OA19316A - Antibodies specific for the protofibril form of beta-amyloid protein - Google Patents

Abstract

Isolated antibodies have been characterized which show specific affinity to a repeating conformational epitope of a protofibril form of the human ß-amyloid peptide as compare to low molecular weight forms of ß-amyloid peptide. These isolated antibodies and related pharmaceutically effective compositions may be useful in the therapeutic and/or prophylactic treatment of Alzheimer's disease by effectively blocking the ability of the protofibril form of ßamyloid peptide to form fibril forms linked with complications associated with Alzheimer's disease. The isolated antibodies of the present invention are also useful in various diagnostic assays and associated kits

Description

The amyloid precursor protein (APP) plays an important rôle in the pathogenesis of Alzheimer's disease (AD).

Proteolytic processing of APP by β- and γ-secretases generate Αβ peptides (Αβ) which normally range in the length from 39 to 43 amino acids in length. The onset of Alzheimer's disease is characterized by the accumulation of oligmeric or aggregated forms of Αβ in the brain. The immunological 15 compositions of the présent invention are useful in treating or preventing Alzheimer's disease, for use as reagents in diagnostic assays, as well as for designing small molécule inhibitors of amyloid déposition. The 13C3-like antibodies of the présent invention maj b.e administered 20 prophylactically to the general population of a mammal, especially a human, in a contemplated pharmaceutically acceptable formulation in an amount and/or dosage régime sufficient to eliminate, reduce or delay onset of the disease. Methods of prophylactic treatment are especially warranted with individuals known to be at a genetic or familiar risk of Alzheimer's disease. Numerous genetic markers of risk for Alzheimer's disease hâve been identified, including but not limited to APP mutations (e.g., the Indian mutation (Val717Phe), the Swedish mutations (Lys670Asn, Met671Leu), the Hendricks mutation (Ala692Gly), the Dutch mutation (Glu693Gln), the Iranian

-1619316 mutation (Thr714Ala), the German mutation (Val715Ala) , and the Florida mutation (Ile716Val), to list a few. Additional mutations which may indicate an increased risk of Alzheimer's disease include mutations in the presenilin 5 genes (PSI and PS2) and ApoE4 . The présent invention also relates to therapeutic intervention via pharmaceutically acceptable compositions comprising a 13C3-like antibody for individuals presently suffering from Alzheimer’s disease can be recognized from characteristic dementia, especially in 10 the presence of risk factors described above or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the Alzheimer's disease symptoms and complications. Either prophylactic- or therapeutic-based treatment methods contemplated herein may 15 be used to address early or date onset Alzheimer's disease.

In view of the importance of oligomeric forms of Αβ in the onset of Alzheimer's disease, the présent invention relates to an isolated antibody that specifically interacts and shows a measured affinity to a repeating conformational 20 epitope of a protofibril form of Αβ peptide. The monomer of wild type Αβ peptide (Αβ₄;_:; the 42 amino acid form) is known in the art and is shown herein as SEQ ID NO:1:

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala 25 Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala (SEQ ID NO:1) .

The isolated antibodies of the présent invention will show affinity for a repeated conformational epitope of the larger molecular weight, oligomeric protofibrillar form of the Αβ 30 peptide while showing minimal affinity for other forms of Αβ peptide, such as low molecular weight monomers and dimers.

-1719316 ) U

The présent invention also relates to an isolated antibody that interacts and shows a measurable affinity to a conformational epitope of a protofibril form of Αβ peptide, whereby the protofibril epitope is represented by an exposed 5 région of a Αβ-protofibril form which comprises the amino terminal portion of an exposed portion of the Αβ peptide.

The présent invention further relates to an isolated antibody that specifically interacts and shows a measurable affinity to a conformational epitope of a protofibril form 10 of Αβ peptide, whereby the protofibril epitope is represented by an exposed région of a Αβ-protofibril form which comprises amino acids 1-20 (SEQ ID NO:2) of an exposed portion of the Αβ peptide, as follows : Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu Val Phe Phe 15 (SEQ ID NO:2).

As exemplified herein, mouse monoclonal antibodies hâve been identified which specifically show spécifie affinity for the protofibril (PF) form of the Αβ peptide, while showing minimal affinity for low molecular weight species of 20 the Αβ peptide. The dimer form of Αβ (-15 kDa) over time polymerizes to form a soluble PF form of Αβ, with a molecular weight of approximately 670 kDa. Mice were immunized with this higher molecular weight PF Αβ. Monoclonal antibodies were screened for specificity to the 25 high molecular weight PF form of the Αβ peptide while showing minimal or no ability to bind lower molecular weight forms of Αβ. This portion of the présent invention is exemplified by the screening, isolation and characterization of the 1303 sériés of monoclonal antibodies raised against 30 the -670 kDa high molecular weight protofibril form of the Αβ peptide (i.e., 1303, 1D1 and 19A6). This sériés of

-1819316 monoclonal antibodies shows the intended specificity in vitro while also reducing Alzheimer's disease-associated plaque formation in a transgenic mouse Alzheimer's disease model. Thus, in a particular embodiment of the invention, 5 the isolated antibody specifically interacts and shows a measurable affinity to a conformational epitope of a protofibril form of Αβ peptide, whereby the protofibril epitope is represented by an exposed région of a Αβprotofibril form which comprises amino acids 4-12 (SEQ ID 10 N0:3) and 9-20 (SEQ ID N0:4) of an exposed portion of the Αβ peptide: Phe Arg His Asp Ser Gly Tyr Glu Val (SEQ ID NO:3); Gly Tyr Glu Val His His Gin Lys Leu Val Phe Phe (SEX ID NO : 4) .

One embodiment of the présent invention relates to an 15 antibody which comprises a V_H (SEQ ID NO: 7) and/or V_L (SEQ ID NO: 5) région as disclosed for 13C3, so as to impart 13C3-like specificity to the PF versus LMW form of the Αβ peptide. An additional embodiment is a 1303-like antibody or biologically relevant fragment thereof which show 20 specificity to the PF form over the LMW form of the Αβ peptide. Thus, the présent invention also relates to biologically active fragments and/or mutants of the 13C3, 1D1, 19A6 or a 13C3-like antibody, including but not necessarily limited to amino acid substitutions (e.g., as a 25 directed form of affinity maturation of the V_H or V_L régions), délétions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide a basis for an antibody or antibody binding portion that results in a similar or improved version of a 13C3, 30 1D1, 19A6 or 13C3-like antibody binding protein. As noted herein, one embodiment of this portion of the invention

-1919316 related to the V_H and/or V_L région of such an antibody comprising the amino acid sequence as set forth in SEQ ID NO: 1 and/or SEQ ID NO:5, respectively. The présent invention notes the existence of codon redundancy which may 5 resuit in differing DNA molécules expressing an identical antibody or portion thereof (e.g., alternative nucleic acid molécules encoding an identical scFv or a VH and/or VL portion of an IgG). For purposes of this spécification, a sequence bearing one or more replaced codons will be defined 10 as a degenerate variation. Another source of sequence variation may occur through RNA editing. Such RNA editing may resuit in another form of codon redundancy, wherein a change in the open reading frame does not resuit in an altered amino acid residue in the expressed protein. Also 15 included within the scope of this invention are mutations either in the DNA sequence or the translated antibody which improve the ultimate physical properties of the expressed antibody. To this end, the présent invention relates to (i) affinity matured versions of a 13C3, 1D1, 19A6 or any other 20 such 13C3-like antioody,- and/or (ii) mutated forms of 13C3,

1D1, 19A6 or any other such 13C3-like antibody, including but not limited to one or more mutations in the CDR1, CDR2 an/or CDR3 régions as generated through known affinity maturation methodology and recombinant DNA techniques known 25 for introducing site spécifie mutations. Thus, the isolated antibodies of the présent invention are antibodies that specifically interact with a conformational epitope of a protofibril form of Αβ peptide. The isolated antibodies of the présent invention will show affinity for such a 30 conformational epitope for the larger molecular weight protofibrillar form of the Αβ peptide while showing minimal affinity for other forms of Αβ peptide, such as fibrils,

-2019316 sheet structures, and low molecular weight oligomers and monomers.

The présent invention also relates to the isolated monoclonal antibody, 13C3. This portion of the invention also relates to a hybridoma which produces the monoclonal antibody, 13C3. A hybridoma which produces the monoclonal antibody 13C3 is available under ATCC Accession No. PTA8830 .

The présent invention also relates to the isolated monoclonal antibody, 1D1. This portion of the invention also relates to a hybridoma which produces the monoclonal antibody, 1D1.

The présent invention also relates to the isolated monoclonal antibody, 19A6. This portion of the invention also relates to a hybridoma which produces the monoclonal antibody, 19A6.

The présent invention also relates to methods of screening for and selecting compounds which may act as an inhibitor of fibril and/or senile plaque formation associated with Alzheimer's disease. Such methodology comprises utilizing an antibody with 13C3-like affinity to the PF form of Αβ peptide in various antibody/peptide/test compound interaction assays in order to select a compound which modulâtes the process of fibril and/or plaque formation. The compound may be a non-proteinaceous organic or inorganic molécule, a peptide (e.g., as a potential prophylactic or therapeutic peptide vaccine), a protein, DNA (single or double stranded) or RNA (such as siRNA or shRNA). It will become évident upon review of the disclosure and teachings of this spécification that any such peptide or small molécule which effectively competes with a 13C3-like

-2119316 antibody for binding to the PF form of Αβ peptide represents a possible lead compound relating to prophylactic or therapeutic treatment of Alzheimer's disease. To this end, interaction assays may be utilized for the purpose of high 5 throughput screening to identify compounds that occupy or interact with the 13C3 epitopes of the PF form of Αβ peptide and displace the antibody.

Various antibody/antigen-based assays known in the art may be used which incorporate and rely on a 13C3-like 10 antibody of the présent invention as an essential reagent in screening for compounds useful in the prophylactic or therapeutic treatment of Alzheimer's disease (e. g., a small inorganic molécule or candidate peptide vaccine), including but not limited to an ELISA assay, a radioimmune assay, a 15 Western blot analysis, any homogenous assay relying on a détectable biological interaction not requiring séparation or wash steps (e.g., see AlphaScreen from PerkinElmer) and/or SPR-based technology (e.g., see BIACore)). Compounds and/or peptide vaccine candidates identified through use of 20 a 13C3-like antibody may be detected by a variety of assays.

