DEGLYCOSYLATED ANTI-AMYLOID BETA ANTIBODIES

Related Applications

This application claims the benefit of U.S. Provisional Application No. 60/843,460 filed September 8, 2006 which is hereby incorporated by reference in its entirety.

Field of the Invention

The invention relates to deglycosylated monoclonal antibodies to amyloid beta peptide.

Description of the Related Art Accumulation of amyloid beta (Abeta) in the brain is a pathological hallmark of

Alzheimer's disease (AD), and the reduction of Abeta has been proposed as a primary therapeutic target for AD (Hardy J and Selkoe DJ 2002 Science 297:353-356). Active immunization with Abeta peptides reduced brain Abeta and improved cognitive performance in an AD mouse model (Schenk D, et al. 1999 Nature 400:173-177). One proposed mechanism of action is the enhancement of Abeta phagocytosis by microglia: antibodies enter the brain, accumulate surrounding the Abeta plaques, and enhance microglial phagocytosis via Fc receptors (FcR) (Bard F, et al. 2000 Nat Med 6:916-919).

In active immunization studies, plasma Abeta levels are significantly elevated; this is called "Abeta sequestration" or "peripheral sink" (DeMattos RB et al. 2001 Proc Natl Acad Sci USA 98:8850-8855; Lemere CA et al. 2003 Neurobiol Dis 14:10-18). The mechanism is not fully clear yet, but it is believed that the presence of anti- Abeta antibodies in the blood enhances Abeta transfer from the brain to the periphery (DeMattos RB et al.

2002 Science 295:2264-2267; DeMattos RB et al. 2002 J Neurochem 81:229-236). We hypothesized that Abeta sequestration is sufficient to lower brain Abeta load, and found that simple Abeta binding agents, which do not evoke any immune reaction, reduced brain

Abeta (Matsuoka Y et al. 2003 J Neurosci 23:29-33). Another group also found that a different Abeta binding agent reduced brain Abeta (Bergamaschini L et al. 2004 J Neurosci

24:4181-4186). In addition, Abeta active immunization reduced Abeta in an AD model mouse lacking FcR (microglial phagocytosis does not occur in these mice) (Das P et al. 2003 J Neurosci 23:8532-8538).

Despite promising results in animal studies, a clinical trial of active immunization was terminated after meningoencephalitis occurred (Orgogozo JM et al. 2003 Neurology

61:46-54). The cause of the meningoencephalitis is not folly known, but immune activation is presumably involved, and T-cell infiltration to the central nervous system (CNS) was documented (Nicoll JA et al. 2003 Nat Med 9:448-452; Ferrer I et al. 2004 Brain Pathol 14:11-20). Passive immunization does not involve T-cell activation, and thus may be safer than active immunization. However, T-cell activation is not the sole cause of neuroinflammation; other neuroinflammatory events such as cytokine release in response to phagocytosis may occur with a passive immunization approach. Indeed, a recent presentation of Phase 1 data with a passive immunotherapy for AD noted the occurrence of focal edema in several treated patients (presented at the 9th International Geneva/Springfield Symposium, April, 2006), possibly an indication of neuroinflammation.

Segue to the Invention

Glycosylation of immunoglobulin (IgG) is critically involved in binding to FcR (Radaev S and Sun PD 2001 J Biol Chem 276:16478-16483) and complement CIq (Winkelhake JL et al. 1980 J Biol Chem 255:2822-2828). While deglycosylated antibodies maintain binding affinity to their targets, they have reduced interaction with FcR and CIq, suggesting that deglycosylated antibodies may cause less neuroinflammation. Recently, chronic administration of deglycosylated antibody in an AD mouse model reduced brain Abeta load and improved cognitive performance; interestingly, histologically-determined microglial activation and hemorrhage were significantly reduced compared to the effects of treatment with intact antibodies (Wilcock DM et al. 2006 J Neurosci 26:5340-5346). However, the relative potency of deglycosylated antibodies in Abeta sequestration and their effects on microglial phagocytosis (as opposed to activation of microglia) and other neuroinflammatory events remain unclear. In this disclosure, we investigated the effects of deglycosylated antibodies on Abeta sequestration, microglial phagocytosis and cytokine release.

Summary of the Invention

The present invention relates to deglycoslyated monoclonal antibodies that bind amyloid beta (Abeta). The invention provides such antibodies, folly human antibodies retaining Abeta-binding ability, and pharmaceutical compositions including such antibodies. The invention further provides for isolated nucleic acids encoding the antibodies of the invention and host cells transformed therewith. Additionally, the invention provides for prophylactic, therapeutic, and diagnostic methods employing the deglycosylated antibodies of the invention.

Brief Description of the Drawings

Figure 1. Schematic diagram of the amyloid precursor protein (APP) and its principal metabolic derivatives, hi the second line, the sequence within amyloid precursor protein that contains the Abeta and TM regions is expanded (SEQ ID NO: 17). Figure 2. Structural characteristics of a model IgGl . The globular domains of the heavy and light chains are shown. Carbohydrate units indicated by black balls are attached to the Fc region between the C _H 2 domains.

Figure 3. Validation of deglycosylation. Deglycosylation was validated by MALDI-

TOF mass spectrometry. Mass spectra of the intact (A) and deglycosylated (B) antibodies are overlaid. Intact IgG peak, 149,491 Da, is shifted toward to lower molecular mass,

146,877 Da, after deglycosylation. Doubly charged ions are observed around 75 kDa

(exactly half of the mass of the primary peaks).

