HK1117543B - Antibodies directed against amyloid-beta peptide and methods using same - Google Patents

Description

Anti-beta-amyloid peptide antibodies and methods of use thereof

Cross Reference to Related Applications

Priority is claimed for this application from U.S. provisional patent application serial No. 60/676,093 filed on 29/4/2005 and U.S. provisional patent application serial No. 60/704,818 filed on 1/8/2005, both of which are hereby incorporated by reference in their entirety.

Technical Field

The present invention relates to anti-beta-amyloid peptide antibodies. The invention also relates to the use of such antibodies in the treatment and/or prevention of diseases, such as alzheimer's disease.

Statement regarding federally sponsored research or development

Not applicable.

Background

Alzheimer's Disease (AD) is a degenerative brain disorder characterized clinically by progressive memory impairment, confusion, gradual physical deterioration, and ultimately death. Approximately fifteen million people worldwide suffer from alzheimer's disease, and it is expected that this number will increase dramatically as the life of humans is extended. Histologically, the disease is characterized by neuritic plaques (neuro-plaques) that occur primarily in the contact cortex, limbic system and basal ganglia. The major component of these plaques is the β -amyloid peptide (a β), which is the cleavage product of β amyloid precursor protein (β APP 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. The variable splicing of transcripts of the APP single gene on chromosome 21 results in several isoforms with different amino acid numbers.

A β appears to have a major role in the neuropathology of Alzheimer's disease. The familial form of the disease has been linked to 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 result in increased production of the 42 amino acid form of a β, the predominant form found in amyloid plaques. Furthermore, immunization of transgenic mice overexpressing disease-associated mutant forms of APP with human A β reduces plaque burden and associated pathology (Schenk et al, 1999, Nature, 400: 173-177; WO99/27944), and peripheral administration of anti-A β antibodies also reduces plaque burden in the brain (Bard et al, 2000, Nature Medicine 6 (8): 916-919; WO 2004/032868; WO 00/72880).

Fc-mediated phagocytosis of microglia and/or macrophages has been reported to be important for plaque removal processes in vivo. Bard et al, proc.natl.acad.sci.usa100: 2023-2028(2003). However, it has also been reported that non-Fc mediated mechanisms are involved in the clearance of beta amyloid peptide in vivo by immunotherapy. Bacskai et al, J.Neurosci.22: 7873-7878 (2002); das et al, j.neurosci.23: 8532-8538(2003).

Thus, antibody therapy provides a promising approach for the treatment and prevention of alzheimer's disease. However, human clinical trials with vaccines including A β 1-42 were suspended due to meningoencephalitis in some patients. Orgozo et al, neurology 61: 7-8 (2003); brain Pathol.14 of Ferrer et al: 11-20(2004). It has been reported that passive immunization with N-terminal specific anti-a β antibodies in transgenic mice exhibiting age-related amyloid plaque development and neurodegeneration and Cerebral Amyloid Angiopathy (CAA) similar to that observed in human AD brain results in a significant reduction of the majority of diffuse amyloid but induces an increase in brain microhemorrhage frequency. Pfeifer et al, Science 298: 1379(2002). It has been suggested that exacerbation of Cerebral Amyloid Angiopathy (CAA) associated microhemorrhage in APP transgenic mice by passive immunization with anti-beta amyloid peptide antibodies is dependent on recognition by the antibody of the deposited form of beta amyloid peptide. Ricke et al, j.neurosci.25: 629-636(2005). In order to reduce the risk of developing inflammation, passive immunization with antibodies against peptide components of amyloid deposits that lack the Fc region has been proposed. WO 03/086310. There remains a need for antibodies against a β and other immunotherapeutic agents with improved efficacy and safety profiles and suitable for use in human patients.

Various publications (including patents and patent applications) are cited in this application. The disclosures of these publications are hereby incorporated by reference in their entirety.

Summary of The Invention

The invention disclosed herein relates to antibodies and polypeptides that bind to the C-terminus of a β peptide. In one aspect, the invention provides methods of treatment of a β_1-40、Aβ_1-42And Abeta_1-43Bound antibody or polypeptide, wherein the antibody or polypeptide binds to A β more than it binds to A β_1-42And Abeta_1-43With higher affinity to a β_1-40Binding, and wherein said antibody or polypeptide binds to A β_1-40Including amino acids 25-34 and 40 above. In some embodiments, the antibody binds to a β_1-40The affinity of the binding is to Abeta_1-42And/or Abeta_1-43At least about 40 times the affinity of the binding. In some embodiments, the antibody is not antibody 2294.

In another aspect, the invention provides an antibody 6G (interchangeable with the term "6G"). The amino acid sequences of the heavy and light chain variable regions of 6G are shown in fig. 1. The Complementarity Determining Region (CDR) portions of antibody 6G, including Chothia and Kabat CDRs, are also shown in figure 1.

In another aspect, the invention also provides 6G antibody variants having the amino acid sequences shown in table 3.

In another aspect, the invention provides an antibody comprising a fragment or region of antibody 6G or a variant thereof shown in table 3. In one embodiment, the fragment is the light chain of antibody 6G. In another embodiment, the fragment is the heavy chain of antibody 6G. In yet another embodiment, the fragment comprises one or more variable regions from a light chain and/or a heavy chain of antibody 6G. In yet another embodiment, the fragment comprises one or more variable regions from a light chain and/or a heavy chain as depicted in figure 1. In yet another embodiment, the fragment comprises one or more CDRs from a light chain and/or a heavy chain of the antibody 6G.

In another aspect, the invention provides a polypeptide (which may or may not be an antibody) comprising any one or more of the following: a) one or more CDRs of antibody 6G or variants thereof shown in table 3; b) CDR H3 from the heavy chain of antibody 6G or the variants thereof shown in table 3; c) CDR L3 from the light chain of antibody 6G or the variants thereof shown in table 3; d) 3 CDRs from the light chain of antibody 6G or variants thereof shown in table 3; e) 3 CDRs from the heavy chain of antibody 6G or variants thereof shown in table 3; f) 3 CDRs from the light chain of antibody 6G or variants thereof shown in table 3 and 3 CDRs from the heavy chain of antibody 6G or variants thereof shown in table 3. The invention also provides polypeptides (which may or may not be antibodies) comprising any one or more of the following: a) one or more (one, two, three, four, five or six) CDRs derived from antibody 6G or variants thereof shown in table 3; b) CDRs derived from antibody 6G heavy chain CDR H3; and/or c) a CDR derived from the CDR L3 of the light chain of antibody 6G. In some embodiments, the CDR is the CDR shown in fig. 1. In some embodiments, the one or more CDRs derived from antibody 6G or variants thereof shown in table 3 are at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least one, at least two, at least three, at least four, at least five, or at least six CDRs of 6G or variants thereof.

In some embodiments, the CDRs are Kabat CDRs. In one embodiment, the CDR is a Chothia CDR. In other embodiments, the CDRs are a combination of Kabat and chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In other words, for any given embodiment comprising more than one CDR, the CDRs can be any CDR selected from Kabat, Chothia, and/or a combination CDR.

In some embodiments, the antibodies of the invention are human antibodies. In other embodiments, the antibodies of the invention are humanized antibodies. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody (or polypeptide) is isolated. In some embodiments, the antibody (or polypeptide) is substantially pure.

The antibody heavy chain constant region may be from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE; and any isotype, e.g., IgG1, IgG2, IgG3, and IgG 4.

In some embodiments, the antibodies or polypeptides described herein have impaired effector function. In some embodiments, the antibody or polypeptide comprises a heavy chain constant region having impaired effector function, wherein the heavy chain constant region comprises an Fc region. In some embodiments, N-glycosylation in the Fc region is removed. In some embodiments, the Fc region comprises a mutation in the N-glycosylation recognition sequence, whereby the Fc region of the antibody or polypeptide is not N-glycosylated. In some embodiments, the Fc region is pegylated. In some embodiments, the heavy chain constant region of the antibody or polypeptide is a human heavy chain IgG2a constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering is performed with reference to the wild type IgG2 sequence). In some embodiments, the antibody or polypeptide comprises an IgG4 constant region comprising the following mutations: E233F234L235 to P233V234a 235. These amino acid positions are based on Kabat numbering.

In another aspect, the invention provides a polynucleotide (which may be isolated) comprising a polynucleotide encoding a fragment or region of antibody 6G or a variant thereof shown in table 3. In one embodiment, the fragment is the light chain of antibody 6G. In another embodiment, the fragment is the heavy chain of antibody 6G. In yet another embodiment, the fragment contains one or more variable regions from a light chain and/or a heavy chain of antibody 6G. In yet another embodiment, the fragment comprises one or more (i.e., one, two, three, four, five, six) Complementarity Determining Regions (CDRs) from the light chain and/or heavy chain of antibody 6G.

In another aspect, the invention is a polynucleotide (which may be isolated) comprising a polynucleotide encoding antibody 6G or a variant thereof shown in table 3. In some embodiments, the polynucleotide comprises SEQ ID NO: 9 and SEQ ID NO: 10 or a polynucleotide as set forth in claim 10.

In another aspect, the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) or polypeptides described herein.

In another aspect, the invention provides vectors (including expression and cloning vectors) and host cells comprising any of the polynucleotides described herein.

In another aspect, the invention is a host cell comprising a polynucleotide encoding any of the antibodies described herein.

In another aspect, the invention is A β_1-40A complex that binds to antibody 6G or a variant thereof shown in table 3.

In another aspect, the invention is A β_1-40A complex that binds to any of the antibodies or polypeptides described herein.

In another aspect, the invention is a pharmaceutical composition comprising an effective amount of any of the antibodies, polypeptides, or polynucleotides described herein and a pharmaceutically acceptable excipient. In some embodiments, the antibody or polypeptide comprises one or more CDRs of antibody 6G.

In another aspect, the invention is a method of making antibody 6G, comprising: culturing a host cell or progeny thereof under conditions that allow production of antibody 6G, wherein the host cell comprises an expression vector encoding antibody 6G; and, in some embodiments, purifying the antibody 6G. In some embodiments, the expression vector comprises SEQ ID NO: 9 and SEQ ID NO: 10, or both.

In another aspect, the invention provides a method of making any of the antibodies or polypeptides described herein by: one or more polynucleotides encoding the antibody (which may be expressed separately as a separate light or heavy chain, or both from one vector) or polypeptide are expressed in a suitable cell, typically followed by recovery and/or isolation of the antibody or polypeptide of interest.

The invention also provides methods of preventing, treating, inhibiting or delaying the progression of Alzheimer's disease and other diseases associated with altered expression of A β or β APP or accumulation of A β peptide, such as Down's syndrome, Parkinson's disease, multi-infarct dementia, mild cognitive impairment, cerebral amyloid angiopathy, depression, Creutzfeldt-Jakob disease, dementia with Lewy bodies, and AIDS. The method comprises administering to the individual an effective amount of a pharmaceutical composition comprising an antibody, polypeptide or polynucleotide of the invention.

The invention also provides a method of delaying the development of symptoms associated with alzheimer's disease or other diseases associated with accumulation of a β peptide in an individual comprising administering to the individual an effective amount of a pharmaceutical composition comprising an antibody, polypeptide or polynucleotide of the invention.

The invention also provides a method of inhibiting the formation of amyloid plaques and/or amyloid accumulation in a subject, comprising administering to the subject an effective dose of a pharmaceutical composition comprising an antibody, polypeptide or polynucleotide of the invention. In some embodiments, the amyloid plaques are in the brain (brain tissue) of the subject. In some embodiments, the amyloid plaques are present in cerebrovascular vasculature. In other embodiments, amyloid accumulation occurs in the circulatory system.

The invention also provides a method of reducing amyloid plaques and/or amyloid accumulation in a subject comprising administering to the subject an effective dose of a pharmaceutical composition comprising an antibody, polypeptide, or polynucleotide of the invention. In some embodiments, the amyloid plaques are in the brain (brain tissue) of the subject. In some embodiments, the amyloid plaques are present in cerebrovascular vasculature. In other embodiments, amyloid accumulation occurs in the circulatory system.

The invention also provides a method of removing or clearing amyloid plaques and/or amyloid accumulation in a subject comprising administering to the subject an effective dose of a pharmaceutical composition comprising an antibody, polypeptide or polynucleotide of the invention. In some embodiments, the amyloid plaques are in the brain (brain tissue) of the subject. In some embodiments, the amyloid plaques are present in cerebrovascular vasculature. In other embodiments, amyloid accumulation occurs in the circulatory system.

In addition, the invention provides a method of inhibiting accumulation of a β peptide in a tissue, comprising contacting the tissue with an antibody or polypeptide of the invention.

The invention also provides a method of reducing a β peptide (e.g., soluble, oligomeric, and deposited forms) in a subject, comprising administering to the subject an effective amount of an antibody, polypeptide, or polynucleotide of the invention. In some embodiments, the accumulation of a β peptide in the brain is inhibited and/or reduced. In some embodiments, the toxic effects of a β peptide are inhibited and/or reduced. Thus, the methods of the invention may be used to treat any disease in which accumulation of A β peptide is present or suspected, for example, Alzheimer's disease, Down's syndrome, Parkinson's disease, multi-infarct dementia, mild cognitive impairment, cerebral amyloid angiopathy, depression, Creutzfeldt-Jakob disease or dementia with Lewy bodies.

The invention also provides a method of improving cognitive function or reversing cognitive decline associated with a β amyloid deposition-related disease (e.g., alzheimer's disease) in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody, polypeptide, or polynucleotide of the invention.

Any of the antibodies, polypeptides or polynucleotides described herein can be used in the methods of the invention. In some embodiments, the antibody is antibody 6G.

The antibodies and polypeptides of the invention may also be used to detect, diagnose and monitor Alzheimer's disease and other diseases associated with altered A β or β APP expression, such as Down's syndrome and AIDS. The method comprises contacting a sample of a patient suspected of having altered expression of a β or β APP with an antibody of the invention and determining whether the level of a β or β APP differs from the level of a control or comparative sample. In some embodiments, serum a β levels are measured before and after administration of anti-a β antibodies; and any increase in serum a β levels was assessed.

Administration of any antibody or polypeptide of the invention may be by any means known in the art, including: intravenous, subcutaneous, by inhalation, intra-arterial, intramuscular, intracardiac, intraventricular, parenteral, intrathecal, and intraperitoneal. Administration may be systemic, e.g., intravenous, or local. In general, this also applies to the polypeptides and polynucleotides of the invention.

In another aspect, the invention provides kits and compositions comprising any one or more of the compositions described herein. These kits are generally in suitable packaging and provided with appropriate instructions for use in any of the methods described herein.

Brief Description of Drawings

FIG. 1 shows the heavy chain variable region amino acid sequence (SEQ ID NO: 1) and the light chain variable region amino acid sequence (SEQ ID NO: 2) of antibody 6G. The Kabat CDRs are shown in bold, and the Chothia CDRs are underlined. The amino acid residues of the heavy and light chain variable regions are numbered sequentially.

Figure 2 shows epitope mapping of antibody 6G by ELISA. A.beta.peptides (1-16, 1-28, 17-40, 17-42, 22-35, 28-40, 28-42, 1-38, 1-40, 1-42, 1-43, and 33-40) were immobilized on ELISA plates. Monoclonal antibody 6G (20nM) was incubated with each immobilized peptide for 1 hour. Antibody 6G bound to immobilized Α β peptide was measured using HRP-conjugated goat anti-human kappa secondary antibody.

Figure 3 shows epitope mapping of antibody 6G by ELISA. Each of the A.beta.peptides (sequences designated SEQ ID NOS: 18-29 from top to bottom) was immobilized on an ELISA plate. Monoclonal antibody 6G was incubated with various immobilized peptides for 1 hour. Antibody 6G bound to immobilized Α β peptide was measured using HRP-conjugated goat anti-human kappa secondary antibody. "NB" means no binding was detected.

Fig. 4 is a schematic diagram showing the epitope on a β that binds to antibody 6G. The relative position of a β in Amyloid Precursor Protein (APP) and the cell membrane portion of APP is shown. "CT 99" refers to the C-terminal 99 amino acids of APP. The amino acid sequence shown is designated as SEQ ID NO: 30.

FIG. 5 shows anti-Abeta_1-16Photograph of immunostaining of APP-expressing cells with the monoclonal antibody (m2324) and antibody 6G of (1). The top panel shows cells observed under a fluorescent microscope after incubation of the cells with m2324 or 6G (5 μ G/ml each) and detection of binding using Cy 3-conjugated goat anti-mouse or anti-human secondary antibody. The bottom panel shows cells observed under a microscope.