The assay may be a simple yes/no assay to détermine whether there is a change in the ability to form the known antibody/antigen complex, or may be made quantitative in nature by utilizing an assay such as an ELISA based assay, a 25 homogenous assay, or an SPR-based assay. To this end, the présent invention relates to any such assay, regardless of the known methodology employed, which measures the ability of a test compound to compete with 13C3-like antibody, to an appropriate peptide or protein mimetic of the amino terminal 30 portion of a 13C3 epitope of the PF form of Αβ peptide.

The antibodies described herein may be used as the basic reagents in a number of different immunoassays to

ΙΟ détermine the presence of a Αβ protofibril form in a tissue sample. Generally speaking, the antibodies can be employed in any type of immunoassay, whether qualitative or quantitative. This includes both the two-site sandwich assay and the single site immunoassay of the non-competitive type, as well as in traditional compétitive binding assays. One embodiment of interest, for ease of détection, and its quantitative nature, is the sandwich or double antibody assay, of which a number of variations exist, ail of which are intended to be encompassed by this portion of the présent invention. For example, in a typical forward sandwich assay, unlabeled antibody is immobilized on a solid substrate, e.g., microtiter plate welis, and the sample to be tested is brought into contact with the bound molécule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex, a second antibody, labeled with a reporter molécule capable of inducing a détectable signal, is then added and incubation is continued allowing sufficient time for binding with the antigen at a different site and the formation of a ternary complex of antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal, which may be quantitated by comparison with a control sample containing' known amounts of antigen. Variations on the forward sandwich assay include the simultaneous assay, in which both sample and antibody are added simultaneously to the bound antibody, or a reverse sandwich assay in which the iabeied antibody and sample to be tested are first combined, incubated and added to the unlabelled surface bound antibody. These techniques are well known to those skilled in the art, and the possibiiity of minor variations wiii be

-2319316 readily apparent. As used herein, sandwich assay is intended to encompass ail variations on the basic two-site technique.

For the sandwich assays of the présent invention, the 5 oniy iimiting factor is that both antibodies hâve different binding specificities for the Αβ protofibril form. Thus, a number of possible combinations are possible. As a more spécifie example, in a typical forward sandwich assay, a primary antibody is either covalently or passively bound to 10 a solid support. The solid surface is usually glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrène, polyvinylchloride or polypropylene. The solid supports may be in the form of tubes, beads, dises or microplates, or any other surfaces suitable for conducting an immunoassay. The binding processes are well known in the art. Following binding, the solid phase-antibody complex is washed in préparation for the test sample. An aliquot of the body fluid containing a Αβ protofibril form to be tested is then added to the solid 20 phase complex and incubated at 25°C for a period of time sufficient to allow binding of any Αβ protofibril form protein présent to the antibody spécifie for the Αβ protofibril form. The second antibody is then added to the solid phase complex and incubated at 25°C for an additional 25 period of time sufficient to allow the second antibody to bind to the primary antibody-antigen solid phase complex. The second antibody is linked to a reporter moiecule, the visible signal of which is used to indicate the binding of the second antibody to any antigen in the sample. By 30 reporter moiecule, as used in the présent spécification is meant a moiecule which by its Chemical nature, provides an analytically détectable signal which allows the détection of

-2419316 antigen-bound antibody. Détection must be at least relatively quantifiable, to allow détermination of the amount of antigen in the sample, this may be calculated in absolute terms, or may be done in comparison with a standard 5 (or sériés of standards) containing a known normal level of antigen.

The most commonly used reporter molécules in this type of assay are either enzymes or fluorophores. In the case of an enzyme immunoassay an enzyme is conjugated to the second 10 antibody, often by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are well known to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase 15 and alkaline phosphatase, among others. The substrates to be used with the spécifie enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a détectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase 20 conjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In ail cases, the enzyme-labeled antibody is added to the 25 first antibody- Αβ protofibril protein complex and allowed to bind to the complex, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the tertiary complex of antibodyantigen-labeled antibody. The substrate reacts with the 30 enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually

-2519316

spectrophotometrically, to give an évaluation of the amount of antigen that is présent in the sérum sample.

Additionally, fluorescent compounds, such as fluorescein or rhodamine, may be chemically coupled to 5 antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a State of excitability in the molécule, followed by émission of the light at a 10 characteristic longer wavelength. The émission appears as a characteristic color visually détectable with a light microscope. As in the enzyme immunoassay (EIA), the fluorescent-labelled antibody is allowed to bind to the first antibody- Αβ protofibril form protein complex. After 15 washing the unbound reagent, the remaining ternary complex is then exposed to light of the appropriate wavelength, and the fluorescence observed indicates the presence of the antigen. Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the présent method. However, other reporter molécules, such as radioisotopes, chemiluminescent or bioluminescent molécules may also be employed. It will be readily apparent to the skilled artisan how to vary the procedure to suit the required use.

In another embodiment, the sample to be tested (e.g., human blood or spinal fluid containing a Αβ protofibril form) may be used in a single site immunoassay wherein it is adhered to a solid substrate either covalently or noncovalently. An unlabeled anti- Αβ protofibril protein 30 antibody is brought into contact with the sample bound on the solid substrate. After a suitable period of incubation, for a period of time sufficient to allow formation of an

-2619316 antibody-antigen binary complex a second antibody, labelled with a reporter molécule capable of inducing a détectable signal, is then added and incubation is continued allowing sufficient time for the formation of a ternary complex of antigen-antibody-labeled antibody. For the single site immunassay, the second antibody may be a general antibody (i.e., zenogeneic antibody to immunoglobulin, particularly anti-(IgM and IgG) linked to a reporter molécule) that is capable of binding an antibody that is spécifie for the Αβ protofibril protein form of interest.

A 13C3-like antibody may take one of numerous forms known in the art. Antibodies may take the form of any type of relevant antibody fragment, antibody binding portion, spécifie binding member, a non-protein synthetic mimic, or any other relevant terminology known in the art which refers to an entity which at least substantially retains the binding specificity/neutralization activity. Thus, the term antibody as used in any context within this spécification is meant to include, but not be limited to, any spécifie binding member, immunoglobulin class a'nd/or isotype (e.g., IgGi, IgG₂, IgG₃, IgG₄, IgM, IgA, IgD, IgE and IgM) ; and biologically relevant fragment or spécifie binding member thereof, including but not limited to Fab, F(ab')2, Fv, and scFv (single chain or related entity). Therefore, it is well known in the art, and is included as review only, that an antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding portion thereof. A heavy chain is comprised of a heavy chain variable région (VH) and a heavy chain constant région (CH1, CH2 and CH3) . A light chain is comprised of a light chain variable région (VL) and a Light chain constant région (CL).

The variable régions of both the heavy and light chains comprise framework. régions (FWR) and complementarity determining régions (CDR). The four FWR régions are relatively conversed while CDR régions (CDR1, CDR2 and CDR3) 5 represent hypervariable régions and are arranged from NH₂ terminus to the COOH terminus as follows: FWR1, CDR1, FWR2,

CDR2, FWR3, CDR3, FWR4. The variable régions of the heavy and light chains contain a binding domain that interacts with an antigen while, depending of the isotype, the 10 constant région(s) may médiate the binding of the immunoglobuiin to host tissues or factors. That said, also included in the working définition of antibody are chimeric antibodies, humanized antibodies, a recombinant antibody, as human antibodies generated from a transgenic 15 non-human animal, as well as antibodies selected from libraries using enrichment technologies available to the artisan. Antibody fragments are obtained using techniques readily known and available to those of ordinary skill in the art, as reviewed beiow. Therefore, an antibody is any 20 such entity or spécifie binding member, which specifically binds the conformational epitope of the protofibril form of Αβ as described herein. Therefore, the term antibody describes an immunoglobuiin, whether natural or partly- or whoiiy synthetically produced; any polypeptide or protein 25 having a binding domain which is, or is substantially homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobuiin isotypes and their isotypie subeiasses;

fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd and diabodies, as discussed without limitation, infra. It is known in the art that it is possible to manipulate monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molécules which retain the specificity of the original antibody Such techniques may evolve introducing DNA encoding the immunoglobulin variable région, or the complementarity determining régions (CDRs), of an antibody to the constant régions, or constant régions plus framework région”, of a different immunoglobulin. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced. Antibodies can be modified in a number of ways, and the term antibody should be construed as covering any spécifie binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, dérivatives, functional équivalents and homologues of antibody including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Such an entity may be a binding fragment encompassed within the term antigenbinding portion or spécifie binding member of an antibody including but not limited to (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge région; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody (v) a dAb fragment, which comprises a VH domain; (vi) an isolated complementarity determining région (CDR); (vii) a 'scAb', an antibody fragment containing VH and VL as well as either CL or CH; and (viii) artificial antibodies based upon protein

-2919316 scaffolds, including but not limited to fibronectin type III polypeptide antibodies (e.g., see U.S. Patent No. 6, 703, 199, issued to Koide on March 9, 2004 and PCT International Application Publication No. WO 02/32925). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein Chain in which the VL and VH régions pair to form monovalent molécules (known as single Chain Fv (scFv)).

In one embodiment, the variable light (VL) région for the isolated 13C3 or 13C3-like antibodies of the présent invention may comprise a 113 amino acid peptide sequence (SEQ ID NO: 5) which is encoded by a 339 base pair nucléotide sequence (SEQ ID NO: 6):

Asp Val Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gin Ala Ser Ile Ser Cys Arg Ser Gly Gin Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu Ile Tyr Thr Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gin Asn Thr Phe Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg (SEQ ID NO: 5)

GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCC ATCTCTTGCAGATCTGGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGG TACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTATACAGTTTCCAACCGATTT TCTGGGGTCCCGGACAGGTTCAGTGGCAGTGGATCAGGGTCAGATTTCACACTCAAGATC AGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAATACATTTGTTCCT TGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG (SEQ ID NO: 6)

In a further embodiment, the variable heavy (VH) région for the isolated 13C3 or 13C3-like antibodies of the présent invention may comprise a 115 amino acid peptide sequence

-3019316 (SEQ ID NO: 7) encoded by a 345 base pair nucléotide sequence (SEQ ID NO: 8):

Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Arg Pro Gly

Val Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr

Asp Tyr Ala Met His Trp Val Lys Gin Ser His Ala Lys Ser Leu

Glu Trp Ile Gly Val Ile Ser Thr Lys Tyr Gly Lys Thr Asn Tyr

Asn Gin Lys Phe Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser

Ser Ser Thr Ala Tyr Met Glu Leu Ala Arg Leu Thr Ser Glu Asp

Ser Ala Ile Tyr Tyr Cys Ala Arg Gly Asp Asp Gly Tyr Ser Trp

Gly Gin Gly Thr Ser Val Thr Val Ser Ser (SEQ ID NO: 7);

CAGGTCCAGCTGCAGCAGTCTGGGCCTGAGCTGGTGAGGCCTGGGGTCTCAGTGAAGATT TCCTGCAAGGGTTCCGGCTACACATTCACTGATTATGCTATGCACTGGGTGAAGCAGAGT CATGCAAAGAGTCTAGAGTGGATTGGAGTTATTAGTACTAAGTATGGTAAGACAAACTAC AACCAGAAGTTTAAGGGCAAGGCCACAATGACTGTTGACAAATCCTCCAGCACAGCCTAT 15 ATGGAGCTTGCCAGATTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGAGGGGAC

GATGGTTATTCCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 8) .