Figure 4. Deglycosylated antibodies do not evoke microglial phagocytosis in primary cultured microglia. Effects of the intact and deglycosylated antibodies in microglial phagocytosis was determined using primary cultured microglia (>97% pure). A) Microglia were treated with Abeta and intact or deglycosylated antibody, and immunostained with a rabbit polyclonal anti-Abeta antibody. Cellular structure was visualized using markers for actin (phalloidin) and the nucleus (Hoechst 33258). B) Abeta levels in the cell lysate, i.e., phagocytosed Abeta, were determined using an ELISA. The intact antibodies significantly enhanced Abeta phagocytosis (***P<0.001, compared to vehicle-treated control), while deglycosylated antibodies did not (${JP<0.001, {{PO.01, compared to intact antibodies and no difference compared to vehicle-treated controls). Bar = 20 μm in Aa for Aa, 10 μm in Ab for Ab-Ad.

Figure 5. Deglycosylated ^' antibodies elevate plasma Abeta to a similar or greater level compared to intact antibodies. Intact and deglycosylated antibodies were intravenously administered to an AD mouse model. Plasma Abeta change was determined using pre-treatment level as the baseline. AU antibodies significantly elevated plasma

Abeta (***P<0.001 compared to the baseline). In the case of 82El, deglycosylated antibody elevated plasma Abeta more significantly (A, JPO.05 compared to the intact antibody), while both intact and deglycosylated 6E10 antibodies showed virtually identical change (B).

Figure 6. Deglycosylated antibody does not influence microglial cytokine production. Effects of intact and deglycosylated antibodies on TNFalpha levels were

examined using primary cultured microglia. Microglia were treated with intact or deglycosylated antibody, without or with Abeta (A and B, respectively), and the level of TNFalpha in culture medium was determined. A) Intact antibody significantly increased TNFalpha levels (***P<0.001), while deglycosylated antibody did not (significant reduction compared to treatment with intact antibody, JJJPO.OOl). B) Abeta treatment elevated TNFalpha level, and intact antibody caused a further increase (***P<0.001). TNFalpha level after treatment with deglycosylated antibody was slightly lower than the vehicle-treated control level (significant reduction compared to treatment with intact antibody, JJ JPO.001).

Table 1. Brief Description of the SEO ID NOs

Detailed Description of the Preferred Embodiment

Accumulation of amyloid beta (Abeta) is a pathological hallmark of Alzheimer's disease, and lowering Abeta is a promising therapeutic approach. Abeta binding agents present in the blood reduce brain Abeta load by enhancing Abeta transfer from the brain to the periphery (Abeta sequestration). Intact antibodies induce Abeta sequestration, but they also evoke immune reactions not involved in sequestration. The glycan portion of immunoglobulin is critically involved in interactions with effectors including the Fc receptor and complement clq; deglycosylation eliminates these interactions, while binding affinity is maintained. In this disclosure, we investigated the efficiency of deglycosylated antibody in Abeta sequestration as well as its effects on microglial phagocytosis and neuroinflammation. After enzymic deglycosylation, undigested antibody (intact antibody) was not detected and deglycosylated antibodies maintained Abeta binding affinity.

Deglycosylated antibodies did not enhance Abeta phagocytosis or cytokine release in primary cultured microglia, whereas intact antibodies significantly enhanced phagocytosis and cytokine levels. Intravenous injection of deglycosylated antibodies in an Alzheimer's transgenic mouse model elevated plasma Abeta level to a similar or greater degree compared to intact antibodies. Deglycosylated and intact antibodies showed comparable short-term kinetics. Overall, deglycosylated antibodies effectively induced Abeta sequestration without provoking neuroinflamrnation. Thus, deglycosylated antibodies are envisioned as being the basis for sequestration therapy for Alzheimer's disease. Amyloid Precursor Protein and Proteolytic Fragments β-Amyloid protein is derived from amyloid precursor protein by sequential cleavages by proteases referred to as β-secretase and γ-secretase (Figure 1). The amyloid precursor protein comprises ubiquitously expressed proteins whose heterogeneity comes from the "alternative splicing" together of different protein coding regions (exons) within the amyloid precursor protein gene and also from post-translational modifications, such as the addition of sugar or phosphate groups to the protein backbone. Alternatively spliced forms of amyloid precursor protein containing 751 or 770 amino acids are widely expressed in cells throughout the body and also occur in neurons. However, neurons express much higher levels of a 695-amino acid splice form. The difference between the 751-, 770-, and 695-residue forms is the retention in the former of an exon that encodes an amino acid sequence that is homologous to certain inhibitors of serine proteases. The existence of this form suggests one normal function for these longer amyloid precursor protein isoforms; indeed, the amyloid precursor protein 751 that is in human platelets has been shown to inhibit factor XIa (a serine protease) in the clotting cascade.

In Figure 1, the amyloid precursor protein (APP) and its principal metabolic derivatives are depicted. The first line depicts the largest of the known (amyloid precursor protein alternate splice forms, comprising 770 amino acids. Regions of interest are indicated at their correct relative positions. A 17-residue signal peptide occurs at the N- terminus (box with vertical lines). Two alternatively spliced exons of 56 and 19 amino acids are inserted at residue 289; the first contains a serine protease inhibitor domain of the Kunitz type (KPl). A single membrane-spanning domain (transmembrane, TM) at amino acids 700 through 723 is indicated (dotted lines). The β-amyloid protein (Aβ) fragment includes 28 residues just outside the membrane plus the first 12 to 14 residues of the TM domain, hi the second line, the sequence within amyloid precursor protein that contains the

β-amyloid protein and TM regions is expanded. The underlined residues represent the β- amyloid proteins 1 to 42 peptide. The green letters below the wildtype sequence indicate the currently known missense mutations identified in certain families with Alzheimer disease or hereditary cerebral hemorrhage with amyloidosis. The 3-digit numbers are codon numbers (amyloid precursor protein 770 isoform). hi the third line, the first arrow indicates the site (after residue 687) of a cleavage by α-secretase that enables secretion of the large, soluble ectodomain of amyloid precursor protein (APP _s -α) into the medium and retention of the 83-residue C-terminal fragment (C83) in the membrane. The C83 fragment can undergo cleavage by the protease called γ-secretase at residue 711 or residue 713 to release the p3 peptides. The fourth line depicts the alternative proteolytic cleavage after residue 671 by β-secretase that causes the secretion of the slightly truncated APP _s -β molecule and the retention of a 99 residue C-terminal fragment (C99). The C99 fragment can also undergo cleavage by γ-secretase to release the β amyloid peptides. Cleavage of both C83 and C99 by γ-secretase releases the β amyloid precursor protein intracellular domain (AICD) into the cytoplasm. Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. See, e.g., Singleton P and Sainsbury D., in Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons, Chichester, New York, 2001.