Figure 6 shows epitope mapping of antibodies 2294 and 6G by ELISA. A.beta.peptides (SEQ ID NOS: 18-26, 31 and 27-29 from top to bottom) were immobilized on ELISA plates. The antibodies were incubated with the various immobilized peptides for 1 hour. Antibody 6G bound to immobilized Α β peptide was measured using HRP-conjugated goat anti-human kappa secondary antibody. Antibody 2294 bound to the immobilized a β peptide was measured using a goat anti-mouse antibody, which is capable of binding both the heavy and light chains, is an HRP conjugated secondary antibody. "NB" means no binding was detected. The numbers in the columns under "2294" and "6G" indicate the absorption value at 450 nm.

Detailed Description

The invention disclosed herein provides antibodies and polypeptides that bind to the C-terminus of a β. The invention also provides polynucleotides encoding these antibodies and/or polypeptides. The invention also provides methods of making and using these antibodies and polypeptides.

The invention also provides methods of treating or preventing a disease associated with beta-amyloid deposition in a subject, such as, for example, alzheimer's disease, Down's syndrome, multi-infarct dementia, mild cognitive impairment, cerebral amyloid angiopathy, depression, Creutzfeldt-Jakob disease, and dementia with Lewy bodies, by administering to the subject an effective amount of a pharmaceutical composition comprising an antibody, polypeptide, or polynucleotide encoding an antibody or polypeptide as described herein.

General techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are fully described in the literature, for example, Molecular Cloning: a Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; oligonucleotide Synthesis (m.j. gait eds., 1984); methods in Molecular Biology, HumanaPress; cell Biology: a Laboratory Notebook (J.E. Cellis, eds., 1998) Academic Press; animal Cell Culture (r.i. freshney, eds., 1987); introduction to Cell and Tissue Culture (J.P.Matherand P.E.Roberts, 1998) Plenum Press; cell and Tissue Culture: laboratory Procedures (A.Doyle, J.B.Griffiths, and D.G.Newell eds., 1993-1998) J.Wiley and Sons; methods in Enzymology (Academic Press, Inc.); handbook of Experimental Immunology (d.m.weir and c.c.blackwell, eds.); gene Transfer vector for Mammarian Cells (eds. J.M.Miller and M.P.Calos, 1987); current protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987); and (3) PCR: the Polymerase Chain Reaction (Mullis et al, 1994); CurrentProtocols in Immunology (J.E.Coligan et al, eds., 1991); short protocols in Molecular Biology (Wiley and Sons, 1999); immunobiology (c.a. janeway and p.travers, 1997); antibodies (p.finch, 1997); antibodies: a practical proproach (D.Catty. eds., IRL Press, 1988-; monoclonal antigens: a practicallappacach (edited by p. shepherd and c. dean, Oxford University Press, 2000); using antibodies: a Laboratory manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); the reagents (M.Zaneti and J.D.Capra, eds., Harwood academic publishers, 1995).

Definition of

An "antibody" is an immunoglobulin molecule that is capable of specifically binding a target (e.g., a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab ', F (ab')₂Fv), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, and any other modified configuration (configuration) of the immunoglobulin molecule comprising an antigen recognition site. The antibodies of the invention include any type of antibody, such as IgG, IgA or IgM (or subtypes thereof), which need not be of any particular type. Immunoglobulins can be assigned to different classes depending on the amino acid sequence of the constant region of the heavy chain of the antibody. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which can be further subdivided into subtypes (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The heavy chain constant regions corresponding to different types of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different types of immunoglobulins are well known.

Herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies (i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts). Monoclonal antibodies are highly specific, pointing to a single antigenic site. Moreover, unlike polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" refers to that characteristic of an antibody being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of that antibody by any particular method. For example, monoclonal antibodies for use according to the invention may be produced by Kohler and Milstein, 1975, Nature 256: 495 or may be prepared by recombinant DNA methods such as those described in U.S. patent No. 4,816,567. Monoclonal antibodies can also be prepared from antibodies using, for example, McCafferty et al, 1990, Nature, 348: 552-554.

As used herein, a "humanized" antibody refers to a form of non-human (e.g., murine) antibody containing minimal non-human immunoglobulin-derived sequences that is a specific chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab ', F (ab')₂Or other antigen binding subsequences of antibodies). Humanized antibodies are primarily human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not present in either the acceptor antibody or the introduced CDR or framework sequences, but are included in the humanized antibody to further improve and optimize the performance of the antibody. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable regions in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those having human immunoglobulin consensus sequences. Optimally, the humanized antibody will also comprise at least a portion of a constant region or domain (Fc) of an immunoglobulin, typically a human immunoglobulin. The antibody may have an Fc region modified as described in WO 99/58572. Other humanized antibody forms have one or more CDRs (one, two, three, four, five, six) that have been altered relative to the original antibody, also referred to as one or more CDRs "derived" from one or more CDRs of the original antibody.

Herein, "human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and/or an antibody produced using any technique known in the art or described herein for producing human antibodies. This definition of human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be made using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses human antibody (Vaughan et al 1996, Nature Biotechnology, 14: 309-. Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated. The process is described in us patent 5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425 and 5,661,016. Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies against the target antigen (which B lymphocytes can be recovered from the individual or can have been immunized in vitro). See, e.g., 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 U.S. Pat. No. 5,750,373.

Herein, the terms "6G" and "antibody 6G" are used interchangeably to refer to a polypeptide having the amino acid sequence of SEQ id no: 11 and the amino acid sequence of the heavy chain shown in SEQ ID NO: 12, or a light chain amino acid sequence as set forth in seq id no. The amino acid sequences of the heavy and light chain variable regions are shown in FIG. 1. The CDR portions of antibody 6G (including Chothia and Kabat CDRs) are shown diagrammatically in FIG. 1. The polynucleotides encoding the heavy and light chains are shown in SEQ ID NOs: 13 and SEQ ID NO: 14 (c). The features of 6G are described in the examples.

The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified, either naturally or by intervention; the modification is, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling element. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It will be appreciated that, since the polypeptides of the invention are antibody-based, the polypeptides may be in single chain form or in multiple chains linked together.

"Polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Polynucleotides may comprise modified nucleotides, for example, methylated nucleotides and their analogs. Modifications to the nucleotide structure, if present, may occur before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling element. Other types of modifications include, for example, "capped", substitutions of one or more naturally occurring nucleotides to an analog, internucleotide modifications, for example, uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamides, cabamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), linkages containing such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators containing (e.g., metals, radioactive metals, boron, metal oxides, etc.), alkylators containing linkages with modifications (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of these polynucleotides. Furthermore, any of the hydroxyl groups typically present in sugars may be replaced by, for example, phosphonic acid groups, phosphate groups, protected by standard protecting groups, or activated in preparation for additional attachment to another nucleotide, or may be conjugated to a solid support. The 5 'and 3' terminal OH groups may be phosphorylated or replaced by an amine or organic end capping group moiety having 1 to 20 carbon atoms. Other hydroxyl groups may also be derivatized as standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2 '-O-methyl-, 2' -O-allyl, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptuloseAnalogues of the analogues, acyclic analogues and abasic nucleosides, such as methyl nucleosides. One or more phosphodiester linkages may be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, those wherein the phosphate ester is substituted with P (O) S ("thioester"), P (S) S ("dithio"), (O) NR₂("amidates"), P (O) R, P (O) OR', CO OR CH₂("formacetal") wherein each R or R' is independently H or substituted or unsubstituted alkyl (1-20C) (optionally containing an ether (-O-) linkage), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

An antibody "variable region" refers to either an antibody light chain variable region or an antibody heavy chain variable region, alone or in combination. The variable regions of both heavy and light chains each consist of 4 Framework Regions (FRs) linked together by three Complementarity Determining Regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al, Sequences of proteins of Immunological Interest (5 th edition, 1991, National institutes Health, Bethesda, Md.)); and (2) crystallography studies based on antigen-antibody complexes (Al-lazikani et Al (1997), J.Molec.biol.273: 927-948)). Herein, a CDR may refer to a CDR determined by one of the methods or by a combination of the two methods.

An antibody "constant region" refers to either the antibody light chain constant region or the antibody heavy chain constant region, alone or in combination.

An "epitope" that "preferentially binds" or "specifically binds" (used interchangeably herein) to an antibody or polypeptide is a term well known in the art, as are methods of determining such specific binding or preferential binding. If a molecule can react with a particular cell or substance more frequently and more rapidly than it can react with other cells or substancesA velocity, longer duration, and/or greater affinity reaction or binding, the molecule is said to exhibit "specific binding" or "preferential binding. An antibody "specifically binds" or "preferentially binds" to a target if it binds to the target with a higher affinity, avidity, more readily, and/or for a longer duration than it binds to other substances. E.g. specifically or preferentially with a certain Abeta_1-40An epitope-binding antibody is an antibody that binds to the epitope in a ratio that it binds to other A.beta.s_1-40Epitope or non-Abeta_1-40Binding of the epitope is of higher affinity, avidity, easier and/or longer duration. It is also understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

Herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of impurities), more preferably at least 90% pure, more preferably at least 95% pure, more preferably at least 98% pure, more preferably at least 99% pure.

"host cell" includes a single cell or cell culture that may be or has been the recipient of a vector for incorporation of a polynucleotide insert. Host cells include progeny of a single host cell that may not necessarily be identical (morphologically or in genomic DNA complementarity) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with a polynucleotide of the invention.

The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxy terminus. The numbering of residues in the Fc region is that of the EU index (EU index) as in Kabat et al, Sequence of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institute of Health, Bethesda, Md., 1991. Immunoglobulin Fc regions generally comprise two constant regions, CH2 and CH 3.

Herein, "Fc region" and "FcR" describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds to IgG antibodies (gamma receptors), including Fc γ R1, Fc γ RII, and Fc γ RIII subtype receptors, including allelic variants and variable splice forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), both of which have similar amino acid sequences, which differ primarily in their cytoplasmic domains. For reviews on FcR see ravatch and Kinet, 1991, ann.rev.immunol.9: 457-92; capel et al, 1994, immunoassays, 4: 25-34; and de Haas et al, 1995, j.lab.clin.med.126: 330-41. "FcR" also includes the neonatal receptor FcRn, which is responsible for 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 a target that occurs in the presence of complement. Binding of the first component of the complement system (C1q) to a molecule that forms a complex with an cognate antigen (e.g., an antibody) will result in initiation of the complement activation pathway. To evaluate complement activation, CDC assays can be performed, such as Gazzano-Santoro et al, j.immunol.methods, 202: 163 (1996).

A "functional Fc region" has at least one effector function of a native sequence Fc region. Exemplary "effector functions" include Clq binding; complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. This effector function generally requires the Fc region to be combined with a binding domain (e.g., an antibody variable region domain), and can be evaluated using various assays known in the art for evaluating effector function of such antibodies.

The "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of an Fc region of a native sequence by at least one amino acid modification, but retains at least one effector function of the Fc region of the native sequence. Preferably, the variant Fc region has at least one amino acid substitution as compared to the Fc region of the native sequence or the Fc region of the parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, preferably from about 1 to about 5 amino acid substitutions, in the Fc region of the native sequence or in the Fc region of the parent polypeptide. Herein, the variant Fc region preferably has at least about 80% sequence identity, most preferably at least about 90% sequence identity, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the Fc region of the native sequence and/or to the Fc region of the parent polypeptide.

Herein, "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (fcrs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay, for example, the assay described in U.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for this assay include Peripheral Blood Mononuclear Cells (PBMC) and NK cells. Alternatively, or in addition, the ADCC activity of the molecule of interest may be in vivo, e.g. in animal models, e.g. Clynes et al 1998 pnas (usa) 95: 652-656.

Herein, an "effective dose" or "effective amount" of a drug, compound or pharmaceutical composition is an amount effective to achieve a beneficial or desired result. For prophylactic applications, beneficial or desired results include, for example, elimination or reduction of the risk of, lessening the severity of, or delaying the onset of a disease, including biochemical, histological, and/or behavioral symptoms of the disease, complications thereof, and intermediate pathological phenotypes manifested during the course of disease progression. For therapeutic applications, beneficial or desired results include clinical results, for example, inhibiting, hindering or reducing amyloid plaque formation, reducing, removing, clearing amyloid plaques, improving cognition, reversing or slowing cognitive decline, sequestering or increasing soluble a β peptide circulating in biological fluids, reducing one or more symptoms (biochemical, histological and/or behavioral) resulting from the disease, including its complications and intermediate pathological phenotypes that occur during disease development, increasing the quality of life of patients with the disease, reducing the dose of other drugs required to treat the disease, enhancing the effect of other drugs, delaying disease progression, and/or prolonging survival of patients. The effective dose may be administered in one or more divided doses. For the purposes of the present invention, an effective dose of a drug, compound or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, prophylactic or therapeutic treatment. It is clinically understood that an effective dose of a drug, compound or pharmaceutical composition may or may not be achieved by combination with other drugs, compounds or pharmaceutical compositions. Thus, an "effective dose" may be identified in the context of administration of one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if it achieves, or can achieve, the desired result in combination with one or more other agents.

Herein, "treatment" or "therapy" is a method for obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: inhibiting, hindering or reducing the formation of amyloid plaques, reducing, removing or clearing amyloid plaques, improving cognition, reversing or slowing cognitive decline, sequestering soluble a β peptide circulating in a biological fluid, reducing a β peptide (including soluble, oligomeric and deposited) in a tissue (e.g., the brain), inhibiting, slowing and/or reducing the accumulation of a β peptide in the brain, inhibiting, slowing and/or reducing the toxic effects of a β peptide in a tissue (e.g., the brain), reducing the symptoms resulting from the disease, increasing the quality of life of a patient with the disease, reducing the dosage required for other drugs to treat the disease, delaying the progression of the disease, and/or prolonging the survival of a patient.

Herein, "delaying" the progression of Alzheimer's disease means delaying, impeding, slowing, arresting, calming and/or delaying the progression of the disease. This delay may be of varying lengths of time depending on the history of the disease and/or the individual to be treated. As will be apparent to those skilled in the art, a sufficient or significant delay may, in fact, comprise prevention, i.e., the individual does not develop the disease. A method of "delaying" the progression of Alzheimer's disease is a method that reduces the likelihood of disease progression and/or reduces the extent of disease within a given time frame, compared to not using the method. The comparison is typically performed based on clinical studies using a statistically significant number of individuals.

"development" of Alzheimer's disease means 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 herein. However, progression also refers to disease progression that may not be detectable initially. For the purposes of the present invention, progression refers to the biological course of a disease state, in which case it can be determined by standard neurological examination, or patient visit, or it can be determined by more specialized tests. Various such diagnostic tests include, but are not limited to, neuroimaging, detecting changes in the levels of specific proteins (e.g., amyloid peptides and Tau) in serum or cerebrospinal fluid, Computerized Tomography (CT), and Magnetic Resonance Imaging (MRI). "development" includes occurrence, recurrence and onset. As used herein, the "onset" or "occurrence" of Alzheimer's disease includes initial onset and/or recurrence.

Herein, "administering in combination" includes administering simultaneously and/or at different times. Co-administration also includes administration in the form of a co-formulation or in the form of separate compositions. Herein, co-administration is meant to encompass any situation in which an anti-a β antibody and another agent are administered to an individual, wherein administration of the anti-a β antibody and the other agent may be simultaneous and/or separate. As discussed further herein, it is understood that the anti-a β antibody and the other agent may be administered at different dosing frequencies or intervals. For example, the anti-a β antibody may be administered weekly, while the other agent may be administered less frequently. It will be appreciated that the anti-a β antibody and the other agent may be administered using the same route of administration or different routes of administration.

"biological sample" includes a variety of sample types obtained from an individual that may be used in diagnostic or monitoring assays. This definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. This definition also includes samples that have been manipulated in any way after they have been obtained, e.g., by treating with reagents, solubilizing or enriching certain components such as proteins or polynucleotides, or embedding in a semi-solid or solid matrix for sectioning purposes. The term "biological sample" includes clinical samples, and also includes cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

The "subject" (alternatively referred to as "individual") is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals (e.g., cows), sport animals, pets (e.g., cats, dogs, horses), primates, mice, and rats.

Herein, a "vector" refers to a construct capable of delivering, preferably expressing, one or more genes or sequences of interest to 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 condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

Herein, "expression control sequence" refers to a nucleic acid sequence that directs the expression of a nucleic acid. The expression control sequence may be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

Herein, "pharmaceutically acceptable carrier" includes any substance that, when combined with an active ingredient, is capable of allowing the ingredient to retain biological activity and which is non-reactive with the immune system of an individual. Examples include, but are not limited to, any standard pharmaceutical carrier, for example, phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents. For aerosol or parenteral administration, the preferred diluent is phosphate buffered saline or normal saline (0.9%). Compositions comprising such carriers can be formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition, A.Gennaro, eds., Mack Publishing Co., Easton, P.A., 1990; and Remington, the science and Practice of Pharmacy, 20 th edition, Mack Publishing, 2000).