In a further embodiment, the framework régions, FWR1, FWR2, FWR3, and FWR4, of the VL chain may be comprised of 20 amino acid set forth in SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, as follows:

Asp Val Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Ser Leu

Gly Asp Gin Ala Ser Ile Ser Cys Arg Ser Gly (SEQ ID NO: 9);

Leu His Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu

Ile Tyr (SEQ ID NO: 10);

Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

Gly Ser Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp

Leu Gly Val Tyr Phe Cys (SEQ ID NO: 11);

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg (SEQ ID NO: 12).

In a further embodiment, the complementarity determining régions, CDR1, CDR2, and CDR3, of the VL chain may be comprised of the amino acids set forth in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, respectively, as follows:

Gin Ser Leu Val His Ser Asn Gly Asn Thr Tyr (SEQ ID NO: 13);

-3119316

Thr Val Ser (SEQ ID NO: 14);

Ser Gin Asn Thr Phe Val Pro Trp Thr (SEQ ID NO: 15).

In a further embodiment, the framework régions, FWR1, FWR2, FWR3, and FWR4, of the VH chain may be comprised of 5 the amino acids set forth in SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18, and SEQ ID N0:19, respectively, as follows:

Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Arg Pro Gly Val Ser Val Lys Ile Ser Cys Lys (SEQ ID NO: 16);

Met His Trp Val Lys Gin Ser His Ala Lys Ser Leu Glu Trp Ile 10 Gly Val (SEQ ID NO: 17);

Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg (SEQ ID NO: 18);

Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser (SEQ ID NO: 19).

In a further embodiment, the complementarity determining régions, CDR1, CDR2, and CDR3, of the VH chain may be comprised of the amino acids set forth in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, respectively, as follows:

Gly Ser Gly Tyr Thr Phe Thr Asp Tyr Ala (SEQ ID NO: 20);

Ile Ser Thr Lys Tyr Gly Lys Thr Asn Tyr Asn Gin Lys Phe Lys Gly Lys (SEQ ID NO: 21) ;

Gly Asp Asp Gly Tyr Ser (SEQ ID NO: 22).

Polyclonal or monoclonal antibodies for use in the disclosed treatment methods may be raised by known techniques. Monospecific murine (mouse) antibodies showing specificity to a conformational epitope of a target of choice may be purified from mammalian antisera containing antibodies reactive against this région, or may be prepared as monoclonal antibodies using the technique of Kohler and

Milstein (1975, Nature 256: 495-497). Monospecific antibody as used herein is defined as a single antibody species or

-3219316 multiple antibody species with homogenous binding characteristics, such as the mouse monoclonal antibodies exemplified herein with the 13C3 sériés of monoclonal antibodies. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas. The splenic antibody producing cells and myeloma cells are fused, selected, and screened for antibody production. Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson (1973, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds, Academie Press) . Monoclonal antibodies are produced in vivo by injecting respective hydridoma cells into pristine primed mice, collecting ascite fluid after an interval of time, and prepared by techniques well known in the art.

Beyond species spécifie monoclonal antibodies described above, the antibodies of the présent invention may also be in the form of a chimeric· antibody, a monoclonal antibody constructed from the variable régions derived from say, the murine source, and constant régions derived from the intended host source (e.g., human; for a review, see Morrison and Oi, 1989, Advances in Immunology, 44: 65-92). For example, the variable light and heavy DNA sequences (e.g. SEQ ID NO: 6 and 8, respectively) from the rodent (e.g., mouse) antibody may be cloned into a mammalian expression vector. These light and heavy chimeric expression vectors are cotransfected into a récipient cell line and selected and expanded by known techniques. This cell line may then be subjected to known cell culture techniques, resulting in production of both the light chain

-3319316 and heavy chain of a chimeric antibody. Such chimej_ic antibodies hâve historically been shown to hâve the antigenbinding capacity of the original rodent monoclonal while significantly reducing immunogenicity problems upon host 5 administration.

A logical improvement to the chimeric antibody is the humanized antibody, which arguably reduces the chance of the patient mounting an immune response against a therapeutic antibody when compared to use of a chimeric or 10 full murine monoclonal antibody The strategy of humanizing a murine Mab is based on replacing amino acid residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grafting of entire complementarity determining régions 15 (Jones et al., 1986, Nature 321: 522-526). This technology is again now well known in the art and is represented by numerous strategies to improve on this technology; namely by implementing strategies including, but not limited to, reshaping (see Verhoeyen, et al., 1988, Science 239: 153420 1536), hyperchimerization (see Queen, et al., 1991, Proc.

Natl. Acad. Sci. 88:2869-2873) or veneering (Mark, et al., 1994, Dérivation of Therapeutically Active Humanized and Veneered anti-CD18 Antibodies Metcalf end Dalton, eds . Cellular Adhesion: Molecular Définition to Therapeutic 25 Potential. New York: Plénum Press, 291-312). These strategies ail involve to some degree sequence comparison between rodent and human sequences to détermine whether spécifie amino acid substitutions from a rodent to human consensus is appropriate. Whatever the variations, the 30 central theme involved in generating a humanized antibody relies on CDR grafting, where these three antigen binding sites from both the light and heavy chain are effectively

-3419316 removed from the rodent expressing antibody clone and subcloned (or grafted) into an expression vector coding for the framework région of the human antibody. For example, utilizing the above techniques a humanized antibody may be expressed wherein the CDR1, CDR2, and CDR3 régions of the variable light chain are set forth in SEQ ID NOS: 13, 14 and 15, respectively, and the CDR1, CDR2, and CDR3 régions of the variable heavy chain are set forth in SEQ ID NOS 20, 21 and 22, respectively. Therefore, a humanized antibody is effectively an antibody constructed with only murine CDRs (minus any additional improvements generated by incorporating one or more of the above mentioned strategies), with the remainder of the variable région and ail of the constant région being derived from a human source.

The présent invention also relates to isolated nucleic acid molécules and associated amino acid sequences which relate to the V_E and/or V_L régions of the 13C3 antibody, and more specifically, an isolated nucleic acid molécule (polynucleotide) encoding a biologically relevant portion of 13C3, or affinity matured version or otherwise mutated version of 13C3, 1D1, 19A6 or other 13C3-like antibody. These nucleic acids are substantially free from other nucleic acids. For most cloning purposes, DNA is a preferred nucleic acid. These DNA molécules may be subcloned into an expression vector and subsequently transfected into a host cell of choice wherein the recombinant host cell provides a source for substantial levels of a relevant portion of the 13C3, 1D1, 19A6 or 13C3, 1D1-, or 19A6-like antibody, or affinity matured version thereof. Such procedures may be used for a variety of utilities, such as generating scFvs or for co-expressing

-3519316 these V_H and V_L chains in a mammalian expression vector system which encodes human Ch and C_L régions, of say, an IgG antibody. The degeneracy of the genetic code is such that, for ail but two amino acids, more than a single codon encodes a particular amino acid. This allows for the construction of synthetic DNA that encodes an antibody of the présent invention where the nucléotide sequence of the synthetic DNA differs significantly from the nucléotide sequences disclosed herein, but still encodes such an antibody. Such synthetic DNAs are intended to be within the scope of the présent invention. If it is desired to express such synthetic DNAs in a particular host cell or organism, the codon usage of such synthetic DNAs can be adjusted to reflect the codon usage of that particular host, thus leading to higher levels of expression of the an antibody of the présent invention. In other words, this redundancy in the various codons which code for spécifie amino acids is within the scope of the présent invention. Therefore, this invention is also directed to those DNA sequences which encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid, as shown below: A=Ala=Alanine: codons GCA, GCC, GCG, GCU; C=Cys=Cysteine : codons -UGC, UGU; D=Asp=Aspartic acid: codons GAC, GAU E=Glu=Glutamic acid: codons GAA, GAG; F=Phe=Phenylaianine: codons UUC, UUU; G=Gly=Giycine: codons GGA, GGC, GGG, GGU; H=His =Histidine: codons CAC, CAU; I=Ile =Isoleucine: codons AUA, AUC; AUU; K=Lys-Lysine: codons AAA, AAG; L=Leu=Leucine : codons UUA, UUG, CUA, CUC, CUG, CUU; M=Met=Methionine: codon AUG; N=Asp=Asparagine : codons GAU, GAC; P=Pro=Proline: codons CCA, CCC, CCG, CCU; Q=Gln=Glutamine: codons CAA, CAG; R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU; S=Ser=Serine: codons AGC, AGU,

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UCA, UCC, UCG, UCU; T=Thr=Threonine: codons ACA, ACC, ACG, ACU; V=Val=Valine: codons GUA, GUC, GUG, GUU;

W=Trp=Tryptophan: codon UGG; Y=Tyr=Tyrosine: codons UAC, UAU. Such recombinant expression vectors may then be stably 5 or transientiy transfected into an appropriate cell line for the génération of alternative antibody form.

The présent invention notes the existence of codon redundancy which may resuit in differing DNA molécules expressing an identical antibody or portion thereof (e.g., 10 alternative nucleic acid molécules encoding an identical scFv or a VH and/or VL portion of an IgG). For purposes of this spécification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Another source of sequence variation may occur through RNA editing.