The transitional term "comprising" is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

The transitional phrase "consisting of excludes any element, step, or ingredient not specified in the claim, but does not exclude additional components or steps that are unrelated to the invention such as impurities ordinarily associated therewith.

The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. As used herein, the term "antibody" means an immunoglobulin molecule or a fragment of an immunoglobulin molecule having the ability to specifically bind to a particular antigen. Antibodies are well known to those of ordinary skill in the science of immunology. As used herein, the term "antibody" means only substantially full-length

antibody molecules and not fragments of antibody molecules. In particular, as used herein, the term "antibody" means only substantially full-length immunoglobulin molecules but not antigen binding active fragments such as the well-known active fragments F(ab') ₂ , Fab, Fv, and Fd. Deglycosylation of Antibodies: The effector function of the antibodies of the invention is impaired by removing N-glycosylation of the Fc region (e.g., in the C _H 2 domain of IgG) of the anti-Abeta monoclonal antibodies. As shown in Fig. 2, IgG antibodies are glycosylated by attachment of carbohydrate units to C _R 2 domains. a) In some embodiments, N-glycosylation of the Fc region is removed by mutating the glycosylated amino acid residue or flanking residues that are part of the glycosylation recognition sequence in 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 the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain for N-glycosylation. Mutating any of the amino acids in the tripeptide sequences in the constant region yields an aglycosylated IgG. b) In other embodiments, glycosylation, or certain types of glycosylation, can be enzymatically removed from the glycoprotein, for example using endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase Fl, endoglycosidase F2, and endoglycosidase F3. c) In certain embodiments, host cells can be chosen so that they do not produce glycosylated antibody chains (e.g., prokaryotic cells), or engineered so that they are glycosylation defective.

As used herein, the terms "Alzheimer's disease" refers to a progressive degenerative disease of the brain of unknown etiology, characterized by diffuse atrophy throughout the cerebral cortex with distinctive lesions called senile plaques and clumps of fibrils called neurofibrillary tangles.

As used herein with respect to polypeptides, the term "substantially pure" means that the polypeptides are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use. In particular, the polypeptides are sufficiently pure and are sufficiently free from other biological constituents of their host cells so as to be useful in, for example, generating antibodies, sequencing, or producing pharmaceutical preparations. By techniques well known in the art, substantially pure polypeptides may be produced in light of the nucleic

acid and amino acid sequences disclosed herein. Because a substantially purified polypeptide of the invention may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the polypeptide may comprise only a small percentage by weight of the preparation. The polypeptide is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems.

As used herein with respect to nucleic acids, the term "isolated" means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art. As used herein, a coding sequence and regulatory sequences are said to be "operably joined" when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.

The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating sequences involved with initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' non-transcribing regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences, as desired.

As used herein, a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification and selection of cells which have been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β- galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques. Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.

Anti-Abeta Monoclonal Antibodies

The present invention derives, in part, from the isolation and characterization of monoclonal antibodies that selectively bind to Abeta. As described more fully below, antibody 82El was raised against aal-16 of human Abeta; the antibody binds Abeta terminating at position 40 or 42. Such antibodies can be produced using well-known hybridoma techniques (Kohler, G. and Milstein, C. 1975 Nature 256:495-497). The paratope of the anti- Abeta Fab fragments associated with the epitope on Abeta are defined by the amino acid (aa) sequences of the immunoglobulin heavy and light chain V-regions described in Table 1 and SEQ ID NO: 1 through SEQ ID NO: 16. In one set of embodiments, the present invention provides a substantially full- length, anti-Abeta monoclonal antibody in isolated form and in pharmaceutical preparations. Similarly, as described below, the present invention provides isolated nucleic acids, host cells transformed with nucleic acids, and preparations including isolated nucleic acids, encoding the substantially full-length anti-Abeta monoclonal antibody. Finally, the present invention provides methods, as described more fully below, employing these antibodies in the in vitro and in vivo diagnosis, prevention and therapy of Alzheimer's disease.

Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Male D., Brostoff J., Roth D.B., and Roitt I. 2006 in Immunology 7 ^th Ed. Mosby Elsevier, Philadelphia PA). The pFc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of a full- length antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of a full-length antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the

paratope (see, in general, Male D., Brostoff J., Roth D.B., and Roitt L, 2006, supra). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FRl through FR4) separated respectively by three complementarity determining regions (CDRl through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.

The complete amino acid sequences of the antigen-binding Fab portions of the anti- Abeta 82El monoclonal antibodies as well as the relevant FR and CDR regions are disclosed herein. SEQ ID NO: 1 discloses the amino acid sequence of the Fd fragment of the anti-Abeta 82El monoclonal antibodies. The amino acid sequences of the heavy chain FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4 regions are disclosed as SEQ ID NO: 2 through SEQ ID NO: 8, respectively. SEQ ID NO: 9 discloses the amino acid sequence of the light chain of the anti-Abeta 82El monoclonal antibodies. The amino acid sequences of the light chain FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4 regions are disclosed as SEQ ID NO: 10 through SEQ ID NO: 16, respectively.