The term "k_on"is intended herein to refer to the binding rate constant (ontate constant) of an antibody to an antigen.

The term "k_off"is intended herein to mean the off rate constant (off constant) at which an antibody dissociates from an antibody/antigen complex.

The term "K_D"is intended herein to refer to the equilibrium dissociation constant for antibody-antigen interactions.

Composition and method for preparing the same

Anti-amyloid beta antibodies and polypeptides

The present invention provides antibodies that bind to the C-terminus of a β peptide. The present invention provides a beta-amino acid derivative_1-40、Aβ_1-42And Abeta_1-43A bound antibody or polypeptide. In some embodiments, the antibody isThe body or polypeptide being such as to bind to Abeta more than it does_1-42And Abeta_1-43Binding of Abeta with higher affinity_1-40. In some embodiments, the antibody binds to a β_1-36、Aβ_1-37、Aβ_1-38And Abeta_1-39And (4) combining. In some embodiments, the antibody binds to a β_22-35And (4) combining. In some embodiments, the antibody binds to a β_28-40And (4) combining. In some embodiments, the antibody or polypeptide binds to a β_1-40Including amino acids 25-34 and 40.

The invention also provides a composition comprising any of the antibodies or polypeptides described herein (e.g., antibody 6G and variants thereof shown in table 3 or a polypeptide derived from antibody 6G and variants thereof shown in table 3); or a polynucleotide as described herein, including pharmaceutical compositions. As used herein, a composition comprises one or more of the following compounds with A beta_1-40And/or one or more antibodies that encode one or more antibodies that bind to a β (which may or may not be an antibody) and/or one or more antibodies that bind to a β_1-40A sequence of the C-terminal bound antibody or polypeptide of (a). These compositions may also contain suitable excipients, for example, pharmaceutically acceptable excipients well known in the art, including buffers.

The antibodies and polypeptides of the invention are characterized by any (one or more) of the following features: (a) and Abeta_1-40、Aβ_1-42And Abeta_1-43Combining; (b) and Abeta_1-40、Aβ_1-42And Abeta_1-43In combination with Abeta_1-40The binding affinity is higher than that of A beta_1-42And Abeta_1-43Affinity of binding; (c) and Abeta_1-40Epitope binding including amino acids 25-34 and 40 above; (d) and Abeta_1-36、Aβ_1-37、Aβ_1-38And Abeta_1-39Bind, but with less affinity than it binds to A β_1-36(ii) affinity; (e) at a K of less than about 1 μ M_DAnd Abeta_22-37Combining; (f) and Abeta_22-35Combining; (g) and Abeta_28-40Combining; (h) does not bind APP expressed in the cells; (i) inhibiting the formation of amyloid plaques in a subject; (j) reducing amyloid in a subjectWhite spots; (k) treating, preventing, ameliorating one or more symptoms of alzheimer's disease or other a β accumulation-related diseases (e.g., Down's syndrome, parkinson's disease, multi-infarct dementia, mild cognitive impairment, cerebral amyloid angiopathy, depression, Creutzfeldt-Jakob disease, dementia with Lewy bodies); (1) improving cognitive function. The antibodies and polypeptides of the invention may also have impaired effector functions as described herein. Unlike other anti-a β antibodies reported, antibodies and polypeptides with impaired effector function may exhibit the desired complete profile. For example, the compositions of the present invention may not cause significant or unacceptable levels of any one or more of the following: bleeding in the cerebrovascular system (cerebral hemorrhage); meningoencephalitis (including altered nuclear magnetic resonance scans); elevated white blood cell count in cerebrospinal fluid; inflammation of the central nervous system.

Accordingly, the present invention provides any of the following, or compositions (including pharmaceutical compositions) comprising any of the following: (a) antibody 6G or variants thereof shown in table 3; (b) fragments or regions of antibody 6G or variants thereof shown in table 3; (c) a light chain of antibody 6G or variants thereof shown in table 3; (d) the heavy chain of antibody 6G or variants thereof shown in table 3; (e) one or more variable regions of the light and/or heavy chain from antibody 6G or variants thereof shown in table 3; (f) one or more CDRs (1, 2, 3,4, 5, or 6 CDRs) of antibody 6G or variants thereof shown in table 3; (g) CDR H3 from antibody 6G heavy chain; (h) CDR L3 from the light chain of antibody 6G or the variants thereof shown in table 3; (i) 3 CDRs from the light chain of antibody 6G or variants thereof shown in table 3; (j) 3 CDRs from the heavy chain of antibody 6G or variants thereof shown in table 3; (k) 3 CDRs from the light chain of antibody 6G or variants thereof shown in table 3 and 3 CDRs from the heavy chain of antibody 6G or variants thereof shown in table 3; and (1) an antibody comprising any one of (b) to (k). The invention also provides polypeptides comprising any one or more of the above items.

The CDR portions of antibody 6G (including Chothia and Kabat CDRs) are shown diagrammatically in FIG. 1. Determination of CDR regions is well known to those skilled in the art. It is understood that in some embodiments, the CDRs may be a combination of Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In some embodiments, the CDRs are Kabat CDRs. In other embodiments, the CDR is a Chothia CDR. In other words, in embodiments having more than one CDR, the CDR can be any one of Kabat, Chothia, a combination CDR, or a combination thereof.

In some embodiments, the invention provides a polypeptide (which may or may not be an antibody) comprising at least one CDR, at least two, at least three, at least four, at least five, or all six CDRs substantially identical to at least one CDR, at least two, at least three, or at least four, at least five, or all six CDRs of 6G or variants thereof shown in table 3. Other embodiments include antibodies having at least two, three, four, five, or six CDRs that are substantially identical to at least two, three, four, five, or six CDRs of or derived from 6G. In some embodiments, the at least one, two, three, four, five, or six CDRs are at least about 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one, two, three, four, five, or six CDRs of 6G or variants thereof shown in table 3. It will be appreciated that for the purposes of the present invention, binding specificity and/or overall activity is generally maintained, but the degree of activity may be different (may be higher or lower) compared to 6G or the variants thereof shown in table 3.

The invention also provides a polypeptide (which may or may not be an antibody) comprising the amino acid sequence of 6G or a variant thereof shown in table 3, wherein the amino acid sequence has any one of: antibody 6G or at least 5 consecutive amino acids, at least 8 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, at least about 25 consecutive amino acids, at least about 30 consecutive amino acids in the sequence of the variant shown in table 3 thereof, wherein at least 3 of these amino acids are from the variable region of 6G (figure 1) or the variant shown in table 3 thereof. In one embodiment, the variable region is from a light chain of 6G. In another embodiment, the variable region is from a heavy chain of 6G. An exemplary polypeptide has contiguous amino acids (length as described above) from the heavy chain variable region and the light chain variable region of 6G. In another embodiment, the 5 (or more) contiguous amino acids are from the Complementarity Determining Region (CDR) of 6G shown in fig. 1. In some embodiments, the contiguous amino acids are from the variable region of 6G.

The binding affinity of the antibodies and polypeptides of the invention may vary, and need not be (but may be) a particular value or range, as in the exemplary embodiments described below. Antibodies and polypeptides of the invention and Abeta_1-40Binding affinity (K) of (2)_D) Can be about 0.10 to about 0.80nM, about 0.15 to about 0.75nM, and about 0.18 to about 0.72 nM. In some embodiments, the binding affinity is about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, about 40pM, or greater than about 40 pM. In one embodiment, the binding affinity is about 2pM to about 22 pM. In other embodiments, the binding affinity is less than about 10nM, about 5nM, about 4nM, about 3nM, about 2nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 150pM, about 100pM, about 90pM, about 80pM, about 70pM, about 60pM, about 50pM, about 40pM, about 30pM, about 10 pM. In some embodiments, the binding affinity is about 10 nM. In other embodiments, the binding affinity is less than about 10nM, less than about 50nM, less than about 100nM, less than about 150nM, less than about 200nM, less than about 250nM, less than about 500nM, or less than about 1000 nM. In other embodiments, the binding affinity is less than about 5 nM. In other embodiments, the binding affinity is less than about 1 nM. In other embodiments, the binding affinity is about 0.1nM or about 0.07 nM. In other embodiments, the binding affinity is less than about 0.1nM or less than about 0.07 nM. In other embodiments, the binding affinity is from about 10nM, about 5nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, largeAny of about 200pM, about 150pM, about 100pM, about 90pM, about 80pM, about 70pM, about 60pM, about 50pM, about 40pM, about 30pM, about 10pM to about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, or about 40 pM. In some embodiments, the binding affinity is any one of about 10nM, about 5nM, about 1nM, about 900pM, about 800pM, about 700pM, about 600pM, about 500pM, about 400pM, about 300pM, about 200pM, about 150pM, about 100pM, about 90pM, about 80pM, about 70pM, about 60pM, about 50pM, about 40pM, about 30pM, about 10 pM. In still other embodiments, the binding affinity is about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, about 40pM, or greater than about 40 pM.

The antibodies and polypeptides of the invention may also be conjugated to A beta_1-36、Aβ_1-37、Aβ_1-38、Aβ_1-39、Aβ_1-42And Abeta_1-43But with less binding affinity than they bind to a β_1-40Binding affinity of (4). In some embodiments, the antibody or polypeptide binds to a β_1-36、Aβ_1-37、Aβ_1-38、Aβ_1-39、Aβ_1-42And Abeta_1-43K of any one or more of_DIs a combination of it with Abeta_1-40K of_DAt least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 80 times, at least about 100 times, at least about 150 times, at least about 200 times, or at least about 250 times.

The invention also provides methods of making any of these antibodies or polypeptides. Antibodies of the invention can be prepared by methods known in the art. For example, the antibody can be prepared by immunizing a mammal with an A β peptide (e.g., A β 25-40 as an immunogen). The polypeptides may be prepared by proteolytic or other degradation of the antibody, or by recombinant methods (i.e., single or fused polypeptides) as described above, or by chemical synthesis. The polypeptides of the antibodies, particularly the shorter polypeptides of no more than about 50 amino acids, can be conveniently prepared by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, antibodies can be prepared by an automated polypeptide synthesizer using a solid phase method. See also U.S. patent 5,807,715; 4,816,567; and 6,331,415.

In another alternative, the antibody may be recombinantly produced using methods well known in the art. In one embodiment, the polynucleotide comprises SEQ ID NO: 9 and SEQ ID NO: 10 encoding the heavy and/or light chain variable region of antibody 6G. In another embodiment, the polypeptide comprising SEQ ID NO: 9 and SEQ ID NO: 10 into one or more vectors for expression or amplification. The sequence encoding the antibody of interest may be maintained in a vector in the host cell, and the host cell may then be expanded and frozen for use. Vectors (including expression vectors) and host cells are further described herein.

The invention also includes single chain variable fragments ("scFv") of the antibodies of the invention, e.g., 6G. Single chain variable fragments can be prepared by linking the light and/or heavy chain variable regions together using short linking peptides. Bird et al (1988) Science 242: 423-426. An example of a linker peptide is (GGGGS)₃Which forms a bridge of about 3.5nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Linkers with other sequences have also been designed and used. Bird et al (1988). The linker in turn can be modified to have additional functions, such as attachment of a drug or attachment of a solid support. The single-chain variants may be prepared recombinantly or synthetically. For synthetic preparation of scFv, an automated synthesizer can be used. For recombinant production of an scFv, a suitable plasmid containing a polynucleotide encoding the scFv can be introduced into a suitable host cell, which can be a eukaryotic cell such as a yeast, plant, insect, or mammalian cell, or a prokaryotic cell such as e. The polynucleotide encoding the scFv of interest may be obtained byBy a standard procedure such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

Other forms of single chain antibodies, such as diabodies, are also encompassed by the invention. Diabody is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but two antigen-binding sites are generated by using linkers that are so short as to not allow pairing of the two domains on the same chain, thereby forcing the domains to pair with complementary domains on the other chain (see, e.g., Holliger, p. et al (1993) proc. natl. acad. sci. usa 90: 6444-.

For example, bispecific antibodies, monoclonal antibodies having binding specificity for at least two different antigens can be prepared using the antibodies disclosed herein. Methods for making bispecific antibodies are known in the art (see, e.g., Suresh et al, 1986, Methods in Enzymology 121: 210). Traditionally, recombinant production of bispecific antibodies has been based on co-expression of two immunoglobulin heavy-light chain pairs, wherein the two heavy chains have different specificities (Millstein and Cuello, 1983, Nature, 305: 537-539).

According to one method of making bispecific antibodies, antibody variable domains (antibody-antigen binding sites) having the desired binding specificities are fused to immunoglobulin constant domain sequences. The fusion preferably has an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2 and CH3 regions. Preferably, there is a first heavy chain constant region (CH1) in at least one of the fusions that contains the site necessary for binding to a light chain. The DNA encoding these immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where the three polypeptide chains used in the construct have unequal ratios that provide optimal yields, this allows for great flexibility in adjusting the mutual proportions of the three polypeptide fragments. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when the expression of at least two polypeptide chains in equal ratios can lead to high yields or when the ratios are not of particular interest.

In one approach, a bispecific antibody consists 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. The asymmetric structure, the immunoglobulin light chain being present only in one half of the bispecific molecule, facilitates the separation of the desired bispecific compound from the unwanted immunoglobulin chain combinations. This method is described in PCT publication No. WO94/04690, published 3/3 of 1994.

Heteroconjugate antibodies (heteroconjugate 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 (U.S. Pat. No. 4,676,980) and to treat HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugated antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents and techniques are well known in the art and are described in U.S. Pat. No. 4,676,980.

Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate (butylimidate).

Humanized antibodies comprising one or more CDRs of antibody 6G or derived from one or more CDRs of antibody 6G can be made using any method known in the art. For example, monoclonal antibodies can be humanized using 4 general procedures. The steps are as follows: (1) determining the nucleotide sequence and predicted amino acid sequence of the starting antibody light and heavy chain variable domains; (2) designing a humanized antibody, i.e., determining which antibody framework regions to use in the humanization process; (3) actual humanization methods/techniques; and (4) transfecting and expressing the humanized antibody. See, for example, U.S. Pat. nos. 4,816,567; 5,807,715, respectively; 5,866,692, respectively; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; 6,180,370; 5,225,539; 6,548,640.

In recombinant humanized antibodies, the Fc γ portion may be modified to avoid interaction with the Fc γ receptor and the complement immune system. Modification of this type was designed by doctor Clark, university of cambridge, department of pathology, and techniques for making such antibodies are described in WO99/58572, published at 11/18 of 1999.

For example, if the antibody is used in clinical trials and human therapy, the constant region can be engineered to more closely resemble a human constant region to avoid an immune response. See, for example, U.S. patent nos. 5,997,867 and 5,866,692.

The invention includes modified forms of antibody 6G, including functionally equivalent antibodies and variants with enhanced or diminished activity and/or affinity that do not significantly affect their properties. For example, the amino acid sequence of antibody 6G can be mutated to obtain a polypeptide having the desired A.beta._1-40The peptide binds to the antibody of affinity. Modification of polypeptides is routine in the art and need not be described in detail herein. Modifications to the polypeptides are exemplified in the examples. Examples of modified polypeptides include polypeptides having conservative amino acid residue substitutions, one or more amino acid deletions or additions that do not significantly adversely alter functional activity, or the use of chemical analogs.

Amino acid sequence insertions include amino and/or carboxy terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as single or multiple amino acid residue insertions within the sequence. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with enzymes or polypeptides that increase the serum half-life of the antibody.

Substitutional variants remove at least one amino acid residue in the antibody molecule and insert a different residue at that position. The most interesting sites for substitution mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in table 1 under the heading "conservative substitutions". If such substitutions result in an alteration in biological activity, more substantial alterations designated "exemplary substitutions" in Table 1 or described further below with reference to amino acid classes can be introduced and the products screened.

Table 1: amino acid substitutions

Substantial modification of the biological properties of an antibody can be achieved by selecting the structure for maintaining (a) the polypeptide backbone in the replacement region, e.g., in a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) substitution where side chain volume has a significantly different effect. Natural residues can be divided into the following groups based on the usual side chain properties:

(1) non-polar: norleucine, Met, Ala, Val, Leu, Ile;

(2) polar uncharged: cys, Ser, Thr, Asn, Gln;

(3) acidic (negatively charged): asp and Glu;

(4) basic (positively charged): lys, Arg;

(5) residues that affect side chain orientation: gly, Pro; and

(6) aromatic: trp, Tyr, Phe, His.

Non-conservative substitutions may be made by changing the members belonging to one of these classes to another class.

Any cysteine residue not involved in maintaining the normal conformation of the antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. In turn, cysteine bonds may be added to the antibody, particularly when the antibody is an antibody fragment such as an Fv fragment, in order to improve its stability.

Amino acid modifications can range from altering or modifying one or more amino acids to completely reproducing the design of a region, e.g., a variable region. Changes in the variable region may alter binding affinity and/or specificity. In some embodiments, no more than 1 to 5 conservative amino acid substitutions are made in a CDR domain. In other embodiments, no more than 1 to 3 conservative amino acid substitutions are made in a CDR domain. In still other embodiments, the CDR domain is CDRH3 and/or CDR L3.

Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, e.g., glycosylation, acetylation, and phosphorylation with different sugars. 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 influence the function of the protein (Boyd et al, 1996, mol. Immunol.32: 1311-1318; Wittwe and Howard, 1990, biochem.29: 4175-4180) and the intramolecular interactions between the different parts of the glycoprotein-which can influence the conformation of the glycoprotein and the three-dimensional surface presented (Hefferis and Lund, supra; Wys and Wagner, 1996, Current Opin. Biotech.7: 409-416). Oligosaccharides may also be used to target a given glycoprotein to certain molecules based on a specific recognition structure. Glycosylation of antibodies has also been reported to affect antibody-dependent cell-mediated cytotoxicity (ADCC). Specifically, CHO cells expressing β (1, 4) -N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing the formation of forked GlcNAc, under the regulation of tetracycline have been reported to have improved ADCC activity (Umana et al, 1999, Mature Biotech.17: 176-.

Glycosylation of antibodies is typically N-linked or O-linked. N-linked glycosylation refers to the attachment of a sugar moiety to the side chain of an asparagine residue. The tripeptide sequences, asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine (where X is any amino acid except proline) are recognition sequences that enzymatically attach the sugar moiety to the asparagine side chain. Thus, potential glycosylation sites will be created when any of these tripeptide sequences are present in a polypeptide. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence to include one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be achieved by adding or substituting one or more serine or threonine residues to the original antibody sequence (for O-linked glycosylation sites).

The glycosylation pattern of an antibody can also be altered without altering the underlying nucleotide sequence. Glycosylation is to a large extent dependent on the host cell used to express the antibody. Since the cell type used to express recombinant glycoproteins, such as antibodies, as potential therapeutic agents is rarely the naive cell, variations in antibody glycosylation patterns can be expected (see, e.g., Hse et al, 1997, J.biol.chem.272: 9062-.

Factors that influence glycosylation during recombinant production of antibodies include, in addition to host cell selection, growth pattern, media formulation, culture density, oxygenation, pH, purification protocols, and the like. Various methods have been proposed to alter the glycosylation pattern obtained in a particular host organism, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation, or certain types of glycosylation, can be removed from glycoproteins by enzymatic methods, e.g., using endoglycosidase h (endo h), N-glycosidase F (see description of example 1), endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. In addition, recombinant host cells can be genetically engineered to be deficient in the processing of certain types of polysaccharides. These and similar techniques are well known in the art.

Other methods of modification include the use of coupling techniques known in the art, including, but not limited to, enzymatic methods, oxidative substitution, and chelation. The labels can be attached, for example, by modification for use in immunoassay assays. Modified 6G polypeptides can be prepared using procedures well established in the art, and can be screened using standard assay assays known in the art, some of which are described below and in the examples.

Other antibody modifications include antibodies modified as described in PCT publication No. WO99/58572, published 11/18/1999. These antibodies, in addition to comprising a binding domain directed to a target molecule, comprise effector domains having amino acid sequences substantially homologous to all or part of a constant domain of a human immunoglobulin heavy chain. These antibodies are capable of binding to a target molecule without triggering significant complement-dependent lysis, or cell-mediated target destruction. In some embodiments, the effector domain is capable of specifically binding FcRn and/or fcyriib. These are typically derived from two or more human immunoglobulin heavy chains C_H2-domain chimeric domain. Antibodies modified in this manner are particularly suitable for use in long-term antibody therapy to avoid inflammatory and other adverse reactions that occur with conventional antibody therapy.

The present invention includes embodiments of affinity saturation. For example, affinity-saturating antibodies can be prepared by procedures known in the art (Marks et al, 1992, Bio/Technology, 10: 779- & 783; Barbas et al, 1994, Proc Nat. Acad. Sci, USA 91: 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; and WO 2004/058184).

The following methods can be used to modulate the affinity of an antibody and to characterize the CDRs. A method for characterising CDRs and/or altering (e.g. increasing) binding affinity of a polypeptide, such as an antibodyThe method is called "library scanning mutagenesis". In general, library scanning mutagenesis is performed as follows. One or more amino acid positions in a CDR are replaced with two or more (e.g., 3,4, 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids using methods known in the art. This results in small libraries of clones (in some embodiments, 1 library for each amino acid position analyzed), each library having a complexity of two or more members (if two or more amino acids are substituted at each position). Typically, the library also includes clones containing the original (non-substituted) amino acid. A small number of clones, e.g., about 20-80 clones, per library (depending on the complexity of the library) are screened for binding affinity to the target polypeptide (or other binding agent), and candidates with increased, identical, reduced or no binding are identified. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using BIAcore surface plasmon resonance analysis that detects differences in binding affinity of about 2-fold or more. When the starting antibody has been raised to a relatively high affinity, e.g., a K of about 10nM or less_DBIAcore is particularly useful when binding occurs. Screening using BIAcore surface plasmon resonance is described in the examples herein.

Binding affinity can be determined using Kinexa Biocensor, the scintillation proximity Assay (scientific proximity Assay), ELISA, ORIGEN Immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and/or yeast display. Binding affinities may also be screened using a suitable bioanalytical assay.

In some embodiments, all 20 substitutions of the natural amino acids are made at each amino acid position of the CDR (in some embodiments, one position at a time) using art-recognized mutagenesis methods, some of which are described herein. This results in small libraries of clones (in some embodiments, one for each amino acid position analyzed), each library having a complexity of 20 members (if all 20 amino acid substitutions occur at each position).

In some embodiments, the library to be screened comprises substitutions that occur at two or more positions, which positions may be located in the same CDR or in two or more CDRs. Thus, the library may comprise substitutions occurring at two or more positions in one CDR. The library may comprise substitutions that occur at two or more positions in two or more CDRs. The library may comprise substitutions occurring at 3,4, 5 or more positions present in two, three, four, five or six CDRs. The substitutions may be made using low redundancy codons. See, e.g., Balint et al (1993) Gene137 (1): 109-18 in table 2.

The CDRs may be CDRH3 and/or CDRL 3. The CDR may be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH 3. The CDRs may be Kabat CDRs, Chothia CDRs, or extended CDRs.

Candidates with improved binding can be sequenced, thereby identifying CDR substitution mutants that result in improved affinity (also referred to as "improved" substitutions). Candidates that bind may also be sequenced, thereby identifying CDR substitutions that remain bound.

Multiple rounds of screening can be performed. For example, candidates with improved binding (each comprising an amino acid substitution at one or more positions in one or more CDRs) can also be used to design a second library comprising at least the original and substituted amino acids at each improved CDR position (i.e., the CDR amino acid position in which the substitution mutant exhibiting improved binding is located). The preparation of the library, and screening or selection is discussed further below.

Library scanning mutagenesis also provides a means for characterizing CDRs, and the frequency of clones with improved binding, identical binding, reduced binding or no binding also provides information about the importance of each amino acid position for the stability of the antibody-antigen complex. For example, if a position of a CDR remains bound when changed to all 20 amino acids, that position may be identified as not likely to be necessary for antigen binding. Conversely, if a position of a CDR remains bound for only a small fraction of substitutions, that position is identified as a position of importance to the CDR function. Thus, library scanning mutagenesis methods can yield information about positions in a CDR that can be changed to many different amino acids (including all 20 amino acids) as well as positions in a CDR that cannot be changed or can be changed to only a few amino acids.

Candidates with improved affinity may be combined in a second library that includes the improved amino acid at that position, the original amino acid, and may also include other substitutions at that position, depending on the complexity of the library that is desired or allowed using the desired screening or selection method. Further, at least two or more amino acids may be randomly assigned to adjacent amino acid positions, if desired. Randomization of adjacent amino acids may allow for additional conformational flexibility in the mutated CDRs, which in turn may allow or facilitate the introduction of a greater number of modifying mutations. The library may also contain substitutions at positions that do not show improved affinity in the first round of screening.

The selection is performed using any method known in the art, including screening using BIAcore surface plasmon resonance analysis, and using any selection method known in the art for selection, including phage display, yeast display, and ribosome display, screening or selecting library members having improved and/or altered binding affinities from the second library.

The invention also includes fusion proteins comprising one or more fragments or regions from an antibody (e.g., 6G) or polypeptide of the invention. In one embodiment, there is provided a polypeptide comprising SEQ ID NO: 2 (fig. 1) and/or at least 10 consecutive amino acids of the light chain variable region of SEQ ID NO: 1 (figure 1) in a heavy chain variable region (figure 1). In other embodiments, there is provided a polypeptide comprising SEQ ID NO: 2 (fig. 1) and/or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the light chain variable region set forth in SEQ ID NO: 1 (fig. 1), at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the heavy chain variable region set forth in fig. 1. In another embodiment, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 1 and/or a 6G light chain variable region and/or a heavy chain variable region. In another embodiment, the fusion polypeptide comprises one or more CDRs of 6G. In yet another embodiment, the fusion polypeptide comprises CDR H3 and/or CDR L3 of antibody 6G. For the purposes of the present invention, a 6G fusion protein contains one or more 6G antibodies and another amino acid sequence that is not linked to 6G in the native molecule, e.g., a heterologous sequence or a homologous sequence from another region. Examples of heterologous sequences include, but are not limited to, a "tag," such as a FLAG tag or a 6His tag. Labels are well known in the art.

The 6G fusion polypeptides can be prepared by methods known in the art, e.g., synthetic or recombinant methods. Typically, the 6G fusion proteins of the invention are produced by preparing and expressing polynucleotides encoding them using recombinant methods described herein, but can also be produced by other means known in the art, including, for example, chemical synthesis.

The invention also provides compositions comprising a 6G antibody or polypeptide conjugated (e.g., linked) to a reagent that facilitates coupling to a solid support (e.g., biotin or avidin). For simplicity, reference is generally made to 6G or antibodies, but it should be understood that these methods are applicable to any of the a β s described herein_1-40In combination with the embodiments. Conjugation generally refers to linking these components together as described herein. Linking (which generally means fixing the ingredients in close relation, at least for administration purposes) can be achieved in a variety of ways. For example, when the reagent and the antibody each have a substituent capable of reacting with each other, the reagent and the antibody may react directly. For example, a nucleophilic group, e.g., amino or sulfhydryl, located on one of the molecules may be capable of reacting with a carbonyl-containing group, e.g., anhydride or acyl, located on the other moleculeHalocarbons, or alkyl groups containing a good leaving group (e.g., halogen).

The antibody or polypeptide of the invention may be linked to a labeling agent (alternatively referred to as a "label"), such as a fluorescent molecule, a radioactive molecule, or any other label known in the art. Labels are known in the art and typically provide (directly or indirectly) a signal.

The invention also provides compositions (including pharmaceutical compositions) and kits comprising antibody 6G, and, as explained in this disclosure, any or all of the antibodies and/or polypeptides described herein.

anti-Abeta antibodies and polypeptides with impaired effector function

The antibodies or polypeptides described herein (including pharmaceutical compositions comprising the antibodies or polypeptides) may have impaired effector function. Herein, an antibody or polypeptide having "impaired effector function" (which may be used interchangeably with "immunologically inert" or "partially immunologically inert") refers to an antibody or polypeptide that does not have any effector function or has one or more reduced activities of effector function (as compared to an antibody or polypeptide having an unmodified or naturally occurring constant region), e.g., does not have or has reduced any one or more of the following activities: a) triggering complement-mediated lysis; b) stimulation of antibody-dependent cell-mediated cytotoxicity (ADCC); and c) activating microglia. The effector functional activity may be reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%. In some embodiments, the antibody binds to beta-amyloid peptide without triggering significant complement-dependent target lysis or cell-mediated target destruction. For example, Fc receptor binding sites on the constant region may be modified or mutated to remove or reduce binding affinity to certain Fc receptors, e.g., Fc γ RI, Fc γ RII, and/or Fc γ RIII. For simplicity, reference to an antibody should be understood to apply to the polypeptide as well. The EU numbering system (Kabat et al, Sequences of Proteins of Immunological Interest; 5 th edition, public health Service, National Institutes of health, Bethesda, Md., 1991) is used to indicate which amino acid residue (or residues) in a constant region (e.g., the constant region of an IgG antibody) is altered or mutated. This numbering may be for a particular type of antibody (e.g., IgG1) or species (e.g., human), but it is understood that similar variations may be made across antibody and species types.

In some embodiments, an antibody that specifically binds to a β peptide comprises a heavy chain constant region with impaired effector function. The heavy chain constant region may have a native sequence or be a variant. In some embodiments, the amino acid sequence of the native heavy chain constant region is mutated, e.g., by amino acid substitution, insertion, and/or deletion, thereby impairing effector function of the constant region. In some embodiments, the N-glycosylation of the heavy chain constant region Fc region can also be altered, e.g., can be removed, in whole or in part, thereby impairing the effector function of the constant region.

In some embodiments, effector function is impaired by removing N-glycosylation of the anti- Α β peptide Fc region (e.g., in the CH2 domain of IgG). In some embodiments, the N-glycosylation of the Fc region is removed by mutating amino acid residues that are glycosylated or flanking residues that form part of the glycosylation recognition sequence of the constant region. The tripeptide sequences, asparagine-X-serine (N-X-S), asparagine-X-threonine (N-X-T) and asparagine-X-cysteine (N-X-C) (where X is any amino acid except proline), are recognition sequences for the enzymatic attachment of the sugar moiety to the asparagine side chain to form N-glycosylation. Mutation of any amino acid in this tripeptide sequence in the constant region results in aglycosylated IgG. For example, the N-glycosylation site N297 of human IgG1 and IgG3 can be mutated to A, D, Q, K or H. See, Tao et al, j.immunology, 143: 2595-2601 (1989); and Jefferis et al, Immunological Reviews 163: 59-76(1998). Human IgG1 and IgG3 with Asn-297 to Gln, His or Lys substitutions have been reported to not bind to human Fc γ RI and not activate complement, with complete loss of Clq binding capacity for IgG1 and a dramatic decrease in IgG 3. In some embodiments, the amino acid N in the tripeptide sequence is mutated to any one of the following amino acids: a, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y. In some embodiments, the amino acid N in the tripeptide sequence is mutated to a conservative substitution. In some embodiments, amino acid X in the tripeptide sequence is mutated to proline. In some embodiments, the amino acid S in the tripeptide sequence is mutated to a, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, Y. In some embodiments, the amino acid T in the tripeptide sequence is mutated to a, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, Y. In some embodiments, the amino acid C in the tripeptide sequence is mutated to a, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, Y. In some embodiments, the amino acids located after the tripeptide are mutated to P. In some embodiments, the N-glycosylation of the constant region is removed enzymatically (e.g., N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3, and endoglycosidase H as shown in example 1). N-glycosylation can also be removed by producing the antibody in a cell line that is deficient in N-glycosylation. Wright et al, JImmunol.160 (7): 3393-402(1998).

In some embodiments, the amino acid residues that interact with oligosaccharides attached to the N-glycosylation sites of the constant region are mutated to reduce binding affinity to Fc γ RI. For example, F241, V264, D265 of human IgG3 can be mutated. See, Lund et al, j.immunology157: 4963-4969(1996).

In some embodiments, effector function is impaired by modification regions such as 233-. PCT WO99/58572 and Armour et al describe antibodies that, in addition to comprising a binding domain directed to a target molecule, comprise effector domains having amino acid sequences substantially homologous to all or part of a human immunoglobulin heavy chain constant region. These antibodies are capable of binding to a target molecule while not triggering significant complement-dependent target lysis or cell-mediated target destruction. Some embodimentsIn one embodiment, the effector domain has reduced affinity for Fc γ RI, Fc γ RIIa, and/or Fc γ RIII. In some embodiments, the effector domain is capable of specifically binding FcRn and/or fcyriib. These are typically derived from two or more human immunoglobulin heavy chains C_H2-domain chimeric domain. Antibodies modified in this way are particularly suitable for long-term antibody therapy to avoid inflammatory and other adverse effects that occur with conventional antibody therapy. In some embodiments, the antibody heavy chain constant region is human heavy chain IgG1 with any of the following mutations: 1) a327a330P331 to G327S330S 331; 2) E233L234L235G236 to P233V234a235 with G236 missing; 3) E233L234L235 to P233V234a 235; 4) E233L234L235G236a327a330P331 to P233V234a235G327S330S331 and G236 is missing; 5) E233L234L235a327a330P331 to P233V234a235G327S330S 331; and 6) N297 to A297 or any other amino acid other than N. These mutations may be combined, for example, any of 1) to 5) may be combined with 6). In some embodiments, the antibody heavy chain constant region is human heavy chain IgG2 with the following mutations: a330P331 to S330S 331; n297 to Q297; and N297G327a330P331 to Q297G327S330S 331. In some embodiments, the antibody heavy chain constant region is human heavy chain IgG4 with any of the following mutations: E233F234L235G236 to P233V234a235 with G236 missing; E233F234L235 to P233V234a 235; P228L235 to S228E 235; n297 to Q297; and E233F234L235G236N297 to P233V234a235G236Q 297.

The antibody constant region may also be modified to disrupt complement activation. For example, complement activation of IgG antibodies after binding to the C1 component of complement can be reduced by mutating amino acid residues within the C1 binding motif (e.g., Clq binding motif) in the constant region. Ala mutations to each of D270, K322, P329 and P331 of human IgG1 have been reported to significantly reduce the ability of the antibody to bind Clq and activate complement. For murine IgG2b, the Clq binding motif consists of residues E318, K320 and K322. Idusogie et al, j.immunology164: 4178-4184 (2000); duncan et al, Nature 322: 738-740(1988).

The Clq binding motifs E318, K320 and K322 identified for murine IgG2b are believed to be common to other antibody isotypes. Duncan et al, Nature 322: 738-740(1988). The Clq binding activity of IgG2b can be abolished by replacing any of the three specific residues with a residue having unsuitable functionality on its side chain. It is not necessary to abolish Clq binding by replacing the ionic residue with Ala. Other alkyl substituted non-ionic residues, e.g., Gly, Ile, Leu or Val, or aromatic non-polar residues such as Phe, Tyr, Trp and Pro may also be used in place of any of the three residues to abolish the Clq binding. In addition, polar non-ionic residues such as Ser, Thr, Cys and Met may also be used instead of residues 320 and 322 and not 318 to abolish Clq binding activity.

The invention also provides antibodies with impaired effector function, wherein the antibodies have a modified hinge region. The binding affinity of human IgG to its Fc receptor can be modulated by modifying the hinge region. Canfield et al, J.Exp.Med.173: 1483-1491 (1991); hezareh et al, J.Virol.75: 12161-12168 (2001); redpath et al, Humanimmunology 59: 720-727(1998). Specific amino acid residues may be mutated or deleted. The modified hinge region may comprise a complete hinge region derived from an antibody of a different antibody type or subclass than that of the CH1 domain. For example, the constant region (CH1) of an IgG class antibody can be linked to the hinge region of an IgG4 class antibody. Alternatively, the novel hinge region may comprise a portion of a natural hinge or repeating units, wherein each unit in the repeat is derived from a natural hinge region. In some embodiments, the native hinge region is altered by converting one or more cysteine residues to neutral residues, e.g., alanine, or by converting residues located at appropriate positions to cysteine residues. U.S. Pat. No. 5,677,425. These changes can be made as described herein using art-recognized protein chemistry and, preferably, genetic engineering techniques.

Polypeptides that specifically bind to a β peptides and are fused to heavy chain constant regions with impaired effector function may also be used in the methods described herein. In some embodiments, the polypeptide comprises a sequence derived from antibody 6G or a variant thereof shown in table 3. In some embodiments, the polypeptide is derived from a single domain antibody that binds to a β peptide. Single domain antibodies can be prepared using methods known in the art. Omidfar et al, Tumour biol.25: 296-305 (2004); herring et al, Trends in Biotechnology 21: 484-489(2003).

In some embodiments, the antibody or polypeptide is F (ab')₂And (3) fragment. In some embodiments, the antibody or polypeptide is a Fab fragment. In some embodiments, the antibody or polypeptide is a single chain antibody scFv. In some embodiments, the antibody or polypeptide is PEGylated F (ab')₂And (3) fragment. In some embodiments, the antibody or polypeptide is a pegylated Fab fragment. In some embodiments, the antibody or polypeptide is a pegylated single chain antibody scFv.

Other methods known in the art for producing antibodies with impaired effector function may also be used.

Antibodies or polypeptides having modified constant regions can be detected in one or more assays to assess the level of decreased biological activity of effector function relative to the starting antibody. For example, an antibody or polypeptide having an altered Fc region can be evaluated for the ability to bind complement or an Fc receptor (e.g., an Fc receptor on a microglia cell) or an antibody or polypeptide having an altered hinge region using the assays disclosed herein as well as any art-recognized assays. PCT WO 99/58572; armour et al, Molecular Immunology 40: 585-593 (2003); reddy et al, j.immunology 164: 1925-1933 (2000); song et al, Infection and Immunity 70: 5177-5184(2002).

Competition assays can be used to determine whether two antibodies bind to the same epitope by recognizing the same or spatially overlapping epitopes or whether one antibody competitively inhibits the binding of the other antibody to the antigen. These assays are known in the art. Typically, antigens are immobilized on multi-well plates and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured. For such competitive assays, the labels commonly used are radioactive labels or enzymatic labels.

Antibodies and polypeptides that specifically bind to a β can be screened for efficacy in clearing amyloid deposits and other beneficial effects, such as improved cognitive ability. For example, the antibody or polypeptide can be administered to an animal with a pathological condition of Alzheimer's disease. Various animal models of Alzheimer's disease are known in the art. Following administration, the level of compact and diffuse amyloid plaques, cognitive behavioral analysis, and microglial activation and microhemorrhage can be detected using methods known in the art and described in detail in example 2. PCT WO 2004/032868; wilcock et al, J.Neurosci.23: 3745-3751 (2003); wilcock et al, J.Neurooil flash 1: 24(2004).

Polynucleotides, vectors and host cells

The invention also provides isolated polynucleotides encoding the antibodies and polypeptides of the invention, including antibodies comprising polypeptide sequences of the light and heavy chain variable regions shown in figure 1, and vectors and host cells comprising the polynucleotides.

Accordingly, the present invention provides polynucleotides (or compositions, including pharmaceutical compositions) comprising a polynucleotide encoding any one of: a) antibody 6G or variants thereof shown in table 3; b) fragments or regions of antibody 6G or variants thereof shown in table 3; c) a light chain of antibody 6G or variants thereof shown in table 3; d) the heavy chain of antibody 6G or variants thereof shown in table 3; e) one or more variable regions of the light and/or heavy chain from antibody 6G or variants thereof shown in table 3; f) one or more CDRs (one, two, three, four, five or six CDRs) of antibody 6G or variants thereof shown in table 3; g) CDR H3 from antibody 6G heavy chain; h) CDR L3 from the light chain of antibody 6G or the variants thereof shown in table 3; i) 3 CDRs from the light chain of antibody 6G or variants thereof shown in table 3; j) 3 CDRs from the heavy chain of antibody 6G or variants thereof shown in table 3; k) 3 CDRs from the light chain of antibody 6G or variants thereof shown in table 3 and 3 CDRs from the heavy chain of antibody 6G or variants thereof shown in table 3; and 1) an antibody comprising any one of (b) to (k). In some embodiments, the polynucleotide comprises SEQ ID NO: 9 and SEQ ID NO: 10 or a polynucleotide as set forth in claim 10.

In another aspect, the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) and polypeptides described herein, e.g., antibodies and polypeptides having impaired effector function. Polynucleotides can be prepared by methods known in the art.

In another aspect, the invention provides a composition (e.g., a pharmaceutical composition) comprising any of the polynucleotides of the invention. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding antibody 6G described herein. In other embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies or polypeptides described herein. In still other embodiments, the composition comprises SEQ ID NO: 9 and SEQ ID NO: 10 or a polynucleotide as set forth in claim 10. The administration of the expression vector and polynucleotide composition will be further described herein.

In another aspect, the invention provides a method of making any of the polynucleotides described herein.

Polynucleotides complementary to any such sequence are also encompassed by the invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic DNA) or RNA molecules. RNA molecules include HnRNA molecules that contain introns and correspond to DNA molecules in a one-to-one manner, as well as mRNA molecules that do not contain introns. Other coding or non-coding sequences may, but need not, be present in the polynucleotides of the invention. The polynucleotide may, but need not, be linked to other molecules and/or support materials.

The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or portion thereof) or may comprise a variant of that sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the naturally-occurring immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally 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 sequence encoding the native antibody or portion thereof.

Two polynucleotide or polypeptide sequences are said to be "identical" if their nucleotide or amino acid sequences are identical in the two sequences if they are aligned for maximum correspondence as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. Herein, a "comparison window" refers to a segment of at least about 20 contiguous positions, typically 30 to about 75, 40 to about 50 contiguous positions, in which a sequence can be compared to a reference sequence having the same number of contiguous positions after optimal alignment has been performed.

The sequence best alignment performed for comparison purposes can be performed using the Megalign program in Lasergene bioinformatics software group (DNASTAR, inc., Madison, WI) and default parameters. This program embodies several alignment schemes described in the following references: dayhoff, M.O. (1978) model of evolutionary changes in proteins-matrix for detecting distant relationships (A model of evolution change in proteins-matrix for detecting differences) Dayhoff, M.O (eds.) Atlas of Protein sequences and Structure, National biomedical research Foundation, Washington DC Vol.5, Suppl.3, pp.345-358; hein J., 1990, United apparatus to Alignment and olefins pp.626-645 Methods in Enzymology vol.183, Academic Press, Inc., San Diego, Calif.; higgins, d.g. and Sharp, p.m., 1989, cabaos 5: 151-153; myers, e.w. and Muller w., 1988, cabaos 4: 11-17; robinson, e.d., 1971, comb. 105; santou, n., Nes, m., 1987, mol.biol.evol.4: 406-425; sneath, p.h.a. and Sokal, r.r., 1973, Numerical taxomones and Practice of Numerical taxomones, Freeman Press, San Francisco, CA; wilbur, w.j. and Lipman, d.j., 1983, proc.natl.acad.sci.usa80: 726-730.

Preferably, the "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein to obtain optimal alignment of the two sequences, the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20% or less, typically 5% to 15% or 10% to 12%, relative to the reference sequence (which does not comprise additions or deletions). The percentage is calculated as follows: the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences is determined to yield the number of matched positions, which are then divided by the total number of positions in the reference sequence (i.e., the size of the window), and the result is multiplied by 100 to yield the percentage of sequence identity.

The variant may also or alternatively be substantially homologous to the native gene or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing to a native DNA sequence (or complementary sequence) encoding a native antibody under moderately stringent conditions.

Suitable "moderately stringent conditions" include a prewash in a solution of 5XSSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridization at 50-65 ℃ in 5XSSC overnight; this was followed by two washes at 65 ℃ with 2X, 0.5X and 0.2XSSC containing 0.1% SDS, respectively, for 20 minutes each.

Herein, "high stringency conditions" or "high stringency conditions" refer to those that utilize: (1) washing is carried out using low ionic strength and high temperature, for example, 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate at 50 ℃; (2) denaturing agents such as formamide, e.g., 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH6.5 and 750mM sodium chloride, 75mM sodium citrate at 42 ℃; or (3) using 50% formamide, 5XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42 ℃ and washing at 42 ℃ in 0.2XSSC (sodium chloride/sodium citrate) and at 55 ℃ in 50% formamide, followed by a high stringency wash at 55 ℃ with 0.1XSSC containing EDTA. One skilled in the art will appreciate how to adjust the temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

One skilled in the art will appreciate that due to the degeneracy of the genetic code, there are many nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides share little homology with the nucleotide sequence of any native gene. However, polynucleotides that differ due to differences in codon usage are specifically contemplated by the present invention. Moreover, alleles of genes comprising the polynucleotide sequences provided herein are also within the scope of the invention. An allele is an endogenous gene that is altered due to one or more nucleotide mutations, such as deletions, additions and/or substitutions. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification, and/or database sequence comparison).

The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Chemical polynucleotide synthesis methods are well known in the art and need not be described in detail herein. One skilled in the art can use the sequences provided herein and a commercial DNA synthesizer to generate the desired DNA sequence.

For the preparation of polynucleotides using recombinant methods, polynucleotides containing the desired sequences can be inserted into a suitable vector, which is then introduced into a suitable host cell for replication and amplification, as will be discussed further herein. The polynucleotide may be inserted into the host cell by any means known in the art. Cells can be transformed by introducing exogenous polynucleotides by direct uptake, endocytosis, transfection, F-mating, or electroporation. Once introduced, the exogenous polynucleotide may be maintained in the cell in the form of a non-integrative vector (e.g., a plasmid) or may be integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well known in the art. See, e.g., Sambrook et al (1989).

Alternatively, PCR allows DNA sequences to be replicated. PCR techniques are well known in the art and are described in U.S. Pat. nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202 and PCR: the Polymerase Chain Reaction, Mullis et al, eds., Birkauswer Press, Boston (1994).

RNA can be obtained by using the isolated DNA in a suitable vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated using methods well known in the art (see, e.g., Sambrook et al (1989)).

Suitable cloning vectors can be constructed using standard techniques, or can be selected from a wide variety of cloning vectors available in the art. Although the cloning vector selected may vary depending on the host cell intended for use, generally useful cloning vectors will be self-replicating, may have a single target for a particular restriction endonuclease, and/or may carry a marker gene, wherein the marker may be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, for example, pUC18, pUC19, Bluescript (such as pBS SK +) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT 28. These and many other cloning vectors are available from suppliers such as BioRad, Strategene and Invitrogen.

Expression vectors are generally replicable polynucleotide constructs comprising a polynucleotide of the invention. This means that the expression vector must be able to replicate in the host cell, either in episomal form or as part of integration into the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the expression vectors disclosed in PCT publication No. WO 87/04462. Carrier ingredients may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional control elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational control elements are also typically required, e.g., a ribosome binding site, a translation start site, and a stop codon.

The vector containing the polynucleotide of interest may be transfected by any of a variety of suitable means, including electroporation, using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; bombardment of particles; lipofection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of vector or polynucleotide introduction often depends on the nature of the host cell.

The invention also provides a host cell comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating the gene encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotic cells (e.g., E.coli or Bacillus subtilis) and yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Kluyveromyces lactis). Preferably, the host cell expresses the cDNA at a level that is about 5-fold, more preferably 10-fold, even more preferably 20-fold higher than the level of the corresponding endogenous antibody or protein of interest (if present) in the host cell. Based on the interaction with A.beta.by immunoassay or FACS_1-40And (3) screening the host cell by specific binding. Cells that overexpress the antibody or polypeptide of interest can be identified.

Diagnostic use of 6G-derived antibodies and anti-A beta antibodies with impaired effector function

The presence or absence of a β can be identified or detected using antibody 6G that binds to the C-terminus of a β. For simplicity, reference is generally made to 6G or antibodies, but it is understood that these methods are applicable to any of the a β binding embodiments (e.g., polypeptides) described herein. Detection generally involves contact of an antibody described herein that binds to a β with a biological sample and the formation of a complex between a β and an antibody that can specifically bind to a β (e.g., 6G). The complex may be formed in vitro or in vivo. The term "detecting" herein includes qualitative and/or quantitative (measured level) detection with or without reference to a control.

A variety of known methods can be used for detection, including, but not limited to, immunoassays using antibodies that bind the polypeptide, e.g., enzyme-linked immunosorbent assays (ELISAs), Radioimmunoassays (RIAs), and the like; and functional assays for the encoded polypeptide, e.g., binding activity or enzymatic assays. In some embodiments, the antibody is detectably labeled. Other embodiments are known in the art and described herein.

The antibodies and polypeptides of the invention may be used to detect, diagnose and monitor diseases, conditions or disorders associated with altered or abnormal A β or β APP expression, for example, Alzheimer's disease and Down's syndrome. Thus, in some embodiments, the invention provides methods comprising contacting a sample of an individual suspected of having altered or aberrant a β expression with an antibody or polypeptide of the invention, and determining whether the a β level is different from a control or comparison sample a β level. In other embodiments, the invention provides methods comprising contacting a sample (specimen) of an individual and determining the expression level of a β.

For diagnostic applications, the antibody may be labeled with a detectable moiety, including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels. Methods for conjugating labels to antibodies are known in the art. In other embodiments of the invention, the antibody of the invention need not be labeled, and its presence can be detected by using a labeled antibody capable of binding to the antibody of the invention.

The antibodies of the invention can be used in any known assay method, for example, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: a Manual of Techniques, pp.147-158(CRCPress, Inc. 1987).

The antibodies may also be used in vivo diagnostic assays, such as in vivo imaging. Typically, the nuclear species is generated using a radionuclide (e.g.,¹¹¹In，⁹⁹Tc，¹⁴C，¹³¹I，¹²⁵i, or³H) The antibody is labeled so that the desired cell or tissue can be located using immunoscintigraphy (immunoscintigraphy).

Antibodies can also be used as staining agents in pathologies using techniques well known in the art.

Anti-a β antibodies with impaired effector function can be used to measure brain amyloid burden, to diagnose individuals at risk for, or who have been diagnosed as having AD, and to assess the progression and disease stage of any treatment. Peripheral administration of monoclonal anti-a β antibodies has been reported to result in a rapid increase in plasma a β, and the magnitude of this increase is highly correlated with amyloid burden in the hippocampus and cortex. DeMattos et al, Science 295: 2264-2267(2002). In some embodiments, an anti-a β antibody with impaired effector function is administered to a subject and a level of a β in plasma is measured, whereby an increase in plasma a β would indicate the presence and/or level of brain amyloid burden in the subject. These methods can be used to monitor the effectiveness of treatment and disease stage and to determine future doses and frequency of administration. Antibodies with impaired effector function may have a better safety profile and provide advantages for these diagnostic applications.

Methods of using anti-A beta antibodies for therapeutic purposes

The antibodies, including polypeptides, polynucleotides and pharmaceutical compositions described herein may be used in methods of treating, preventing and inhibiting Alzheimer's disease and other diseases associated with altered expression of A β or β APP or accumulation or deposition of A β peptides (collectively "A β -related diseases"), e.g., Down's syndrome, Parkinson's disease, multi-infarct dementia, mild cognitive impairment, cerebral amyloid angiopathy, vascular disorders caused by deposition of A β peptides in blood vessels (e.g., stroke and HCHWA-D), depression, Creutzfeldt-Jakob disease, dementia with Lewy bodies, and the like. The method comprises administering the antibody, polypeptide, or polynucleotide, or pharmaceutical composition to an individual. In prophylactic applications, the pharmaceutical composition or medicament is administered to a patient susceptible to Alzheimer's disease or at risk of developing Alzheimer's disease (or other A β -related disease) in an amount sufficient to eliminate or reduce said risk, reduce the severity of the disease, or delay the onset of the disease, wherein the disease comprises biochemical, histological, and/or behavioral symptoms of the disease, complications thereof, and intermediate pathological phenotypes occurring during the course of disease development. In therapeutic applications, the composition or medicament is administered to a suspected patient of such a disease or to a patient already suffering from such a disease in an amount sufficient to cure or at least partially inhibit the symptoms of the disease (biochemical, histological and/or behavioral), including its complications and intermediate pathological phenotypes present during the development of the disease.

The invention also provides a method of delaying the development of symptoms associated with alzheimer's disease (or other a β -related diseases) in an individual comprising administering to the individual an effective dose of a pharmaceutical composition comprising an antibody, polypeptide, or polynucleotide described herein. Symptoms associated with alzheimer's disease include, but are not limited to, abnormalities in memory, problem solving, language, computation, visuospatial perception, judgment, and behavior.

The invention also provides a method of inhibiting or reducing the formation of amyloid plaques and/or a β accumulation in a subject, comprising administering to the subject an effective dose of a pharmaceutical composition comprising an antibody, polypeptide, or polynucleotide described herein. In some embodiments, the amyloid plaques are in the brain of the subject. In some embodiments, the amyloid plaques are in the cerebral vasculature of the subject. In other embodiments, a β accumulation is present in the circulatory system of the subject.

The invention also provides a method of reducing amyloid plaques and/or reducing or slowing a β accumulation in a subject comprising administering to the subject an effective dose of a pharmaceutical composition comprising an antibody, polypeptide, or polynucleotide described herein. In some embodiments, the amyloid plaques are in the brain of the subject. In some embodiments, the amyloid plaques are in the cerebral vasculature of the subject. In other embodiments, a β accumulation is present in the circulatory system of the subject.

The invention also provides a method of removing or clearing amyloid plaques and/or a β accumulation in a subject comprising administering to the subject an effective dose of a pharmaceutical composition comprising an antibody, polypeptide, or polynucleotide described herein. In some embodiments, the amyloid plaques are in the brain of the subject. In some embodiments, the amyloid plaques are in the cerebral vasculature of the subject. In other embodiments, a β accumulation is present in the circulatory system of the subject.

The invention also provides a method of reducing a β peptide in a tissue (e.g., brain), inhibiting and/or reducing accumulation of a β peptide in a tissue (e.g., brain), and inhibiting and/or reducing the toxic effects of a β peptide in a tissue (e.g., brain) in an individual, comprising administering to the individual an effective dose of a pharmaceutical composition comprising an antibody, polypeptide, or polynucleotide described herein. The a β polypeptide may be in soluble, oligomeric, or deposited form. Oligomeric forms of a β may consist of 2 to 50 a β polypeptides, which may be full-length 1-40 and 1-42 peptides and/or mixtures of any truncated forms of these peptides.

The invention also provides a method of improving cognition or reversing cognitive decline associated with a β amyloid deposition-related disease (e.g., alzheimer's disease) in a subject comprising administering to the subject an effective dose of a pharmaceutical composition comprising an antibody, polypeptide, or polynucleotide described herein.

The methods described herein (including prophylaxis or treatment) can be performed by a single direct injection at a single site or multiple sites at a single time point or multiple time points. It can also be administered to multiple sites at about the same time. The frequency of administration can be determined and adjusted during the course of treatment and is based on 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 suitable. A variety of formulations and devices are known in the art for achieving sustained release.

Patients, subjects, or individuals include mammals, e.g., humans, cows, horses, dogs, cats, pigs, and sheep animals. The subject is preferably a human, which may or may not have a disease or may not presently exhibit symptoms. In the case of Alzheimer's disease, virtually anyone is at risk for developing Alzheimer's disease if he or she lives long enough. Thus, the methods of the invention can be prophylactically administered to the general population without any assessment of the risk of the individual patient. The methods of the invention are useful for individuals who do have a known genetic risk of Alzheimer's disease. Such individuals include individuals having relatives with the disease, and individuals determined to be at risk by analysis of genetic or biochemical markers. Genetic markers of risk for Alzheimer's disease include mutations in the APP gene, particularly the mutations at positions 717 and 670 and 671, referred to as the Hardy and Swedish mutations, respectively (see Hardy (1997) Trends neurosci.20: 154-9). Other markers of risk are mutations in presenilin genes PS1 and PS2, and ApoE4, family history of AD, hypercholesterolemia, or atherosclerosis. Individuals currently suffering from alzheimer's disease can be identified by the presence of characteristic dementias and the risk factors mentioned above. In addition, a variety of diagnostic tests are available for identifying individuals with AD. These assays include measuring levels of CSF tau and Α β 42. Elevated tau and reduced a β 42 levels indicate the presence of AD. Individuals with Alzheimer's disease can also be diagnosed by the ADRDA (Association for Alzheimer's disease and related disorders) criteria. In asymptomatic patients, treatment may be initiated at any age (e.g., 10, 20, 30 years of age). Typically, however, treatment need not begin until the patient reaches the age of 40, 50, 60, or 70. Treatment typically requires multiple administrations over a period of time. Monitoring of the treatment can be performed over time using a variety of methods known in the art. In the case of a potential Down syndrome patient, treatment may be initiated by administering the therapeutic agent to the mother either before birth or shortly after birth.

Pharmaceutical compositions that can be used in the above methods include any of the antibodies, polypeptides, and/or polynucleotides described herein. In some embodiments, the antibody is antibody 6G or a variant thereof shown in table 3. In some embodiments, the antibody is an antibody that is capable of specifically binding to a β peptide and comprises a constant region with impaired effector function.

Administration and dosage

The antibody is preferably administered to the mammal in a carrier, preferably a pharmaceutically acceptable carrier. Suitable carriers and their formulation are described in Remington's Pharmaceutical Sciences, version 18, a.gennaro, eds, Mack Publishing co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20 edition MackPublishing, 2000. Typically, a suitable amount of a pharmaceutically acceptable salt is administered in the formulation to render the formulation isotonic. Examples of the carrier include saline, Ringer's solution and glucose solution. The pH of the solution is preferably from about 5 to about 8, more preferably from about 7 to about 7.5. Other carriers include sustained release preparations, e.g., semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be appreciated by those skilled in the art that certain carriers may be more preferred depending on, for example, the route of administration and the concentration of antibody administered.

The antibody can be administered to the mammal by injection (e.g., systemically, intravenously, intraperitoneally, subcutaneously, intramuscularly, intraportally, intracerebroventricularly, and intranasally) or by other methods that ensure that the antibody is delivered to the bloodstream in an effective form, such as infusion. The antibody may also be administered to effect local therapeutic action by isolated perfusion techniques, e.g., isolated tissue perfusion. Intravenous injection is preferred.

Effective dosages and schedules for administering the antibodies can be determined empirically, and it is within the skill of the art how to make such determinations. It will be apparent to those skilled in the art that the dosage of antibody that must be administered will vary depending upon, for example, the mammal receiving the antibody, the route of administration, the particular type of antibody used, and other drugs being administered to the mammal. Guidance on how to select appropriate doses of Antibodies can be found in literature on the therapeutic use of Antibodies, such as, for example, Handbook of Monoclonal Antibodies, Ferrone et al, Nos. Publications, Park Ridge, N.J., 1985, ch.22 and pp.303-357; smith et al, Antibodies in Human Diagnosis and Therapy, edited by Haber et al, Raven Press, New York, 1977, pp.365-389. A typical daily dosage of antibody used alone, depending on the factors described above, may be from about 1. mu.g/kg body weight to no more than 100mg/kg body weight per day or more. Generally, any of the following dosages may be used: administering at least about 50mg/kg body weight; at least about 10mg/kg body weight; at least about 3mg/kg body weight; at least about 1mg/kg body weight; at least about 750 μ g/kg body weight; at least about 500 μ g/kg body weight; at least about 250 μ g/kg body weight; at least about 100 μ g/kg body weight; at least about 50 μ g/kg body weight; at least about 10 μ g/kg body weight; at least about 1 μ g/kg body weight; or a larger dose. The antibody may be administered at a lower dose or less frequently at the beginning of the treatment to avoid potential side effects, e.g., transient amyloid cerebrovascular disease (CAA).

In some embodiments, more than one antibody may be present. 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.

The antibody may also be administered to the mammal in combination with an effective amount of one or more other therapeutic agents. The antibody and the one or more therapeutic agents may be administered sequentially or simultaneously. The amount of antibody and therapeutic agent will depend on, for example, the type of drug used, the pathological condition being treated, and the schedule and route of administration, but generally the amount will be less than each when used alone.

After administration of the antibody to a mammal, the physiological condition of the mammal can be monitored in various ways well known to those skilled in the art.

The above principles of administration and dosage may be adapted according to the polypeptides described herein.

Polynucleotides encoding the antibodies or polypeptides described herein may also be used to deliver and express the antibodies or polypeptides to desired cells. Obviously, expression vectors can be used to direct antibody expression. The expression vector may be administered systemically, intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraventricularly, orally, enterally, parenterally, intranasally, transdermally, or by inhalation. For example, administration of the expression vector includes local or systemic administration, including injection, oral, gene gun or catheterized administration, and topical administration. Those skilled in the art are familiar with the administration of expression vectors to obtain expression of foreign proteins in vivo. See, for example, U.S. patent nos. 6,436,908; 6,413,942, respectively; and 6,376,471.

Therapeutic compositions comprising polynucleotides encoding the antibodies of the invention may also be delivered in a targeted manner. 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 (J.A. Wolff, ed.) (1994); wu et al, J.biol.chem. (1988) 263: 621 of the first and second substrates; 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. therapeutic compositions containing the polynucleotides are administered topically in a gene therapy regimen in a range of about 100ng to about 200mg of DNA. A concentration range of about 500ng to about 50mg, about 1. mu.g to about 2mg, about 5. mu.g to about 500. mu.g, and about 20. mu.g to about 100. mu.g of DNA can also be used during a gene therapy regimen. Therapeutic polynucleotides and polypeptides of the invention may be delivered using gene delivery vectors. Gene delivery vectors can be of viral and 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 these coding sequences can be induced using endogenous mammalian promoters or heterologous promoters. Expression of the coding sequence may be constitutive or regulated.

Viral-based vectors for delivering a desired polynucleotide and effecting expression in a desired cell are well known in the art. Exemplary virus-based vectors include, but are not limited to, recombinant retroviruses (see, for example, PCT publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. No. 5,219,740; 4,777,127; GB patent No. 2,200,651; and EP0345242), alphavirus-based vectors (e.g., Sindbis virus vector, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross river virus (Ross river virus) (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC zuelandii encephatis virus) (ATCC VR-923; ATCC VR-1250; ATCC CVR 9; ATCC VR-532)), and adeno-associated virus (AAV) vectors (AAV) (see, for example, PCT publication Nos. WO 94/12692; WO 3512692; WO 3556355638/36938; WO 35563655/95; WO 353627/95). Also, for example, Curiel, hum. Gene Ther, (1992) 3: 147 the DNA ligated to the killed adenovirus.

Non-viral delivery vectors and methods may also be used, including, but not limited to, DNA alone condensed with killed adenovirus-linked or unlinked polycations (see, e.g., Curiel, hum. gene Ther. (1992) 3: 147); ligand-linked DNA (see, e.g., Wu, j.biol.chem. (1989) 264: 16985); eukaryotic cell delivery vector 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 nucleic acid charge neutralization (nuclear charging dissociation) or fusion with cell membranes. Naked DNA may also be used. Examples of methods for introducing naked DNA are described in PCT publication No. WO90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can serve as gene delivery vehicles are described in U.S. Pat. nos. 5,422,120; PCT publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Philip, mol.cell Biol. (1994) 14: 2411, and Woffendin, proc.natl.acad.sci. (1994) 91: 1581 other methods are described.

Medicine box

The invention also provides products and kits containing materials useful for treating pathological conditions such as Alzheimer's disease or other A β -related diseases (e.g., Down's syndrome, Parkinson's disease, multi-infarct dementia, mild cognitive impairment, cerebral amyloid angiopathy, vascular disorders due to deposition of A β peptide in blood vessels (e.g., stroke and HCHWA-D)), or for detecting or purifying A β or β APP. The product comprises a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The container may be formed using various materials such as glass or plastic. The container contains a composition having an active agent that can be effective in treating a pathological condition or detecting or purifying a β or β APP. The active agent in the composition is an antibody, preferably comprising a monoclonal antibody specific for a β or β APP. In some embodiments, the active agent comprises antibody 6G or any antibody or polypeptide derived from antibody 6G. In some embodiments, the active agent comprises an anti-a β antibody or polypeptide described herein having impaired effector function. In some embodiments, the anti-a β antibody or polypeptide comprises a heavy chain constant region, wherein the constant region has impaired effector function. The label on the container indicates that the composition is for use in treating a pathological condition such as alzheimer's disease or for detecting or purifying a β or β APP, and may also indicate a use for in vivo or in vitro applications, such as those described above.

The invention also provides kits comprising any of the antibodies (e.g., 6G), polypeptides, polynucleotides described herein. In some embodiments, a kit of the invention comprises a container as described above. In other embodiments, the kits of the invention comprise the container described above and a second container comprising a buffer. It can also include other materials that may be desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any of the methods described herein (e.g., methods for treating alzheimer's disease, and methods for inhibiting or reducing accumulation of a β peptide in the brain). In kits for detecting or purifying a β or β APP, the antibody is typically labeled with a detectable label, e.g., a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator, or an enzyme.

In some embodiments, the invention provides compositions (see description herein) for use in any of the methods described herein, whether for use as a medicament and/or for use in the manufacture of a medicament.

The following examples are provided to illustrate, but not to limit, the present invention.

Examples

Example 1: binding affinity assay for antibody 6G and variants thereof

A. General procedure

The following general method was used in this and other examples.

Expression vectors for clone characterization

Similar to Barbas (2001) Phage display: a Laboratory manual, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press pg2.10.vector pComb3X), antibody Fab fragments were expressed under the control of the IPTG inducible 1acZ promoter, but the changes included the addition and expression of the following additional domains: human kappa light chain constant domain and IgG2a human immunoglobulin CHI constant domain, Ig γ 2 chain C region, protein accession number P01859; immunoglobulin kappa light chain (homo sapiens), protein accession number CAA 09181.

Small scale Fab preparation

Fab was expressed in 96-well plates at small scale as follows. Starting from E.coli transformed with the Fab library, colonies were picked to inoculate a master plate (agar LB + ampicillin (50. mu.g/mL) + 2% glucose) and a working plate (2 mL/well, 96 wells/plate containing 1.5mL LB + ampicillin (50. mu.g/mL) + 2% glucose). Both plates were grown at 30 ℃ for 8 to 12 hours. The master plate was stored at 4 ℃ and cells from the working plate were pelleted at 5000rpm and resuspended with 1mL LB + ampicillin (50. mu.g/mL) +1mM IPTG to induce Fab expression. After an expression time of 5 hours at 30 ℃, the cells were harvested by centrifugation and then resuspended in 500. mu.L of buffer HBS-EP (100mM HEPES buffer pH7.4, 150mM NaCl, 0.005% P20). HBS-EP resuspended cells were lysed by one round of freezing (-80 ℃) thawing (37 ℃). The cell lysate was centrifuged at 5000rpm for 30 minutes to separate the cell debris and the supernatant containing the Fab. The supernatant was then injected into a BIAcore plasma resonance instrument to obtain affinity information for each Fab. The Fab-expressing clones were rescued from the master plate to sequence the DNA and produce and characterize Fab in detail on a large scale, as described below.

Large Scale Fab preparation

To obtain detailed kinetic parameters, fabs were expressed and purified from large cultures. Flasks containing 200ml LB + ampicillin (50. mu.g/ml) + 2% glucose were inoculated with 5ml overnight cultures from selected Fab expressing E.coli clones. Clones were incubated at 30 ℃ until an OD of 1.0 was obtained_550nmThen, induction was carried out by replacing the medium with 200ml of LB + ampicillin (50. mu.g/ml) +1mM IPTG. After 5h expression time at 30 ℃ the cells were pelleted by centrifugation and then resuspended in 10ml PBS (pH 8). Cells were lysed by two cycles of freeze-thawing (at-80 ℃ and 37 ℃ respectively). The cell lysate supernatant was loaded onto a Ni-NTA Superflow agarose (Qiagen, Valencia, Calif.) column equilibrated with PBS (pH8) and then washed with 5 column volumes of PBS (pH 8). Each Fab was eluted in different fractions using PBS (pH8) +300mM imidazole. Fab-containing fractions were pooled and dialyzed against PBS, followed by affinity characterization after quantification by ELISA.

Preparation of full antibodies

To express whole antibodies, the heavy and light chain variable regions were cloned in mammalian expression vectors, transfected into HEK293 cells using lipofectamine to achieve transient expression. Antibodies were purified using protein a using standard methods.

The vector pdb.6g.hfc2a is an expression vector containing the antibody 6G heavy chain, suitable for transient and stable expression of this heavy chain. The vector pdb.6g.hfc2a has a nucleotide sequence corresponding to the following regions: the murine cytomegalovirus promoter region (nucleotides 1-612); a synthetic intron (nucleotide 619-1507); the DHFR coding region (nucleotide 707-1267); human growth hormone signal peptide (nucleotide 1525-1602); a 6G heavy chain variable region; the human heavy chain IgG2a constant region containing the following mutations: A330P331 to S330S331 (amino acid numbering is performed with reference to the wild type IgG2a sequence; see Eur.J.Immunol. (1999) 29: 2613-2624); SV40 late polyadenylation signal; SV40 enhancer region; a bacteriophage f1 region and a beta lactamase (AmpR) coding region.

The vector peb.6g.hk is an expression vector comprising a 6G antibody light chain, which is suitable for transient expression of the light chain. The vector peb.6g.hk has a nucleotide sequence corresponding to the following regions: the murine cytomegalovirus promoter region (nucleotides 1-612); the human EF-1 intron (nucleotide 619-1142); human growth hormone signal peptide (nucleotides 1173-1150); antibody 6G light chain variable region; a human kappa chain constant region; SV40 late polyadenylation signal; SV40 enhancer region; a bacteriophage f1 region and a beta lactamase (AmpR) coding region.

BIAcore test

BIAcore3000 was used^TMSurface Plasmon Resonance (SPR) system (BIAcore, INC, Piscaway NJ), the affinity of the 6G monoclonal antibody was determined. One method of determining affinity is to immobilize 6G on a CM5 chip and measure A.beta._1-40Binding kinetics of peptides or other a β peptides to the antibody. The CM5 chip was activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antibody 6G or a variant thereof was diluted in 10mM sodium acetate pH4.0 or pH5.0 and injected at a concentration of 0.005mg/mL on the activated chip. Using different flow times through the chip channels to obtainA series of antibody densities were obtained: 1000-2000 or 2000-3000 Response Units (RUs). The chip was blocked with ethanolamine. Regeneration studies showed that a solution containing 2 volumes of PIERCE elution buffer and 1 volume of 4M NaCl effectively removed bound Α β peptide while maintaining the activity of 6G on the chip for up to 200 injections. HBS-EP buffer (0.01M HEPES, pH7.4, 0.15NaCl, 3mM EDTA, 0.005% surfactant P20) was used as the running buffer for all BIAcore experiments. Purifying the A beta_1-40Serial dilutions (0.1-10X estimated K) of synthetic peptide or other A.beta.peptide samples_D) Injection was performed at 100. mu.L/min for 1 min, allowing a dissociation time of 10 min. A kinetic binding rate (k.k.sub.g.for L.sub.G.sub.6.99-110) was obtained by using BIA evaluation program to simulate the 1: 1 Langmuir binding model (Karlsson, R.Ros, H.Fagerstam, L.Petersson, B. (1994). Methods Enzymology 6.99-110) with this data_on) And dissociation rate (k)_off). With k_on/k_offCalculation of equilibrium dissociation constant (K)_D) The value is obtained.

Alternatively, by mixing A beta_1-40Peptides or other a β peptides were immobilized on SA chips and the binding kinetics of Fab of 6G and Fab of 6G variants to the immobilized a β peptides were measured, thereby determining affinity. Affinity of Fab fragments of 6G Fab fragments and variants thereof by Surface Plasmon Resonance (SPR) System (BIAcore 3000)^TMBIAcore, inc., Piscaway NJ). SA chips (streptavidin) were used according to the supplier's instructions. The biotinylated A.beta.peptide was diluted in HBS-EP (100mM HEPES, pH7.4, 150mM NaCl, 3mM EDTA, 0.005% P20) and injected onto the chip at a concentration of 0.005 mg/mL. Two sets of antigen densities were obtained using different flow times through each chip channel: 100-400 Response Units (RU) were used for detailed kinetic studies and 500-2000 RU for concentration studies and screening. Regeneration studies showed that 100mM phosphoric acid (which may also be followed by a solution containing 2 volumes of 50mM NaOH and 1 volume of 70% ethanol) effectively removed bound Fab while maintaining the activity of the a β peptide on the chip for more than 200 injections. HBS-EP buffer was used as the running buffer for all BIAcore experiments. Serial dilutions of purified Fab samples (0.1-10X estimated KD) were injected at 100. mu.L/min for 2 minutes, allowing a10 minute dissociation time. By ELISA and/orThe concentration of the Fab protein was determined by SDS-PAGE electrophoresis using standard Fab of known concentration (determined by amino acid analysis). Kinetic binding rates (k.k.sub.g.for proteins) were obtained simultaneously by using the BIA evaluation program by simulating the 1: 1 Langmuir binding model (Karlsson, R.Ros, H.Fagerstam, L.Petersson, B. (1994). Methods Enzymology 6.99-110) with the data_on) And dissociation rate (k)_off). With k_on/k_offCalculation of equilibrium dissociation constant (K)_D) The value is obtained.

ELISA assay

The binding of antibody 6G and variants to the non-biotinylated a β peptide was measured using ELISA. NUNC maxisorp plates were coated with 2.5. mu.g/ml A.beta.peptide in PBS pH7.4 at 4 ℃ for more than 1 hour. The plates were blocked with 1% BSA in PBS buffer, pH 7.4. Primary antibodies (from cell supernatants, sera containing anti-a β antibodies, or purified whole antibodies or fabs at desired dilutions) were incubated with the immobilized a β peptide for 1 hour at room temperature. After washing, the plates were incubated with a 1: 5000 dilution of a secondary antibody (HRP-conjugated goat anti-human kappa chain antibody, MP Biomedicals, 55233). After washing, bound secondary antibodies were measured by addition of TMB substrate (KPL, 50-76-02, 50-65-02). The HRP reaction was stopped by adding 1M phosphoric acid and absorbance was measured at 450 nm.

Binding of antibody 6G and variants to biotinylated a β peptide was measured using ELISA. NUNCmaxisorp plates were coated with 6. mu.g/ml streptavidin (Pierce, 21122) in PBS pH7.4 at 4 ℃ for more than 1 hour. The plate was blocked with 1% BSA in PBS buffer pH 7.4. After washing, biotinylated A β peptides were incubated for 1 hour at room temperature in PBS pH 7.4. Primary antibodies (from cell supernatants, sera containing anti-a β antibodies, or purified whole antibodies or fabs at desired dilutions) were incubated with the immobilized a β peptide for hours at room temperature. After washing, the plates were incubated with a 1: 5000 dilution of a secondary antibody (HRP-conjugated goat anti-human kappa chain antibody, MP Biomedicals, 55233). After washing, bound secondary antibodies were measured by addition of TMB substrate (KPL, 50-76-02, 50-65-02). The HRP reaction was stopped by adding 1M phosphoric acid and absorbance was measured at 450 nm.

B. Antibodies 6G and variants andAβ_1-40、Aβ_1-42binding affinity to other A beta peptides

Figure 1 shows the amino acid sequences of the heavy and light chain variable regions of antibody 6G. 6G antibodies and A β determined using Biacore as described above_1-40、Aβ_1-42And Abeta_22-37The binding affinities of (a) are shown in table 2 below.

TABLE 2 binding affinities of antibody 6G Fab fragments

The amino acid sequence of the 6G variant is shown in table 3 below. All amino acid substitutions for the variants shown in table 3 are described with respect to the 6G sequence. The relative binding of the 6G variants is also shown in table 3. The ELISA was performed as described above using non-biotinylated A.beta.immobilized on the surface of the ELISA plate_1-40Or Abeta_1-42Binding was determined.

TABLE 3 amino acid sequence and binding data for antibody 6G variants

Example 2 characterization of the epitope on A.beta.peptide that binds to antibody 6G

To determine the epitope on the a β peptide recognized by antibody 6G, an ELISA binding assay was used. Various a β peptides (Global Peptide Services, CO) were immobilized on ELISA plates. Binding of 6G whole antibody (20nM) to immobilized a β was determined by ELISA as described above. Table 5 below shows A.beta._1-40、Aβ_1-42And Abeta_1-43The amino acid sequence of (a). As shown in FIG. 2, antibody 6G binds to A.beta.peptides 17-40, 17-42, 22-35, 28-40, 1-38, 1-40, 1-42, 1-43, and 28-42; but bind much less strongly to 28-42 than other a β peptides. Antibody 6G does not bind to A.beta.peptides 1-16, 1-28 and 33-40. Thus, antibody 6G binds to the C-terminus of various truncated A.beta.peptides, e.g., 22-35, 1-38, 1-40, 1-42, and 1-43.

Table 4 below shows the pass k using the Biacore test_off(1/s) measured Abeta_1-40Comparison of binding affinities to other a β peptides and 6G. Antibodies 6G vs. A β compared to other peptides_1-40Having the highest binding affinity to truncated Abeta_1-40(e.g., 1-36, 1-37, 1-38, and 1-39), A.beta._1-42And Abeta_1-43With significantly lower affinity. This suggests that the side chain or backbone of amino acid 40 (valine) of A.beta.is involved in 6G and A.beta._1-40Combination of (1); moreover, binding is significantly reduced in the absence of the amino acid (e.g., about 10 to about 50-250 fold reduction in affinity). With carboxyl-terminally amidated Abeta_1-40Binding with lower affinity, indicating 6G to A.beta._1-40The binding of (A) is related to but independent of A β_1-40Free C-terminus of (a). And Abeta_1-42And Abeta_1-43The lower affinity binding of (a) may be due to a β_1-40And Abeta_1-42Or Abeta_1-43Due to conformational differences between the monomeric forms of (a). It has been demonstrated that A.beta.in solution_1-42The monomer has a structure of_1-40Different conformations of the monomers. See, A.beta.shown under accession number 1IYT in Protein database Protein DataBank (pbd archive)_1-42The monomer structure coordinates of (a); and A.beta.as shown in the protein database (pbd archive) under accession numbers 1BA6 and 1BA4_1-40The monomer structure coordinates of (a).

TABLE 4

Peptides as analytes were flowed on CM5 chips with 6G monoclonal antibodies (ligands) chemically immobilized by amines

Peptide amidated at the carboxy terminus of # C

Epitope mapping of antibody 6G was performed by ELISA assay. Biotinylated 15-mer or 10-mer A.beta.peptides, which had glycine added to the C-terminus, were immobilized on streptavidin-coated plates. Antibody 6G (2.5. mu.g/ml to 10. mu.g/ml) was incubated with the immobilized peptide and binding was determined as described above. As shown in FIG. 3, antibody 6G binds to A β peptides having amino acids 20-34, 21-35, 22-36, 23-37, 24-38, 25-39, and 25-34 and a glycine at the C-terminus; but not to the a β peptide having amino acids 19-33, 26-40, 27-41, 24-33 and 26-35 and having a glycine at the C-terminus of the peptide. This suggests that the epitope of antibody 6G includes amino acids from positions 25 to 34.

Based on the data presented above, the epitope bound by antibody 6G appears to include amino acids 25-34 and 40. Fig. 4 is a schematic diagram showing the epitope of antibody 6G.

TABLE 5 amino acid sequence of amyloid beta peptide

B. Antibody 6G does not bind APP

To determine whether 6G binds Amyloid Precursor Protein (APP), binding of 6G to cells transfected with wild-type APP was determined. HEK293 cells were transfected with cDNA encoding wild-type human amyloid precursor protein. 48 hours after transfection, cells were incubated on ice with monoclonal anti-Abeta_1-16(m2324) or 6G (5. mu.g/ml in DMEM with 10% FCS) for 45 min. Cells were then washed 3 times for 5 minutes in PBS and fixed with 4% PFA. The cells were washed again 3 times in PBS and antibody binding was detected under a fluorescent microscope with a secondary Cy3 conjugated goat anti-mouse antibody from Jackson Immunoresearch (1: 500 dilution).

As shown in FIG. 5, an antibody recognizing the N-terminal epitope of A.beta.Aβ_1-16The antibody showed significant binding to the cell-expressed APP precursor protein. In contrast, 6G does not bind APP-expressing cells.

Example 3 characterization of the epitope on A.beta.peptide to which antibody 2294 binds

Antibody 2294 is prepared by administering A.beta._1-40Murine antibodies produced by immunized mice. Such antibodies are described in US2004/0146512 and WO 04/032868.

Antibodies 2294 and Abeta_1-40、Aβ_1-42Or Abeta_22-37Binding affinity of (c) was measured using Biacore as described above. Table 6 below shows the affinity of antibody 2294Fab fragment for various a β peptides.

TABLE 6 binding affinity of antibody 2294Fab fragment

Antibody 2294 was epitope mapped by ELISA assay. Biotinylated 15-mer or 10-mer A.beta.peptides, which had glycine added to the C-terminus, were immobilized on streptavidin-coated plates. NUNC maxisorp plates were coated with 6. mu.g/ml streptavidin (Pierce, 21122) in PBS pH7.4 at 4 ℃ for more than 1 hour. The plate was blocked with 1% BSA in PBS buffer pH 7.4. After washing, biotinylated A β peptides were incubated for 1 hour at room temperature in PBS pH 7.4. Antibody 2294 (2.5. mu.g/ml to 10. mu.g/ml) was incubated with the immobilized A.beta.peptide for 1 hour at room temperature. After washing, the plates were incubated with a 1: 5000 dilution of a secondary antibody (HRP-conjugated goat anti-human kappa chain antibody, MP Biomedicals, 55233). After washing, bound secondary antibodies were measured by addition of TMB substrate (KPL, 50-76-02, 50-65-02). The HRP reaction was stopped by adding 1M phosphoric acid and absorbance was measured at 450 nm. As shown in fig. 6, antibody 2294 binds to a β peptides having amino acids 20-34, 21-35, 22-36, 23-37, 24-38, 25-39, 26-40, and 25-34, and a glycine at the C-terminus; but not to the a β peptide having amino acids 19-33, 27-41, 24-33 and 27-35 and a glycine at the C-terminus of the peptide. This suggests that the epitope for antibody 2294 includes amino acids from position 26 to 34.

To further determine the epitope on a β peptide recognized by antibody 2294, an ELISA binding assay was used. Various a β peptides (Global Peptide Services, CO) were immobilized on ELISA plates. 2294 binding of whole antibody (20nM) to immobilized A.beta.was determined by ELISA as described above. Antibody 2294 binds to A β peptides 17-40, 17-42, 28-40, 1-38, 1-40, 1-42, and 1-43. Antibody 2294 does not bind to A β peptides 1-16, 1-28, 28-42, 22-35, and 33-40. Thus, antibody 2294 binds to the C-terminus of various truncated A β peptides, e.g., 1-38, 1-40, 1-42, and 1-43.

Table 7 below shows the A.beta.as determined by the Biacore test_1-40Comparison with other a β peptides and 2294 binding. Antibodies 2294 (Whole antibody) with A.beta.in contrast to other peptides_1-40Has the strongest combination with the truncated Abeta_1-40(e.g., 1-36, 1-37, 1-38, and 1-39), A.beta._1-42And Abeta_1-43With significantly lower binding. This suggests that the side chain or backbone of amino acid 40 (valine) of A.beta.is involved in 2294 and A.beta._1-40Combination of (1); moreover, binding is significantly reduced in the absence of this amino acid.

TABLE 7

"-" indicates no binding; "+" indicates very low binding; "+ +" indicates moderate binding; "+ + + +" indicates strong binding; and "+++" indicates very strong binding.

Based on the above data, the epitope bound by antibody 2294 appears to include amino acids 26-34 and 40. As shown in figure 6, antibody 2294 binds to an epitope very similar to the epitope bound by antibody 6G. However, binding of antibody 6G is less dependent on amino acid 40 than antibody 2294.

Between 2294, 6G, 2H6 and 2289 using the Biacore testAntibody binding competition experiments were performed. Antibody 2H6 is related to A.beta._33-40Bound antibody described in U.S. provisional patent application 60/653,197 filed on 14/2/2005. Antibody 2289 is an antibody that binds to A β 16-28 and is described in U.S. publication No. 2004/0146512 and PCT WO 04.032868. Competition experiments were performed using the Biacore assay. Antibodies 2294, 6G, 2H6, and 2289 were immobilized within different channels of a CM5 chip. The CM5 chip channels were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antibodies 2294, 6G, 2H6, and 2289 were each diluted in 10mM sodium acetate, pH4.0, and injected at a concentration of 0.005mg/mL onto the activated chip. Each channel was blocked with ethanolamine. A.beta.1-40 peptide (150. mu.M) was flowed on the chip for 2 minutes. Then, 0.6. mu.M of antibody 2294 (to be tested for competition for binding) was flowed on the chip for 1 minute. HBS-EP buffer (0.01M HEPES, pH7.4, 0.15NaCl, 3mM EDTA, 0.005% surfactant P20) was used as the running buffer for all these BIAcore tests. In measuring A beta_1-40After binding, all channels on the chip were regenerated by washing 2 times for 6 seconds with a mixture of Pierce elution buffer (product No. 21004, Pierce Biotechnology, Rockford, IL) and 4M NaCl (2: 1). Competitive binding was then performed for antibody 6G, 2H6, and then for antibody 2289. The presence of p-A β between 2294 and 6G and between 2294 and 2H6 was observed_1-40But no competition was observed between 2294 and 2289 and between 6G and 2289. As a positive control, observation of competition between the immobilized antibody and the same antibody flowing on the chip was used. The data indicate that antibody 2294 competes with 2H6 and 6G for a β_1-40And (4) combining.

Example 4 binding affinity of antibody 2294Fc region to murine Fc γ receptor

The binding affinity of the Fc region of this antibody to Fc γ receptors was measured using the BIAcore assay described above. Briefly, purified murine Fc γ receptors (from R & D Systems) were immobilized on BIAcore CM5 chips by amine chemistry. Serial dilutions of the monoclonal antibody (from 2nM to the maximum concentration shown in table 8) were injected. HBS-EP (0.01M HEPES, pH7.4, 0.15M NaCl, 3mM EDTA, 0.005% surfactant P20) was used as running buffer and sample buffer. Binding data were analyzed using either a 1: 1 Langmuir interaction model for high affinity interactions or a steady state affinity model for low affinity interactions.

Table 8 below shows the passage K_D(nM) measured binding affinity of antibody 2294 to murine Fc γ RI, Fc γ RIIb, and Fc γ RIII. Deglycosylated antibodies have a constant region with N-glycosylation removed. As shown in table 8, deglycosylated 2294 had reduced affinity for all murine Fc γ receptors tested compared to the corresponding antibody without removal of N-glycosylation.

TABLE 8 passage K_D(nM) binding affinity of the antibody to murine Fc gamma receptor

NB: there was no significant binding when the antibody was used at the maximum concentration determined.

Example 5 antibodies 2294 and deglycosylated antibodies 2294 in Alzheimer's disease

Effect on reduction of Abeta deposition and cognition in animal models

Deglycosylated antibody 2294 was prepared by incubating purified antibody 2294 with peptide-N-glycosidase F (Prozyme, 0.05U per mg of antibody) in 20mM Tris-HCl pH8.0 for 7 days at 37 ℃. The completeness of deglycosylation was verified by MALDI-TOF-MS and protein gel electrophoresis. The deglycosylated antibody was purified by protein A chromatography and endotoxin removed by Q-Sepharose. Deglycosylated 2294 and A β were determined using the BIAcore assay described above_1-40Was found to deglycosylated 2294 for A β_1-40The same binding affinity as for intact antibody 2294.

The effect of antibody 2294 and deglycosylated 2294 on the reversal of cognitive deficits, histological symptoms and microhemorrhages (microhemorrhages) was examined in transgenic mouse APP Tg 2576. Administration, histology and behavioral analysis of the antibodies were performed as follows.

Antibody administration. Transgenic mice overexpressing the "Swedish" mutant amyloid precursor protein (APP Tg2576 with K670N/M671; Hsiao et al, Science 274: 99-102(1996)) were used in this experiment. The Alzheimer's disease-like phenotype that occurs in these mice has been well characterized. Holcomb et al, nat. med.4: 97-100 (1998); holcomb et al, behav.gen.29: 177-185 (1999); and McGowanE, neurobiol.dis.6: 231-244(1999). For this 16-week treatment study, 20-month-old APP transgenic mice were divided into 4 groups. The first group received intraperitoneal anti- Α β antibody 2294 injections weekly for 16 weeks (n ═ 4). The second group received injections of intraperitoneal deglycosylated anti-a β antibody 2294 weekly for 16 weeks (n ═ 5). The third group received weekly injections of intraperitoneal anti-AMN antibody (2906; mouse monoclonal anti Drosophila (Drosophila) amnesiac protein IgG1) for 16 weeks (n ═ 6). Non-transgenic littermates were treated with anti-AMN antibody (n ═ 4) or 2294(n ═ 2) for 16 weeks.

And (5) analyzing the behaviors. After 16 weeks of antibody treatment, mice from this study received two days radial arm water maze training as previously described. Wilcock et al, J.Neurooil flash 1: 24, (2004) the device is a 6-arm labyrinth as described earlier. Gordon et al, neurobiol. aging 22: 377-385(2001). On day 1, 15 trials were conducted in three 5 trials each. The test was performed sequentially for each batch of 4 mice (i.e., each of the 4 mice was tested 1, then the same mouse was tested 2, etc.). After each 5 test batches, a second group of mice was tested such that there was an extended rest period before the mice were subjected to the second batch of 5 tests. The target arm was different for each mouse in a group to minimize odor cues. The starting arm was varied for each trial, while the target arm was kept constant for two days for a given individual. The platform was alternately visible and then hidden for the first 11 trials (platform hidden for the last 4 trials). On day 2, mice were trained in exactly the same manner as on day 1, except that the platform was hidden in all experiments. The number of errors (incorrect arm entries) was measured in a1 minute time frame. Mice that failed to select arms within 20 seconds were rated as one mistake, but no mice in this study had to be rated as wrong in this way. Because of the number of mice in this study, the experimenter would not know the treatment group identity of each mouse. Since the dependency measurements in the radial arm water maze task are quantitative rather than estimation, the possibility of bias for the experimenter is reduced. To minimize the effect of variation for each single trial, errors per mouse were averaged over 3 consecutive trials, 5 data points were obtained per day, and these data points were analyzed by ANOVA statistics using StatView (sasinstite inc., NC).

And (4) histological analysis. On the day of sacrifice, mice were weighed, overdosed with Nembutal (Abbott laboratories, North Chicago, Ill.) at 100mg/kg, and then perfused intracardially with 25ml of 0.9% sodium chloride. Taking out brain rapidly, and placing left half brain at 100mM KPO₄(pH7.2) for histopathology in freshly prepared 4% paraformaldehyde for 24 hours. To obtain ultra-low temperature protection, the half-brain was then incubated in 10%, 20% and 30% sucrose for 24 hours in succession. Horizontal sections of 25 μ thick were collected using a sliding microtome and stored at 4 ℃ in Dulbecco's phosphate buffered saline with sodium azide (pH7.2) to prevent microbial growth. A series of 8 equally spaced tissue sections 600 μ apart were randomly picked across the entire brain and total A β (rabbit polyclonal anti-total A β; Biosource, Camarillo, CA, 1: 10,000) was stained using free-floating immunohistochemistry as described earlier. Gordon et al, exp. neuron.173: 183-195 (2002); wilcock et al, J.Neurosci.24: 6144-6151(2004). A second series of tissue sections separated by 600 μm were stained with 0.2% Congo red in NaCl-saturated 80% ethanol. In addition, another set of sections was mounted and stained for hematite ferretin using 2% potassium ferrocyanide in 2% hydrochloric acid for 15 minutes, followed by counterstaining in 1% neutral red solution for 10 minutes. Quantification of Congo Red Using Image-ProPlus (Media Cybernetics, Silver Spring, MD)Staining and Α β immunohistochemistry to analyze the percentage area occupied by positive staining. One area of the frontal cortex and three areas of the hippocampus were analyzed (to ensure that there was no regional bias in hippocampal values). Initial analysis of Congo red gave a total value. A second analysis was performed after manual editing to delete all parenchymal amyloid deposits to yield the percentage area restricted to vascular Congo red staining. To estimate the substantial area of Congo red, the value of vascular amyloidosis was subtracted from the total percentage. For hemosiderin staining, the number of prussian blue positive sites was counted on all sections and the average number of sites per section was calculated. Differences between animals were observed qualitatively on sections at low magnification. 8 equally spaced sections were examined and the number of positive spectra determined and averaged to the value per section. To evaluate possible treatment-related differences, the values for each treatment group were analyzed by one-way ANOVA followed by Fisher's LSD mean comparisons.

Serum a β peptide levels were measured using ELISA. Sera were collected 1 day after the last antibody administration, diluted and incubated with 5. mu.g/ml antibody 6E10 (vs. A. beta.)_1-17A bound anti-amyloid beta antibody; signet, Dedham, MA) in PBS buffer ph7.4 (MaxiSorp; nunc, Roskilde, Denmark). Secondary antibody biotinylated 4G8 (with A β) at 1: 5000 dilution_17-24A bound anti-amyloid beta antibody; signet). Streptavidin-horseradish peroxidase conjugate (Amersham Biosciences) was used, followed by detection with TMB substrate (KPL, Gaithersburg, MD). Use of scaled-up Abeta from 6 to 400pM_1-40(American peptide) A calibration curve was prepared.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.

Biological material preservation

The following materials were deposited at the American type culture Collection, 10801 Universal boulevard, Manassas, Virginia20110-2209, USA (ATCC):

the vector peb.6g.hk is a polynucleotide encoding the 6G light chain variable region and the light chain kappa constant region; the vector pDB.6G.hFc2a is a polynucleotide encoding the 6G heavy chain variable region and the heavy chain IgG2a constant region containing the mutations A330P331 to S330S331 (amino acid numbering is based on Kabat numbering, with reference to wild-type IgG2a sequence; see Eur.J.Immunol. (1999) 29: 2613-2624).

These deposits were made in accordance with the provisions of the budapest treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure and the rules for its implementation (budapest treaty). This ensures that a viable culture of the deposit is maintained for 30 years from the date of deposit. The deposit is available under the Budapest treaty under the ATCC and is subject to a protocol between Rinat Neuroscience and ATCC which ensures that progeny of a culture of the deposit are permanently and indefinitely available to the public after issuance of the relevant U.S. patent or after publication of any U.S. or foreign patent application to the public (subject to antecedent), and to those identified as eligible by the U.S. patent and trademark office leader under 35 USCSelection 122 and the office code therefor (including 37CFR Section 1.14 and specifically referenced 886 OG 638).

The assignee of the present application has agreed that if a culture of a deposited material is killed or lost or damaged when cultured under appropriate conditions, the deposited material will be replaced with another portion of the same material immediately upon notification. The availability of the deposited material is not to be construed as an admission that the invention is not entitled to practice it in contravention of the rights granted under the authority of any government in accordance with its patent laws.

Antibody sequences

Claims (25)

1. A monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 3. SEQ ID NO: 4 and SEQ ID NO: 5, wherein the light chain variable region comprises the three CDRs set forth in SEQ ID NOs: 6. SEQ ID NO: 7 and SEQ ID NO: three CDRs shown in fig. 8.

2. The antibody of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1.

3. The antibody of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

4. The antibody of claim 3, wherein the heavy chain amino acid sequence is as set forth in SEQ ID NO: 11, and the light chain amino acid sequence is as set forth in SEQ ID NO: shown at 12.

5. The antibody of claim 1, wherein the antibody binds to a β_1-40Is that it binds to Abeta_1-42Or Abeta_1-43At least about 40 times greater than the affinity of (a).

6. The antibody of claim 5, wherein the Fab fragment of the antibody binds to A β with an affinity of about 10nM or less_1-40。

7. The antibody of claim 6, wherein the Fab fragment of the antibody binds to A β with an affinity of about 5nM or less_1-40。

8. The antibody of claim 7, wherein the isotype of the antibody is selected from the group consisting of: IgG1, IgG2, IgG3, and IgG 4.

9. The antibody of claim 8, wherein the antibody comprises a heavy chain constant region comprising an Fc region, wherein the heavy chain constant region has impaired effector function.

10. The antibody of claim 9, wherein N-glycosylation in the Fc region is removed.

11. The antibody of claim 9, wherein the heavy chain constant region of the antibody is a human IgG2a constant region comprising the amino acid mutations a330P331 to S330S331, wherein the amino acid positions are based on Kabat numbering with reference to a human wild-type IgG2a sequence.

12. The antibody of claim 9, wherein the heavy chain constant region of the antibody is a human IgG4 constant region comprising the amino acid mutations E233F234L235 to P233V234a235, wherein the amino acid positions are based on Kabat numbering with reference to the sequence of human wild-type IgG 4.

13. The antibody of claim 1, wherein the antibody is a human antibody.

14. The antibody of claim 1, wherein the antibody is a humanized antibody.

15. The monoclonal antibody fragment of claim 4, wherein the fragment has or retains the binding specificity of the monoclonal antibody.

16. The fragment of claim 15, wherein said fragment is Fab, Fab ', F (ab')₂Or Fv.

17. A polynucleotide comprising a nucleotide sequence encoding SEQ ID NO: 1 and SEQ id no: 2, or a variable region of the antibody light chain.

18. A vector comprising the polynucleotide of claim 17.

19. A host cell comprising the polynucleotide of claim 17.

20. A method of making an antibody comprising culturing the host cell of claim 19 under conditions such that the antibody is produced; and isolating the antibody from the host cell or culture.

21. A pharmaceutical composition comprising an effective amount of the antibody of any one of claims 1-14 or the fragment of any one of claims 15 or 16, and a pharmaceutically acceptable excipient.

22. Use of the antibody of any one of claims 1-14 in the manufacture of a medicament for treating alzheimer's disease in a subject.

23. Use of the antibody of any one of claims 1-14 in the manufacture of a medicament for inhibiting amyloid plaque formation in a subject.

24. Use of the antibody of any one of claims 1-14 in the manufacture of a medicament for reducing amyloid plaques in a subject.

25. Use of the antibody of any one of claims 1-14 in the manufacture of a medicament for delaying the development of a symptom associated with alzheimer's disease in a subject.