Such RNA editing may resuit in another form of codon redundancy, wherein a change in the open reading frame does not resuit in an altered amino acid residue in the expressed protein. Also included within the scope of this invention are mutations either in the DNA sequence or the translated 20 antibody which improve the ultimate physical properties of the expressed antibody. To this end, the présent invention relates to (i) affinity matured versions of a 13C3-like antibody, including but not limited to 13C3, 19A6 and 1D1, and/or (ii) mutated forms of a 13C3-like antibody, including but not limited to 13C3, 19A6 and/or 1D1, including but not limited to one or more mutations in the CDR1, CDR2 an/or CDR3 régions as generated through known affinity maturation methodology and recombinant DNA techniques known for introducing site spécifie mutation. Such isolated or purified nucleic acid molécules will represent the VH and/or VL portions of a 13C3-like antibody. These nucleic acids are substantially free from other nucleic acids. For most

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cloning purposes, DNA is a preferred nucleic acid. These DNA molécules may be subcloned into an expression vector and subsequently transfected into a host cell of choice wherein the recombinant host cell provides a source for substantial 5 levels of a relevant portion of a 13C3-iike antibody, or affinity matured version thereof. Such procedures may be used for a variety of utilities, such as generating scFvs or for co-expressing these VH and VL chains in a mammalian expression vector system which encodes human CH and CL 10 régions, of say, an IgG antibody.

The présent invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain nucleic acid molécules encoding the respective heavy and/or light régions of a 13C3-like 15 antibody. These nucleic acid molécules, in whole or in part, can be linked with other DNA molécules (i.e., DNA molécules which encompass immunoglobuiin genes used for génération of a recombinant human antibody) that are not naturally linked, to form recombinant DNA molécules which

-20 encode a respective human recombinant antibody. These vectors may be comprised of DNA or RNA. For most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bactériophage, cosmids, yeast artificial chromosomes, and other forms of episomal or integrated DNA. It is within the purview of the skilled artisan to détermine an appropriate vector for a particular gene transfer, génération of a recombinant human antibody or other use. Methods of subcloning nucleic acid molécules of interest into expression vectors, transforming or 30 transfecting host cells containing the vectors, and methods of making substantially pure protein comprising the steps of introducing the respective expression vector into a host

-3819316 cell, and cultivating the host cell under appropriate conditions are well known. The antibody (such as an IgG recombinant human antibody) so produced may be harvested from the host cells in conventional ways. Any known expression vector may be utilized to practice this portion of the invention, including any vector containing a suitable promoter and other appropriate transcription regulatory éléments. The resulting expression construct is transferred into a prokaryotic or eukaryotic host cell to produce recombinant protein. Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their rnRNAs in an appropriate host. Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of réplication for autonomous réplication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes rnRNAs to be initiated at high frequency. Techniques for such manipulations can be found described in Sambrook, et al. (1989, Molecular Cloning. A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) are well known and available to the artisan of ordinary skill in the art. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or

-3919316 viruses. Commercially available martini ali an expression vectors which may be suitable, include but are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIanp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO pSV2-neo (ATCC 37593) pBPV-l(8-2) (ATCC 3/110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565). Also, a variety of bacterial expression vectors are available, including but not limited to pCR2.1 (Invitrogen), pETl la (Novagen), lambda gtl 1 (Invitrogen), and pKK223-3 (Pharmacia). In addition, a variety of fungal cell expression vectors may be used, including but not limited to pYES2 (Invitrogen) and Pichie expression vector (Invitrogen) . Also, a variety of insect cell expression vectors may be used, including but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen) .

Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such -as yeast, mammalian cells including, but not limited to, cell Unes of bovine, porcine, monkey and rodent origin; and insect cells. Mammalian species which may be suitable,-26 include but are not limited to, L cells L-M(TK-) (ATCCCCL1.3) , L cells L-M (ATCC CCL 1.2), Saos-2 (ATCCHTB-85) , 293 (ATCCCRL1573) , Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL1650), COS-7(ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC

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CRL 1616)·, BS-C-1 (ATCC CCL 26), MRC-5 (ATCCCCL171) and CPAE (ATCC CCL 209).

Yet another improvement over re-engineered antibodies as reviewed above is the génération of fully human 5 monoclonal antibodies. The first involves the use of genetically engineered mouse strains which possess an immune system whereby the mouse antibody genes hâve been inactivated and in turn repiaced with a répertoire of functional human antibody genes, while leaving other components of the mouse immune system unchanged. Such genetically engineered mice allow for the natural in vivo immune response and affinity maturation process which results in high affinity, fully human monoclonal antibodies This technology is again now well known in the art and is 15 fully detailed in various publications, including but not limited to U.S. Patent Nos. 5,939, 598; 6,075,181;

6,114,598; 6,150,584 and related family members (assigned to Abgenix, disclosing their XenoMouse technology); as well as U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;

5,789,650; 5, 877, 397; 5,661,016; 5,814,318; 5,874,-299; and

5,⁷70,429 (assigned to GenPharm International and available through Medarex, under the umbreila of the UltraMab Human Antibody Development System). See also a re^view from Kellerman and Green (2002, Curr. Opinion in Biotechnology 25 13: 593-597).

Finally, techniques are available to the artisan for the sélection of antibody fragments from libraries using enrichment technologies, including but not limited to phage display, ribosome display (Hanes and Pluckthun, 1997, Proc.

Nat. Acad. Sci. 94: 4937-4942), bacterial display (Georgiou, et al., 1997, Nature Biotechnology 15: 29-34) and/or yeast display (Kieke, et al., 1997, Protein Engineering 10: 1303

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1310) may be utilized as alternatives to previously discussed technologies to select single chain antibodies which specifically bind to target cytokine. Single-chain antibodies are selected from a library of single chain antibodies produced directly utilizing filamentous phage technology. Phage display technology is known in the art (e.a., see technology from Cambridge Antibody Technology (CAT)) as disclosed in U.S. Patent Nos. 5,565,332;

5,733,743; 5,871,907; 5,872,215; 5,885,793; 5,962,255;

6,140,471; 6,225,447; 6,291650; 6,492,160; 6,521,404;

6,544,731; 6,555,313; 6,582,915; 6,593, 081, as well as other U. S. family members, or applications which rely on priority filing GB 9206318, filed 24 May 1992; see also Vaughn, et al. 1996, Nature Biotechnology 14: 309-314).

Single chain antibodies may also be designed and constructed using available recombinant DNA technology, such as a DNA amplification method (e.g., PCR), or possibly by using a respective hybridoma cDNA as a template. Single-chain antibodies can be mono-or bispecific; bivalent or tetravalent. A nucléotide sequence encoding a single-chain antibody can be constructed using manual or automated nucléotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.

The présent invention further relates to an antibodybased pharmaceutical composition comprising an effective amount a 13C3-like antibody, or an affinity matured version, which provides a prophylactic or therapeutic treatment choice to inhibit fibril and/or senile plaque formation associated with Alzheimer's disease. The antibody-based pharmaceutical composition of the présent invention may be formulated by any number of strategies known in the art

-4219316 (e.g., see McGoff and Scher, 2000, Solution Formulation or Proteins/Peptides : In McNally, E.J., ed. Protein Formulation and Delivery. New York, NY: Marcel Dekker; pp. 139-158;

Akers and Defilippis, 2000, Peptides and Proteins as Parentéral Solutions. In: Pharmaceutical Formulation

Development of Peptides and Proteins. Philadelphia, PA: Talyor and Francis; pp. 145-177; Akers, et al., 2002, Pharm. Biotechnol. 14:47-127). A pharmaceutically acceptable composition suitable for patient administration will contain an effective amount of the antibody in a formulation which both retains biological activity while also promoting maximal stability during storage within an acceptable température range. The pharmaceutical compositions can also include, depending on the formulation desired, pharmaceutically acceptable diluents, pharmaceutically acceptable carriers and/or pharmaceutically acceptable excipients, or any such vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphatebuffered saline, Ringer's solutions, dextrose solution, and Hank's solution. The amount-of an excipient that is useful in the pharmaceutical composition or formulation of this invention is an am.ount that serves to uniformly distribute the antibody throughout the composition so that it can be uniformly dispersed when it is to be delivered to a subject in need thereof. It may serve to dilute the antibody to a concentration which provides the desired bénéficiai palliative or curative results while at the same time minimizing any adverse side effects that might occur from too high a concentration. It may also hâve a preservative

-4319316 effect. Thus, for the antibody having a high physiological activity, more of the excipient will be employed. On the other hand, for any active ingrédient(s) that exhibit a lower physiological activity, a lesser quantity of the excipient will be employed. In general, the amount of excipient in the composition will be between about 50% weight (w) and 99.9% w. of the total composition. If the antibody exhibits a particularly low physiological activity, the amount of excipient could be as little as 1% w. On the 10 other hand, for an antibody that has a particularly high physiological activity, the amount of excipient may be between about 98.0% and about 99.9% w. In addition, the antibody or antibodies may by administered in the form of a Chemical dérivative (a molécule that contains additional

Chemical moieties which are not normally a part of the base molécule). Such moieties may improve the solubility, halflife, absorption, etc. of the biological agent. Alternatively, these moieties may attenuate undesirable side effects of the antibody. Pharmaceutical compositions can 20 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 aggregates (such as oil droplets or liposomes) . Additionally, these carriers can function as immunostimulating agents (i. e., adjuvants). For parentéral administration, agents of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically 30 acceptable diluent with a pharmaceutical carrier which can be a stérile liquid such as water oils, saline, glycerol, or éthanol. Additionally, auxiliary substances, such as

-4419316 wetting or emulsifying agents, surfactants, pH buffering substances and the like can be présent in compositions. Other components of pharmaceutical compositions are those of Petroleum, animal, vegetable, or synthetic origin, for 5 example, peanut oil, soybean oil, and minerai oil.

In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

The antibody formulation may be in liquid form or solid 10 form. A solid formulation is generally lyophilized and brought into solution prior to administration for either single or multiple dosing. The formulations should not be exposed to extreme température or pH so as to avoid thermal dénaturation. Thus, it is essential to formulate an 15 antibody composition of the présent invention within a biologically relevant pH range. A solution buffered to maintain a proper pH range during storage is indicated, especially for liquid formulations stored for longer periods of time between formulation and administration. To date, 20 both liquid and solid formulations require storage at lower températures (usually 2-8”C) in order to retain stability for longer periods. Formulated antibody compositions, especially liquid formulations, may contain a bacteriostat to prevent or minimize proteolysis during storage, including 25 but not linuted to effective concentrations (usually - <1% w/v) of benzyi alcohol, phénol, m-cresol, chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat may be contraindicated for some patients. Therefore, a lyophilized formulation may be reconstituted in a solution either 30 containing or not containing such a component. Additional components may be added to either a buffered liquid or solid antibody formulation, including but not limited to sugars as

-4519316 a cryoprotectant (including but not necessarily limited to polyhydroxy hydrocarbons such as sorbitol, mannitol, glycerol and dulcitol and/or disaccharides such as sucrose, lactose, maltose or trehalose) and, in some instances, a relevant sait (including but not limited to NaCl, KC1 or LiCl). Such antibody formulations, especially liquid formulations slated for long term storage, will rely on a useful range of total osmolarity to both promote long term stability at température of 2-8°C, or higher, while also making the formulation useful for parentéral injection. An effective range of total osmolarity (the total number of molécules in solution) is from about 200 mOs/L to about 800 mOs/L. It will be apparent that the amount of a cyroprotectant, such as sucrose or sorbitol, will dépend upon the amount of sait in the formulation in order for the total osmolarity of the solution to remain within an appropriate range. Therefore a sait free formulation may contain from about 5% to about 25% sucrose, with a preferred range of sucrose from about 7% to about 15%, with an especially preferred- sucrose concentration in a sait free formulation being from 10% to 12%. Altematively, a sait free sorbitol-based formulation may contain sorbitol within a range from about -3% to about 12%, with a preferred range from about 4% to 7%, and an especially preferred range is from about 5% to about 6% sorbitol in a salt-free formulation. Salt-free formulations will of course warrant increased ranges of the respective cryoprotectant in order to maintain effective osmolarity levels. These formulation may also contain a divalent cation (including but not necessarily limited to MgC12, CaClj and MnClj); and a non-32 ionic surfactant (including but not necessarily limited to Polysorbate-80 (Tween 80®), Polysorbate-60 (Tween 60®),

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Polysorbate-40 (Tween 40®) and Polysorbate-20 (Tween 20®), polyoxyethylene alkyl ethers, including but not limited to Brij 58®, Brij 35®, as well as others such as Triton X-100®, Triton X 114®, NP40®, Span 85 and the Pluronic sériés of non5 ionic surfactants (e.g., Pluronic 121)). Any combination of such components, including probable inclusion of a bacteriostat, may be useful to fill the antibody-containing formulations of the présent invention. The antibody composition of the présent invention may also be a Chemical 10 dérivative, which describes an antibody that contains additional Chemical moieties which are not normaily a part of the immunogloblulin molécule (e.g., pegylation). Such moieties may improve the solubility, half-life, absorption, etc. of the base molécule. Alternatively the moieties may 15 attenuate undesirable side effects of the base molécule or decrease the toxicity of the base molécule.

Numerous examples of various carriers, diluents, excipients and the such are known in the art and are disclosed in references cited herein, as well as Remington’s 20 Pharmaceutical Sciences (18th ed. ; Mack Publishing Company, Easton, Pa., 1990), the contents of which are incorporated herein by reference. Briefiy, it will be appreciated that suitable carriers, excipients, and other agents may be incorporated to formulate the pharmaceutical compositions to 25 provide improved transfer, delivery, tolérance, and the like. The methods of incorporating the biological agent and/or additional active ingrédient(s) into the carrier are known to a person of ordinary skill in the art and dépend on the nature of the biological agent and the nature of the 30 carrier selected by a person practicing the current invention. Ionie binding, gel encapsulation or physical trapping inside the carrier, iontophoresis and soaking the

-4719316 carrier in a solution of the biological agent are suitable examples contemplated in formulating a pharmaceutical composition to be used to practice of the disclosed treatment methods. Alternatively, the carrier may be little more than a diluent for the biological agent. These formulations may include for example, powders, pastes, ointments, jelly, waxes, oils, lipids, anhydrous absorption bases, oil-in-water or water-in-oil émulsions, émulsions carbowax (polyethylene glycols of a variety of molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. The dosage regimen utilizing the compounds of the présent invention is selected in accordance with a variety of factors including type, species, âge, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the rénal, hepatic and cardiovascular function of the patient; and the particular biological agent thereof employed. A physician or veterinarian of ordinary skill can readily détermine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal précision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a considération of the distribution, equilibrium, and élimination of a drug. Any of the foregoing formulations may be appropriate in treatments and thérapies in accordance with the présent invention, provided that the active ingrédient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible.

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The pharmaceutical compositions of the présent invention may be administered to the host in any manner, strategy and/or combination available in the art in amounts sufficient to offer a therapeutic treatment against Alzheimer's disease. These compositions may be provided to the individual by a variety of routes known in the art, especially parentéral routes, including but in no way limited to parentera। routes such as intravenous (IV), intramuscular (IM); or subcutaneous (SC) administration, with IV administration being the norm within the art of therapeutic antibody administration. These compositions may be administered as separate or multiple doses (i.e., administration of the antibody at staggered times by maintaining the stérile condition of the formulation through the treatment régime). The dosage regimen utilizing the compounds of the présent invention is selected in accordance with a variety of factors including type, species, âge, weight, sex and medical condition of the patient (such as a human patient); the severity of the condition to be treated; the route of administration; the rénal, hepatic and cardiovascular function of the patient; and the particular antibody thereof employed. A physician or veterinarian of ordinary skill ~can readily détermine and prescribe the effective therapeutic amount of the antibody. Optimal précision in achieving concentrations of antibody within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug¹ s availability to target sites. This involves a considération of the distribution, equilibrium, and élimination of a drug. Antibodies described herein may be used alone at appropriate dosages. Altematively, co-administration or sequential administration of other agents may be désirable. It will be

-4919316 possible to présent a therapeutic dosing régime for the antibodies of the présent invention in conjunction with administration of alternative prophylactic or therapeutic régimes.

upon many

An effective dosage régime will vary depending ' different factors, including means of administration, target site, physiological State of the patient, whether the patient is human or an animal, other médications administered, and whet^her treatment is prophylactic or therapeutic. For administration of a 13C3like antibody, the dosage ranges from about 0. 0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg of the host body weight. In the case of Alzheimer's disease, amyloid deposits 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.

Another aspect regarding delivery and dosage régimes for a 13C3-like antibody composition of the présent invention relates to drug delivery via parentéral routes, which may include non-injectable and injectable devices. Typically, injectable compositions are prepared as either liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The préparation also can be emulsified or encapsulated in liposomes or microparticles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above (see Langer, 1990, Science 249: 1527-1523; and Hanes, 1997, Advanced Drug Delivery Reviens 28: 97-119). The agents of this invention can be administered in the form of a depot injection or implant préparation which can be formulated in such a manner

-5019316 as to permit a sustained or pulsatile release of the active ingrédient.

Spécifie embodiments include PLGA microspheres, as discussed herein and as further known in the art, as well as polymer-based non-degradable vehicles comprising poly (ethylene-co-vinyl acetate; PEVAc). Additionally, controlled-release and localized delivery of antibody-based therapeutic products is reviewed in Grainger, et al., 2004, Expert Opin. Biol. Ther. 4(7): 1029-1044), hereby incorporated by reference in its entirety. Suitable microcapsules capable of encapsulating the antibody may also include hydroxymethylcellulose or gelatin-microcapsules and polymethyl méthacrylate microcapsules prepared by coacervation techniques or by interfacial polymerization. See PCT publication WO 99/24061 entitled Method for Producing IGF-1 Sustained-Release Formulations, wherein a protein is encapsulated in PLGA microspheres, this reference which is hereby incorporated herein by reference in its entirety. In addition, microemulsions or colloïdal drug delivery Systems such as liposomes and albumin microspheres, may also be used. Other preferred sustained-release compositions employ a bioadhesive to retain the antibody at the site of administration. As noted above, the susbainedrelease formulation may comprise a biodégradable polymer into which the antibody is disposed, which may provide for non-immediate release. Non-injectable devices may be described herein as an implant, pharmaceutical depot implant, depot implant, non-injectable depot or some such similar term. Common depot implants may include, but are not limited to, solid biodégradable and nonbiodegradable polymer devices (such as an extended polymer or coaxial rod shaped device), as well as numerous pump

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Systems also known in the art. Injectable devices are split into bolus injections (release and dissipation of the drug subséquent to injection), and repository or depot injections, which provide a storage réservoir at the site of 5 injection, allowing for sustained-reiease of the biological agent over time. A depot implant may be surgically tethered to the point of delivery so as to provide an adéquate réservoir for the prolonged release of the antibody over time. Such a device will be capable of carrying the drug formulation in such quantifies as therapeutically or prophylactically required for treatment over the preselected period. The depot implant may also provide protection to the formulation from dégradation by body processes (such as proteases) for the duration of treatment.

As known in the art, the term sustained-reiease refers to the graduai (continuous or discontinuous) release of such an agent from the block polymer matrix over an extended period of time. Regardless of the spécifie device, the sustainedreiease of the 13C3-like antibody composition will resuit in a local, biologically effective concentrations of the antibody. A sustained release of the biological agent(s) will be for a period of a single day, several days, a week or more; but most likely for a month or more, or up to about six months, depending on the formulation. Natural or synthetic polymers known in the art will be useful as a depot implant due to characteristics such as versatile dégradation kinetics, safety, and biocompatibility. These copolymers can be manipulated to modify the pharmacokinetics of the active ingrédient, shield the agent from enzymatic attack, as well as degrading over time at the site of attachment or injection. The artisan will understand that there are ample teachings in the art to manipulate the

-5219316 properties of these copolymers, including the respective production process, catalysts used, and final molecular weight of the sustained-release depot implant or depot injection. Natural polymers include but are not limited to 5 proteins (e.g., collagen, albumin or gelatin);

polysaccharides (cellulose, starch, alginates, chitin, chitosan, cyclodextrin, dextran, hyaluronic acid) and lipids. Biodégradable synthetic polymers may include but are not limited to various polyesters, copolymers of ΒΙΟ glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-556), polylactides ([PLAJ; U.S.

Pat. No. 3,773,919 and EP 058,481), polylactate polyglycolate (PLGA) such as polylactide-co-glycolide (see, for example, U.S. Pat. Nos. 4,767,628 and 5,654,008), 15 polyglycolide (PG), polyethylene glycol (PEG) conjugates of poly (oc-hydroxy acids), polyorthoesters, polyaspirins, polyphosphagenes, vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA, PEGT-PBT copolymer (polyactive), méthacrylates, poly(N-isopropylacrylamide) , PEO-PPO-PEO (pluronics), PEO20 PPO-PAA copolymers, PLGA-PEO-PLGA, polyorthoesters (POE), or any combinations thereof, as described above (see, for example, U.S. Pat. No. 6,991,654 and U.S. Pat. Appl. No.

20050187631, each of which is incorporated herein by reference in its entirety, hydrogels (see, for example, 25 Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277;

Langer, 1982, Chem. Tech. 12:98-105, non-degradable ethylene-vinyl acetate (e.g. ethylene vinyl acetate disks and poly(ethylene-co-vinyl acetate)), degradable lactic acid-glycolic acid copolyers such as the Lupron Depot™, 30 poly-D-(-)-3-hydroxybutyric acid (EP 133,988), hyaluronic acid gels (see, for example, U.S. Pat. No. 4,636,524), alginic acid suspensions, polyorthoesters (POE), and the

-5319316 like. Polylactide (PLA) and its copolymers with glycolide (PLGA) hâve been well known in the art since the commercialization of the Lupron Depot™, approved in 1989 as the first parentéral sustained-release formulation utilizing 5 PLA polymers. Additional exampies of products which utilize

PLA and PLGA as excipients to achieve sustained-release of the active ingrédient include Atridox (PLA; periodontal disease), Nutropin Depot (PLGA; with hGH), and the Trelstar Depot (PLGA; prostate cancer). Other synthetic polymers 10 included but are not limited to poly(c-caprolactone) , poly3hydroxybutyrate, poly(p-malic acid) and poly(dioxanone)J ; polyanhydrides, polyuréthane (see WO 2005/013936), polyamides, cyclodestrans, polyorthoesters, n-vinyl alcohol, polyethylene oxide/polyethylene terephthalate, polyphosphazene, polyphosphate, polyphosphonate, polyorthoester, polycyanoacrylate, polyethylenegylcol, polydihydropyran, and polyacytal. Non-biodegradable devices include but are not limited to various cellulose dérivatives (carboxymethyl cellulose, cellulose acetate, cellulose 20 acetate propionate, ethyl cellulose, hydroxypropyl methyl cellulose) silicon-based implants (polydimethylsiloxane), acrylic polymers, (polymethacrylate, polymethylmethacrylate, polyhydroxy(ethylmethylacrylate), as well as polyethyleneco-(vinyl acetate), poloxamer, polyvinylpyrroiidone, 25 poloxamine, polypropylene, polyamide, polyacetal, polyester, poly ethylene-chlorotrifluoroethylene, polytetrafluoroethylene (PTFE or Teflon™), styrene butadiene rubber, polyethylene, polypropylene, polyphenylene oxide-polystyrène, poly-a-chloro-p-xylene, polymethylpentene, polysulfone and other related biostable polymers. Carriers suitable for sustained-release depot formulations include, but are not Limited to, micospheres,

-5419316 films, capsules, particles, gels, coatings, matrices, wafers, pills or other pharmaceutical delivery compositions. Examples of such sustained-release formulations are described above. See also U.S. Patent Nos. 6,953,593; 6,946,146; 6,656,508; 6,541,033; and 6,451,346, the contents of each which are incorporated herein by reference. The dosage form must be capable of carrying the drug formulation in such quantities and concentration as therapeutically required for treatment over the pre-selected period, and must provide sufficient protection to the formulation from dégradation by body processes for the duration of treatment. For example, the dosage form can be surrounded by an exterior made of a material that has properties to protect against dégradation from metabolic processes and the risk of, e.g., leakage, cracking, breakage, or distortion. This can prevent expelling of the dosage form contents in an uncontrolled manner under stresses it would be subjected to during use, e.g., due to physical forces exerted upon the drug release device as a resuit of normal joint articulation and other movements by the subject or for example, in convective drug delivery devices, physical forces associated with pressure generated within the réservoir. The drug réservoir or other means for holding or containing the drug must also be of such material as to avoid unintended reactions with the active agent formulation, and is preferably biocompatible (e.g., where the dosage form is implanted, it is substantially non-reactive with respect, to a subject’s body or body fluids). Generally, the respective biological agent(s) is administered to an individual for at least 12 hours to at least a week, and most likely via an implant designed to deliver a drug for at least 10, 20, 30, 100 days or at least 4 months, or at least 6 months or more,

-5519316

ΙΟ as required. The 13C3-like antibody can be delivered at such relatively low volume rates, e.g., from about 0.001 ml/day to 1 ml/day so as to minimize tissue disturbance or trauma near the site where the formulation is released. The formulation may be released at a rate of, depending on the spécifie biological agent (s), at a low dose, e.g., from about 0.01 pg/hr or 0.1 pg/hr, 0.25 pg/hr, 1 pg/hr, generally up to about 200 pg/hr, or the formulation is delivered at a low volume rate e.g., a volume rate of from about 0.001 ml/day to about 1 ml/day, for example, 0.01 micrograms per day up to about 20 milligrams per day. Dosage dépends on a number of factors such as potency, bioavailability, and toxicity of the active ingrédient (e.g., IgG antibody) used and the requirements of the subject.

These and other objects, advantages and features of the présent invention will become apparent to those persons skilled in the art upon reading the details of the methodology and compositions as more fully set forth below.

EXAMPLES EXAMPLE 1: PREPARING THE PROTOFIBRILLAR FORM OF AMYLOID BETA (Αβ42)

Αβ42 synthetic peptides (American Peptide Company, Inc., CA) were prepared according to the method described by Fezoui et al. (Fezoui, et al. Amyloid 7(3): 166-178.

(2000)) Briefly, lyophilized Αβ42 was dissolved in 2mM NaOH at a Img/ml concentration (pH~10.5) followed by sonication and lyophilization. NaOH-treated Αβ was dissolved in water at a concentration of Img/ml and fîltered with a 0.22pm ULTRAFREE-MC filter (Millipore, MA). A 0.5mg/ml peptide solution was buffered at the final concentration of 50mM phosphate; lOOmM sodium chloride and incubated for 4 hr. at

-5619316 room température. To separate the protofibrillar form from the low-molecular weight proteins, the supematant was fractionated using size-exclusion chromatography. Purifîed SEC fractions were then stored at 4C°.

Various forms of the Αβ42 protein are represented as showing its ability as a monomer or dimer to associate together to form a high-moiecuiar weight oiigomer (protofibril) (Figure 1). Further aggregation of the soluble protofibrils créâtes an insoluble form of the 10 protein, whereas the protofibrils can disassociate back to a lower-molecular weight form.

To purify the protofibrillar form of Αβ from the lowmolecular weight proteins, sampies were fractionated with an AKTA chromatography system using a Superdex 75 size15 exclusion column. Figure 2A shows that without incubating the ΑΠ 42 synthetic peptides at room température, there is no aggregation of oligomers to form the protofibrils. Figure 2B illustrâtes that after a 4 hr. incubation of the Αβ42 synthetic peptides, subséquent SEC purification shows a 20 definitive protofibril fraction.

EXAMPLE 2: GENERATING MONOCLONAL ANTIBODIES WITH SPECIFICITY FOR PROTOFIBRILLAR Αβ

The 1303, 19A6, and 1D1 antibodies were created by immunizing Balb/c mice with the fibrilliar Αβ protein using 25 a protocol known in the art. -(Harlow, et al. Cold Spring Harbor Laboratory.(1988)) Spleens were removed and fused with SP2 myeloma cells in several 96 well plates. Fusion cultures were monitored for growth and supernatants were screened for their ability to bind the protofibrilliar 30 fraction by antibody-capture immunoassays.

-5719316

EXAMPLE 3: CHARACTERIZATION OF MONOCLONAL ANTIBODIES WITH SPECIFICITY FOR PROTOFIBRILLAR Αβ

Antibody capture assays were used to further characterize the monoclonal antibodies produced from the 5 hybridomas (13C3, 19A6, and 1D1). To microtiter plates,

50ul of a 2ug/ml protofibrillar Αβ42 protein solution was added to each well and the plates were incubated at 4°C overnight. After incubation, the residual antigen solution was removed and washed with PBS solution. Serial dilutions 10 of the hybridoma supernatants were added to the plates containing the bound antigen and incubated for 1 hour at room température. This primary antibody solution was removed and the wells were again washed with PBS solution. An enzyme-labeled secondary antibody was next added and 15 incubated for 1 hour at room température. After removai of the secondary antibody solution, a chromogenic substrate spécifie for the conjugated enzyme, was added to the reaction and the détection of the captured antibody yielded quantitative results.

Additionally, changing the secondary reagent to isotype-specific anti-immunoglobulin antibodies, the particular immunoglobulin isotype of each monoclonal was identified. In these experiments, commercially available anti-Ap42 antibodies were used to compare the binding specificity of 13C3, 19A6, and 1D1 monoclonal antibodies.

Figures 3A and B illustrate the protofibrillar (PF) and the low-molecular weight (LMW) forms of the Αβ42 peptide used to test the specificity of the 13C3 antibody in antibody capture immunoassays. Specifically, Figure 3A 30 illustrâtes the plot generated from the ELISAs showing that the 13C3 antibody is spécifie for the protofibrillar form(PF) of Αβ42 and does not recognize the low-molecular

-5819316 weight(LMW) forms of the protein. Figure 3B illustrâtes the ELISA data with the commercially available 4G8 antibody, showing that it recognizes both the low-molecular weight and the protofibrillar forms of the Αβ42 protein.

EXAMPLE 4: SPECIFICITY OF MONOCLONAL ANTIBODIES TO THE PROTOFIBRILLAR FORM OF Αβ42 USING SURFACE PLASMON RESONANCE (blACORE).

The purified monocionai antibodies iisted in Table 1 (below), were immobilized to a BIAcore sensor chip in accordance with publish protocols. (Nice, et al. BioEssays 21: 339-352 (1999)). The high sensitivity of the BIAcore optical response quantifies a change in reflectivity and a baseline response for the ligand alone is generated. The interaction analysis is performed as the analytes, the LMW form or the PF form of Αβ42, are injected in solution over the sensor chip and the change in surface plasmon résonance generates a response identifying the specificity of each antibody's ability to bind LMW and PF Αβ42. Both the 13C3 and the 19A6 antibodies ali bound to the PF form of Αβ 42 with higher specificity than the LMW form. Of ail the antibodies used in this experiment, the commerciallyavailable antibodies showed higher specificity for the LMW Αβ42 over the PF form of Αβ 42, as indicated the ratio of PF binding/ LMW binding.

Table 1

Name	Epitope	Isotype	Source	BIACORE Binding Analysis
LMW	PF	Ratio (PF/LMW)
1D1	strtjôlüfe i	igGt	Ravetdi	23.2	122.3	6.3
13C3	structure \	IgGt	Rayetoh	£5	12(4,5	5.0
19À6		ig<33	Ravetch
3D6	Αβ 1-5	lgG2b	Elan Pharmaceuticals	424.6	402.7	0.9
4G8	Αβ 17-22	lgG2b	Senetek Inc.	228.6	340.3	1.5
6E10	Αβ 3-8	lgG2b	Senetek Inc.	400.1	541.1	1.4
82E1	Αβ 1-17	lgG1	IBL	69.9	68.4	1.0

The Surface Plasmon Résonance Analysis shown by sensorgram that 13C3 (Figure 4B) does not bind the LMW forms

-5919316 of Αβ 42 protein. However, the 4G8 (Figure 4A) shows a standard association/disassociation curve for the LMW Αβ42 protein. The antibody isotype control IgGl (Figure C) does not bind the LMW Αβ as well. Automated BIAcore Systems, which use the détection principle of Surface Plasmon Résonance, were used in these experiments. The binding specificity data for the 19A6 antibody showed that 19A6 had a binding ratio of 5.8, which is similar to that of 13C3 at a ratio of 5.3.

EXAMPLE 5: EPITOPE MAPPING OF THE 1303 ANTIBODY

Mapping the epitopes of 13C3, 1D1 and 19A6 was conducted using the RepliTope Microarrays system (JPT Peptide Technologies GmbH) according to published protocol. (Korth, et al. 390: 74 (1997)). Each spot on the microarray contains a 13 amino acid peptide of Αβ42 where each shift in position on the microarray represents an amino acid shift (fom N-term to C-term) , i.e. SEQ ID NO: 23, SEQ ID NO: 24...SEQ ID NO: 51; and SEQ ID NO: 52. Listed below are the peptides and their exact amino acid sequence, corresponding to their position .on the slide array. Once the peptides are fixed to the RepliTope Microarray, the samples are incubated with the 13C3 antibody and then subsequently labeled with a secondary that is conjugated to a chemiluminescence tag of choice. The spots that yield a signal represent the epitope binding sites on the protein by the antibody.

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His (SEQ ID NO: 23)

Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His (SEQ ID NO: 24)

Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin (SEQ ID NO: 25)

Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys (SEQ ID NO: 26)

Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu (SEQ ID NO: 27)

-6019316

His 28) Asp 29)

Asp Ser

Ser Gly

Gly Tyr

Tyr Glu

Glu Val

Val His

His His

His Gin

Gin Lys

Lys Leu

Leu Val

Val Phe

(SEQ (SEQ

ID ID

NO NO

5

Ser 30)

Gly

Tyr

Glu

Val

His

His

Gin

Lys

Leu

Val

Phe

Phe

(SEQ

ID

NO

Gly 31)

Tyr

Glu

Val

His

His

Gin

Lys

Leu

Val

Phe

Phe

Ala

(SEQ

ID

NO

10

Tyr 32)

Glu

Val

His

His

Gin

Lys

Leu

Val

Phe

Phe

Ala

Glu

(SEQ

ID

NO

Glu 33)

Val

His

His

Gin

Lys

Leu

Val

Phe

Phe

Ala

Glu

Asp

(SEQ

ID

NO

Val 34)

His

His

Gin

Lys

Leu

Val

Phe

Phe

Ala

Glu

Asp

Val

(SEQ

ID

NO

15

His 35)

His

Gin

Lys

Leu

Val

Phe

Phe

Ala

Glu

Asp

Val

Gly

(SEQ

ID

NO

His 36)

Gin

Lys

Leu

Val

Phe

Phe

Ala

Glu

Asp

Val

Gly

Ser

(SEQ

ID

NO

20

Gin 37)

Lys

Leu

Val

Phe

Phe

Ala

Glu

Asp

Val

Gly

Ser

Asn

(SEQ

ID

NO

Lys 38)

Leu

Val

Phe

Phe

Al a

Glu

Asp

Val

Gly

Ser

Asn

Lys

(SEQ

ID

NO

Leu 39)

Val

Phe

Phe

Ala

Glu

Asp

Val

Gly

Ser

Asn

Lys

Gly

(SEQ

ID

NO

25

Val 40)

Phe

Phe

Ala

Glu

Asp

Val

Gly

Ser

Asn

Lys

Gly

Ala

(SEQ

ID

NO

Phe 41)

Phe

Ala

Glu

Asp

Val

Gly

Ser

Asn

Lys

Gly

Ala

Ile

(SEQ

ID

NO

30

Phe 42)

Ala

Glu

Asp

Val

Gly

Ser

Asn

Lys

Gly

Ala

Ile

Ile

(SEQ

ID

NO

Ala 43)

Glu

Asp

Val

Gly

Ser

Asn

Lys

Gly

Ala

Ile

Ile

Gly

(SEQ

ID

NO

Glu 44)

Asp

Val

Gly

Ser

Asn

Lys

Gly

Ala

Ile

Ile

Gly

Leu

(SEQ

ID

NO

35

Asp 45)

Val

Gly

Ser

Asn

Lys

Gly

Ala

Ile

Ile

Gly

Leu

Met

(SEQ

ID

NO

Val 46)

Gly

Ser

Asn

Lys

Gly

Ala

Ile

Ile

Gly

Leu

Met

Val

(SEQ

ID

NO

40

Gly 47)

Ser

Asn

Lys

Gly

Ala

Ile

Ile

Gly

Leu

Met

Val

Gly

(SEQ

ID

NO

Ser 48)

Asn

Lys

Gly

Ala

Ile

île

Gly

Leu

Met

Val

Gly

Gly

(SEQ

ID

NO

Asn 49)

Lys

Gly

Ala

Ile

Ile

Gly

Leu

Met

Val

Gly

Gly

Val

(SEQ

ID

NO

45

Lys 50)

Gly

Ala

Ile

Ile

Gly

Leu

Met

Val

Gly

Gly

Val

Val

(SEQ

ID

NO

Gly 51)

Ala

Ile

Ile

Gly

Leu

Met

Val

Gly

Gly

Val

Val

Ile

(SEQ

ID

NO

50

Ala 52)

Ile

Ile

Gly

Leu

Met

Val

Gly

Gly

Val

Val

Ile

Ala

(SEQ

ID

NO

-6119316

Figure 5A illustrâtes a dot blot from a RepliTope Microarray experiment identifying the epitopes of the antibodies, 13C3, 1D1 and 4G8 on the Αβ 1-42 peptide. The bound antibody is represented by a chemiluminescent signal. Figures 3B illustrâtes the Αβ 1-42 amino acid sequence showing the polypeptide seqments of the 13C3 epitopes as they occur in the sequence. The 1D1 antibody shows the same epitopes as the 13C3 whereas the commercial 4G8 antibody identifies a different epitope. EXAMPLE 6: CHARACTERIZATION OF 13C3 SPECIFICITY

Figure 6 illustrâtes fractions from size-exclusion chromatography of the supernatants from the 7PA2 cell line, a secreting Αβ oligomer cell line. Antibody capture assays were used to further characterize the binding of the 13C3 antibody with the protofibrillar and low-molecular weight fractions from the SEC-purified 7PA2. To microtiter plates, lOOul of a 1:200 dilution of each fraction was added to each weli and the plates were incubated at 4°C overnight. After incubation, the residual antigen solution was removed and washed with PBS solution. Serial dilutions of the 13C3 supernatants were added to the plates containing the bound antigen and incubated for 1 hour at room température. This primary antibody solution was removed and the wells were again washed with PBS solution. An enzyme-labeled secondary antibody was next added and incubated for 1 hour at room température. After removal of the secondary antibody solution, a chromogenic substrate spécifie for the conjugated enzyme, was added to the reaction and the détection of the captured antibody yielded quantitative results. This assay identified that the 13C3 antibody specifically recognizes only the protofibrillar fraction whereas the 4G8 antibody recognizes ail fractions. The 7PA2

-6219316 cell line was provided by Dennis J. Selkoe, M.D. at Harvard Medical School. . .

EXAMPLE 7 CHARACTERIZATION OF 13C3 REACTIVITY BY EM.

The method of staining was performed using a standard protocol. (Brenner, et al. Biochim. Biophys. Ada 34, 103110 (1959)). A small volume (10 microliters) of a 0.2mg/ml protofibrillar solution was applied to carbon-coated formvar arids (400 mesh) for 2 min. Then the grids were blocked in 1%BSA and incubated with the 1303 antibody followed by a subséquent incubation with a secondary antibody conjugated to colloïdal gold. The samples were negatively stained by placing on 2 successive drops of 2% phosphotungstic acid for 30 sec each. Excess stain was drawn off with filter paper, the grids were air dried, and observed on a JEOL 100CX transmission électron microscope at 80kV. Images were recorded on large format Kodak 4489 négatives and digitized on a fiat bed scanner.

IEM (Immuno-Electron Microscopy) images showing the binding specificity of the anti-Αβ antibody clone 1303 to Αβ42 fibers (Figures 7B and 70), whereas the isotype control antibody, IgGl shows no binding (Figure 7A) . The secondary antibody is conjugated to a colloïdal gold particle. EXAMPLE 8: 1303 TREATMENT OF~A MOUSE MODEL 0F HUMAN AD

The 1303 monoclonal antibody was used to treat Αβ plaques in an Alzheimer's Disease mouse model, TgCRNDS. The mouse contains the human APP695 cDNA transgene, which accelerates the déposition of Αβ amyloid plaques in the mouse brain, appearing within 1 month of âge. A sample group of 5 TgCRND8 mice five weeks in âge were give immunizations of the 1303 monoclonal antibody at a concentration of 10mg/ kg of mouse once a week for the duration of seven weeks. A second group of 5 TgCRND8 mice,

-6319316 were given the treatment course, however an isotype control IcfGl antibody was administered. Experiments were repeated with treatments at twice a week instead of once a week.

Both control and experimental animais were sacrificed at 12 weeks of âge. Histological préparations of the brains revealed réductions in Αβ plaques in 13C3 treated mice.

Serial sections of cryopreserved brains from TgCRND8 mice were treated with 13C3 or IgGl monoclonal antibodies. Figures 8A and 8B illustrate différences in the number of Αβ amyloid plaques between each respective antibody.

Statistical T-tests show that the 1303 antibody treatment at once a week reduces Αβ amyloid plaques in the Alzheimer Disease model (Figure 9A) . However, twice a week treatments (Figure 9B) show the same level of plaque réduction.

Ail of the above TgCRND8 mice were obtained from Dr. David Westaway of the University of Toronto.

EXAMPLE 9: MOLECULAR CHARACTERIZATION OF THE VARIABLE REGIONS OF MAB 1303

The IgG heavy chain variable région and the IgG Kappa light chain région were cloned from the 1303 hybridoma. Both heavy and light chain sequences (Figure 10) were analyzed using VBASE2 (http://www.vbase2.org), a database of germ-line variable genes from the immunoglobulin loci of human and mouse extracted from the , EMBL-Bank and Ensembl data libraries. (Retter et al. Nucleic Acids Res. 33:D671-4 (2005)). Results for the analysis identified that both the heavy and light chain variable régions were from a newly identified immunoglobulin but had 73% and 81% identity, respectively, to other immunoglobulin variable régions in the database. Also identified in these sequences against these databases were the Frame Work Régions (FWR) and the Compiementarity Determining Régions (CDR). Results were

-6419316 only slightly varied when sequences were analyzed against VBASE, KABAT, and IMGT/LIGM database

EXAMPLE 10: ACUTE PERIPHERAL ADMINISTRATION OF 1303 IN APP TRANSGENIC MICE DOES NOT LEAD TO AN INCREASE IN PLASMA Αβ UNLIKE REFERENCE ANTIBODY 3D6 ADMINISTRATION

APP transgenic mice (Thy APPSL, âge 10-14 weeks) were injected intraperitoneally at the dose of 10 mg/kg (i.e., 300 pg/mouse) with antibodies 13C3, a control IgGl (DM4, not recognizing Αβ) and a reference anti-Αβ antibody 3D6 recognizing ali conformers of Αβ. Plasma Αβ was quantified at time zéro pre-injection, 6h, 24 h and 7days post injection in the same mice. Quantification of plasma Αβ was performed with an immunoassay using anti-Αβ antibody pairs not interfering with 13C3 or 3D6 binding to Αβ.

Administration of 3D6, an antibody against ail conformers of Αβ, ieads to a large increase in plasma Αβ, likely by protecting Αβ molécules from dégradation. This effect was used to suggest the potential peripheral sink hypothesis as mechanism of action of anti-Αβ immunotherapy (Demattos et al., 2001, PNAS 17:8850) . Unlike 3D6, 13C3 administration does not lead to any increase in plasma Αβ levels. This is consistent with the properties of 13C3, an antibody that is spécifie for the protofibrillar forms of Αβ and is not recognizing the soluble mono- or oligomeric forms of Αβ peptide. These forms are the likely ones présent in plasma.

EXAMPLE 11: 13C3 RECOGNIZES HUMAN AMYLOID NEURITIC PLAQUES (AGGREGATED) IN AD BRAINS BUT NOT THE DIFFUSE Αβ DEPOSITS UNLIKE THE REFERENCE 3D6 ANTI-Αβ ANTIBODY

Immunohistochemistry studies were performed with 13C3 and 3D6 antibodies on human Alzheimer-diagnosed brain sections using standard techniques. Antibody immunostaining was detected with a DAB chromogen (Fig. 12). 13C3 labels amyloid deposits with a typical morphology of mature amyloid -65 neuritic plaques (also called dense plaques) with a very dense core surrounded by a lighter halo or for the larger plaques a very strong staining. In adjacent brain sections, 3D6 stains many more objects than 13C3 as seen at lower 5 magnification (Fig. 12, left panels). Further characterization at higher magnification (Fig. 12, right panels) indicated that 3D6 labels the same mature amyloid neuritic plaques as 13C3 and, in addition, numerous diffuse amyloid deposits that hâve been classically described using 10 anti-Αβ immunolabelling. The diffuse plaques are not of fibrillar nature as described in the literature as they cannot be detected by thioflavin S and other histological markers of fibrils (Mann, 1989, Ann. Med. 21:133) . To rule out différences in sensitivity of the two antibodies, 15 similar experiments were conducted with a higher concentration (20 pg/ml) of 13C3 and again diffuse deposits could not be detected. This data is consistent with the properties of 13C3, an antibody that is spécifie for the protofibrillar forms of Αβ and is not recognizing the 20 soluble mono- or oligomeric forms of Αβ peptide unlike 3D6.

INDUSTRIAL APPLICABILITY

The invention has applications in uhe treatment and diagnosis of Alzheimer's disease.

Ail publications cited in the spécification, both 25 patent publications and non-patent publications, are indicative of the level of skill of those skilled in the art to which this invention pertains. Ail these publications are herein fully incorporated by reference to the same extent as if each individual publication were specifically 30 and individually indicated as being incorporated by reference.

-6619316

Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the présent invention. It is 5 therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the présent invention as defined by the following claims.

Claims (29)

1. An isolated antibody that specifically interacts and shows a measurable affinity to a conformational epitope of a protofibril form of Αβ peptide, whereby the protofibril epitope is represented by an exposed région of a Αβ-protofibril form 5 comprising the amino acid sequence as set forth in SEQ ID NO:2, wherein said antibody shows minimal or no affinity for monomer or dimer forms of Αβ peptide.

2. The antibody of claim 1 which is a monoclonai antibody.

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

10

4. An isolated antibody that specifically interacts and shows a measurable affinity to a conformational epitope of a protofibril form of Αβ peptide, whereby the protofibril epitope is represented by an exposed région of a Αβ-protofibril form comprising an amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:4, wherein said antibody shows minimal or no affinity for monomer or 15 dimer forms of Αβ peptide.

5. The antibody of claim 4 which is a monoclonal antibody.

6. The monoclonal antibody of claim 5 which is designated 13C3.

7. The monoclonal antibody of claim 5 which is a humanized monoclonal antibody or a human monoclonal antibody.

20

8. The antibody of claim 4 further comprising a variable light chain comprised of the amino acid sequence as set forth in SEQ ID NO:5, or a variable heavy chain comprised of the amino acid sequence as set forth in SEQ ID NO:7.

9. The antibody of claim 4 further comprising a variable light chain comprising a CDR1 région as set forth in SEQ ID NO: 13, a CDR2 région as set forth in

25 SEQ ID NO: 14, and a CDR3 as set forth in SEQ ID NO: 15, or a variable heavy chain comprised of a CDR1 région as set forth in SEQ ID NO:20, a CDR2 région as set forth in SEQ ID NO:21, and a CDR3 as set forth in SEQ ID NO:22.

10. A method of producing a monoclonal antibody which specifically binds in vitro to a repeating conformational epitope of a protofibril form of β-amyloid peptide while showing greater affinity to a protofibril form of β-amyloid peptide than to a low molecular weight form of β-amyloid peptide, comprising:

(a) immunizing a mammal with the protofibril form of β-amyloid peptide;

(b) harvesting B-cells of said mammal;

(c) creating hybridomas from the harvested B-cells, wherein said hybridomas produce antibodies; and, (d) selecting hybridomas which produce antibodies specifically binding to the protofibril form of β-amyloid peptide while showing minimal affinity to monomer or dimer forms of β-amyloid peptide.

11. A method for quantifying the amount of a protofibril form of β-amyloid peptide in a tissue or fluid sample, comprising:

(a) obtaining the tissue or fluid sample from a subject;

(b) contacting the tissue or fluid sample with an antibody or fragment thereof that specifically binds to the protofibril form of β-amyloid peptide while showing minimal affinity to low molecular weight forms of β-amyloid peptide; and, (c) quantifying the amount of protofibril form of β-amyloid peptide in the sample.

12. The method of claim 11 wherein the antibody is monoclonal antibody selected from the group consisting of 13C3, 1D1 and 19A6.

13. A kit for detecting protofibril form of β-amyloid peptide while showing greater affinity to a protofibril form of β-amyloid peptide than to a low molecular weight form of β-amyloid peptide, comprising:

(a) an antibody or a fragment thereof, capable of specifically binding in vitro to a repeating conformational epitope of a protofibril form of β-amyloid peptide while showing minimal affinity to low molecular weight forms of β-amyloid peptide; and,

(b) a reagent that binds, directly, or indirectly, to said antibody or the fragment thereof.

14. The kit of claim 13 wherein the antibody is monoclonal antibody selected from the group consisting of 13C3, 1D1 and 19A6.

15. A pharmaceutical composition comprising a variable région fragment which specifically interacts with the protofibrillar form of b-amyloid, wherein said spécifie interaction is characterized by a ratio of the affinity of said variable région fragment for the protofibrillar Ab form to the affinity for other Ab forms greater than about 2.

16. The method of claim 15 wherein the antibody is a monoclonal antibody.

17. The method of claim 16 wherein the monoclonal antibody is an antibody selected from the group consisting of 13C3, 19A6 and 1D1.

18. The method of claim 16 wherein the monoclonal antibody is a humanized monoclonal antibody or a human monoclonal antibody.

19. A hybridoma which sécrétés an antibody selected from the group consisting of 13C3, 19A6 and 1D1.

20. An isolated nucleic acid molécule encoding a variable heavy chain fragment of monoclonal antibody 13C3 wherein the variable heavy chain fragment comprises the amino acid sequence as set forth in SEQ ID NO:7.

21. An isolated nucleic acid molécule encoding a variable heavy chain fragment of monoclonal antibody 13C3 wherein the nucleic acid molécule comprises the nucieotide sequence as set for in SEQ ID NO:8.

22. An expression vector for the expression of a variable heavy chain fragment of monoclonal antibody 13C3 in a recombinant host cell wherein said expression vector contains the nucleic acid molécule of claim 21.

23. A host cell which expresses a variable heavy chain fragment of monoclonal antibody 13C3 wherein said host cell contains the expression vector of claim 22.

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24. An isolated nucleic acid molécule encoding a variable light chain fragment of monoclonal antibody 13C3 wherein the variable light chain fragment comprises the amino acid sequence as set forth in SEQ ID NO:5.

25. An isolated nucleic acid molécule encoding a variable light chain fragment 5 of monoclonal antibody 13C3 wherein the nucleic acid molécule comprises the nucléotide sequence as set for in SEQ ID NO:6.

26. An expression vector for the expression of a variable light chain fragment of monoclonal antibody 13C3 in a recombinant host cell wherein said expression vector contains the nucleic acid molécule of claim 25.

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27. A host cell which expresses a variable light chain fragment of monocional antibody 13C3 wherein said host cell contains the expression vector of claim 26.

28. An isolated variable heavy chain fragment of monoclonal antibody 13C3 which comprises the amino acid sequence set forth in SEQ ID NO:7.

29. An isolated variable light chain fragment of monoclonal antibody 13C3 15 which comprises the amino acid sequence set forth in SEQ ID NO:5.