It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. Thus, for example, PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of full-length antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions of the anti-Abeta 82El monoclonal antibodies have been replaced by homologous human or non-human sequences. Thus, those skilled in the art may alter the anti-Abeta 82El monoclonal antibodies by the construction of CDR grafted or chimeric antibodies containing all, or part thereof, of the disclosed heavy and light chain V- region CDR aa sequences (Jones, P.T. et al. 1986 Nature 321:522-525; Verhoeyen, M. et al. 1988 Science 39:1534-1536; and Tempest, P.R. et al. 1991 Biotechnology 9:266-271),

without destroying the specificity of the antibodies for the Abeta epitope. Such CDR grafted or chimeric antibodies can be effective in prevention and treatment of Alzheimer's disease. hi some embodiments, the chimeric antibodies of the invention are fully human monoclonal antibodies including at least the heavy chain CDR3 region of the anti-Abeta 82El monoclonal antibodies. As noted above, such chimeric antibodies may be produced in which some or all of the FR regions of the anti-Abeta 82El monoclonal antibodies have been replaced by other homologous human FR regions, hi addition, the Fc portions may be replaced so as to produce IgA or IgM as well as IgG antibodies bearing some or all of the CDRs of the anti-Abeta 82El monoclonal antibodies. Of particular importance is the inclusion of the anti-Abeta 82El monoclonal antibodies heavy chain CDR3 region and, to a lesser extent, the other CDRs of the anti-Abeta 82El monoclonal antibodies. Such fully human or chimeric antibodies will have particular utility in that they will not evoke an immune response against the antibody itself. It is also possible, in accordance with the present invention, to produce chimeric antibodies including non-human sequences. Thus, one may use, for example, murine, ovine, equine, bovine or other mammalian Fc or FR sequences to replace some or all of the Fc or FR regions of the anti-Abeta 82El monoclonal antibodies. Some of the CDRs may be replaced as well. Again, however, it is preferred that at least the heavy chain CDR3 of the anti-Abeta 82El monoclonal antibodies, be included in such chimeric antibodies and, to a lesser extent, it is also preferred that some or all of the other CDRs of the anti-Abeta 82El monoclonal antibodies be included. Such chimeric antibodies bearing non-human immunoglobulin sequences admixed with the CDRs of the human anti-Abeta 82El monoclonal antibodies are not preferred for use in humans and are particularly not preferred for extended use because they may evoke an immune response against the non-human sequences. They may, of course, be used for brief periods or in immunosuppressed individuals but, again, fully human monoclonal antibodies are preferred. Alternatively, chimeric antibodies bearing non-human mammalian Fc and FR sequences but inclμding at least the heavy chain CDR3 of the anti-Abeta 82El monoclonal antibodies maybe used for brief periods or in immunosuppressed subjects are contemplated as alternative embodiments of the present invention.

For inoculation or prophylactic uses, the antibodies of the present invention are preferably substantially full-length antibody molecules including the Fc region. Such

substantially full-length antibodies will have longer half-lives than smaller fragment antibodies (e.g., Fab) and are more suitable for intravenous, etc. administration.

It is possible to determine, without undue experimentation, if an altered or chimeric antibody has the same specificity as the anti-Abeta monoclonal antibodies by ascertaining whether the former blocks the latter from binding to amyloid beta peptide. If the monoclonal antibody being tested competes with the anti-Abeta monoclonal antibody as shown by a decrease in binding of the anti-Abeta monoclonal antibody, then it is likely that the two monoclonal antibodies bind to the same, or a closely spaced, epitope. Still another way to determine whether a monoclonal antibody has the specificity of the anti-Abeta monoclonal antibodies is to pre-incubate the anti-Abeta monoclonal antibody with amyloid beta peptide with which it is normally reactive, and then add the monoclonal antibody being tested to determine if the monoclonal antibody being tested is inhibited in its ability to bind amyloid beta peptide. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or a functionally equivalent, epitope and specificity as the anti- Abeta monoclonal antibodies of the invention. Screening of anti-Abeta monoclonal antibodies also can be carried out by determining whether the mAb binds to the amyloid beta peptide (e.g., by Western blot or immunoprecipitation).

By using the antibodies of the invention, it is now possible to produce anti-idiotypic antibodies which can be used to screen other monoclonal antibodies to identify whether the antibody has the same binding specificity as an antibody of the invention, hi addition, such antiidiotypic antibodies can be used for active immunization (Herlyn, D. et al. 1986 Science 232:100-102). Such anti-idiotypic antibodies can be produced using well-known hybridoma techniques (Kohler, G. and Milstein, C. 1975 Nature 256:495-497). An anti- idiotypic antibody is an antibody which recognizes unique determinants present on the monoclonal antibody produced by the cell line of interest. These determinants are located in the hypervariable region of the antibody. It is this region which binds to a given epitope and, thus, is responsible for the specificity of the antibody. An anti-idiotypic antibody can be prepared by immunizing an animal with the monoclonal antibody of interest. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an antibody to these idiotypic determinants. By using the anti-idiotypic antibodies of the immunized animal, which are specific for the monoclonal antibodies of the invention, it is possible to identify other clones with the same idiotype as the antibody of the hybridoma used for immunization. Idiotypic identity

between monoclonal antibodies of two cell lines demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant. Thus, by using anti-idiotypic antibodies, it is possible to identify other hybridomas expressing monoclonal antibodies having the same epitopic specificity. Nucleic Acids Encoding Anti-Abeta Antibodies

Given the disclosure herein of the amino acid sequences of the heavy chain Fd and light chain variable domains of the anti-Abeta 82El monoclonal antibodies, one of ordinary skill in the art is now enabled to produce nucleic acids which encode this antibody or which encode the various chimeric antibodies described above. It is contemplated that such nucleic acids will be operably joined to other nucleic acids forming a recombinant vector for cloning or for expression of the antibodies of the invention. The present invention includes any recombinant vector containing the coding sequences, or part thereof, whether for prokaryotic or eukaryotic transformation or transfection. Such vectors may be prepared using conventional molecular biology techniques, known to those with skill in the art, and would comprise DNA coding sequences for the immunoglobulin V-regions of the anti- Abeta 82El monoclonal antibodies, including framework and CDRs or parts thereof, and a suitable promoter either with (Whittle, N. et al. 1987 Protein Eng 1:499-505 and Burton, D.R. et al. 1994 Science 266:1024-1027) or without (Marasco, W. A. et al. 1993 Proc Natl Acad Sci USA 90:7889-7893 and Duan, L. et al. 1994 Proc Natl Acad Sci USA 91:5075- 5079) a signal sequence for export or secretion. Such vectors may be transformed or transfected into prokaryotic (Huse, W.D. et al. 1989 Science 246:1275-1281; Ward, S. et al. 1989 Nature 341:544-546; Marks, J.D. et al. 1991 J M?/ Biol 222:581-597; and Barbas, CF. et al. 1991 Proc Natl Acad Sci USA 88:7978-7982) or eukaryotic (Whittle, N. et al. 1987 Protein Eng 1:499-505 and Burton, D.R. et al. 1994 Science 266:1024-1027) cells by conventional techniques, known to those with skill in the art.

The expression vectors of the present invention include regulatory sequences operably joined to a nucleotide sequence encoding one of the antibodies of the invention. As used herein,. the term "regulatory sequences" means nucleotide sequences which are necessary for or conducive to the transcription of a nucleotide sequence which encodes a desired polypeptide and/or which are necessary for or conducive to the translation of the resulting transcript into the desired polypeptide. Regulatory sequences include, but are not limited to, 5' sequences such as operators, promoters and ribosome binding sequences, and 3 ' sequences such as polyadenylation signals. The vectors of the invention may optionally

include 5' leader or signal sequences, 5' or 3' sequences encoding fusion products to aid in protein purification, and various markers which aid in the identification or selection of transformants. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. The subsequent purification of the antibodies may be accomplished by any of a variety of standard means known in the art.

A preferred vector for screening monoclonal antibodies, but not necessarily preferred for the mass production of the antibodies of the invention, is a recombinant DNA molecule containing a nucleotide sequence that codes for and is capable of expressing a fusion polypeptide containing, in the direction of amino- to carboxy-terminus, (1) a prokaryotic secretion signal domain, (2) a polypeptide of the invention, and, optionally, (3) a fusion protein domain. The vector includes DNA regulatory sequences for expressing the fusion polypeptide, preferably prokaryotic, regulatory sequences. Such vectors can be constructed by those with skill in the art and have been described by Smith, G.P. et al. (1985 Science 228:13151317); Clackson, T. et al. (1991 Nature 352:624-628); Kang et al. (1991 in Methods: A Companion to Methods in Enzymologv, vol. 2, R.A. Lerner and D.R. Burton, ed. Academic Press, NY, pp 111-118); Barbas, CF. et al. (1991 Proc Natl Acad Sci USA 88:7978-7982); Roberts, B.L. et al. (1992 Proc Natl Acad Sci USA 89:2429-2433).

A fusion polypeptide may be useful for purification of the antibodies of the invention. The fusion domain may, for example, include a poly-His tail which allows for purification on Ni ⁺ columns or the maltose binding protein of the commercially available vector pMAL (New England BioLabs, Beverly, MA). A currently preferred, but by no means necessary, fusion domain is a filamentous phage membrane anchor. This domain is particularly useful for screening phage display libraries of monoclonal antibodies but may be of less utility for the mass production of antibodies. The filamentous phage membrane anchor is preferably a domain of the cpIII or cpVIII coat protein capable of associating with the matrix of a filamentous phage particle, thereby incorporating the fusion polypeptide onto the phage surface, to enable solid phase binding to specific antigens or epitopes and thereby allow enrichment and selection of the specific antibodies or fragments encoded by the phagemid vector. The secretion signal is a leader peptide domain of a protein that targets the protein to the membrane of the host cell, such as the periplasmic membrane of Gram-negative bacteria. A preferred secretion signal for E. coli is a pelB secretion signal. The leader sequence of the pelB protein has previously been used as a secretion signal for fusion

proteins (Better, M. et al. 1988 Science 240:1041-1043; Sastry, L. et al. 1989 Proc Natl Acad Sd USA 86:5728-5732; and Mullinax, RX. et al., 1990 Proc Natl Acad Sd USA 87:8095-8099). Amino acid residue sequences for other secretion signal polypeptide domains from E. coli useful in this invention can be found in Neidhard, F.C. (ed.), 1987 in Escherichia coli and Salmonella Typhimurium: Typhimurium Cellular and Molecular Biology, American Society for Microbiology, Washington, D. C.

To achieve high levels of gene expression in E. coli, it is necessary to use not only strong promoters to generate large quantities of mRNA, but also ribosome binding sites to ensure that the mRNA is efficiently translated. In E. coli, the ribosome binding site includes an initiation codon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotides upstream from the initiation codon (Shine J. and Dalgarno L. 1975 Nature 254:34-38). The sequence, which is called the Shine-Dalgarno (SD) sequence, is complementary to the 3' end of E. coli 16S rRNA. Binding of the ribosome to mRNA and the sequence at the 3' end of the mRNA can be affected by several factors: the degree of complementarity between the SD sequence and 3' end of the 16S rRNA; the spacing lying between the SD sequence and the AUG; and the nucleotide sequence following the AUG, which affects ribosome binding. The 3' regulatory sequences define at least one termination (stop) codon in frame with and operably joined to the heterologous fusion polypeptide. In preferred embodiments with a prokaryotic expression host, the vector utilized includes a prokaryotic origin of replication or replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such origins of replication are well known in the art. Preferred origins of replication are those that are efficient in the host organism. A preferred host cell is E. coli. For use of a vector in E. coli, a preferred origin of replication is CoIEI found in pBR322 and a variety of other common plasmids. Also preferred is the pl5A origin of replication found on pACYC and its derivatives. The CoIEI and pl5A replicons have been extensively utilized in molecular biology, are available on a variety of plasmids and are described by Sambrook et al., 1989, in Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory Press.

In addition, those embodiments that include a prokaryotic replicon preferably also include a gene whose expression confers a selective advantage, such as drag resistance, to a

bacterial host transformed therewith. Typical bacterial drug resistance genes are those that confer resistance to ampicillin, tetracycline, neomycin/kanamycin or chloramphenicol. Vectors typically also contain convenient restriction sites for insertion of translatable DNA sequences. Exemplary vectors are the plasmids pUC18 and pUC19 and derived vectors such as those commercially available from suppliers such as Invitrogen (San Diego, CA).

When the antibodies of the invention include both heavy chain and light chain sequences, these sequences may be encoded on separate vectors or, more conveniently, may be expressed by a single vector. The heavy and light chain may, after translation or after secretion, form the heterodimeric structure of natural antibody molecules. Such a heterodimeric antibody may or may not be stabilized by disulfide bonds between the heavy and light chains.

A vector for expression of heterodimeric antibodies, such as the substantially full- length antibodies of the invention or the chimeric antibodies of the invention, is a recombinant DNA molecule adapted for receiving and expressing translatable first and second DNA sequences. That is, a DNA expression vector for expressing a heterodimeric antibody provides a system for independently cloning (inserting) the two translatable DNA sequences into two separate cassettes present in the vector, to form two separate cistrons for expressing the first and second polypeptides of a heterodimeric antibody. The DNA expression vector for expressing two cistrons is referred to as a dicistronic expression vector.

Preferably, the vector comprises a first cassette that includes upstream and downstream DNA regulatory sequences operably joined via a sequence of nucleotides adapted for directional ligation to an insert DNA. The upstream translatable sequence preferably encodes the secretion signal as described above. The cassette includes DNA regulatory sequences for expressing the first antibody polypeptide that is produced when an insert translatable DNA sequence (insert DNA) is directionally inserted into the cassette via the sequence of nucleotides adapted for directional ligation.

The dicistronic expression vector also contains a second cassette for expressing the second antibody polypeptide. The second cassette includes a second translatable DNA sequence that preferably encodes a secretion signal, as described above, operably joined at its 3' terminus via a sequence of nucleotides adapted for directional ligation to a downstream DNA sequence of the vector that typically defines at least one stop codon in the reading frame of the cassette. The second translatable DNA sequence is operably joined

at its 5' terminus to DNA regulatory sequences forming the 5' elements. The second cassette is capable, upon insertion of a translatable DNA sequence (insert DNA), of expressing the second fusion polypeptide comprising a secretion signal with a polypeptide coded by the insert DNA. The antibodies of the present invention may additionally, of course, be produced by eukaryotic cells such as CHO cells, mouse hybridomas, immortalized B-lymphoblastoid cells, and the like. In this case, a vector is constructed in which eukaryotic regulatory sequences are operably joined to the nucleotide sequences encoding the antibody polypeptide or polypeptides. The design and selection of an appropriate eukaryotic vector is within the ability and discretion of one of ordinary skill in the art. The subsequent purification of the antibodies may be accomplished by any of a variety of standard means known in the art.

In another embodiment, the present invention provides host cells, both prokaryotic and eukaryotic, transformed or transfected with, and therefore including, the vectors of the present invention.

Diagnostic and Pharmaceutical Anti-Abeta Antibody Preparations

The invention also relates to a method for preparing diagnostic or pharmaceutical compositions comprising the monoclonal antibodies of the invention, the pharmaceutical compositions being used for immunoprophylaxis or immunotherapy of Alzheimer's disease. The pharmaceutical preparation includes a pharmaceutically acceptable carrier. Such carriers, as used herein, means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.

A preferred embodiment of the invention relates to monoclonal antibodies whose heavy chains comprise in CDR3 the polypeptide having SEQ ID NO: 7, and/or whose light chains comprise in CDR3 the polypeptide having SEQ ID NO: 15 and conservative variations of these peptides. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as

isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies having the substituted polypeptide also bind amyloid beta peptide. Analogously, another preferred embodiment of the invention relates to polynucleotides which encode the above noted heavy chain polypeptides and to polynucleotide sequences which are complementary to these polynucleotide sequences. Complementary polynucleotide sequences include those sequences that hybridize to the polynucleotide sequences of the invention under stringent hybridization conditions.

The anti-Abeta antibodies of the invention may be labeled by a variety of means for use in diagnostic and/or pharmaceutical applications. There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the monoclonal antibodies of the invention, or will be able to ascertain such, using routine experimentation. Furthermore, the binding of these labels to the monoclonal antibodies of the invention can be done using standard techniques common to those of ordinary skill in the art.

Another labeling technique which may result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically altered by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies.

The materials for use in the assay of the invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a monoclonal antibody of the invention that is, or can be, detectably labeled. The kit may also have containers containing buffer(s) and/or a container comprising a reporter-means, such as a biotin-

binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic or fluorescent label.

In vitro Detection and Diagnostics

The monoclonal antibodies of the invention are suited for in vitro use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier, hi addition, the monoclonal antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize the monoclonal antibodies of the invention are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (imrnunometric) assay. Detection of antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation. The monoclonal antibodies of the invention can be bound to many different carriers and used to detect the presence of amyloid beta peptide. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble or insoluble for puiposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such, using routine experimentation.

For purposes of the invention, amyloid beta peptide may be detected by the monoclonal antibodies of the invention when present in biological fluids and tissues. Any sample containing a detectable amount of amyloid beta peptide can be used. A sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum or the like; a solid or semi-solid such as tissues, feces, or the like; or, alternatively, a solid tissue such as those commonly used in histological diagnosis. In vivo Detection of Abeta

In using the monoclonal antibodies of the invention for the in vivo detection of antigen, the detectably labeled monoclonal antibody is given in a dose which is diagnostically effective. The term "diagnostically effective" means that the amount of detectably labeled monoclonal antibody is administered in sufficient quantity to enable

detection of the site having the amyloid beta peptide antigen, for which the monoclonal antibodies are specific.

The concentration of detectably labeled monoclonal antibody which is administered should be sufficient such that the binding to Abeta is detectable compared to the background. Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.

As a rule, the dosage of detectably labeled monoclonal antibody for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. The dosage of monoclonal antibody can vary from about 0.01 mg/kg to about 500 mg/kg, preferably 0.1 mg/kg to about 200 mg/kg, most preferably about 0.1 mg/kg to about 10 mg/kg. Such dosages may vary, for example, depending on whether multiple injections are given, on the tissue being assayed, and other factors known to those of skill in the art. For in vivo diagnostic imaging, the type of detection instrument available is a major factor in selecting an appropriate radioisotope. The radioisotope chosen must have a type of decay which is detectable for the given type of instrument. Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough such that it is still detectable at the time of maximum uptake by the target, but short enough such that deleterious radiation with respect to the host is acceptable. Ideally, a radioisotope used for in vivo imaging will lack a particle emission but produce a large number of photons in the 140-250 keV range, which may be readily detected by conventional gamma cameras.

For in vivo diagnosis, radioisotopes may be bound to immunoglobulin either directly or indirectly by using an intermediate functional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions are the bifunctional chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetra-acetic acid (EDTA) and similar molecules. Typical examples of metallic ions which can be bound to the monoclonal antibodies of the invention are ¹¹¹ In, ⁹⁷ Ru, ⁶⁷ Ga, ⁶⁸ Ga ₅ ⁷² As, ⁸⁹ Zr and ²⁰¹ Tl.

The monoclonal antibodies of the invention can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional method for visualizing

diagnostic imaging can be utilized. Usually gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI. Elements which are particularly useful in such techniques include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr and ⁵⁶ Fe.

The monoclonal antibodies of the invention can be used in vitro and in vivo to monitor the course of therapies for Alzheimer's disease. Thus, for example, by measuring the increase or decrease in the number of amyloid beta plaques or changes in the concentration of amyloid beta peptide present in the body or in various body fluids (e.g., in serum), it would be possible to determine whether a particular therapeutic regimen aimed at ameliorating Alzheimer's disease. Prophylaxis and Therapy of Alzheimer's Disease

The monoclonal antibodies can also be used in prophylaxis and as therapy for Alzheimer's disease. The terms, "prophylaxis" and "therapy" as used herein in conjunction with the monoclonal antibodies of the invention denote both prophylactic as well as therapeutic administration and both passive immunization with substantially purified polypeptide products. Thus, the monoclonal antibodies can be administered to high-risk subjects (humans) in order to lessen the likelihood and/or severity of Alzheimer's disease, or administered to subjects (humans) already evidencing active Alzheimer's disease, hi the present invention, substantially full-length antibody molecules that bind Abeta are used to treat Alzheimer's disease, and fully human or chimeric antibodies are otherwise preferred. As used herein, a "prophylactically effective amount" of the monoclonal antibodies of the invention is a dosage large enough to produce the desired effect in which the likelihood of Alzheimer's Disease is decreased. A prophylactically effective amount is not, however, a dosage so large as to cause adverse side effects, such as neuroinflammation, and the like. Generally, a prophylactically effective amount may vary with the subject's age, condition, and sex, as well as the possibility of the disease in the subject and can be determined by one of skill in the art. The dosage of the prophylactically effective amount may be adjusted by the individual physician in the event of any complication. A prophylactically effective amount may vary from about 0.01 mg/kg to about 500 mg/lcg, preferably from about 0.1 mg/kg to about 200 mg/kg, most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more administrations daily, for one or several days.

As used herein, a "therapeutically effective amount" of the monoclonal antibodies of the invention is a dosage large enough to produce the desired effect in which the symptoms of Alzheimer's disease are ameliorated. A therapeutically effective amount is

not, however, a dosage so large as to cause adverse side effects, such as neuroinflammation and the like. Generally, a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the extent of the disease in the subject and can be determined by one of skill in the art. The dosage of the therapeutically effective amount may be adjusted by the individual physician in the event of any complication. A therapeutically effective amount may vary from about 0.01 mg/kg to about 500 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.

The monoclonal antibodies of the invention can be administered by injection or by gradual infusion over time. The administration of the monoclonal antibodies of the invention may, for example, be intravenous. Techniques for preparing injectate or infusate delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties of the antibodies, such as the paratope binding capacity (see, for example, Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publishing). Those of skill in the art can readily determine the various parameters and conditions for producing antibody injectates or infusates without resort to undue experimentation.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and the like.

