippocampus was manually outlined and the total pixel area occupied by the hippocampus 82 was calculated. The percent amyloid burden was measured as: (the fraction of the hippocampal area containing A,6 deposits immunoreactive with mAO 3D6) x 100. Similarly, the percent neuritic burden was measured as: (the fraction of the 5 hippocampal area containing dystrophic neurites reactive with mAO 8E) x100. The C-Imaging System (Compix, Inc., Cranberry Township, PA) operating the Simple 32 Software Application program was linked to a Nikon Microphot-FX microscope through an Optronics camera and used to quantitate the percentage of 10 the retrospenial cortex occupied by GFAP-positive astrocytes and MAC-1-and MHC II-positive microglia. The image of the immunoreacted section was stored in a video buffer and a monochrome-based threshold was determined to select and calculate the total pixel area occupied by immunolabeled 15 cells. For each section, the retrosplenial cortex (RSC) was manually outlined and the total pixel area occupied by the RSC was calculated. The percent astrocytosis was defined as: (the fraction of RSC occupied by GFAP-reactive astrocytes) X 100. Similarly, percent microgliosis was defined as: (the fraction 20 of the RSC occupied by MAC-1- or MHC II-reactive microglia) X 100. For all image analyses, six sections at the level of the dorsal hippocampus, each separated by consecutive 240 pm intervals, were quantitated for each animal. In all cases, the treatment status of the animals was unknown to the 25 observer. Although the foregoing invention has been described in detail for purposes of clarity of understanding, it will be obvious that certain modifications may be practiced within the scope of the appended claims. All publications and patent 30 documents cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were so individually denoted.

0 010 0 01 0101DI010 0:i 01C0101i0 0 0 0 01 0 0 03 0 E 0 C) IO 00 0 0 M C-IO 0 :) 1o0 00 0D LI) -Y) I fI (\JD 0 0E 00000T 0 o Cl0 C i1 I 0- 0 0 0 C m E0 Mr-c E 010~ 0 0Lo c.JcI0 :: cp) 0 E) :3o 0.0 c:)~~ C \JC CD -0 0 0~ 0Tl*I 00l 00 0: 0C00O 0 00 1 c) 0U 0 01

Claims (17)

1. An antibody to AP, or an active fragment or derivative of the antibody, for use in preventing or treating a disease characterized by amyloid deposit in a patient, wherein said antibody induces an immune response by binding to AP in the patient. 5

2. An antibody of claim 1, which is a monoclonal antibody.

3. An antibody of claim 2 which is a humanized antibody or a human antibody.

4. An antibody of claim 3, wherein the isotype is human IgG1 .

5. An antibody of any preceding claim which is a Fab, Fab', F(ab')2 or 10 Fv fragment, or which is a single chain antibody.

6. An antibody of any preceding claim which binds specifically to the aggregated form of AP peptide without binding to the dissociated form.

7. An antibody of any of claims 1-5 which binds specifically to the dissociated form of AP peptide without binding to the aggregated form. 15

8. An antibody of any of claims 1-5 which binds to both aggregated and dissociated forms of AP peptide.

9. An antibody of any of the preceding claims, wherein the amyloid deposit comprises aggregated AP peptide.

10. An antibody of any of the preceding claims, wherein the patient is a 20 human.

11. An antibody of any of the preceding claims, wherein the disease is Alzheimer's disease.

12. An antibody of any of the preceding claims, wherein the patient is asymptomatic optionally wherein the patient is under 50, and/or has inherited risk factors 25 indicating susceptibility to Alzheimer's disease.

13. An antibody of any of the preceding claims, wherein the antibody is administered orally, subcutaneously, intramuscularly, topically or intravenously.

14. An antibody of any preceding claims which is administered at a dosage from about 0.01 to 5 mg/kg of the host body weight. 30

15. An antibody according to any of the preceding claims which is administered by a first immunization followed by boosters at 6-weekly intervals, or which is administered by a first immunization followed by boosters 1, 2 and 12 months later, or which is administered every two months for life. 85

16. An in vitro method of monitoring Alzheimer's disease or susceptibility thereto in a patient, comprising: detecting an antibody that specifically binds to AP peptide in a sample from the patient, wherein an antibody as recited in any of claims 1-8 effective to treat or prevent Alzheimer's disease has been administered to the patient, and 5 the level of the detected antibody determines the future treatment regime of the patient.

17. An in vitro method of assessing efficacy of an Alzheimer's treatment method in a patient, comprising determining a value for an amount of antibody specific for AP peptide in tissue sample from a patient who has been treated with an antibody as recited in any of claims 1 10 8; comparing the value with a control value determined from a population of patient experiencing amelioration of, or freedom from, symptoms of Alzheimer's disease due to treatment with the antibody as recited in any of claims 1-8; wherein a value in the patient at least equal to the control value indicates a positive 15 response to treatment. Janssen Alzheimer Immunotherapy Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON