CN1849139A - Methods of treating alzheimer's disease using antibodies directed against amyloid beta peptide and compositions thereof - Google Patents

Abstract

Monoclonal antibodies directed against amyloid beta peptide and methods of using same for treatment and prevention of Alzheimer's disease and Down's syndrome are described.

Description

Methods of treating Alzheimer's disease using antibodies against amyloid beta peptide and compositions thereof

Cross-references to related applications

This application claims U.S. Provisional Application Serial No. 60/417,232, filed October 9, 2002, U.S. Provisional Application Serial No. 60/447,611, filed February 13, 2003, U.S. Provisional Application Serial No. 60/447,611, filed April 22, 2003 60/464,754 and U.S. Provisional Application Serial No. 60/480,353, filed June 20, 2003, which are incorporated by reference in their entirety.

technical field

The present invention generally relates to the detection and treatment of diseases associated with the expression of amyloid beta peptide (Aβ), such as Alzheimer's disease and Down's syndrome. The present invention relates more particularly to antibodies against A[beta] and its precursor, [beta]APP. Accordingly, the present invention provides immunotherapeutic compositions and methods for the detection and treatment of diseases associated with the overexpression (or accumulation) of Aβ and βAPP.

Background of the invention

Alzheimer's disease (AD) is a degenerative brain disorder characterized clinically by progressive memory loss, confusion, progressive physical deterioration, and finally death. Approximately 15 million people worldwide are living with Alzheimer's disease, and this number is expected to rise dramatically as life expectancy increases. Histologically, the disease is characterized by neuritic plaques first found in the association cortex, limbic system, and basal ganglia. The main component of these neuritic plaques is amyloid β peptide (Aβ), the cleavage product of β-amyloid precursor protein (βAPP or APP). APP is a type I transmembrane glycoprotein containing a large ectopic N-terminal domain, a transmembrane domain and a small cytoplasmic C-terminal tail. Alternative splicing of a single APP gene transcript on chromosome 21 produces several isoforms with different numbers of amino acids.

Aβ appears to play an important role in the neuropathology of Alzheimer's disease. Familial forms of the disease are associated with mutations in the APP and presenilin genes (Tanzi et al., 1996, Neurobiol. Dis. 3: 159-168; Hardy, 1996, Ann. Med. 28: 255-258). Disease-associated mutations in these genes lead to increased production of the 42 amino acid form of Aβ, which is the predominant form in amyloid plaques. Furthermore, immunization of transgenic mice overexpressing disease-associated mutant APP with human Aβ reduced plaque burden and associated pathology (Schenk et al., 1999, Nature 400:173-177), and peripheral administration of antibodies against Aβ It can also reduce the burden of plaques in the brain (Bard et al., 2000, Nature Medicine 6(8):916-919).

Therefore, antibody therapy offers a promising way to treat and prevent Alzheimer's disease. There remains a need to improve the efficacy of antibodies and other immunotherapeutics directed against A[beta] and suitable for use in human patients.

Throughout this application, various publications, including patents and patent applications, are referenced. The disclosures of these publications are therefore incorporated by reference in their entirety.

Summary of the invention

The present invention provides isolated monoclonal antibodies that bind to Aβ peptide (SEQ ID NO: 1) (Table 6). More specifically, antibodies that bind to amino acids 1-16, 16-28, or 28-40 of the A[beta] peptide are provided. Preferably, the antibody competitively inhibits the binding of a monoclonal antibody having the amino acid sequence shown in SEQ ID NO: 4, 6, 8 or 10 (Tables 9 and 11), or competitively inhibits the binding of a monoclonal antibody designated as 8A1.2A1, 3C6. Binding of monoclonal antibodies produced by hybridomas of 1F9 or 10B10.2E6. In some embodiments, the binding affinity of the monoclonal antibody to the Aβ peptide is about 200 nM or less, about 100 nM or less, about 60 nM or less, preferably about 30 nM or less, more preferably is about 3 nM or less, about 2 nM or less, and about 1 nM or less. In some embodiments, the Fab fragment of the monoclonal antibody binds to the Aβ peptide with an affinity of about 200 nM or less, about 100 nM or less, about 60 nM or less, about 30 nM or less, about 3 nM or Less, about 2 nM or less, and about 1 nM or less. In a preferred embodiment, the antibody binds to the same Aβ epitope as the monoclonal antibody having the amino acid sequence shown in SEQ ID NO: 4, 6, 8 or 10 or named 8A1.2A1, 3C6 .1F9 or 10B10.2E6 hybridoma production. The invention also provides a monoclonal antibody produced by a hybridoma designated 8A1.2A1, 3C6.1F9 or 10B10.2E6. The monoclonal antibodies described herein can optionally be conjugated to a therapeutic agent and/or labeled with a detectable marker.

In another aspect, the present invention provides an isolated antibody that preferentially binds to amino acids 28-40 of Aβ peptide (SEQ ID NO: 1) (Table 6). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody preferentially binds to an epitope comprising amino acids 39 and/or 40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the antibody binds to an epitope comprising amino acids 39 and/or 40 of the Aβ peptide (SEQ ID NO: 1), but does not exhibit significant crossover to the Aβ _1-42 and/or Aβ _1-43 peptides reaction. In some embodiments, the antibody binds to an epitope comprising amino acids 36-40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the antibody binds to an epitope comprising amino acids 36-40 of the Aβ peptide (SEQ ID NO: 1), but does not exhibit significant cross-reactivity with the Aβ _1-42 and/or Aβ _1-43 peptides. In some embodiments, the antibody binds Aβ peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less, about 100 nM or less, about 60 nM or less, about 30 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less. In some embodiments, the Fab fragment of the antibody binds to Aβ peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less, about 100 nM or less, about 60 nM or less, about 30 nM or less , about 3 nM or less, about 2 nM or less, about 1 nM or less. In some embodiments, the antibody Fab fragment that preferentially binds to an epitope comprising amino acid 39 and/or 40 of Aβ peptide (SEQ ID NO: 1) binds to Aβ peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less, about 100 nM or less, about 60 nM or less, about 30 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less. In some embodiments, the antibody Fab fragment that preferentially binds to an epitope comprising amino acids 36-40 of Aβ peptide (SEQ ID NO: 1) binds to Aβ peptide (SEQ ID NO: 1) with an affinity of about 200 nM or greater Small, about 100 nM or less, about 60 nM or less, about 30 nM or less, about 3 nM or less, about 2 nM or less, about 1 nM or less. In some embodiments, the antibody competitively inhibits the monoclonal antibody comprising the amino acid sequence shown in SEQ ID NO: 4 and/or 6, or the monoclonal antibody produced by the hybridoma designated 8A1.2Al, from the Aβ _1-40 peptide ( SEQ ID NO: 1) Binding. In some embodiments, the antibody binds to an epitope bound by a monoclonal antibody comprising the amino acid sequence set forth in SEQ ID NO: 4 and/or 6, or a monoclonal antibody produced by a hybridoma designated 8A1.2Al. In some embodiments, the antibodies are human antibodies. In some embodiments, the antibody comprises the amino acid sequence set forth in SEQ ID NO: 4 and 6, or is produced by a hybridoma designated 8A1.2A1.

In another aspect, the present invention is a humanized antibody derived from a monoclonal antibody comprising the amino acid sequence shown in SEQ ID NO: 4 and/or 6, or a monoclonal antibody produced by a hybridoma named 8A1.2A1. In some embodiments, the humanized antibody has one or more monoclonal antibodies comprising the amino acid sequence shown in SEQ ID NO: 4 and/or 6, or a monoclonal antibody produced by a hybridoma designated 8A1.2A1 CDR. In another aspect, the present invention provides a humanized antibody that binds to the same epitope as a monoclonal antibody comprising the amino acid sequence shown in SEQ ID NO: 4 and/or 6, or a monoclonal antibody produced by a hybridoma named 8A1.2A1 . Generally, the humanized antibody of the present invention comprises one or more (one, two, three, four, five, six) CDRs which are related to those shown in SEQ ID NO: 4 and/or 6. The amino acid sequences of the monoclonal antibody, or the CDRs of the monoclonal antibody produced by the hybridoma named 8A1.2A1 are identical to and/or derived from them.

In another aspect, the invention is a chimeric antibody comprising variable regions derived from the variable regions of the heavy and light chains of monoclonal antibodies and constant regions derived from the constant regions of the heavy and light chains of human antibodies comprising The amino acid sequence shown in SEQ ID NO: 4 and/or 6, or produced by a hybridoma named 8A1.2A1.

In some embodiments, the antibodies disclosed herein have an affinity of about 100 pM or less, about 50 pM or less, about 25 pM or less, about 15 pM or less, about 10 pM or less, about 5 pM or less, Or about 2pM or less.

In addition, the present invention provides compounds that bind to βAPP (SEQ ID NO: 2) (Table 7) and competitively inhibit compounds designated 25E12.1F9.1H8 (BP26), 24H4.2E10.1F5 (BP27), 1F10.8E6.2A2 ( BP80), 13E12.1C5 (BP81), or 14D9.1G8 (BP82) hybridoma-produced monoclonal antibodies that bind to isolated monoclonal antibodies.

Compositions comprising one or more antibodies of the invention are also provided. In some embodiments, the composition comprises at least two antibodies, a first antibody directed against amino acids 16-28 of the Aβ peptide and a second antibody directed against amino acids 28-40 of the Aβ peptide. Optionally, the composition also includes a physiologically acceptable carrier.

The invention also provides isolated polynucleotides encoding the monoclonal antibodies described herein, as well as vectors comprising such polynucleotides and host cells comprising such vectors. Such an expression system can be used in a method of producing an immunoreactive polypeptide, such as an antibody of the invention, comprising culturing a host cell, and recovering the polypeptide produced from the cultured host cell. In some embodiments, the polynucleotide or vector of the present invention comprises the nucleotide sequence shown in SEQ ID NO: 3 and/or 5 (Table 8). In some embodiments, the polynucleotide or vector of the present invention comprises the nucleotide sequence shown in SEQ ID NO: 7 and/or 9 (Table 10). The invention also provides host cells containing the vectors described herein. In another aspect, the invention also provides a method of producing an immunoreactive polypeptide comprising culturing a host cell as described herein and recovering the polypeptide thus produced. The invention also provides immunoreactive polypeptides produced by culturing the host cells described herein, and recovering the polypeptides so produced.

The invention also provides pharmaceutical compositions comprising an effective amount of any polypeptide (including any antibody) or polynucleotide described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an antibody that preferentially binds to amino acids 16-28 of the Aβ peptide. In some embodiments, the pharmaceutical composition comprises an antibody that preferentially binds to amino acids 28-40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the pharmaceutical composition comprises an antibody that preferentially binds to an epitope comprising amino acids 39 and/or 40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, an antibody that binds to an epitope comprising amino acids 39 and/or 40 of Aβ peptide (SEQ ID NO: 1) does not significantly cross-react with Aβ _1-42 and/or Aβ _1-43 peptide. In some embodiments, the antibody that binds to an epitope comprising amino acids 36-40 of the Aβ peptide (SEQ ID NO: 1) does not significantly cross-react with the Aβ _1-42 and/or Aβ _1-43 peptides. In some embodiments, the antibody that binds to amino acids 28-40 of Aβ peptide (SEQ ID NO: 1) binds Aβ peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less, 100 nM or less, 60 nM or less, 30 nM or less, 3 nM or less, 2 nM or less, 1 nM or less. In some embodiments, the Fab fragment of the antibody binds to Aβ peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less, 100 nM or less, 60 nM or less, 30 nM or less, 3 nM or less, 2nM or less, 1nM or less. In some embodiments, the Fab fragment of the antibody that preferentially binds to an epitope comprising amino acid 39 and/or 40 of Aβ peptide (SEQ ID NO: 1) binds to Aβ peptide (SEQ ID NO: 1) with an affinity of about 200 nM or Less, 100 nM or less, 60 nM or less, 30 nM or less, 3 nM or less, 2 nM or less, 1 nM or less. In some embodiments, the antibody Fab fragment that preferentially binds to an epitope comprising amino acids 3-40 of Aβ peptide (SEQ ID NO: 1) binds to Aβ peptide (SEQ ID NO: 1) with an affinity of about 200 nM or greater Small, 100 nM or less, 60 nM or less, 30 nM or less, 3 nM or less, 2 nM or less, 1 nM or less. In some embodiments, the antibody competitively inhibits the binding of a monoclonal antibody comprising the amino acid sequence set forth in SEQ ID NO 4 and/or 6, or a monoclonal antibody produced by a hybridoma designated 8A1.2A1. In some embodiments, the antibody binds to an epitope of the Aβ peptide (SEQ ID NO: 1) that hybridizes to an antibody comprising the amino acids set forth in SEQ ID NO: 4 and/or 6, or to an antibody designated 8A1.2A1 Monoclonal antibodies produced by tumors. In other embodiments, the pharmaceutical compositions comprise human antibodies, human or chimeric antibodies derived from any of the antibodies described herein.

The invention also provides hybridomas designated 8A1.2A1, 3C6.1F9 or 10B 10.2E6.

The present invention also provides methods for preventing, treating, inhibiting, or delaying the development of Alzheimer's disease and other diseases associated with altered expression of Aβ or βAPP, or accumulation of Aβ peptides, such as Down's syndrome, Parkinson's disease, Multi-infarct dementia. The method comprises administering to a subject a pharmaceutical composition comprising an effective dose of an antibody (including polypeptide) or polynucleotide of the invention. In some embodiments, the pharmaceutical composition comprises an antibody that preferentially binds to amino acids 16-28 of the Aβ peptide. In some embodiments, the antibody preferentially binds to amino acids 28-40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the antibody preferentially binds to an epitope comprising amino acids 39 and/or 40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the antibody preferentially binds to an epitope comprising amino acids 36-40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the antibody that binds to an epitope comprising amino acids 39 and/or 40 of the Aβ peptide (SEQ ID NO: 1) does not exhibit significant cross-reactivity with the Aβ _1-42 and/or Aβ _1-43 peptides . In some embodiments, the antibody that binds to an epitope comprising amino acids 36-40 of the Aβ peptide (SEQ ID NO: 1) does not exhibit significant cross-reactivity with the Aβ _1-42 and/or Aβ _1-43 peptides. In some embodiments, the antibody that binds to amino acids 28-40 of Aβ peptide (SEQ ID NO: 1) has an affinity for binding to Aβ peptide (SEQ ID NO: 1) of about 200 nM or less, 100 nM or less, 60 nM or less, 30 nM or less, 3 nM or less, 2 nM or less, 1 nM or less. In some embodiments, the Fab fragment of the antibody binds to Aβ peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less, 100 nM or less, 60 nM or less, 30 nM or less, 3 nM or less, 2 nM or less, 1 nM or less. In some embodiments, the antibody Fab fragment that preferentially binds to an epitope comprising amino acid 39 and/or 40 of Aβ peptide (SEQ ID NO: 1) binds to Aβ peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less, 100 nM or less, 60 nM or less, 30 nM or less, 3 nM or less, 2 nM or less, 1 nM or less. In some embodiments, the Fab fragment of the antibody that preferentially binds to an epitope comprising amino acids 36-40 of Aβ peptide (SEQ ID NO: 1) binds to Aβ peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less, 100 nM or less, 60 nM or less, 30 nM or less, 3 nM or less, 2 nM or less, 1 nM or less. In some embodiments, the antibody competitively inhibits the binding of a monoclonal antibody comprising the amino acid sequence set forth in SEQ ID NO: 4 and/or 6, or a monoclonal antibody produced by a hybridoma designated 8A1.2A1. Antibodies can be chimeric, human, or humanized antibodies, and can be fragments of antibodies. Examples of antibody fragments include, but are not limited to, Fab, F(ab') ₂ and Fv fragments. Antibodies can also be single chain, bispecific or multispecific antibodies comprising one or more antibody fragments. Antibodies can be further linked to, or administered with, a therapeutic agent.

The present invention also provides a method for delaying the development of Alzheimer's disease or other disease-related symptoms associated with the accumulation of Aβ peptides in a subject, comprising administering to the subject an antibody (including polypeptide) or polynucleotide comprising the present invention The effective dose of the pharmaceutical composition.

The present invention also provides a method of inhibiting the formation of amyloid plaques in a subject, comprising administering to the subject an effective dose of a pharmaceutical composition comprising an antibody (including polypeptide) or polynucleotide of the present invention. In some embodiments, the amyloid plaques are located in the subject's brain.

The invention also provides a method of reducing amyloid plaques in a subject comprising administering to the subject a pharmaceutical composition comprising an effective amount of an antibody (including polypeptide) or polynucleotide of the invention. In some embodiments, the amyloid plaques are located in the subject's brain.

The present invention also provides methods for removing or clearing amyloid plaques in a subject, comprising administering to the subject an effective dose of a pharmaceutical composition comprising an antibody (including polypeptide) or polynucleotide of the present invention. In some embodiments, the amyloid plaques are located in the subject's brain.

Furthermore, the present invention provides a method of inhibiting the accumulation of A[beta] peptide in tissue comprising contacting the tissue with a monoclonal antibody of the present invention.

The invention also provides methods of reducing A[beta] peptides (including soluble and precipitated forms) in the brain of an individual comprising administering to the individual an effective amount of an antibody of the invention. In some embodiments, the accumulation of Aβ peptide in the brain is inhibited and/or reduced. In some embodiments, the toxic effects of the Aβ peptide are inhibited and/or reduced. Therefore, the method of the present invention can be used to treat any disease in which Aβ peptide accumulation exists or is suspected, such as Alzheimer's disease, Down's syndrome, Parkinson's disease, multi-infarct dementia.

In some embodiments of the methods described herein, the composition is administered by systemic injection. In some embodiments, the composition is administered by intraperitoneal injection.

Compositions administered in the methods of the invention described above also include compositions comprising antibodies that bind to epitopes including amino acids 42 and/or 43 of Aβ _1-43 (SEQ ID NO: 12), binding to Aβ _1-43 (SEQ ID NO: 12) An antibody that binds to the C-terminus of ₄₃ (SEQ ID NO: 12) but does not show significant cross-reactivity with Aβ _1-42 (SEQ ID NO: 11) and/or Aβ _1-40 (SEQ ID NO: 1), and includes Aβ _1-42 (SEQ ID NO: 11) 41 and/or antibody binding to the epitope of amino acid 42, or bind to the C-terminus of Aβ _1-42 (SEQ ID NO: 11) but bind to Aβ _1-43 (SEQ ID NO : 12) and/or Aβ _1-40 (SEQ ID NO: 1) did not show significant cross-reactive antibody compositions.

The antibody of the present invention can be further used for detecting, diagnosing and monitoring Alzheimer's disease and other diseases associated with altered expression of Aβ or βAPP, such as Down's syndrome. The method comprises contacting a patient sample suspected of having altered expression of Aβ or βAPP with an antibody of the invention and determining whether the level of Aβ or βAPP is different from a control or comparative sample.

In further embodiments, the invention provides articles of manufacture and materials containing materials useful for the treatment of pathological diseases such as Alzheimer's disease or other diseases associated with altered expression of Aβ or βAPP, or for the detection or purification of Aβ or βAPP kit. Finished goods include labeled containers. Suitable containers include, eg, bottles, vials, test tubes. Containers can be made of different materials such as glass or plastic. The container contains a composition containing an active agent effective for treating a pathological disease or detecting or purifying Aβ or βAPP. The active agent in the composition is an antibody and preferably comprises an A[beta] or [beta]APP specific monoclonal antibody or any other composition of the invention. As noted above, the label on the container indicates that the composition is used for treating a pathological disease (such as Alzheimer's disease) or detecting or purifying A[beta] or [beta]APP, and may also be indicated for in vivo or in vitro use.

The kit of the invention comprises a container as described above and another container containing a buffer. Other materials meeting commercial and user needs may further be included, including other buffers, diluents, filters, needles, syringes, and packaged with instructions for any of the methods described herein.

The invention also provides any such composition (eg, antibody) for any of the uses described herein, whether as a medicament and/or for the preparation of a medicament.

Brief description of the drawings

Figure 1 is a bar graph illustrating that monoclonal antibodies directed against A[beta] do not cross-react with [beta]APP.

Figure 2 is a bar graph illustrating the capture of soluble A[beta] by all monoclonal antibodies directed against A[beta].

Figure 3 is a bar graph illustrating the preferential binding of antibodies produced by hybridoma 8A1.2A1 to epitopes comprising amino acids 39 and/or 40 of Aβ _1-40 .

Figure 4 shows quantification of total A[beta], thioflavine-S and MHC-II staining of the frontal cortex and hippocampus after intracranial injection of antibodies 2286, 2324, 2289 or a control antibody against amnesia.

Figure 5 shows that anti-Aβ2286F _(ab')2 fragment does not activate microglia, nor does it remove dense amyloid deposits as efficiently as intact anti-Aβ2286 IgG, panels AD show CD45 immunohistochemistry in the hippocampus. EH panels show the immunohistochemistry of total Aβ in the hippocampus. IL images showing Thioflavin-S staining in the hippocampus. Anti-Aβ2286 IgG (A, E, and I), anti-Aβ2286F _(ab′)2 fragment (B, F, and J), control (anti-amnesia) IgG (C, G, and K), or control (anti-amnesia ) F _(ab')2 fragments (D, H and L) injected mice. Magnification = 4Ox, graticule = 120 [mu]m.

Figure 6 shows the quantification of CD45 and total Aβ by immunohistochemistry and Thioflavin-S staining after intracranial injection of anti-Aβ2286 antibody and anti-Aβ2286 F _(ab')2 fragment. Panel A shows the ratio of right to left CD45 immunohistochemistry. Panel B shows the right to left ratio of Aβ immunohistochemistry. Panel C shows the ratio of right side to left side of thioflavin-S staining. Solid bars show values for frontal cortex and open bars show values for hippocampus. On the X-axis, IgG-Cont refers to the intact IgG of the control (anti-amnesia), F(ab')2-Cont refers to the F _(ab')2 fragment of the control (anti-amnesia), IgG-Aβ refers to the complete anti-Aβ IgG; F(ab')2-Aβ refers to anti-Aβ F _(ab')2 fragment. "***" indicates P<0.001 compared with two control antibody groups, and "*" indicates P<0.05 compared with two control antibody groups. Lines on the bar graphs represent P values for comparisons between the specific two groups indicated.

Figure 7 shows serum levels of A[beta] (upper panel) and concentrations of anti-A[beta] antibodies in serum (bottom panel) following systemic injection of antibody 2286. Each point in the graph represents the A[beta] serum level or anti-A[beta] antibody concentration for one mouse treated under the indicated conditions. Lines in the graph represent mean A[beta] serum levels or anti-A[beta] antibody concentrations in mice treated under the indicated conditions.

Figure 8 shows the binding of Antibody 2286 and Antibody 2324 to different A[beta] peptide variants.

Detailed description of the invention

The present invention provides Aβ peptide and βAPP specific monoclonal antibodies. The anti-Aβ antibodies disclosed herein bind with high affinity and do not cross-react with βAPP, making them particularly useful for the detection and treatment of Alzheimer's disease and other diseases associated with altered expression of Aβ (such as Down syndrome) Methods. In one embodiment, the invention provides antibodies directed against the C-terminal portion of the A[beta] peptide. In one embodiment, the C-terminal portion of the Aβ peptide to which the antibody is directed includes amino acids 28-40. In some embodiments, the antibody preferentially binds to an epitope comprising amino acids 39 and/or 40 of A[beta _]1-40 . In some embodiments, the antibody preferentially binds to an epitope comprising amino acids 36-40 of A[beta _{] 1-40} . In some embodiments, the antibody binds to the C-terminal portion of _Aβ1-40 with an affinity of about 200 nM or less, 150 nM or less, 100 nM or less, 60 nM or less, 30 nM or less, 3 nM or less, 2nM or less, 1nM or less. In some embodiments, the antibody preferentially binds to an epitope comprising amino acids 39 and 40 _{of Aβ1-40} with an affinity of about 200 nM or less, 150 nM or less, 100 nM or less, 60 nM or less, 30 nM or Less, 3nM or less, 2nM or less, 1nM or less. In some embodiments, the antibody preferentially binds to an epitope comprising amino acids 36-40 of Aβ _1-40 with an affinity of about 200 nM or less, 150 nM or less, 100 nM or less, 60 nM or less, 30 nM or less Small, 3nM or less, 2nM or less, 1nM or less. The therapeutic utility of antibodies directed against the C-terminal portion of the Aβ peptide is based on the surprising discovery that antibodies can remove both Aβ and Thioflavin-S precipitates (toxic fibrous form precipitates) in the brain tissue of animal models of Alzheimer's disease. indications).

Contrary to other reports, it was found that targeting amino acids 28-40 of Aβ (in some embodiments, including amino acids 39 and 40 of Aβ (SEQ ID NO: 1) or amino acids 36-40 of Aβ (SEQ ID NO: 1) epitope) effectively removes fibril deposits in animal models of Alzheimer's disease. As reviewed by Schenk (October 2002, Nat Rev Neurosci.3(10):824-8), not all anti-Aβ antibodies are effective in attenuating pathology in the brain, and those that can alleviate symptoms are limited to the first 16 amino acids of Aβ ( Bacskai et al., 2002, J Neurosci.22(18):7873-8; Bard et al., 2000, Nature Med.6:916-919), or amino acids 16-28 (DeMattos et al., 2001, Proc.Natl Acad.Sci, 98(15):8850-55; DeMattos et al., 2002, Science, 295(5563):2264-7; Dodart et al., 2002 Nat. Neuroscience, 5(5):452-7). In contrast, antibodies directed against the carboxyl terminus (eg amino acids 33-42) do not reduce amyloid burden in the brain (Bacskai 2002, supra, Bard 2000, supra).

definition

All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the assigned meanings.

As used herein, "antibody" includes intact immunoglobulins or antibody molecules, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), and immunoglobulin fragments (e.g., Fab, F(ab') ₂ or Fv), as long as they can exhibit any of the desired specific binding properties described herein. Antibodies are generally proteins or polypeptides that exhibit specific binding to a specific antigen.

As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, each antibody comprising the population is identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies directed against a single antigenic site are highly specific. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes). The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared according to the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or according to recombinant DNA methods (as described in U.S. Patent No. 4,816,567). For example, monoclonal antibodies can also be isolated from phage libraries generated using the technique described by McCafferty et al., 1990, Nature, 348:552-554.

As used herein, a "human" antibody refers to a specific chimeric immunoglobulin, immunoglobulin chain, or fragment thereof that contains minimal sequence derived from a non-human immunoglobulin (e.g., Fv, Fab, Fab', F(ab ') ₂ or other antigen-binding sequence of an antibody) of a non-human antibody (eg, murine) version. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the complementarity-determining regions (CDRs) of the recipient have been replaced by a non-human species (donor antibody) such as mouse, rat or Rabbit) Residue substitutions in CDRs with desired specificity, affinity and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies can comprise residues found neither in the recipient antibody nor in the imported CDR or framework sequences, which are included to further improve and optimize antibody performance. In general, a humanized antibody comprises substantially completely at least one, and usually two, variable domains, wherein the CDRs in the variable domains correspond to all or substantially all of the CDR regions of a non-human immunoglobulin, and all or substantially all of the CDR regions are non-human immunoglobulin CDRs, and all or substantially All FR regions are sequences consistent with human immunoglobulins. Humanized antibodies preferably also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), usually a human immunoglobulin. Antibodies with Fc regions modified as described in WO99/58572 are preferred. Other forms of humanized antibodies have one or more (one, two, three, four, five, six) CDRs altered relative to the original antibody, also known as "derived from" the original antibody. One or more CDRs of one or more CDRs.

Each variable region of the heavy and light chains consists of four framework regions (FR) joined by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are tightly bound by the FRs and together with the CDRs of the other chains contribute to the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on sequence variability across species (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991, National Institutes of Health, Bethesda MD); (2) antigen-based methods - Methods for crystallographic studies of antibody complexes (Chothia et al. (1989) Nature 342:877). As used herein, CDR refers to CDRs defined by one method or a combination of two methods.

As used herein, a "human antibody" refers to an antibody having an amino acid sequence corresponding to a portion of an antibody produced in a human, and/or an antibody prepared using any of the techniques for preparing human antibodies known in the art or disclosed herein. The definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. An example of this is an antibody comprising a murine light chain and a human heavy chain polypeptide. Human antibodies can be produced using a variety of techniques well known in the art. In one embodiment, the human antibody is obtained from a phage library expressing human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human antibodies can also be prepared by introducing human immunoglobulin loci into transgenic animals, eg, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach has been described in US Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016. Alternatively, human antibodies can be produced by immortalizing human B lymphocytes that produce antibodies to the target antigen (such B lymphocytes can be recovered from the individual or immunized in vitro). See, eg, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J. Immunol., 147(1):86-95 and US Patent No. 5,750,373.

"Chimeric antibody" refers to a heavy and light chain in which a portion of each amino acid sequence is homologous to the corresponding sequence in an antibody derived from or belonging to a particular species, but other segments of the chains are homologous to corresponding portions of another antibody those antibodies. Typically, in these chimeric antibodies, the variable regions of the two heavy and light chains mimic those of antibodies derived from one mammal, while the constant regions are homologous to sequences derived from antibodies from another species. An obvious advantage of this chimeric type is that variable regions can be readily obtained from currently known sources, for example using readily available hybridomas or B cells from non-human host organisms, and compared with those derived from, for example, human cells. The constant region of the formulation binds. The variable region has the advantage of being easy to prepare, and its specificity is not affected by its source. When injecting antibodies, the constant region of human origin is less likely to cause an immune response in human subjects than the constant region from a non-human source. . However, the present definition is not limited to this particular instance.

A "functional Fc region" possesses at least one effector function of a native sequence Fc region. Representative "effector functions" include C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor body, BCR) and so on. Such effector function generally requires an Fc region associated with a binding domain (eg, an antibody variable domain), and can be assessed using a variety of assays known in the art for assessing such antibody effector function.

A "native sequence Fc region" comprises the same amino acid sequence as an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, yet retains at least one effector function of a native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent polypeptide, for example about one to about ten amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide, Preferably about one to about five amino acid substitutions are made. The variant Fc region herein preferably has at least about 80% sequence identity, most preferably at least about 90% sequence identity, more preferably at least about 95% sequence identity with the native sequence Fc region and/or the parent polypeptide Fc region sequence identity.

As used herein, "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated response in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer cells (NK), Neutrophils and macrophages) recognize bound antibodies on target cells, which subsequently leads to lysis of the target cells. ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay (as described in US Patent No. 5,500,362 or 5,821,337). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively, or in addition, ADCC activity of a molecule of interest may be assessed in vivo, for example in an animal model as disclosed by Clynes et al., 1998, PNAS (USA), 95:652-656.

As used herein, "human effector cell" refers to a leukocyte that expresses one or more FcRs and can perform effector functions. Preferably, the cells express at least FcγRIII and can perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, preferably PBMC and NK cells. Effector cells can be isolated from natural sources such as blood or PBMCs.

As used herein, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, preferred FcR binding IgG antibodies (gamma receptors), include receptors of the FcyRI, FcyRII and FcyRIII subclasses, including allelic variants and alternative splicing of these receptors. FcyRII receptors include FcyRIIA ("activating receptor") and FcyRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. In Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9: 457-92; Capel et al., 1994, Immunomethods, 4: 25-34 and de Haas et al., 1995, J. Lab. Clin. Med., 126: 330-41 review of FcR. "FcR" also includes the neonatal receptor FcRn responsible for the transfer of maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587 and Kim et al., 1994, J. Immunol., 24:249).

"Complement-dependent cytotoxicity" and "CDC" refer to the lysis of target cells in the presence of complement. The complement activation pathway is activated by the binding of the first component of the complement system (Clq) to molecules complexed with cognate antigens, such as antibodies. To assess complement activation, a CDC assay as described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996) can be performed.

As used herein, an "affinity matured" antibody refers to an antibody that has one or more alterations in one or more of its CDRs that result in an increased affinity of the antibody for the antigen compared to a parent antibody without the alterations. Preferred affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies by methods known in the art (Marks et al., 1992, Bio/Technology, 10: 779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA91: 3809-3813; Schier et al., 1995, Gene, 169: 147-155; Yelton et al., 1995, J. Immunol., 155: 1994-2004; Jackson et al., 1995, J. Immunol., 154(7): 3310-9, Hawkins et al., 1992, J. Mol. Biol., 226:889-896).

As used herein, "immunospecific" binding of an antibody means that it occurs at the antigen-binding site of the antibody and the specific antigen recognized by the antibody (i.e., in an ELISA or other immunoassay, the antibody reacts with the protein, but not with the detectable Antigen-specific binding interactions between unrelated protein responses).

An epitope to which an antibody or polypeptide "specifically binds" or "preferentially binds" (used interchangeably herein) is a well-understood term in the art, as are methods for determining such specific or preferential binding. A molecule is considered to exhibit " specific binding" or "preferential binding". An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, with greater ease, and/or with greater duration than it binds to other substrates. For example, an antibody that specifically or preferentially binds to _Aβ1-40 with greater affinity, avidity, greater affinity than the antibody binds to other _Aβ1-40 epitopes or non- _Aβ1-40 epitopes Antibodies that bind the epitope readily and/or with a longer duration. It will also be understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds a first target may or may not specifically or preferentially bind a second target. Likewise, "specific binding" or "preferential binding" does not necessarily require (although includes) exclusive binding. Typically, but not necessarily, references to combinations denote preferential combinations.

As used herein, "polypeptide" includes proteins, protein fragments and peptides, whether isolated from natural sources, produced by recombinant techniques, or chemically synthesized. Polypeptides of the invention generally comprise at least about 6 amino acids.

As used herein, a "vector" is a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic concentrating reagents, DNA or RNA expression vectors encapsulated in liposomes Vectors and certain eukaryotic cells (such as producer cells).

As used herein, "expression control sequence" refers to a nucleotide sequence that directs the transcription of a nucleotide. The expression control sequence may be a promoter, such as a constitutive or inducible promoter, or an enhancer. Expression control sequences are operably linked to the nucleotide sequence to be transcribed.

As used herein, "nucleic acid" or "polynucleotide" refers to a polymer of deoxyribonucleic acid or ribonucleotides in either single- or double-stranded form, and includes, unless otherwise limited, analogues of naturally occurring nucleotides. A known analogue of a natural nucleotide that hybridizes to a nucleotide in the same manner.

As used herein, a "pharmaceutically acceptable carrier" includes any substance that, when combined with an active ingredient, allows the ingredient to retain its biological activity and is nonreactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline, water, emulsions (eg, oil/water emulsions), and various wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or physiological saline (0.9%).

By well-known conventional methods (see, for example, "Remington's Pharmaceutical Sciences" 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990 and Remington "The Science and Practice of Pharmacy" 20th edition. MackPublishing, 2000) to formulate compositions comprising such carriers.

As used herein, "adjuvant" includes those adjuvants commonly used in the art to promote an immune response. Examples of adjuvants include, but are not limited to, helper peptides, aluminum salts (such as aluminum hydroxide gel (alum) or aluminum phosphate), Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, MI), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), AS-2 (Smith-Kline Beecham), QS-21 (Aquilla Biopharmaceuticals), MPL or 3d-MPL (Corixa Corporation, Hamilton, MT), LEIF, Calcium, iron or zinc salts, insoluble suspensions of acylated tyrosine, acylated sucrose, cationically or anionically derivatized polysaccharides, polyphosphazenes, biodegradable microspheres, monophosphatidyl lipid A and quilA, muramyl tripeptide phosphatidylethanolamine, or immunostimulatory complexes including cytokines (such as GM-CSF or interleukin-2, -7 or -12) and immunostimulatory DNA sequences. In some embodiments, as in the case of polynucleotide vaccines, an adjuvant such as a helper polypeptide or cytokine may be provided by a polynucleotide encoding the adjuvant.

As used herein, an "effective dose" or "effective amount" of a drug, compound or pharmaceutical composition is an amount sufficient to achieve a beneficial or desired result. For prophylactic use, beneficial or desirable results include those that eliminate or reduce risk, lessen severity, or delay onset of disease, including biochemical, histological, and/or behavioral signs of disease, other Transitional pathological phenotypes that arise during onset and disease progression. For therapeutic use, beneficial or desirable outcomes include clinical outcomes such as inhibition or suppression of amyloid plaque formation, reduction, removal, or clearance of amyloid plaques, improved recognition or reversal of cognitive decline, sequestration in Soluble Aβ peptides circulating in biological fluids, attenuating one or more of the disease-causing (biochemical, histological, and/or behavioral) symptoms, including its onset and transitional pathological phenotypes that occur during disease progression , improve the quality of life of those suffering from the disease, reduce the dose of other drugs needed to treat the disease, enhance the effect of other drugs, delay the progression of the disease and/or prolong the survival of the patient. An effective dose can be administered in one or more doses. For purposes of the present invention, an effective dosage of a drug, compound or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, prophylactic or therapeutic treatment. An effective dose of a drug, compound or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound or pharmaceutical composition, as understood in clinical terms. Thus, an "effective dose" can be considered in the sense of administering one or more therapeutic agents, and an effective amount of a single agent administered if possible or capable of achieving the desired result in conjunction with one or more other agents.

As used herein, "treatment" is a method of obtaining a beneficial or desired result, including a clinical result. For purposes of the present invention, favorable or anticipated clinical outcomes include, but are not limited to, one or more of the following: inhibition or suppression of amyloid plaque formation, inhibition or suppression of amyloid plaque formation, reduction in , remove, or clear amyloid plaques, improve recognition or reverse cognitive decline, sequester soluble Aβ peptides circulating in biological fluids, reduce Aβ peptides (both soluble and precipitated) in tissues such as the brain ), inhibiting and/or reducing the accumulation of Aβ peptides in the brain, inhibiting and/or reducing the toxic effects of Aβ peptides in tissues (such as the brain), attenuating the symptoms caused by the disease, improving the quality of life of those patients suffering from the disease , reducing the dose of other drugs needed to treat the disease, delaying the progression of the disease and/or prolonging the patient's survival.

As used herein, "delaying" the progression of Alzheimer's disease means postponing, arresting, slowing, delaying, stabilizing and/or stalling the progression of the disease. The length of this delay varies, depending on the medical history and/or treatment of the individual. As will be apparent to those skilled in the art, sufficient or significant delay actually includes prevention, since the individual has not yet developed the disease. A method of "delaying" the development of Alzheimer's disease is a method of reducing the likelihood of disease development in a given time frame and/or reducing the extent of the disease in a given time frame compared to not using the method. Such comparisons are usually based on clinical studies using a statistically significant number of subjects.

"Development" of Alzheimer's disease refers to the onset and/or progression of Alzheimer's disease in an individual. The development of Alzheimer's disease can be detected using standard clinical techniques described here. However, development also refers to an initially undetectable disease process. For the purposes of this invention, progression refers to the biological course of a disease state, as determined by standard neurologic examination or patient consultation, or as may be the case by more specialized testing. Various such diagnostic tests include, but are not limited to, neuroimaging, detection of changes in serum or cerebrospinal fluid levels of specific proteins (such as amyloid peptide and Tau), computed tomography (CT), and magnetic resonance imaging (MRI). "Development" includes occurrence, relapse and onset. As used herein, "onset" or "occurrence" of Alzheimer's disease includes initial onset and/or relapse.

As used herein, an "at-risk" individual is one who is at risk of developing Alzheimer's disease. Individuals at risk may or may not have detectable disease, and may or may not have exhibited detectable disease prior to the methods of treatment described herein. "At risk" means that an individual has one or more so-called risk factors, which are measurable parameters related to the development of Alzheimer's disease. Individuals with one or more of these risk factors are more likely to develop Alzheimer's disease than individuals without these risk factors. These risk factors include (but are not limited to) age, type, race, diet, previous medical history, presence of antecedent diseases, genetic (ie, hereditary) factors, and environmental exposures.

As used herein, "a" or "an" means at least one, unless otherwise stated.

Antibody

The invention provides isolated monoclonal antibodies (including human, humanized or chimeric antibodies of the invention) that bind to the Aβ peptide (SEQ ID NO: 1). More specifically, antibodies that bind to amino acids 1-16, 16-28, or 28-40 of the Aβ peptide are provided. In some embodiments, the antibody preferentially binds to an epitope comprising amino acids 39 and/or 40 of the Aβ peptide (SEQ ID NO: 1). An antibody that binds to an Aβ peptide comprising amino acids 11-40 of SEQ ID NO: 11-40 but does not bind (as understood by those skilled in the art) to an Aβ peptide comprising amino acids 11-38 of SEQ ID NO, It is an antibody that preferentially binds to an epitope including amino acids 39 and/or 40 of Aβ peptide (SEQ ID NO: 1). In some embodiments, the antibody binds to an epitope comprising amino acids 36-40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the binding affinity of the antibody to amino acids 28-40 of Aβ peptide (SEQ ID NO: 1) is about 200 nM or less, 60 nM or less, 30 nM or less, 3 nM or less, or 1 nM or less smaller. In some embodiments, the antibody preferentially binds an epitope comprising amino acids 39 and/or 40 of the Aβ peptide (SEQ ID NO: 1) with an affinity of about 60 nM or less, 30 nM or less, 3 nM or less. In some embodiments, the antibody preferentially binds an epitope comprising amino acids 39 and/or 40 of Aβ peptide (SEQ ID NO: 1) with an affinity of about 3 nM or less. Preferably, the antibody competitively inhibits the binding of a monoclonal antibody having the amino acid sequence shown in SEQ ID NO: 4, 6, 8 and/or 10, or competitively inhibits the binding of a monoclonal antibody designated 8A1.2A1, 3C6.1F9 or 10B10.2E6 Binding of monoclonal antibodies produced by hybridomas. In some embodiments, the monoclonal antibody binds to the Aβ peptide with an affinity of about 60 nM or less, preferably about 30 nM or less, and more preferably about 3 nM or less. In a preferred embodiment, the antibody is hybridized to a monoclonal antibody having the amino acid sequence shown in SEQ ID NO: 4, 6, 8, and/or 10, or a hybridization compound designated by 8A1.2A1, 3C6.1F9, or 10B10.2E6. Monoclonal antibodies produced by tumors bind to the same Aβ epitope. Monoclonal antibodies can optionally be conjugated to a therapeutic agent and/or labeled with a detectable label.

In some embodiments and as described herein (known in the art), affinity is measured using the corresponding Fab fragment of the antibody.

In addition, the present invention provides binding to βAPP (SEQ ID NO: 2) and competitively inhibits compounds designated 25E12.1F9.1H8 (BP26), 24H4.2E10.1F5 (BP27), 1F10.8E6.2A2 (BP80), Isolated monoclonal antibodies bound to monoclonal antibodies produced by hybridomas of 13E12.1C5 (BP81) or 14D9.1G8 (BP82).

In another aspect, the present invention provides a humanized antibody derived from a monoclonal antibody, wherein the monoclonal antibody has the amino acid sequence shown in SEQ ID NO: 4 and/or 6, or is produced by a hybridoma named 8A1.2A1. A humanized form of an antibody may or may not have the same CDRs as the monoclonal antibody from which it is derived. Those skilled in the art are familiar with the determination of CDR regions. In some embodiments, the invention provides antibodies comprising at least one CDR that is associated with at least one CDR, at least two, at least three, at least four, at least five monoclonal antibodies from which it is derived (or in some embodiments In the scheme, there is substantial homology to all six CDRs of the monoclonal antibody, or the CDRs derived from the monoclonal antibody). Other embodiments include having at least two, three, four, five or six CDRs homologous to at least two, three, four, five or six bases of a monoclonal antibody, or derived from a monoclonal antibody. Antibody CDR Antibody. For the purposes of the present invention, it is understood that binding specificity and/or overall activity (which may be based on clearance of Aβ precipitates) is generally retained, although compared to the monoclonal antibody produced by the hybridoma designated 8A1.2A1 , the range of activity may change. The invention also provides methods of making any of these antibodies. Methods of making antibodies are well known in the art and described herein.

By recognizing identical or spatially overlapping epitopes, competition assays can be used to determine whether two antibodies bind to the same epitope. Typically, the antigen is immobilized on a multiwell plate, and the ability of the unlabeled antibody to block the binding of the labeled antibody is measured. Common labels used in such competition assays are radiolabels or enzymatic labels.

One method of determining the affinity of antibodies for A[beta] peptides is to measure the affinity of antibody monofunctional Fab fragments. To obtain monofunctional Fab fragments, antibodies, eg, IgG can be cleaved with papain or expressed recombinantly. The affinity of the anti-Aβ Fab fragment of the monoclonal antibody can be determined by Surface Plasmon Resonance (SPR) (BIAcore 3000TM, BIAcore, Inc., Piscaway, NJ). SA chips (streptavidin) were used according to the manufacturer's instructions. Biotinylated Aβ peptide 1-40 (SEQ ID NO: 1) can be diluted in HBS-EP (100mM HEPES pH7.4, 150mM NaCl, 3mM EDTA, 0.005% P20) and injected at a concentration of 0.005mg/mL on chip. Using variable flow times on each chip channel, two ranges of antigen density can be achieved: 10-20 response units (RU) for detailed kinetic studies, and 500-600 RU for concentrations. Regeneration studies showed that a mixture of Pierce elution buffer and 4M NaCl (2:1) effectively removed bound Fab while retaining the activity of Aβ peptide on the chip after more than 200 injections. For all BIAcore assays, HBS-EP buffer can be used as running buffer. Serially diluted (0.1-10x estimated KD) purified Fab samples were injected at 100 μL/min for 2 min and a dissociation time of 30 h min was allowed. Using known concentrations of Fab standards (determined by amino acid analysis), the concentration of Fab protein can be determined by ELISA and/or SDS-PAGE electrophoresis. Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data to a 1:1 Langmuir binding model (Lofas & Johnsson, 1990) using the BIA evaluation program. Equilibrium dissociation constant (KD) values were calculated as koff/kon.

The invention provides antibodies in monomeric, dimeric and multivalent forms. For example, the antibodies disclosed herein can be used to prepare bispecific antibodies, monoclonal antibodies that have binding specificities for at least two different antigens. Methods for making bispecific antibodies are well known in the art (see, eg, Suresh et al., 1986, Methods in Enzymology 121:210). Traditionally, recombinant products of bispecific antibodies are based on the co-expression of heavy chain-light chain pairs of two immunoglobulins with different specificities (Millstein and Cuello, 1983, Nature 305, 537-539 ).

According to one method of making bispecific antibodies, antibody variable domains (antibody-antigen combining sites) with the desired binding specificity are fused to immunoglobulin constant domain sequences. The fusion is preferably to an immunoglobulin heavy chain constant region domain comprising at least part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) in at least one fusion containing the site necessary for light chain binding. DNA encoding the immunoglobulin heavy chain fusion and the immunoglobulin light chain (if desired) is inserted into separate expression vectors and co-transfected into a suitable host organism. This allows great flexibility in adjusting the mutated ratios of the three polypeptide fragments in embodiments when different ratios of the three polypeptide chains used in the construction provide optimal yields. However, when the expression of at least two polypeptide chains in the same ratio results in high yields, or when the ratio is not particularly critical, the coding sequences for two or all three polypeptide chains may be inserted into one expression vector.

In one approach, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. binding specificity) composition. This asymmetric structure, in which immunoglobulin light chains are only half of the bispecific molecule, facilitates the separation of desired bispecific compounds from combinations of unwanted immunoglobulin chains. PCT Publication No. WO94/04690, published March 3, 1994, describes this method.

Heteroconjugated antibodies comprising two covalently linked antibodies are also within the scope of the invention. Such antibodies have been used to target immune system cells to unwanted cells (US Patent No. 4,676,980), and in the treatment of HIV infection (PCT Application Publication Nos. WO91/00360 and WO92/200373, EP03089). Heteroconjugated antibodies can be prepared using any convenient cross-linking method. Suitable crosslinking agents and techniques are well known in the art and are described in US Patent No. 4,676,980.

In certain embodiments, the immunoreactive molecule is an antibody fragment. Various techniques have been developed to generate antibody fragments. These fragments can be obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-117 and Brennan et al., 1985, Science 229:81), or directly by recombinant host cells produce. For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio/Technology 10:163-167). In another embodiment, the leucine zipper GCN4 is used to facilitate the assembly of F(ab')2 molecules to form F(ab')2. According to another approach, the Fv, Fab or F(ab')2 is isolated directly from recombinant host cell culture.

The monoclonal antibodies of the invention may be provided in the form of pharmaceutical compositions, optionally together with a carrier.

Antibodies can also be embedded in microcapsules, e.g. by coacervation techniques, or interfacial polymerization (e.g. hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g. lipid plastids, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in a marcoemulsion. This technology has been published in "Remington's Pharmaceutical Sciences" 18th Edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990 and Remington's "The Science and Practice of Pharmacy" 20th Edition. Mack Publishing, 2000 . As described in US Pat. No. 5,739,277, to increase the serum half-life of antibodies, salvage receptor binding epitopes can be incorporated into antibodies (especially antibody fragments). As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (eg, IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the serum half-life of an IgG molecule in vivo.

The antibodies disclosed herein can also be formulated as immunoliposomes. Antibody-containing liposomes are prepared by methods known in the art, such as Epstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688, Hwang et al., 1980, Proc. Methods described in US Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation are disclosed in US Patent No. 5,013,556. Very useful liposomes can be generated by the reverse evaporation method using a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. In addition, Fab' fragments of antibodies of the invention can be conjugated to liposomes as described by Martin et al., 1982, J. Biol. Chem. 257:286-288, via a disulfide interchange reaction.

In some embodiments, the antibodies of the invention are single-chain (ScFv), mutants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabody linear antibodies, single-chain antibodies, and immune Any modified configuration of a globulin molecule.

Antibody production

Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, 1975, Nature 256:495. In the hybridoma approach, typically a mouse, hamster or other suitable host animal is immunized with an immunizing agent to elicit lymphocytes that produce or can produce antibodies that specifically bind the immunogen. Alternatively, lymphocytes can be immunized in vitro.

Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in US Patent No. 4,816,567. DNA encoding the monoclonal antibody is isolated and sequenced using conventional methods, such as using polynucleotide probes that bind specifically to genes encoding the heavy and light chains of the monoclonal antibody. Once the DNA is isolated, it is placed into an expression vector, which is then transfected into host cells (such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins) to synthesize monoclonal antibodies in recombinant host cells.

For example, the DNA can be modified by covalently linking the entire coding sequence of the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. "Chimeric" or "hybrid" antibodies having the binding specificities of the monoclonal antibodies described herein are prepared in this manner. Typically, this non-immunoglobulin polypeptide is substituted for the constant domain of an antibody of the invention or the variable domain of one of the antigen binding sites of an antibody of the invention to generate a chimeric diabody comprising One antigen-binding site specific for a surface epitope of Aβ (or βAPP) and another antigen-binding site specific for a different antigen.

The invention also includes humanized antibodies. Therapeutic antibodies often elicit side effects, in part by eliciting an immune response to the administered antibody. This can lead to decreased drug efficacy, rejection of cells bearing the target antigen, and undesired inflammatory responses. To avoid the above problems, recombinant anti-Aβ humanized antibodies were produced. Antibody polynucleotide sequences (such as SEQ ID NO: 3 and/or 5) can be used for genetic manipulation to produce "humanized" antibodies, or to improve the affinity or other properties of antibodies. The general principles of antibody humanization include retaining the basic sequence of the antigen-binding portion of the antibody while exchanging the human antibody sequence for the non-human remainder of the antibody. There are four general steps in humanizing monoclonal antibodies. They are: (1) determine the nucleotide sequence and predicted amino acid sequence of the variable domains of the light and heavy chains of the starting antibody; (2) design the humanized antibody, that is, determine the antibody used in the humanization process framework regions; (3) efficient humanization methods/techniques; and (4) transfection and expression of humanized antibodies. For example, if the antibody is used in clinical trials and therapy in humans, the constant regions can be engineered to more closely resemble human constant regions to avoid immune responses. See, eg, US Patent Nos. 5,997,867 and 5,866,692.

In recombinant humanized antibodies, the Fcγ portion can be modified to avoid interaction with the Fcγ receptors and the complement immune system. Dr. Mike Clark, Department of Pathology, University of Cambridge, designed this modification and described techniques for making such antibodies in WO99/58572, published 18 November 1999.

Several "humanized" antibodies comprising antigen-binding sites derived from non-human immunoglobulins have been described, including rodent V regions and their associated complementarity determining regions (CDRs) fused to human constant domains. ) chimeric antibody. See, for example, Winter et al., Nature 349:293-299 (1991); Lobuglio et al., Proc. Nat. Acad. Sci. USA 86:4220-4224 (1989), Shaw et al., J Immunol. ) and Brown et al. Cancer Res. 47:3577-3583 (1987). Other references describe rodent CDRs grafted into human supporting framework regions (FRs) prior to fusion to appropriate human antibody constant domains. See, eg, Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988) and Jones et al., Nature 321:522-525 (1986). Another reference describes rodent CDRs supported by recombined veneered rodent framework regions. See, eg, European Patent Publication No. 519,596. These "humanized" molecules are designed to minimize deleterious immune responses against rodent anti-human antibody molecules that limit the duration and effectiveness of therapeutic applications of therapeutic agents in human recipients. Other methods of humanizing antibodies that may also be used are disclosed in Daugherty et al., Nucl. Acids Res., 19:2471-2476 (1991) and U.S. Pat. in public.

In another alternative, however, fully human antibodies can be obtained through the use of commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce more desirable (eg, fully human antibodies), or stronger immune responses, can also be used to produce humanized or human antibodies. Examples of this technology are Xenomouse ^™ from Abgenix (Fremont, CA) and HuMAb-Mouse&commat and TC Mouse ^™ from Medarex (Princeton, NJ).

In another alternative, antibodies can be produced recombinantly by phage display technology. See, eg, US Patent Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150 and Winter et al., Annu. Rev. Immunol. 12:433-455 (1994). Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to generate human antibodies in vitro from the repertoire of immunoglobulin variable (V) domain genes from unimmunized donors and antibody fragments. According to this technique, the antibody V domain gene is cloned in-frame into the major or minor coat protein gene of a filamentous phage (such as M13 or fd) and displayed as a functional antibody fragment on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional characteristics of the antibody also results in selection of the gene encoding the antibody exhibiting those characteristics. Thus, phages mimic some of the characteristics of B cells. Phage display can be performed in a variety of formats, reviewed in, eg, Johnson, Kevin S and Chiswell, David J., Current Opinion in Structural Biology 3, 564-571 (1993). Several sources of V-gene fragments are available for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a diverse set of anti-oxazolone antibodies from a small random combinatorial library of v genes derived from the spleens of immunized mice. According to the technique described in Mark et al., J.Mol.Biol.222:581-597 (1991) or Griffith et al., EMBO J.12:725-734 (1993), can construct the V gene derived from unimmunized human donor. repertoire, and essentially separate a pool of distinct antibodies against antigens, including self-antigens. In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). During subsequent antigen challenge, some of the changes introduced will confer higher affinity, and B cells displaying high-affinity surface immunoglobulins are preferentially replicated and differentiated. This natural process can be mimicked by employing a technique known as "chain shuffling" (Marks et al., Bio/Technol. 10:779-783 (1992)). In this approach, acquisition by phage display can be enhanced by sequentially replacing the heavy and light chain V region genes with repertoires of naturally occurring variants (repertoires) of V domain genes obtained from unimmunized donors. The affinity of the "primary" human antibody. This technique enables the generation of antibodies and antibody fragments with affinities in the pM-nM range. Waterhouse et al., Nucl. Acids Res. 21:2265-2266 (1993) have described a strategy for making very large phage antibody repertoires (also known as "mather-of-all libraries"). Gene shuffling is also used to derive human antibodies from rodent antibodies, where the human antibody has similar affinities and specificities to the starting rodent antibody. According to this method, also known as "epitope imprinting", the repertoire of human V domain genes is used to replace the heavy or light chain V domain genes of rodent antibodies obtained by phage display technology to create Rodent-human chimera. Selection on antigen results in the isolation of human variable regions that can repair a functional antigen binding site, ie the epitope governs (imprints) the choice of partner. When this process is repeated to replace the remaining rodent V domain, human antibodies are obtained (see PCT Patent Application PCT WO 9306213 published April 1, 1993). Unlike the traditional humanization of rodent antibodies by CDR grafting, this technique provides fully humanized antibodies without framework or CDR residues of rodent origin. Obviously, while the above discussion refers to humanized antibodies, the general principles discussed can be applied to antibodies tailored for use in dogs, cats, primates, horses and cattle.

Chimeric or hybrid antibodies can also be prepared using known methods of synthetic protein chemistry, including those involving cross-linking reagents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate.

Single-chain Fv fragments can also be generated as described in Iliades et al., 1997, FEBS Letters, 409:437-441. Coupling of such single-chain fragments using various linker peptides is described in Kortt et al., 1997, Protein Engineering, 10: 423-433. Various techniques for recombinant production and manipulation of antibodies are well known in the art.

Variant and Modified Immunoreactive Polypeptides and Antibodies

The present invention also provides a polypeptide comprising the amino acid sequence of the antibody of the present invention, such as a monoclonal antibody having the amino acid sequence shown in SEQ ID NO: 4 and/or 6, or a monoclonal antibody produced by a hybridoma named 8A1.2A1 Antibody. The immunologically active polypeptides described herein can be used in any of the compositions, kits and methods described herein. A polypeptide has one or more of the binding properties described herein, and in some embodiments, exhibits any one or more of the additional functional characteristics described herein. In some embodiments, the polypeptide comprises the light and/or heavy chain variable regions of one or more monoclonal antibodies. In some embodiments, the polypeptide comprises one or more (one, two, three, four, five, or six) light and/or heavy chain CDRs of a monoclonal antibody. In some embodiments, the polypeptide comprises three CDRs of the light and/or heavy chain of the monoclonal antibody. In some embodiments, the amino acid sequence of the monoclonal antibody comprised by the polypeptide has any of the following: at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least about 10 contiguous amino acids of the monoclonal antibody sequence , at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous amino acids, wherein at least 3 amino acids are from the variable region of the monoclonal antibody. In one embodiment, the variable region is derived from the light chain of a monoclonal antibody. In another embodiment, the variable region is from the heavy chain of a monoclonal antibody. In another embodiment, 5 (or more) contiguous amino acids are from a complementarity determining region (CDR) of a monoclonal antibody.

As used herein, a polypeptide "variant" is a polypeptide that differs from the native protein by one or more substitutions, deletions, additions and/or insertions such that the immunoreactivity of the polypeptide is not substantially reduced. In other words, the ability of the variant to specifically bind antigen may be enhanced or unchanged, or reduced by less than 50%, preferably less than 20%, compared to the native protein. Polypeptide variants preferably exhibit at least about 80%, more preferably at least about 90% and most preferably at least about 95% identity (determined as described herein) to the identified polypeptide.

Amino acid sequence variants of antibodies are prepared by introducing appropriate nucleotide changes in the antibody DNA, or by peptide synthesis. These variants include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of SEQ ID NO: 4, 6 or 8 described herein. Any combination of deletions, insertions and substitutions can be used to obtain the final construct in order for the final construct to have the desired characteristics. Amino acid changes may also alter the post-translational processing of antibodies, such as altering the number and location of glycosylation sites.

A method for identifying certain residues or regions of an antibody that are preferred locations for mutagenesis is so-called "alanine scanning mutagenesis" and is described in Cunningham and Wells, 1989, Science, 244:1081-1085 . A residue or population of target residues (e.g. charged residues such as arg, asp, his, lys and glu) are identified and neutral or negatively charged amino acids (most preferably alanine or polyalanine) Acid) substitutions to affect the interaction of amino acids with antigens. Those amino acid positions that exhibit functional sensitivity to the substitution are then pinpointed by introducing additional or additional variants at or to the site of the substitution. Thus, when predetermining the site to introduce an amino acid sequence variant, it is not necessary to predetermine the nature of the mutation itself. For example, to analyze the effect of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region, and the expressed antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions of one residue in length to polypeptides containing several hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal inserts include antibodies with N-terminal methionyl residues, or antibodies fused to epitope tags. Other insertional variants of antibody molecules include N- or C-terminal fusions of the antibody to enzymes or polypeptides, which fusions increase the serum half-life of the antibody.

Substitution variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted in its place. Sites of greatest interest for substitution mutagenesis included hypervariable regions, but FR changes were also considered. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions". If such substitutions result in a change in biological activity, more substantial changes, designated "representative substitutions" in Table 1, or further described below with reference to amino acid types, can be introduced and the expression products screened.

Table 1: Conservative Substitutions initial residue preferred replacement permutation of examples Ala(A) Val Val, Leu, Ile Arg(R) Lvs Lys, Gln, Asn Asn(N) Gln Gln, His, Asp, Lys, Arg Asp(D) Glu Glu, Asn Cys(C) Ser Ser, Ala Gln(Q) Asn Asn, Glu Glu(E) Asp Asp, Gln Gly(G) Ala Ala His(H) Arg Asn, Gln, Lys, Arg Ile (I) Leu Leu, Val, Met, Ala, Phe, Norleucine Leu(L) Ile Norleucine, Ile, Val, Met, Ala, Phe Lys(K) Arg Arg, Gln, Asn Met(M) Leu Leu, Phe, Ile Phe(F) Tyr Leu, Val, Ile, Ala, Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr Tyr, Phe Tyr(Y) Phe Trp, Phe, Th, Ser Val(V) Leu Ile, Leu, Met, Phe, Ala, Norleucine

By selecting substitutions that differ significantly in their effect on maintaining (a) the polypeptide backbone structure of the substituted region, such as a folded or helical conformation; (b) the charge and hydrophobicity of the molecule at the target site; or (c) side chain volume, Substantial modification of the biological characteristics of antibodies can be achieved. Based on general side chain characteristics, naturally occurring residues are divided into the following groups: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral and hydrophilic: Cys, Ser, Thr; (3) acidic: Asp, Glu; (4) basic: Asn, Gln, His, Lys, Arg; (5) residues that affect the orientation of the chain: Gly, Pro; and ( 6) Aromatic: Trp, Tyr, Phe.

Exchanging members of one of these groups with members of another group results in non-conservative substitutions.

Any cysteine residues unrelated to maintaining the correct conformation of the antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and avoid aberrant crosslinking. Conversely, cysteine linkages can be added to antibodies to increase their stability, especially when the antibody is an antibody fragment (eg, an Fv fragment).

A particularly preferred type of substitutional variant comprises substituting one or more hypervariable region residues of the parent antibody. Generally, the resulting variant selected for further development will have improved biological characteristics compared to the parent antibody from which it was generated.

Most preferred are antibodies modified as described in WO 99/58572, published November 18, 1999. In addition to the binding domain for the target molecule, these antibodies comprise an effector domain having an amino acid sequence substantially homologous to all or a portion of a human immunoglobulin heavy chain constant domain. These antibodies can bind a target molecule without initiating significant complement-dependent lysis, or cell-mediated destruction of the target. Preferably, the effector domain can specifically bind FcRn and/or FcγRIIb. These are generally based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this way are preferred for long-term antibody therapy to avoid inflammation and other side effects of conventional antibody therapy.

Glycosylation variants of an antibody are variants that alter the glycosylation pattern of the antibody. "Alteration" means deletion of one or more carbohydrate moieties found in an antibody, addition of one or more carbohydrate moieties to an antibody, alteration of the combination of glycosylation (glycosylation pattern), expansion of glycosylation, and the like. Glycosylation variants can be prepared, for example, by removing, altering and/or adding one or more glycosylation sites in the antibody-encoding nucleic acid sequence.

Antibodies are glycosylated at conserved positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains of immunoglobulins affect protein function (Boyd et al., 1996, Mol. Immunol. 32: 1311-1318; Wittwe and Howard, 1990, Biochem. 29: 4175-4180) and molecules between glycoprotein moieties Intramolecular interactions can affect the conformation of glycoproteins and the three-dimensional surfaces presented (Hefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416). Oligosaccharides are also used to target a given glycoprotein to certain molecules based on specific recognition structures. Glycosylation of antibodies has been reported to affect antibody-dependent cellular cytotoxicity (ADCC). Specifically, CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosamine transferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisected GlcNAc, have been reported to have enhanced ADCC activity (Umana et al., 1999, Mature Biotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of the asparagine residue. The tripeptide sequences—asparagine-X-serine and asparagine-X-threonine—are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain, where X is anything but proline any amino acid. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, although 5-hydroxyproline or 5-hydroxylysine are also used, but are most common is linked to serine or threonine.

Typically, addition of glycosylation sites to antibodies is accomplished by altering the amino acid sequence so that it contains one or more of the tripeptide sequences described above (N-linked glycosylation sites). The alteration may also be the addition or substitution of one or more serines or threonines to the sequence of the original antibody (O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of antibodies can be prepared by a variety of methods well known in the art. These methods include (but are not limited to) isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or by oligonucleotide-mediated (site-directed) targeting of variants made earlier or non-variant versions of antibodies prepared by mutagenesis, PCR mutagenesis and cassette mutagenesis.

The glycosylation pattern of an antibody can also be altered without altering the underlying nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Because cell types used to express recombinant glycoproteins (e.g., antibodies) as potential therapeutics are rarely native cells, changes in antibody glycosylation patterns can be expected (see, e.g., Hse et al., 1997, J. Biol. Chem. 272 : 9062-9070).

In addition to the choice of host cells, factors affecting glycosylation during recombinant production of antibodies include growth regime, media formulation, culture density, oxygenation, pH, purification protocol, etc. Various methods have been proposed to achieve altered glycosylation patterns in particular host organisms, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (US Pat. Nos. 5,047,335, 5,510,261 and 5,278,299). Glycosylation, or certain types of glycosylation, can be removed enzymatically from a glycoprotein, for example using endoglycosidase H (Endo H). In addition, recombinant host cells can be genetically engineered to be deficient in certain types of polysaccharide processing. These and similar techniques are well known in the art.

A polypeptide contains a signal (or leader) sequence at the N-terminus of the protein, wherein the signal sequence mediates the transfer of the protein either during or after translation. The polypeptide may be conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide (eg, poly-FE), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide can be conjugated to an immunoglobulin Fc region.

Portions and other variants having less than about 100 amino acids, typically about 50 amino acids, can also be produced synthetically, using techniques well known to those of ordinary skill in the art. Such polypeptides can be synthesized, for example, using any commercially available solid phase technique, such as the Merrifield solid phase synthesis method in which amino acids are sequentially added to a growing chain of amino acids. See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963. Equipment for automated peptide synthesis is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, CA) and can be performed according to the manufacturer's instructions.

Using FMOC chemistry with HPTU (O-Benzotriazole N, N, N', N'-tetramethyluronium hexafluorophosphate) (O-Benzotriazole N, N, N', N'-tetramethyluronium hexafluorophosphate) activated, Peptides can be synthesized on a Perkin Elmer/Applied Biosystems Division 430A Peptide Synthesizer. A Gly-Cys-Gly sequence can be added to the amino terminus of a polypeptide to provide a means of conjugating or labeling the polypeptide to an immobilization surface. Polypeptides can be cleaved from the solid support using a cleavage mixture of trifluoroacetic acid:ethylthiol:thioanisole:water:phenol (40:1:2:2:3). After 2 hours of cleavage, the peptides were precipitated in cold methyl tert-butyl ether. The precipitated peptide was then dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized prior to C18 reverse phase HPLC purification. Peptides can be eluted using a gradient of 0% to 60% acetonitrile (with 0.1% TFA) in water. After the purified fraction is lyophilized, the polypeptide can be characterized by amino acid analysis using electrospray or other types of mass spectrometry.

fusion protein

In some embodiments, the polypeptide is a fusion protein comprising multiple polypeptides described herein, or comprising at least one polypeptide described herein and an unrelated sequence. Additional fusion partners can be added.

The fusion partner, for example an immunological fusion partner, assists in providing a T helper epitope, preferably a T helper epitope recognized by humans. As another example, a fusion partner can act as an expression enhancer, assisting in the expression of a protein at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners can be chosen to enhance the solubility of the protein or to target the protein to a desired intracellular compartment. Further fusion partners also include affinity tags to facilitate protein purification.

Fusion proteins are generally prepared using standard techniques, including chemical conjugation. Preferably, the fusion protein is expressed as a recombinant protein in an expression system, which increases the level of production compared to the non-fusion protein. Briefly, DNA sequences encoding polypeptide elements can be assembled independently and ligated into a suitable expression vector. With or without a peptide linker, the 3' end of a DNA sequence encoding one polypeptide element is ligated to the 5' end of a DNA sequence encoding a second polypeptide element such that the reading frames of the sequences are in phase. This allows translation into a single fusion protein that retains the biological activity of both polypeptide elements.

A peptide linker sequence can be used to separate the first and second polypeptide elements by a distance sufficient to ensure that each polypeptide can fold into its secondary and tertiary structures. This polypeptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Selection of an appropriate peptide linker can be based on the following factors: (1) the ability to adopt a flexible, extendable conformation; (2) the absence of a secondary structure that can interact with functional epitopes on the first and second polypeptides; ability; and (3) lack of hydrophobic or charged residues that can react with a functional epitope of the polypeptide. Preferred peptide linkers contain Gly, Asn and Ser residues. Other near-neutral amino acids such as Thr and Ala can also be used in linker sequences. Amino acid sequences useful as linkers include Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; published sequence. Linker sequences are generally 1-50 amino acids in length. A linker sequence is not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent steric interference.

The linked DNA sequences are operably linked to appropriate transcriptional or translational regulatory elements. Regulatory elements responsible for DNA expression are located 5' to the DNA sequence encoding the first polypeptide. Similarly, stop codons and transcription termination signals required to terminate translation occur 3' to the DNA sequence encoding the second or final polypeptide.

Fusion proteins comprising a polypeptide of the invention and an unrelated immunogenic protein are also provided. Preferably, the immunogenic protein elicits a memory response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, eg, Stoute et al., 1997, New Engl. J. Med. 336:86-91).

In a preferred embodiment, the immunological fusion partner is derived from protein D, a surface protein of the Gram-negative bacterium Haemophilus influenzae B (WO 91/18926). Preferably, the protein D derivative comprises about the first third of the protein (eg, the N-terminal first 100-110 amino acids), and the protein D derivative may be lipidated. In certain preferred embodiments, the N-terminus includes the first 109 residues of the lipoprotein D fusion partner to provide the polypeptide with additional foreign T-cell epitopes and to increase expression levels in E. coli (Thus, function as an expression enhancer). The lipid tail ensures optimal presentation of antigens to antigen-presenting cells. Other fusion partners include the nonstructural protein NS1 (hemagglutinin) of influenza virus. Typically the N-terminal 81 amino acids are used, although different fragments including the T-helper epitope can be used.

In another embodiment, the immunological fusion partner is the protein known as LYTA or a portion thereof (preferably, the C-terminal portion). LYTA is derived from Streptococcus pneumoniae which synthesizes an N-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytA gene, Gene 43: 265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is responsible for the affinity for choline or some choline analogs such as DEAR. This property has been exploited to develop the E. coli CLYTA expression plasmid for fusion protein expression. Purification of hybrid proteins containing a C-LYTA fragment at the amino terminus has been described (see, Biotechnology 10:795-798, 1992). In preferred embodiments, repeat portions of LYTA can be incorporated into fusion proteins. A repeat is found in the C-terminal region starting from residue 178. A particularly preferred repeat incorporates residues 188-305.

In general, polypeptides (including fusion proteins) and polynucleotides as described herein are isolated. An "isolated" polypeptide or polynucleotide is one that has been removed from its original environment. For example, a naturally occurring protein can be isolated if it is independent of some or all of the coexisting species in the natural system.

Preferably, such polypeptides are at least about 90% pure, more preferably at least about 95% pure, and most preferably at least 99% pure. For example, a polynucleotide is considered isolated if it is cloned into a vector that is not part of its natural environment.

Polynucleotides and Vectors

The invention further provides isolated polynucleotides encoding the monoclonal antibodies described herein and vectors comprising the polynucleotides, as well as host cells comprising the vectors. Such expression systems can be used in methods of producing immunoreactive polypeptides, such as antibodies of the invention, wherein host cells are cultured and the polypeptide produced by the cultured host cells is recovered. A polynucleotide encoding an antibody of the invention can also be delivered to a host subject so that the antibody is expressed by cells of the host subject. Examples of strategies for the delivery of polynucleotides to the central nervous system of a host subject and the expression of anti-senilin antibodies in the central nervous system of a host subject are described in PCT Application No. W098 published on October 15, 1998 /44955.

The invention also includes polynucleotides that are complementary to any such sequence. A polynucleotide can be single-stranded (coding or antisense) or double-stranded, and can be a DNA (genomic, cDNA, or synthetic) or RNA molecule. RNA molecules include HnRNA molecules that contain introns in a one-to-one manner corresponding to DNA molecules, and mRNA molecules that do not contain introns. A polynucleotide of the invention may, but need not, have additional coding or non-coding sequences, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

A polynucleotide may comprise native sequences (ie, endogenous sequences encoding antibodies or portions thereof), or may comprise variants of such sequences. A polynucleotide variant contains one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not reduced compared to the naturally immunologically active molecule. Generally, the immunoreactivity effect of an encoded polypeptide can be assessed as described herein. Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to the polynucleotide encoding a native antibody or portion thereof.

Two polynucleotide or polypeptide sequences are said to be "identical" if their nucleotide or amino acid sequences are identical when aligned for maximum correspondence as described below. Typically, a comparison between two sequences is accomplished by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A "comparison window" as used herein refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, where optimal alignment of two sequences allows comparison of the sequences with the same number of contiguous positions. Reference sequences for comparison.

Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI) using default parameters. This procedure includes several arrangements described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change inproteins-Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure (National Biomedical Research Foundation, Washington DC Vol.5, Suppl.3), pages 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, pages 626-645, Methods in Enzymology vol.183, Academic Press, Inc ., San Diego, CA; Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5: 151-153; Myers, E.W. and Muller W., 1988, CABIOS 4: 11-17; Robinson, E.D., 1971, Comb. Theor .11: 105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4: 406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press , San Francisco, CA; Wilbur, W.J. and Lipman, DJ., 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions with a reference sequence (containing no deletions or additions) for the optimal alignment of the two sequences For comparison, the portion of the polynucleotide or polypeptide sequence comparison window may contain 20% or fewer additions or deletions (ie, gaps), typically 5-15%, or 10-12%. The number of matching positions is divided by the total number of positions in the reference sequence (i.e., the window size) by determining the number of identical nucleic acid base or amino acid residue positions present in the two sequences that yields the number of matching positions and multiplying the result by 100 to generate a sequence % uniformity, to calculate the percentage.

A variant is also (or alternatively) substantially homologous to a native gene, or a portion thereof, or its complement. Such polynucleotide variants hybridize under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or the complement).

Suitable "moderately stringent conditions" include pre-washing in 5×SSC, 0.5% SDS, 1.0 mMEDTA (pH 8.0) solution; Wash twice in 2×SSC, 0.5× and 0.2×SSC with 0.1% SDS for a total of 20 minutes.

As used herein, "highly stringent conditions" or "high stringency conditions" are (1) washing with low ionic strength and high temperature, for example at 50°C, 0.015M sodium chloride/0.0015M sodium citrate/0.1% dodecyl In sodium alkyl sulfate; (2) Use denaturant during hybridization, such as at 42°C under formamide conditions, for example containing 0.1% bovine serum albumin/0.1% fico/0.1% polyvinylpyrrolidone/50mM containing 750mM chlorine NaCl in sodium phosphate buffer (pH 6.5), 75 mM sodium citrate in 50% (v/v) formamide; or (3) 50% formamide, 5×SSC (0.75M NaCl, 0.075M Sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5×Denhardt solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS and 10% dextran sulfate, in Washes in 0.2X SSC (sodium chloride/sodium citrate) at 42°C and in 50% formamide at 55°C followed by a high stringency wash consisting of 0.1X SSC with EDTA at 55°C. The skilled artisan will recognize how adjustments to temperature, ionic strength, etc. may be necessary to accommodate factors such as probe length.

Those of ordinary skill in the art will appreciate that, because of the degeneracy of the genetic code, there are many nucleotide sequences encoding the polypeptides described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage are specifically contemplated in the present invention. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered by one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard techniques (eg, hybridization, amplification, and/or database sequence comparison).

Polynucleotides can be prepared using any technique known in the art. Antibody-encoding DNA can be obtained from cDNA libraries prepared from tissues expressing antibody mRNA. Antibody-encoding genes can also be obtained from genomic libraries or by oligonucleotide synthesis. Libraries can be screened using probes (eg, binding partners or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein it encodes. Representative libraries include a human liver cDNA library (human liver 5' extended positive strand cDNA, Clontech Laboratories, Inc.) and a mouse kidney cDNA library (mouse kidney 5' extended positive strand cDNA, Clontech Laboratories, Inc.). A cDNA or genomic library can be screened with the selected probes using standard procedures as described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", New York: Cold Spring Harbor Laboratory Press, 1989. Alternatively, the gene encoding the antibody can be isolated using the PCR method (Sambrook et al., supra; Dieffenbach et al., "PCR Primer: A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1995).

The oligonucleotide sequences selected as probes should be sufficiently long and unambiguous to minimize false positives. The oligonucleotides are preferably labeled so that they can be detected after hybridization to DNA in the library being screened. Labeling methods are well known in the art and include the use of radioactive labels (eg ³² P-labeled ATP), biotin or enzymatic labels. Hybridization conditions including medium stringency and high stringency are provided in Sambrook et al. (supra).

Sequences identified by such library screening methods can be compared and aligned with other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at the amino acid or nucleotide level) can be determined within defined regions of a molecule or across the full length of the sequence by computer software programs that employ various algorithms to measure homology.

Nucleic acid molecules with protein coding sequences can be obtained by screening cDNA or genomic libraries of choice and, if necessary, using conventional primer extension procedures as described by Sambrook et al. (supra) to detect mRNAs that may not have been reverse transcribed into cDNA precursors and processing intermediates.

Polynucleotide variants may generally be prepared by any method known in the art, including chemical synthesis, such as solid-phase phosphoramidite chemical synthesis. Modifications can also be introduced into polynucleotide sequences using standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis (see, Adelman et al., DNA 2:183, 1983). Alternatively, RNA molecules can be produced by transcribing the DNA sequence in vitro or in vivo, provided the DNA encoding the antibody or a portion thereof is incorporated into a vector with a suitable RNA polymerase promoter such as T7 or SP6. As described herein, certain portions may be used to prepare encoded polypeptides. Additionally or alternatively, the moiety (nucleic acid) may be administered to the patient for in vivo (e.g., by transfecting antigen-presenting cells, such as dendritic cells, with a cDNA construct encoding the polypeptide, and administering the transfected cells to the patient ) to produce the encoded polypeptide.

Any polynucleotide can be further modified to increase stability in vivo. Possible modifications include (but are not limited to) the addition of flanking sequences at the 5' and/or 3' ends, the use of phosphorothioate or 2' O-methyl groups instead of phosphodiesterase linkages in the backbone, and/or Or include acetyl-, methyl-, thio-, and other modified forms of unusual bases such as inosine, queosine, and wybutosine, as well as adenine, cytidine, guanine, thymine, and uracil.

Using established recombinant DNA techniques, a nucleotide sequence can be joined to a variety of other nucleotide sequences. For example, polynucleotides can be cloned into a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives, and cosmids. Particularly relevant vectors include expression vectors, replication vectors, probe production vectors and sequencing vectors. In general, a vector contains an origin of replication functional in at least one organism, conventional restriction endonuclease sites, and one or more selectable markers. Other elements depend on the desired use and will be apparent to those of ordinary skill in the art.

In certain embodiments, a polynucleotide may be formulated to allow its entry into, and expression within, mammalian cells. Such formulations are particularly useful for therapeutic purposes, as described below. One of ordinary skill in the art understands that there are a variety of methods by which expression of a polynucleotide in a target cell can be achieved, and any suitable method can be employed. For example, polynucleotides can be incorporated into viral vectors such as, but not limited to, adenoviruses, adeno-associated viruses, retroviruses, or vaccinia or poxviruses (eg, fowlpox virus). Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. Retroviral vectors may additionally transfer or incorporate genes as selectable markers (to aid in the identification or selection of transduced cells) and/or targeting moieties (such as genes encoding ligands for receptors on specific target cells) to allow the vector to acquire the target toward specificity. Targeting can also be achieved using antibodies by methods known to those of ordinary skill in the art.

Other formulations for therapeutic purposes include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes . A preferred colloidal system for delivery vehicles in vitro and in vivo is liposomes (ie, artificial membrane vehicles). The preparation and use of such systems are well known in the art.

pharmaceutical composition

The invention provides antibodies, polypeptides and/or polynucleotides for incorporation into pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises an antibody that preferentially binds to amino acids 28-40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the pharmaceutical composition comprises a monoclonal antibody that preferentially binds to an epitope comprising amino acids 39 and/or 40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the pharmaceutical composition comprises a monoclonal antibody that preferentially binds to an epitope comprising amino acids 36-40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the antibody binds to amino acids 28-40 of Aβ peptide (SEQ ID NO: 1) with an affinity of about 60 nM or less, about 30 nM or less, or about 3 nM or less. In some embodiments, the antibody preferentially binds to an epitope comprising amino acids 39 and/or 40 of Aβ peptide (SEQ ID NO: 1) with an affinity of about 60 nM or less, about 30 nM or less, or about 3 nM or more few. In some embodiments, the antibody preferentially binds an epitope comprising amino acids 36-40 of the Aβ peptide (SEQ ID NO: 1) with an affinity of about 60 nM or less, about 30 nM or less, or about 3 nM or less. In some embodiments, the aforementioned antibodies do not exhibit significant cross-reactivity with Aβ _1-41 and/or Aβ _1-42 . In some embodiments, the antibody competitively inhibits the binding of a monoclonal antibody produced by a hybridoma comprising the amino acid sequence set forth in SEQ ID NO 4 and/or 6, or designated 8A1.2A1. In some embodiments, the antibody binds to the same epitope on the Aβ peptide (SEQ ID NO: 1) as the antibody comprising the amino acid sequence shown in SEQ ID NO 4 or 6 or the monoclonal antibody produced by the hybridoma designated 8A1.2A1. In other embodiments, the pharmaceutical compositions comprise human, humanized or chimeric antibodies derived from any of the antibodies described herein. Pharmaceutical compositions comprise one or more such compounds and optionally a physiologically acceptable carrier. Pharmaceutical compositions within the scope of this invention may also contain other compounds, which may or may not be biologically active. In a preferred embodiment, the composition comprises at least two antibodies, a first antibody directed against amino acids 16-28 of the Aβ peptide and a second antibody directed against amino acids 28-40 of the Aβ peptide.

Pharmaceutical compositions may contain DNA encoding one or more of the aforementioned polypeptides, so that the polypeptides are produced in situ. As noted above, the DNA may be present in any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. A variety of gene delivery techniques are well known in the art, as described in Rolland, 1998, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198 and references cited therein. Appropriate nucleic acid expression systems contain the DNA sequences necessary for expression in the patient (eg, suitable promoters and termination signals).

In preferred embodiments, the DNA can be introduced using a viral expression system (eg, vaccinia or other poxviruses, retroviruses, or adenoviruses), which involves the use of non-pathogenic (defective), replication-competent viruses. Suitable systems are described, for example, in Fisher-Hoch et al., 1989, Proc.Natl.Acad.Sci.USA 86:317-321; Flexner et al., 1989, Ann.N.Y.Acad Sci.569:86-103; Flexner et al., 1990, Vaccine 8:17-21; U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487, WO 89/01973, U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242, WO 91/02805, Berkner-Biotechniques 6:616-6827, etc.; , 1991, Science 252:431-434; Kolls et al, 1994, Proc.Natl.Acad.Sci.USA 91:215-219; Kass-Eisler et al, 1993, Proc.Natl.Acad.Sci.USA 90:11498- 11502, Guzman et al., 1993, Circulation 88: 2838-2848 and Guzman et al., 1993, Cir. Res. 73: 1202-1207. Techniques for introducing DNA into such expression systems are well known to those of ordinary skill in the art. DNA can also be naked as described in reviews by Ulmer et al., 1993, Science 259: 1745-1749 and Cohen, 1993, Science 259: 1691-1692. Uptake of naked DNA can be increased by coating the DNA on biodegradable beads that are efficiently delivered to cells.

Although any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of the present invention, the type of carrier will vary depending on the mode of administration. Compositions of the invention may be formulated for any suitable mode of administration including, for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, ethanol, fat, wax or buffer. For buccal administration, any one of the above carriers or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose and magnesium carbonate may be used. Biodegradable microspheres (such as polylactic acid and polyglycolic acid) can also be used as carriers of the pharmaceutical composition of the present invention. Suitable biodegradable microspheres are disclosed, for example, in US Patent Nos. 4,897,268 and 5,075,109.

Such compositions may also contain buffers (such as neutral buffered saline or phosphate buffered saline), carbohydrates (such as glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids (such as glycine ), antioxidants, chelating agents (such as EDTA or glutathione), adjuvants (such as aluminum hydroxide) and/or preservatives. Alternatively, compositions of the invention may be formulated as lyophilized powders. Compounds can also be encapsulated within liposomes using well known techniques.

The compositions described herein may be administered as part of a sustained release formulation (ie, a formulation such as a capsule or sponge that results in slow release of the compound after administration). Such formulations are generally prepared using well known techniques and administered, for example, by oral, rectal or subcutaneous implantation or by implantation at the desired target site. Sustained release formulations may contain the polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained in a depot surrounded by a rate controlling membrane. The carrier used in such formulations is biocompatible, and also biodegradable, preferably the formulation provides a relatively constant level of release of the active ingredient. The amount of active ingredient contained in a sustained release formulation will depend upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

Treatment and Prevention

Antibodies (including polypeptides), polynucleotides and pharmaceutical compositions of the present invention can be used to treat, prevent and inhibit Alzheimer's disease and other diseases associated with Aβ or βAPP expression changes, or Aβ peptide accumulation (such as Down syndrome syndrome, Parkinson's disease and multi-infarct dementia). Such methods comprise administering to a subject an immunoreactive molecule, antibody (including polypeptide), polynucleotide or pharmaceutical composition. In prophylactic applications, a pharmaceutical composition or medicament is administered to a patient susceptible to or at risk of developing Alzheimer's disease, wherein the amount of the pharmaceutical composition or medicament is sufficient to eliminate or reduce the risk, alleviate Severity or delayed onset of disease (including biochemical, histological and/or behavioral symptoms of disease, its complications and intermediate pathological phenotypes present during disease development). In therapeutic applications, a composition or medicament is administered to a patient suspected of or already suffering from such a disease, wherein the amount of the pharmaceutical composition or medicament is sufficient to cure or at least partially control the disease (including its complications and intermediates in the progression of the disease) Phenotype) (biochemical, histological and/or behavioral) symptoms.

The present invention also provides a method of delaying the development of Alzheimer's disease-related symptoms in a patient, comprising administering to the subject an effective dose of a pharmaceutical composition comprising an antibody (including polypeptide) or polynucleotide of the present invention. Symptoms associated with Alzheimer's disease include, but are not limited to, abnormalities in memory, problem solving, language, calculation, vision, judgment, and behavior.

The present invention also provides a method of inhibiting or suppressing the formation of amyloid plaques in a subject, comprising administering to the subject an effective dose of the pharmaceutical composition of the present invention. In some embodiments, the amyloid plaques are located in the subject's brain.

The invention also provides a method of reducing amyloid plaques in a subject comprising administering to the subject an effective amount of a pharmaceutical composition of the invention. In some embodiments, the amyloid plaques are located in the subject's brain.

The invention also provides a method of removing or clearing amyloid plaques in a subject comprising administering to the subject an effective amount of a pharmaceutical composition of the invention. In some embodiments, the amyloid plaques are located in the subject's brain.

The present invention also provides for reducing Aβ peptides in tissues (such as the brain), inhibiting and/or reducing the accumulation of Aβ peptides in tissues (such as the brain), and inhibiting and/or reducing the toxic effects of Aβ peptides in tissues (such as the brain) of a subject. A method comprising administering to a subject an effective dose of a pharmaceutical composition of the present invention.

The methods described herein (including prophylactic or therapeutic methods) can be accomplished by unidirectional injection at a single time point or multiple time points at a single site or multiple sites. Administration to multiple sites at approximately the same time is also possible. The frequency of dosing can be determined and adjusted during the course of treatment according to the desired result to be achieved. In some cases, sustained continuous release formulations of the antibodies (including polypeptides), polynucleotides and pharmaceutical compositions of the invention may be appropriate. Various formulations and devices for achieving sustained release are well known in the art.

Patients, subjects or individuals include mammals such as humans, cattle, horses, dogs, cats, pigs and sheep. Preferably the subject is a human being and may or may not be afflicted with or about to exhibit symptoms of a disease. As far as Alzheimer's disease is concerned, virtually anyone is at risk of developing Alzheimer's disease if he or she lives long enough. Thus, the method can be administered prophylactically to the general population without any assessment of risk to the subject patient. The methods of the invention are useful for individuals known to be at genetic risk for Alzheimer's disease. Such individuals include those whose relatives have already experienced the disease, as well as those whose risk has been determined by analysis of genetic or biochemical markers. Genetic hallmarks of risk for Alzheimer's disease include mutations in the APP gene, particularly at position 717 and at positions 670 and 671, known as the Hardy and Swedish mutations, respectively (see Hardy (1997) Trends Neurosci. 20: 154- 9). Other risk markers are mutations in the presenilin genes, PS1 and PS2, and ApoE4, family history of AD, hypercholesterolemia, or atherosclerosis. Individuals to be afflicted with Alzheimer's disease can be identified on the basis of the characteristic dementia and the presence of the aforementioned risk factors. In addition, several diagnostic tests are available to identify individuals with AD. These include measuring levels of CSF tau and Aβ42. Elevated Tau and decreased Aβ42 levels imply the presence of AD. Individuals with Alzheimer's disease can also be diagnosed by ADRDA (Alzheimer's Disease and Related Disorders Association) criteria. In asymptomatic patients, treatment can be initiated at any age (eg, 10, 20, 30). Often, patients do not necessarily start treatment until they are 40, 50, 60 or 70 years old. Often multiple doses of treatment are given over a period of time. Treatment can be monitored by a variety of methods known in the art over time. For potential Down syndrome patients, treatment can be initiated before birth, or shortly after birth, by administering a therapeutic agent to the mother.

Pharmaceutical compositions that can be used in the above methods include (but are not limited to) antibodies that preferentially bind to amino acids 28-40 of Aβ peptide (SEQ ID NO: 1), preferentially bind to amino acids including Aβ peptide (SEQ ID NO: 1) 39 and/or 40 An antibody that binds to an epitope of an amino acid at position 28-40 of Aβ peptide (SEQ ID NO: 1) with a binding affinity of about 60 nM or less, 30 nM or less or 3 nM or less antibody, preferentially associated with Aβ Antibodies that bind to an epitope at amino acid 39 and/or 40 of the peptide (SEQ ID NO: 1) with a binding affinity of about 60 nM or less, 30 nM or less, or 3 nM or less, and antibodies encoding any of the antibodies described herein and polypeptide polynucleotides. In other embodiments, antibodies that bind to an epitope comprising amino acids 39 and/or 40 of the Aβ peptide (SEQ ID NO: 1 ) but do not show significant cross-reactivity with Aβ _1-42 and Aβ _1-43 can be used Antibodies, Fab fragments of antibodies with a binding affinity of about 200 nM or less, or 1 nM or less, to the Aβ _1-40 peptide (SEQ ID NO: 1), competitively inhibit the amino acid sequence comprising SEQ ID NO: 4 and 6 An antibody that binds to the Aβ _1-40 peptide (SEQ ID NO: 1), an antibody that binds to the same epitope as a monoclonal antibody comprising the amino acid sequences of SEQ ID NO: 4 and 6, and any of the above-mentioned characteristics Combined Antibodies.

Administration and Dosage

Preferably, the antibody is administered to the mammal in a carrier, preferably a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington, Pharmaceutical Sciences, 18th ed., A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990 and Remington, The Science and Practice of Pharmay, 20th ed. Mack Publishing, 2000. In general, appropriate amounts of pharmaceutically acceptable salts are used in the formulation to render the formulation isotonic. Examples of carriers include saline solution, Ringer's solution and dextran solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. Further carriers include sustained-release formulations, such as semipermeable matrices of solid-phase hydrophobic multimers containing antibodies, where the matrices are in the form of shaped objects such as films, liposomes or microparticles. It will be apparent to those skilled in the art that certain carriers are more preferred depending, for example, on the route of administration and the concentration of antibody administered.

Antibodies can be administered to mammals by injection (eg, systemic, intravenous, intraperitoneal, subcutaneous, intramuscular, intraportal) or by other methods such as infusion to ensure delivery to the bloodstream in an effective form. Antibodies can also be administered by isolated perfusion techniques (eg, isolated tissue perfusion) to exert a localized therapeutic effect. Intravenous injection is preferred.

Effective dosages and regimens for administering antibodies can be determined empirically, and making such determinations is within the skill of the art. Those skilled in the art will understand that the dose of antibody that must be administered will vary depending on, for example, the mammal receiving the antibody, the route of administration, the particular type of antibody used, and other drugs administered to the mammal. Guidance for selecting appropriate antibody doses can be found in the literature on the therapeutic use of antibodies, eg, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, 1985, ch, 22 and pp. 303-357; Smith et al. , "Antibody in Human Diagnosis and Therapy", edited by Haber et al., Raven Press, New York, 1977, pages 365-389.

Usual daily dosages of antibodies alone are about 1 μg/Kg daily up to 100 mg/Kg body weight, or more, depending on the factors mentioned above. Generally, any of the following doses can be used: administering a dose of at least about 50 mg/Kg body weight, a dose of at least about 10 mg/Kg body weight, a dose of at least about 3 mg/Kg body weight, a dose of at least about 1 mg/Kg body weight, at least about 750 μg A dose per Kg body weight, a dose of at least about 500 μg/Kg body weight, a dose of at least about 250 μg/Kg body weight, a dose of at least about 100 μg/Kg body weight, a dose of at least about 50 μg/Kg body weight, a dose of at least about 10 μg/Kg body weight , a dose of at least about 1 μg/Kg body weight, or more.

In some embodiments, more than one antibody may be presented. Such compositions may contain at least one, at least two, at least three, at least four, at least five different antibodies (including polypeptides) of the invention.

Antibodies can also be administered to a mammal in combination with one or more effective amounts of other therapeutic agents. Antibodies can be administered sequentially or simultaneously with one or more other therapeutic agents. The amounts of antibody and therapeutic agent depend on, for example, the type of drug used, the pathological condition being treated, and the dosing regimen and route, but are generally less than each used alone.

Following administration of antibodies to a mammal, the physiological condition of the mammal can be monitored in a variety of ways well known to the skilled artisan.

The dosing principles and dosages described above are applicable to the polypeptides described herein.

Polynucleotides encoding antibodies (including polypeptides) of the invention can also be used to deliver and express antibodies or polypeptides in cells of interest. Obviously, expression vectors can be used for direct expression of antibodies. The expression vector can be administered systemically, intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraventricularly, orally, enterally, parenterally, intranasally, intradermally or by inhalation. For example, administration of expression vectors includes local or systemic administration, including injection, oral administration, gene gun or administration with a catheter, and topical administration. Those skilled in the art are familiar with the administration of expression vectors to obtain in vivo expression of foreign proteins. See, eg, US Patent Nos. 6,436,908, 6,413,942, and 6,376,471.

Targeted delivery of therapeutic compositions comprising polynucleotides encoding antibodies of the invention may also be used. Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al., Trends Biotechnol, (1993) 11:202; Chiou et al., "Gene Therapeutics Methods And Applications Of Direct Gene Transfer" (ed. J.A. Wolff) (1994); Wu et al., J. Biol. Chem. (1998) 263: 621; Wu et al., J. Biol. Chem. (1994) 269: 542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990) 87: 3655; Wu et al., J. Biol. Chem. (1991) 266:338. In a gene therapy protocol, the topical administration of a polynucleotide-containing therapeutic composition ranges from about 100 ng to about 200 mg of DNA. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg can also be used in gene therapy protocols. Therapeutic polynucleotides and polypeptides of the invention can be delivered using gene delivery means. Gene delivery vehicles can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185 and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences is induced using endogenous mammalian or heterologous promoters. Expression of coding sequences may be constitutive or regulatory.

Viral-based vectors for delivery and expression of polynucleotides of interest in cells of interest are well known in the art. Representative viral-based delivery vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. /02805; U.S. Patent Nos. 5,219,740, 4,777127; GB Patent No. 2,200,651 and EP0345242), alphavirus-based vectors (e.g., Sindbis virus vectors, Simonleick Forest virus (ATCC VR-67, ATCC VR-1247), Ross River virus (ATCC VR-373, ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923, ATCC VR-1250, ATCC VR-1249ATCC VR-532) and adeno-associated virus (AAV) vectors (see, e.g. PCT Publication Nos. WO 94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984 and WO 95/00655). Also available as Curiel, Hum. Gene Ther. (1992) 3:147 Said administration is linked to the DNA of an inactivated adenovirus.

Non-viral delivery tools and methods can also be utilized, including, but not limited to, polycation-enriched DNA linked or unlinked to inactivated adenovirus (see, e.g., Curiel, Hum. GeneTher. (1992) 3:147), Ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985), eukaryotic cell delivery tool cells (see, e.g., U.S. Pat. No. 5,814,482, PCT Publication Nos. WO 95/07994, WO 96 /17072, WO 95/30763 and WO97/42338) and nuclear charge neutralization or fusion with cell membranes. Naked DNA can also be used. Representative naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Patent No. 5,580,859. Liposomes useful as gene delivery vehicles are described in U.S. Patent No. 5,422,120, PCT Publication Nos. WO 95/13796, WO 94/23697, WO 91/14445 and EP0524968. Additional methods are described in Philip, Mol. Cell Biol. (1994) 14:2411 and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

Diagnostic Use of Antibodies

The antibodies of the present invention are useful in the detection, diagnosis and monitoring of Alzheimer's disease and other diseases associated with altered expression of Aβ or βAPP, such as Down's syndrome. The method comprises contacting a patient sample suspected of having altered expression of Aβ or βAPP with an antibody of the invention and determining whether the level of Aβ or βAPP is different from a control or comparative sample.

For diagnostic applications, antibodies are typically labeled with a detectable moiety including, but not limited to, radioisotopes, fluorescent labels, and various enzyme-substrate labels. Methods of conjugating labels to antibodies are well known in the art. In other embodiments of the invention, the antibodies of the invention need not be labeled and their presence can be detected using a labeled antibody that binds the antibodies of the invention.

The antibody of the present invention can be applied to any known assay method, such as competition binding assay, direct and indirect sandwich assay, and immunoprecipitation assay. Zola, Monoclonal Antibodies: A Technical Handbook, pp. 147-158 (CRC Press, Inc. 1987).

Antibodies can also be used in in vivo diagnostic assays, such as in vivo imaging. Typically, antibodies are labeled with a radionuclide (eg, ¹¹¹ In, ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ¹²⁵ I, or ³ H) in order to localize target cells or tissues using immunoscintiography.

Antibodies can also be used as staining reagents in pathology, according to techniques well known in the art.

Reagent test kit

In a further embodiment, the invention provides articles of manufacture and kits comprising materials useful for the treatment of pathological diseases such as Alzheimer's disease, Down's syndrome, or for the detection or purification of Aβ or βAPP. Or other diseases associated with altered expression of Aβ or βAPP. The finished product consists of a labeled container. Suitable containers include, for example, bottles, vials and test tubes. Containers can be made of different materials such as glass or plastic. The container contains a composition having an active agent effective for treating a pathological disease or detecting or purifying Aβ or βAPP. The active agent in the composition is an antibody, and preferably comprises a monoclonal antibody specific for Aβ or βAPP. In some embodiments, the active agent is an antibody that binds to an epitope in amino acids 28-40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the antibody preferentially binds to an epitope spanning amino acids 38-40 of the Aβ peptide (SEQ ID NO: 1). In some embodiments, the antibody preferentially binds to amino acids 28-40 of the Aβ peptide (SEQ ID NO: 1) with an affinity of about 60 nM or less, about 30 nM or less, about 3 nM or less. In some embodiments, the antibody preferentially binds an epitope comprising amino acids 39 and/or 40 of the Aβ peptide (SEQ ID NO: 1 ) with an affinity of about 60 nM or less, about 30 nM or less, 3 nM or less. In some embodiments, the antibody competitively inhibits the binding of a monoclonal antibody comprising the amino acid sequence set forth in SEQ ID NO 4 and/or 6, or a monoclonal antibody produced by a hybridoma designated 8A1.2A1. In some embodiments, the antibody is identical to an antibody containing the amino acid sequence shown in SEQ ID NO4 and/or 6, or a monoclonal antibody produced by a hybridoma named 8A1.2A1 that binds to the Aβ peptide (SEQ ID NO: 1). gauge. In some embodiments, the active agent comprises any humanized, chimeric, or human antibody described herein. The label on the container indicates that the composition is for treating a pathological disease such as Alzheimer's disease or detecting or purifying A[beta] or [beta]APP, and may also refer to in vivo or in vitro use, as described above.

In some embodiments, kits of the invention comprise the containers described above. In other embodiments, a kit of the invention comprises the container described above and another container containing a buffer. Other materials, including other buffers, diluents, filters, needles, syringes, and other materials with instructions for performing any of the methods described herein (such as methods for treating Alzheimer's disease, and A packaging insert for instructions for methods of inhibiting or reducing accumulation of Aβ peptides in the brain). In kits for the detection or purification of Aβ or βAPP, antibodies are typically labeled with a detectable marker, such as a radioisotope, fluorescent compound, bioluminescent compound, chemiluminescent compound, metal chelator or enzyme.

Example

The following examples are presented to illustrate the invention and to assist those of ordinary skill in making and using the invention. These examples are not meant to otherwise limit the scope of the invention in any way.

Example 1: Generation and Characterization of Monoclonal Antibodies Against Aβ

The data presented in this example demonstrates that high affinity, specific antibodies against Aβ can be generated and provide beneficial therapeutic agents targeting Aβ-related diseases. Mice were immunized with 50-100 μg of Aβ _1-40 peptide in Ribi adjuvant (50 μl per footpad, 100 μl total per mouse) at 10 consecutive weekly intervals as in Geerligs HJ et al., 1989, J. Immunol. Methods 124: 95-102, Kenney JS et al., 1989, J. Immunol. Methods 121: 157-166 and Wicher K et al., 1989, Int. Arch. Allergy Appl. Immunol. 89: 128-135.

Splenocytes were obtained from immunized mice and fused with NSO myeloma cells at a ratio of 10:1 in the presence of polyethylene glycol 1500. Hybridoma cells (hybrids) were seeded in 96-well plates in DMEM containing 20% horse serum and 2-oxaloethyl/pyruvate/insulin (Sigma), and hypoxanthine/aminopterin/thymidine selection was initiated. On day 8, 100 μl of DMEM containing 20% horse serum was added to all wells. Supernatants of hybridoma cells were screened by using an antibody capture immunoassay. Determination of antibody class can be performed using a class-specific secondary antibody.

A panel of monoclonal antibody producing cell lines was selected for their characterization. These cell lines and information describing the corresponding antibodies are listed in Table 2.

Table 2: Characteristics of Monoclonal Antibodies Antigen Aβ Monoclonal Antibody Producing Cell Lines isotype gauge ELISA Affinity Kd(nM) cross reactivity direct capture 2286 8A1.2A1 IgG1 Aβ28-40 yes yes 2.7 no 2287 11A4.1E5 IgG2a Aβ16-28 yes yes 59 no 2288 23E9.1A1 IgG2b Aβ1-16 yes yes ND no 2289 3C6.1F9 IgG2b Aβ16-28 yes yes 2.9 no 2290 14E10.1F3 IgG2a Aβ16-28 yes yes 9.2 no 2294 13E11.1A12 IgG2b Aβ28-40 yes yes 38 no 2324 10B10.2E6 IgG2b Aβ-1-16 yes ND 0.9 no

Binding to A[beta] and [beta]APP of different origins as well as control peptides was tested in a sandwich assay. The Aβ peptides tested were: β-amyloid peptides 1-42 and 1-40 obtained from Calbiochem (San Diego, CA), and β-amyloid peptide 1-43 obtained from Bachem (Torrance, CA). Peptides were immobilized on plates, antibodies added, and their binding detected using GAMIgG(Fc)HRP and absorbance read at 490nm. The cross-reactivity described in Figure 1 demonstrates that mAbs against Aβ do not cross-react with βAPP.

Capture assays were performed to confirm that the antibodies capture soluble Aβ peptides. In this assay, Aβ peptides were immobilized on assay plates, mAbs were added directly or after pre-incubation with 10 μg/ml Aβ, and binding was detected using GAMIgG(Fc) HRP and absorbance reading at 490 nm. Controls were mouse anti-βAPP and monoclonal antibody 6E10 detecting amino acid residues 1-17 of the human β-amyloid peptide (Signet, Dedham, MA). Data showing capture of soluble Aβ are shown in Figure 2.

The ability of candidate therapeutic antibodies to effectively reduce plaque burden in the central nervous system in vivo can be assayed ex vivo as described by Bard et al., 2000, Nature Medicine 6(8):916-919.

Example 2: Characterization of epitopes on Aβ polypeptides bound by antibodies against Aβ

To determine the epitopes on the Aβ polypeptide recognized by the monoclonal antibodies, surface plasmon resonance (SPR, Biacore 3000) binding assays were used. Aβ _1-40 polypeptide (SEQ ID NO: 1 ) conjugated with biotin (Global Peptide Services, CO) was immobilized to streptavidin-coated plates. Aβ antibodies (100 nM) bound to immobilized Aβ _1-40 in the absence or presence of different soluble Aβ peptide fragments (1000 nM, from American Peptide Company Inc., CA). It is necessary to replace the Aβ peptides of the monoclonal antibodies 2324, 2289 and 2286 (more specifically, these antibodies were isolated from their respective hybridoma cell lines 10B10.2E6, 3C6.1F9 and 8A1.2A1) that bind to Aβ _1-40 These are Aβ _1-16 , Aβ _1-28 and Aβ _1-40 , respectively ( FIG. 3 ). Soluble Aβ _1-40 inhibited the binding of all three antibodies to Aβ _1-40 . However, the _Aβ1-38 peptide inhibited the binding of _Aβ1-40 to MAb 2324 and 2289, but not to MAb 2286, suggesting that the epitope bound by MAb 2286 includes amino acids 39 and/or 40 of the _Aβ1-40 peptide (image 3).

Furthermore, _Aβ1-42 and _Aβ1-43 peptides did not inhibit the binding of MAb 2286 to _Aβ1-40 , although they readily inhibited the binding of _Aβ1-40 to MAbs 2324 and 2289 (Figure 3). These results show that MAb2286 preferentially binds to Aβ _1-40 rather than Aβ _1-42 and Aβ _1-43 .

To further evaluate the discrete amino acid residues involved in the binding of Mab 2286 to the β-amyloid peptide, the last 5 amino acids (amino acid residues 36-40 of Aβ _1-40 ) were generated by site-directed mutagenesis, respectively, with alanine (alanine Amino acid scanning mutagenesis) substituted different _Aβ1-40 variants. These _Aβ1-40 variants (sequences shown in Table 6) were expressed in E. coli as glutathione S-transferase (GST) fusion proteins (AmershamPharmacia Biotech, Piscataway, NJ USA) and subsequently expressed in glutathione Affinity purification was performed on agarose beads (Sigma-Aldrich Corp., St. Louis, MO, USA). As controls, wild type (WT) _Aβ1-40 (SEQ ID NO: 1) and _Aβ1-41 (SEQ ID NO: 13) were also expressed as GST fusion proteins. Then, Aβ _1-40 , Aβ _1-41 and 5 different variants (SEQ ID NO: 14-18) were immobilized on the assay plate (0.25 μg per well) and mixed with Mab 2286 or Mab 2324 (for Aβ respectively). _1-40 amino acid 28-40 or 1-16 epitope, 2nM per antibody) incubation. After 10 consecutive washes, the assay plate was incubated with biotin-conjugated goat anti-mouse (H+L) antibody (Vector Laboratories, Burllingame CA, USA), followed by HRP-conjugated streptavidin (Amersham Biosciences Corp., NJ, USA) incubation. The absorbance of the plate was read at 450nm.

As shown in Figure 8, Mab 2324 directed against the Aβ N-terminal epitope recognized all variants with equal intensity and served as an internal positive control for protein concentration and protein integrity on the plate. Mab2286 does not recognize _Aβ1-41 (or _Aβ1-42 as shown in Figure 3), and mutation of V40 at the C-terminus to Ala did not affect binding, suggesting that the aminocarboxy-terminal part of the protein may be directly associated with the Mab 2286 epitope , while the side chain of V40 may be less important. The _Aβ1-40 variants V39A, G38A, G37A and V36A showed reduced binding to Mab 2286, suggesting that the Mab 2286 epitope extends by at least 5 amino acids at the C-terminus of _Aβ1-40 . Mutations of V and G to A are very conservative and are unlikely to produce important conformational changes in the protein, therefore, the large effect of these mutations on Mab 2286 binding may be due to the ability of the antibody to distinguish amino acids between the aforementioned amino acids in Aβ ability, and these data demonstrate that this antibody has very high specificity.

Example 3: Generation and Characterization of Monoclonal Antibodies Against βAPP

Mice were immunized with APP as described in Example 1. A panel of monoclonal antibody producing cell lines was selected for characterization. These cell lines and information describing the corresponding antibodies are listed in Table 3.

Table 3: Monoclonal Antibody Characteristics Antigen APP monoclonal producer cell line isotype ELISA Affinity Kd(nM) cross reaction direct capture 2312 25E12.1F9.1H8 (BP26) IgG1 yes ND ND none 2313 24H4.2E10.1F5(BP27) IgG1 yes ND ND none 2334 1F10.8E6.2A2 (BP80) IgG2b yes ND ND none 2335 13E12.1C5 (BP81) IgG2b yes ND ND none 2336 14D9.1G8 (BP82) IgG1 yes ND ND none

Example 4: Humanization of Monoclonal Antibody 2286

Mouse antibody 2286 was humanized by implanting the heavy chain CDRs (Kabat and/or Chothia) into human germline receptor sequences VH3 and VH4, and the light chain Kabat CDRs into human germline receptor sequence 08. The humanized heavy and light chains of antibody 2286 are shown in Table 4 below.

Table 4: Amino acid sequences of heavy and light chains of humanized antibodies Humanized 2286 light chain variable domain (germline framework O8) DIQMTQSPSSLSASVGDRVTITC SASQGISNYLN WYQQKPGKAPKLLIY YTSSLHS GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYRKLPYT FGGGTKVEIKR (SEQ ID NO: 13) Humanized 2286 heavy chain variable domain (germline framework VH3) EVQLVESGGGLVQPGGSLRLSCAASgfdf srYWMN WVRQAPGKGLEWVS EINPDSSTINYTPSLKD RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR QMGY WGQG TTLTVSS (SEQ ID NO: 14) Humanized 2286 heavy chain variable domain (germline framework VH4) QVQLQESGPGLVKPSETLSLTCTVSgfdf srYWMN WIRQPPGKGLEWIG EINPDSSTINYTPSLKD RVTISKDTSKNQFSLKLSSVTAADTAVYYCAR QMGY WGQGTLV TVSS (SEQ ID NO: 15)

*CDR boundaries of antibody heavy and light chains are determined by Kabat names (marked by underlining) except for CDRH1, where Kabat and Chothia (lower case) names in CDRH1 are used to define CDR boundaries.

Equilibrium dissociation constant ( _KD ) values for anti-Aβ monoclonal antibody Fab fragments were determined by the "steady state" method using the BIAcore3000 ^™ Surface Plasmon Resonance (SPR) system described above (BIAcore, INC, Piscaway NJ). Kinetic association rates (kon) and dissociation rates ( _Koff ) were obtained simultaneously by fitting the data to a 1:1 Langmuir binding model (Lofas & Johnsson, 1990) using a computational BIA evaluation program. The affinities of the humanized antibody and mouse antibody 2286 are shown in Table 5 below.

Table 5: Binding Affinities of Humanized Antibodies K0ff(S ^-1 ) Kon(M ^-1 、S ^-1 ) K _D (nM) mouse2286Fab 0.044 5.8×10 ⁴ 76 Humanized 2286Fab (O8; VH3) ND ND 500 Humanized 2286Fab (O8; VH4) ND ND 500

Example 5: Antibodies against Aβ peptides reduce histological symptoms in an animal model of Alzheimer's disease

This example demonstrates that the monoclonal antibodies of the invention provide effective therapeutic agents for the treatment and prevention of Alzheimer's disease. Surprisingly, these data show that antibodies against the C-terminus of Aβ (i.e. amino acids 28-40) are as effective as antibodies against the N-terminus (amino acids 1-16) in a mouse model of Alzheimer's disease Clears Aβ, Thioflavin-S and increases MHC-II staining. Since antibodies targeting the N-terminus of Aβ may be beneficial due to their increased ability to recognize precursors and/or interfere with aggregation of amyloid deposits, these results provide a promising new therapeutic strategy for the treatment of Alzheimer's disease.

To evaluate the therapeutic effect of anti-Aβ antibodies in vivo, monoclonal antibodies 2324, 2286 and 2289, more specifically, antibodies isolated from their respective hybridoma cell lines 10B10.2E6, 8A1.2A1 and 3C6.1F9, were injected with Transgenic mice overexpressing "Swedish" mutant amyloid precursor protein (APP; Tg2576; K670N/M671L; Hsiao et al., 1996, Science 274: 99-102). The Alzheimer's disease-like phenotype that occurs in these mice has been well described (Holcomb LA et al., 1998, Nat.Med.4:97-100, Holcomb LA et al., 1999, Behav.Gen.29 : 177-185; and McGowan E, 1999, Neurobiol. Dis. 6: 231-244).

Antibodies were injected intracranially into 16-month-old Tg2576 transgenic mice following the following experimental procedures, which are not strictly necessary to describe or to make the invention workable. Antibodies injected were monoclonal antibodies 2324 (1.2 μg in a 2 μl volume), 2286 (2 μg in a 2 μl volume) and 2289 (2 μg in a 2 μl volume) and a control monoclonal against a Drosophila protein called “amnesia” Antibodies (2 μg in a 2 μl volume), specifically antibodies isolated from their respective hybridoma cell lines 10B10.2E6, 8A1.2A1 and 3C6.1F9, were injected intracranially. Histopathology of the mouse frontal cortex and hippocampus was evaluated 3 days after injection. A three day time point was chosen from the time course work where another antibody showed complete clearance of starch at this time interval and its microglial activity was maximal compared to 1 day or 7 days. Data are expressed as the ratio of injected and non-injected sides stained for Aβ, Thioflavin-S and MHC-II.

Although the three antibodies used in this study were directed against different parts of the Aβ peptide (amino acids 1-16, 16-28, and 28-40, respectively), they Both removed A[beta] and Thioflavin-S deposits in the hippocampus and cortex (the latter detecting the toxic fibril form of A[beta] deposits) in large proportions (Fig. 4, 40-80%). They also activated microglia, as assessed by MHC-II staining (Figure 4). There were no consistent differences between every pair of the three Aβ antibodies in their ability to remove Aβ, Thioflavin-S or increase MHC-II staining in these mice. These results of the present invention are unexpected in view of previously published studies showing that antibodies directed against the N-terminus (i.e. amino acids 1-16) but not the C-terminus (i.e. amino acids 28-40) are critically important for the clearance of Aβ precipitates (Solomon , B et al., 1996, Proc.Natl.Acad.Sci.USA 93:452-455, Solomon, B et al., 1997, Proc.Natl.Acad.Sci.USA 94:4109-4112.).

Example 6: Potential role of antibody 2286 Fc domain in microglial activation and amyloid clearance

To investigate the potential role of the Fc domain of anti-Aβ antibody 2286 in microglial activation and amyloid clearance, the F(ab')2 fragment of antibody 2286, intact antibody Comparisons were made to a control monoclonal antibody directed against the Drosophila amnesia protein in the animal model described in Example 6, wherein the antibody was administered intracranially.

Preparation of F(ab')2 fragments

The F(ab')2 fragment of anti-Aβ monoclonal antibody 2286 and a control monoclonal against Drosophila amnesia protein were prepared using the immunopure IgG1 Fab and F(ab')2 preparation kit (Pierce Biotechnology, Rockford, IL) Antibody. The following are the instructions provided with the kit. Briefly, 0.5 ml of 1 mg/ml IgG was added to a gentle eluate of 0.5 ml of mouse IgG1. This was used to equilibrate the immobilized ficin column, allowed to enter the column, and digested at 37°C for 20 hours. 4 ml of the eluate was obtained and used on an equilibrated immobilized protein A column to separate F(ab')2 from Fc fragments and undigested IgG. Four 1 ml product fractions were obtained. Only the second and third eluting fractions were found to contain F(ab')2 fragments, as determined by gel electrophoresis, and presented similar intensities on the gel. The two eluted fractions containing F(ab')2 fragments were pooled and concentrated to a volume of approximately 200 μl using a Centricon centrifugal filter device (Millipore Corp. Bedford, MA). Preliminary experiments found that injection of the F(ab')2 fraction concentrated directly from the column resulted in seizures when injected into some mice. Therefore, the initial concentrate was diluted with 4 ml of fresh PBS and reconcentrated to dilute residual elution buffer components that could cause seizures. No seizures or neurotoxicity were found in the mice included here. Concentrated products were subjected to electrophoresis on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bradford assays were performed using Bradford Protein Assay Reagent Concentrate (Bio-Rad, Hercules, CA) to establish the concentration of F(ab')2 fragments.

The F(ab')2 fragment of anti-Aβ mAb 2286 and a control mAb directed against Drosophila amnesia protein were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). The gel showed a very pure product uncontaminated by undigested IgG or Fc fragments as a single band at approximately 105 kDa, which is the molecular weight of an F(ab')2 fragment. Intact IgG molecules yield an intense band at about 150 kDa, the correct molecular weight for an IgG molecule, and a less intense band at about 110 kDa. After confirming its purity by SDS-PAGE, we then performed a Bradford assay to evaluate the recovery of F(ab')2 in the purified fractions. Because we solubilized anti-Aβ F(ab')2 fragments in a smaller volume than used for the starting material, the concentration of F(ab')2 fragments injected intracranially was 1.2 μg/μl, whereas the concentration of intact antibody The concentration was 1 μg/μl, which resulted in an excess of anti-Aβ Fv domain in the F(ab')2 solution.

Antibody Fraction Research

Twenty 19.5-month-old Tg2576APP transgenic mice were divided into 4 groups, and all groups received intracranial injections into the frontal cortex and hippocampus. The first group received anti-Aβ antibody 2286 at a concentration of 2 μg/2 μl at each site. The second group received anti-Aβ F(ab')2 fragment prepared from anti-Aβ antibody 2286 at a concentration of 2.2 μg/2 μl at each site. As a non-specific control for intact IgG injections, a third group received IgG directed against the Drosophila amnesia protein. The last group received a control F(ab')2 fragment prepared from IgG against the Drosophila amnesia protein to control for non-specific effects of F(ab')2 injection. All mice survived 72 hours after surgery.

surgical procedure

On the day of surgery, mice were weighed, anesthetized with isoflurane, and placed in a stereotaxic apparatus (51603 Dual Operations Laboratory Standards, Stoelting, Wood Dale, IL). Make a central incision to expose the skull and make two burr holes using a dental drill in the right frontal cortex and hippocampus at the following coordinates: Cortex: AP+1.5mm, L-2.0mm; Hippocampus: AP-2.7mm, L-2.5mm , starting from the bregma. A 26 gauge injection needle attached to a 10 J.l Hamilton (Reno, NV) pipette was descended 3 mm ventral to bregma and injected 2 μl over a 2 minute period. The incision is washed with saline and closed with surgical staples.

tissue preparation

On the day of sacrifice, the mice were weighed, overdosed with 100 mg/kg pentobarbital (sodium pentobarbital solution, Abbott laboratories, North Chicago IL), intracranially perfused with 25 ml of 0.9% sodium chloride, and then 50 ml of freshly prepared 4% paraformaldehyde (pH=7.4). Brains were quickly removed and fixed by immersion in freshly prepared 4% paraformaldehyde for 24 hours. The brains were then incubated sequentially for 24 hours in 10, 20 and 30% sucrose to cyroprotect them. 25 μm thick horizontal sections were then collected using a slide microtome and stored in DPBS buffer containing sodium azide at 4°C to prevent microbial growth.

Immunohistochemical method

Six to eight ~100 μm sections spanning the point of injection were selected for free-floating immunization against total Aβ (rabbit antiserum mainly reacted with the N-terminus of the Aβ peptide, 1:10000) and CD45 (Serotec, Raleigh NC, 1:3000) Histochemical staining was performed as previously described (Gordon et al., 2002). For immunostaining, some sections did not react with the primary antibody to evaluate non-specific immunohistochemical reactions. Adjacent sections were mounted on slides and stained with 4% Thioflavin-S (Sigma-Aldrich, St Louis MO) for 10 minutes. It should be noted that a limited number of sections included injection sites. The procedure described below is to measure a few reliable markers, rather than obtain a large number of markers with fewer slices.

data analysis

Immunohistochemical reaction products were measured on all stained sections in the injected regions of the frontal cortex and hippocampus and in the corresponding contralateral regions of the brain using a Video V150 image analysis system (Oncor, San Diego, CA). Data are presented as ratios of Aβ, Thioflavin-S and CD45 on the injected side and the non-injected side. Normalizing each injection point to the corresponding contralateral site reduces the effect of inter-animal variability and allows reliable measurement of drug effects with fewer mice. To assess possible treatment-related differences, ratios for each treatment group were analyzed by ANOVA using StatView software version 5.0.1 (SAS Institute Inc., NC), followed by Fischer's LSD mean comparisons.

result

Seventy-two hours after intracranial injection in the frontal cortex and hippocampus, the only antibody that activated microglia was the intact anti-Aβ antibody 2286. The frontal cortex showed a higher degree of activation than the hippocampus, however, in both regions activation was significantly greater than in groups receiving control anti-amnesia protein IgG, F(ab')2 or anti-Aβ2286F(ab')2 Strong (Figure 5A, C and D, Figure 6A; P<0.01 or higher in all comparisons). Following injection of anti-Aβ antibody 2286, the activation pattern in the hippocampus was similar to that when anti-Aβ antibody 44-352, a β-amyloid amino acid 1-16 binding monoclonal antibody (Biosource, Camarillo, CA) was used. In the granule cell layer of the dentate gyrus, there were areas of very intense activation, with more diffuse activation filling the remainder of the dentate gyrus (Fig. 5A). Interestingly, anti-AβF(ab')2 fragments did not produce microglial activation in the frontal cortex and hippocampus (Fig. 5B, Fig. 6A).

In the two anti-amnesia protein controls, Aβ immunohistochemistry showed typical staining patterns in 19.5-month-old APP transgenic mice (Fig. 5G and H). This pattern was qualitatively the same as that observed in 16-month-old mice, although quantitatively higher than in 3.5-month-old mice. Anti-Aβ antibody and anti-Aβ F(ab')2 groups significantly reduced total Aβ immunohistochemistry to a similar extent 72 hours after injection in the frontal cortex and hippocampus. In the frontal cortex, the decrease was about 60% (Fig. 6B). In the hippocampus, the reduction was about 65% (Fig. 5E and F, Fig. 6B).

Thioflavin-S staining detects only dense, fibrillar amyloid deposits. Mice that received intracranial injections of control anti-amnesia protein IgG or control F(ab')2 were similar to the typical staining observed in APP transgenic mice of this age. In the hippocampus, the majority of thioflavin-S-positive plaques were located in the lateral molecular layer of the dentate gyrus near the horn of Ammons and the hippocampal groove (Fig. 5K and L). In frontal cortex and hippocampus, anti-Aβ antibody IgG significantly reduced Thioflavin-S positive dense plaques by about 90% (Fig. 6C). In the hippocampus, there were no, or very few, remaining Thioflavin-S positive deposits (Fig. 5I). In contrast, anti-AβF(ab')2 fragments did not remove dense amyloid plaques as effectively as intact IgG molecules. In the frontal cortex, Thioflavin-S staining was not significantly reduced when compared to the control antibody group (Fig. 6C). In the hippocampus, there was a significant difference (P<0.05) between the anti-AβF(ab')2 group and the control group, however, this decrease was also significantly lower than that observed with intact IgG molecules (Fig. 5J , Figure 6C, P<0.02 or higher).

As expected, CD45 and Thioflavin-S values were compared in a single large regression analysis for all groups receiving anti-AβIgG or anti-AβF(ab')2 regardless of their subsequent treatment , using the log-transformed value of CD45, there was a significant difference between increased levels of microglial activation detected by CD45 immunohistochemistry and clearing of dense plaques detected by thioflavin-S staining in the frontal cortex Correlation (P<0.001, R=0.57). This correlation was also observed in the hippocampus (P<0.02, R=0-427).

Example 7: Increased Serum Aβ Concentration After Peripheral Injection of Antibody 2286

This experiment was performed to test the efficacy of monoclonal antibody 2286 after systemic passive immunization of a transgenic animal model of Alzheimer's disease. Nineteen-month-old Tg2576 transgenic mice (Hsiao et al., 1996, Science 274:99-102) were injected intraperitoneally (IP) with monoclonal antibody 2286 or anti-amnesia antibody (IgG1 control). The antibody was injected once a week at a dose of 10 mg per Kg of body weight for 1, 2 or 3 months, after which the Aβ serum concentration and anti-Aβ antibody titer in the serum were measured.

Serum concentrations of Aβ were determined by using a capture assay in which anti-Aβ antibodies (Clone6E 10, Signet Laboratories Inc., Dedham, MA) were immobilized on assay plates and compared with diluted serum samples from treated mice. Incubation. After 10 consecutive washes, the assay plate was incubated with a secondary biotin-linked anti-Aβ antibody (Clone 4G8, Signet Laboratories Inc., Dedham MA, USA), followed by the addition of HRP-linked streptavidin (Amersham Biosciences Corp. ., NJ, USA). The absorbance at 450 nm of the assay plate was determined and the concentration of Aβ in the serum samples was determined by normalizing using a known concentration of synthetic Aβ _1-40 (American Peptide Company Inc., Sunnyvale CA, USA) as a standard. To measure the titer of the 2286 antibody in serum samples, antibody-Aβ complexes were dissociated by low pH and incubated with assay plates pre-coated with synthetic Aβ _1-40 (0.25 μg/well). After 10 consecutive washes, the assay plate was incubated with biotin-linked goat anti-mouse (H+L) antibody (Vector Laboratories, BurllingameCA, USA), followed by HRP-linked streptavidin (Amersham Biosciences Corp., NJ). , USA) and measure the absorbance at 450 nm. Concentrations of anti-A[beta] antibodies were calculated from standard curves generated in the same assay using known concentrations of Affinity Purified 2286.

As shown in Figure 7, following peripheral administration of 2286, a rapid increase in serum A[beta] concentration was observed in Tg2576 mice, but no increase in anti-amnesia antibodies. Anti-Aβ antibody titers in serum samples showed a significant positive correlation between antibody concentrations in serum and serum Aβ concentrations in treated transgenic mice (by INSTAT PRISM v.4, GraphPad Software Inc., San Diego, CA analysis data, r ² =0.5125, F=26.28, P<0.0001). These data suggest that mAb 2286 may have an altered Aβ balance between the CNS and plasma, and that administration of mAb 2286 facilitates the clearance of Aβ from the CNS. To test this possibility, brain amyloid load was measured to determine whether increases in serum Aβ concentrations correlated with decreases in brain amyloid load in treated mice.

Amino acid sequences of table 6 beta amyloid peptides and variants 1-40(WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG GVV (SEQ ID NO: 1) 1-42(WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 11) 1-43(WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT (SEQ ID NO: 12) 1-41(WT) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVI (SEQ ID NO: 13) V36A(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMAGGVV (SEQ ID NO: 14) G37A(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMGAGVV (SEQ ID NO: 15) G38A(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGAVV (SEQ ID NO: 16) V39A(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGAV (SEQ ID NO: 17 V40A(1-40) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVA (SEQ ID NO: 18)

Table 7 Human amyloid beta (A4) precursor protein (APP) (SEQ ID NO: 2):

MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGK

WDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQ

CKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKET

CSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDDSDVW

WGGADTDYADGSEDKVVEVAEEEEVAEVEEEEADDDEDDEDGDEVEEEAEEP

YEEATERTTSATTTTTTTESVEEVVREVCSEQAETGPCRAMISRWYFDVTEGKC

APFFYGGCGGNRNNFDTEEYCMAVCGSAMSQSLLKTTQEPLARDPVKLPTTAA

STPDAVDKYLETPGDENEHAHFQKAKERLEAKHRERMSQVMREWEEAERQA

KNLPKADKKAVIQHFQEKVESLEQEAANERQQLVETHMARVEAMLNDRRRRLA

LENYITALQAVPPRPRHVFNMLKKYVRAEQKDRQHTLKHFEHVRMVDPKKAA

QIRSQVMTHLRVIYERMNQSLSLLYNVPAVAEEIQDEVDELLQKEQNYSDDVL

ANMISEPRISYGNDALMPSLTETKTTVELLPVNGEFSLDDLQPWHSFGADSVPA

NTENEVEPVDARPAADRGLTTRPGSGLTNIKTEEISEVKMDAEFRHDSGYEVHH

QKLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIHHGVVEVD

AAVTPEERHLSKMQQNGYENPTYKFFEQMQN

Table 8 monoclonal antibody 2286 nucleic acid sequence:

Heavy chain [variable and constant domain 1 (CH1)]; SEQ ID NO: 3:

gaggtgaagcttctcgagtctggaggtgggcctggtgcagcctggaggatccctgaaactctcctgtgcagcctcaggattcga

ttttagtagatactggatgaattgggtccggcaggctccagggaaagggctagaatggattggagaaattaatccagatagca

gtacgataaactatacgccatctctaaaggataaattcatcatctccagagacaacgccaaaaatacgctgtacctgcaaatga

gcaaagtgagatctgaggacacagccctttattactgtgcaagacaaatgggctactggggccaaggcaccactctcacagt

ctcctcagccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctggg

atgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttccc

agctgtcctgcagtctgacctctacactctgagcagctcagtgactgtcccctccagcacctggcccagcgagaccgtcacct

gcaacgttgcccacccggccagcagcaccaaggtggacaagaaaattgtgcccagggattgt

Light chain; SEQ ID NO: 5:

gatatccagatgacacagactacatcctccctgtctgcctctctggggagacagagtcaccatcagttgcagtgcaagtcaggg

cattagcaattatttaaactggtttcagcagaaaccagatggaactgttaaactcctgatctatttacacatcaagtttacactcagg

agtcccatcaaggttcagtggcagtgggtctgggacagattattctctcaccatcagcaacctggaacctgaagatattgccac

ttactattgtcagcagtataggaagcttccgtacacgttcggagggggggaccaagctggaaataaaacgggctgatgctgcac

caactgtatccatcttcccaccactccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctacccc

aaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagca

aagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgagg

ccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt

Table 9 Monoclonal Antibody 2286 Amino Acid Sequence:

Heavy chain [variable domain and constant domain 1 (CH1)]; SEQ ID NO: 4:

EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMNWVRQAPGKGLEWIGEIN

PDSSTINYTPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCARQMGYWGQ

GTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSL

SSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR

DC

Light chain; SEQ ID NO: 6:

DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWFQQKPDGTVKLLIYYTSSLH

SGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYRKLPYTFGGGTKLEIKRAD

AAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWT

DQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

Table 10 Monoclonal antibody 2324 nucleic acid sequence:

Heavy chain [variable and constant domain 1 (CH1)]; SEQ ID NO: 7:

gttactctgaaagagtctggccctgggatattgaagccctcacagaccctcagtctgacttgttctttctctgggttttcactgagc

acttctggtatgggtgtaggctggattcgtcagtcttcagggaagggtctggagtggctggcaacatttggtgggatgatgat

aagtactataacccatccctgaagagccagctcacaatctccaaggatacctccagaaaccaggtattcctcaagatcaccagt

gtggacactgcagatactgccacttactactgtgctcgaagggggggtacgacatagagactactttgactactggggccaagg

caccactctcacagtctcctcagccaaaacaacaccccccatcagtctatccactggcccctgggtgtggagatacaactggttc

ctccgtgactctgggatgcctggtcaagggctacttccctgagtcagtgactgtgacttggaactctggatccctgtccagcagt

gtgcacaccttcccagctctcctgcagtctggactctacactatgagcagctcagtgactgtcccctccagcacctggccaagt

cagaccgtcacctgcagcgttgctcacccagccagcagcaccacggtggacaaaaacttgagcccagcgggcccatttca

acaatcaaccccc

Light chain; SEQ ID NO: 9:

gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagcatt

gtacatagtaatggaaacacctatttgaatggtacctgcagaaaccaggccagtctccaaaactccttatctacaaagtttcca

accgattttctggggtcccagacaggttcagtggcagtggatcagggacagaatttcacactcaagatcagcagagtggaggct

gaggatctggggagttattactgctttcaaggttcacgtgttcctctcacgttcggtgctgggaccaagctggagctgaaacggg

ctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaac

aacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactga

tcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagcta

tacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt

Table 11 Monoclonal antibody 2324 amino acid sequence:

Heavy chain [variable domain and constant domain 1 (CH1)]; SEQ ID NO: 8:

VTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQSSGKGLEWLAHIW

DDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCARRGVRHRDY

DYWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTV

WNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTV

KKLEPSGPISTINP

Light chain; SEQ ID NO: 10:

DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYK

VSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSRVPLTFGAGTKL

ELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNG

VLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRN

EC

Preservation of Biological Material

The following material is deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA (ATCC): Material Antibody number ATCC deposit number Preservation date 8A1.2A1 2286 PTA-5199 May 15, 2003

Deposited in accordance with the Budapest Treaty on the Deposit of Microorganisms for International Consensus on Patent Procedure and the Regulations therein (Budapest Treaty). This guarantees the maintenance of viable cultures for 30 years from the date of deposit. Deposits are available from the ATCC pursuant to the Budapest Treaty, with the consent of Rinat Neuroscience Corp and the ATCC, which guarantees that the public will have access to the deposits since publication of the relevant U.S. patent or disclosure of any U.S. or foreign patent application to the public, whichever comes first. Perpetual and unrestricted access to progeny of the deposited cultures and guaranteed subject to designation by the U.S. Patent and Trademark Office pursuant to 35USC Section 122 and rules approved therein (including 37CFR Section 1.14 and, inter alia, since 886OG 638) Progeny of the deposit may be obtained.

The agent of the present application has agreed that if a culture of the deposited material dies or is lost or destroyed when cultivated under suitable conditions, it will be promptly replaced by another copy of the same material on notice. Access to deposited material does not constitute a license to practice the invention in violation of any government's authorization under the patent laws.

The foregoing description is considered sufficient to enable one skilled in the art to practice the invention. The present invention is not limited in scope by the deposited constructs, as the deposited embodiment is intended only as an example of certain aspects of the invention and any functionally equivalent constructs thereof are within the scope of the present invention. The deposit of material herein neither constitutes an admission that the written description herein is insufficient to practice any aspect of the invention, including its best mode, nor does it constitute a limitation of the scope of the claims to the specific expressions set forth in the specification. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and are intended to fall within the scope of the appended claims.

Claims (50)

1. A method for treating Alzheimer's disease, comprising administering to a subject an effective amount of a pharmaceutical composition comprising a peptide that preferentially binds to Aβ _1-40 peptide (SEQ ID NO: 1) at positions 28-40 Amino acid conjugated antibody and pharmaceutically acceptable carrier. 2. The method of claim 1, wherein the antibody preferentially binds to an epitope comprising amino acids 39 and/or 40 of the A[beta] _1-40 peptide (SEQ ID NO: 1). 3. The method of claim 1 or 2, wherein the antibody does not show significant cross-reactivity with the A[beta] _1-42 and A[beta _{] 1-43} peptides. 4. The method of claim 1, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less. 5. The method of claim 1, wherein the Fab fragment of the antibody binds the A[beta _]1-40 peptide (SEQ ID NO: 1) with an affinity of about 1 nM or less. 6. The method of claim 2, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less. 7. The method of claim 2, wherein the Fab fragment of the antibody binds the A[beta _]1-40 peptide (SEQ ID NO: 1) with an affinity of about 1 nM or less. 8. The method of claim 1, wherein the antibody competitively inhibits the binding of a monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 4 and 6 to the A[beta _{] 1-40} peptide (SEQ ID NO: 1). 9. The method of claim 1, wherein the antibody binds to the same epitope as a monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 4 and 6. 10. The method of claim 1, wherein the antibody is a monoclonal antibody. 11. The method of claim 1, wherein the antibody is a humanized antibody. 12. The method of claim 1, wherein the antibody is a human antibody. 13. A method of inhibiting the formation of amyloid plaques in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a peptide that preferentially binds to Aβ _1-40 (SEQ ID NO: 1) 28 -An antibody bound to amino acid at position 40 and a pharmaceutically acceptable carrier. 14. The method of claim 13, wherein the antibody preferentially binds to an epitope comprising amino acids 39 and/or 40 of the A[beta] _1-40 peptide (SEQ ID NO: 1). 15. The method of claim 13, wherein the antibody does not exhibit significant cross-reactivity with the A[beta _{] 1-42} and A[beta] _1-43 peptides. 16. The method of claim 13, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less. 17. The method of claim 13, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 1 nM or less. 18. The method of claim 14, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less. 19. The method of claim 14, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 1 nM or less. 20. The method of claim 13, wherein the antibody competitively inhibits the binding of the monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 4 and 6 to the A[beta _{] 1-40} peptide (SEQ ID NO: 1). 21. The method of claim 13, wherein the antibody binds to the same epitope as a monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 4 and 6. 22. The method of claim 13, wherein the antibody is a monoclonal antibody. 23. The method of claim 13, wherein the antibody is a humanized antibody. 24. The method of claim 13, wherein the antibody is a human antibody. 25. The method of claim 13, wherein the amyloid plaques are located in the subject's brain. 26. A method of reducing amyloid plaques in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a peptide that preferentially binds to Aβ _1-40 peptide (SEQ ID NO: 1) 28- Antibody combined with amino acid at position 40 and pharmaceutically acceptable carrier. 27. The method of claim 26, wherein the antibody preferentially binds to an epitope comprising amino acids 39 and/or 40 of the A[beta] _1-40 peptide (SEQ ID NO: 1). 28. The method of claim 26, wherein the antibody does not exhibit significant cross-reactivity with the A[beta _{] 1-42} and A[beta] _1-43 peptides. 29. The method of claim 26, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less. 30. The method of claim 26, wherein the Fab fragment of the antibody binds the A[beta _]1-40 peptide (SEQ ID NO: 1) with an affinity of about 1 nM or less. 31. The method of claim 26, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less. 32. The method of claim 27, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 1 nM or less. 33. The method of claim 26, wherein the antibody competitively inhibits the binding of the monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 4 and 6 to the A[beta _{] 1-40} peptide (SEQ ID NO: 1). 34. The method of claim 26, wherein the antibody binds to the same epitope as a monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 4 and 6. 35. The method of claim 26, wherein the antibody is a monoclonal antibody. 36. The method of claim 26, wherein the antibody is a humanized antibody. 37. The method of claim 26, wherein the antibody is a human antibody. 38. The method of claim 26, wherein the amyloid plaques are located in the subject's brain. 39. A method for delaying the development of symptoms associated with Alzheimer's disease in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising preferentially binding to the Aβ _1-40 peptide (SEQ ID NO: 1) Antibody combined with 28-40 amino acids and a pharmaceutically acceptable carrier. 40. The method of claim 39, wherein the antibody preferentially binds to an epitope comprising amino acids 39 and/or 40 of the A[beta] _1-40 peptide (SEQ ID NO: 1). 41. The method of claim 39, wherein the antibody does not exhibit significant cross-reactivity with the A[beta _{] 1-42} and A[beta] _1-43 peptides. 42. The method of claim 39, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less. 43. The method of claim 39, wherein the Fab fragment of the antibody binds the A[beta _]1-40 peptide (SEQ ID NO: 1) with an affinity of about 1 nM or less. 44. The method of claim 40, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 200 nM or less. 45. The method of claim 40, wherein the Fab fragment of the antibody binds the A[beta _{] 1-40} peptide (SEQ ID NO: 1) with an affinity of about 1 nM or less. 46. The method of claim 39, wherein the antibody competitively inhibits the binding of a monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 4 and 6 to the A[beta _{] 1-40} peptide (SEQ ID NO: 1). 47. The method of claim 39, wherein the antibody binds to the same epitope as a monoclonal antibody comprising the amino acid sequence of SEQ ID NO: 4 and 6. 48. The method of claim 39, wherein the antibody is a monoclonal antibody. 49. The method of claim 39, wherein the antibody is a humanized antibody. 50. The method of claim 39, wherein the antibody is a human antibody.

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