Deglycosylated anti-amyloid beta antibodies induce amyloid beta sequestration with reduced microglial phagocytosis and cytokine release Deglycosylated antibodies maintain affinity to Abeta

To deglycosylate antibodies, we treated two mouse monoclonal anti-Abeta antibodies (Table 2) with the deglycosylation enzyme, peptide-N4-(acetyl-beta- glucosaminyl)-asparagine amidase. Deglycosylation of antibody was validated by MALDI-

TOF mass spectrometry (Fig. 3), which established deglycosylation with no residual undigested antibody. We determined the affinity of deglycosylated and intact antibodies using BIAcore. Clone 82El intact and deglycosylated antibodies had K _D S of 0.9 and 1.0 nM, respectively; clone 6E10 intact and deglycosylated antibodies had K _D S of 7.4 and 9.2 nM, respectively. Thus, deglycosylation of antibodies did not affect their affinity for Abeta to a major degree. Table 2. Preparation and Validation of Deglycosylated Antibodies

Affinity to Abeta [KD]

Clone Epitope Subclass Selectivity Intact Deglycosylated

82El Abeta 1-5 IgGl Abeta specific; 0.9 nM LO nM

(not cross reactive with APP)

6E10 Abeta 3-8 IgGl Cross reactive with APP 7.4 nM 9.2 nM

Validation of deglycosylation of antibodies

To validate deglycosylation of antibodies, intact and deglycosylated antibodies were subjected to matrix-assisted laser desorption/Ionization time-of-flight (MALDI-TOF) mass spectrometry. A sample of 1 μl was mixed with 1 μl of 5 mg/ml sinapinic acid in ethanol and water (3:2 ratio) solution (Fluka, St Louis, MO), dried at room temperature, and subjected to MALDI-TOF mass spectrometry (AXIMA-CFR mass spectrometer, Shimadzu, Kyoto, Japan) using positive linear mode with nitrogen laser (? = 337 nm) and 20 kV extraction potential. Mass spectra were calibrated using singly charged ions of a mixture of insulin, cytochrome c, apomyoglobin, aldolase, and albumin, which have average [M+H] ⁺ m/z = 5730.6, 12,361, 16,952, 39,212, and 66,430, respectively. In parallel to the measurements, calibration was validated through desorption from three different MALDI target spots. The molecular masses of the intact and deglycosylated antibodies were determined to be 149,491 and 146,877 Da, respectively (Fig. 3-A and 3-B, respectively). After deglycosylation, the undigested (un-reacted) IgG molecule was not detected. Doubly charged ion signals were observed around m/z 75,000 Da (exactly half of the molecular mass of the primary peaks). Determination of the affinity of intact and deglycosylated antibodies for Abeta

The affinity of the intact and deglycosylated antibodies, clones 6E10 and 82El, was determined using a BIAcore 3000 biosensor (Biacore, Inc., Uppsala, Sweden). Testing antibodies were immobilized on CM5 sensor chips (Biacore) using 10 mM glycine buffer,

pH 1.7, as a regeneration buffer. The KD value was determined using various Abeta 1-40 at 0.8, 1.5, 2.9, 5.8, 11.5 and 23 nM in 0.01 M HEPES buffer consisting of 150 niM NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P20, pH 7.4, as a running buffer at a flow rate of 20 μl/min. Forty μl samples were injected. The dissociation curve was obtained while running buffer for 120 seconds. Data collected represent the value of the observed response units (RU) obtained in the sample cells subtracted from the RU obtained from a reference cell. The data were analyzed using a BIAevaluation 4.1 software (Biacore). In contrast to intact antibodies, deglycosylated antibodies did not enhance microglial phagocytosis. We tested effects of deglycosylation on microglial phagocytosis using primary cultured microglia. Immunostaining using microglia- and astrocyte-specific markers determined that the purity of our microglia was over 97% (i.e., astrocyte contamination was less than 3%). We treated microglia with Abeta and intact or deglycosylated antibody, and assessed microglial phagocytosis. Microglia rapidly changed morphology when Abeta was added (Fig. 4Aa vs. Ab); microglial phagocytosis of Abeta was evident (Fig. 4Ab). Intact antibody significantly enhanced microglial phagocytosis (Fig. 4Ac), whereas deglycosylated antibody did not (Fig. 4Ad). ELISA results indicated that intact antibodies, both clones 82El and 6E10, significantly enhanced Abeta phagocytosis (Fig. 4B, ***P<0.001 compared to vehicle control). In contrast, deglycosylated antibodies had no effect on Abeta phagocytosis (Fig. 4B, JJJPO.OOl and % JPO.01 compared to intact 82El and 6E10, respectively).

Abeta and anti-Abeta antibodies (82El and 6El 0) form complexes that may interfere with Abeta ELISA quantification. In this study, we used our 12B2/1C3 ELISA (Horikoshi Y et al. 2004 Biochem Biophys Res Commun 319:733-737). Abeta was captured by a C terminus antibody (1C3, epitope 38-42) and detected by HRP-coupled 12B2, which binds to an epitope within Abeta amino acid residues 17-28. The study antibodies, clones 6E10 and 82El, bind to epitopes within 1-5 and 3-8 amino acid residues, respectively. Thus, the epitopes of the study antibodies and the ELISA antibodies do not. overlap. ELISA data indicated Abeta levels of 100 ± 7% when various amounts of study antibodies were added to the Abeta peptide, confirming that Abeta quantification using 12B2/1C3 ELISA was not compromised by the study antibodies.

Deglycosylated antibodies elevate plasma Abeta levels to a similar or greater extent than intact antibodies

We investigated the potency of deglycosylated antibodies in Abeta sequestration.

Deglycosylated and intact antibodies (50 μg IgG/mouse) were intravenously administered to an AD mouse model and plasma Abeta level was determined (Fig. 5). At the age we used for this study, 13 week-old mice, no plaque was detectable. Both intact and deglycosylated antibodies elevated plasma Abeta (***P <0.001 compared to the baseline level). Although deglycosylated 82El antibody elevated plasma Abeta significantly more than the intact antibody (Fig. 5A, JPO.05), intact and deglycosylated 6E10 antibodies yielded virtually identical results (Fig. 5B).

Deglycosylated antibodies have short-term pharmacokinetics similar to the intact antibodies

To determine the blood level and brain entry of the administered intact and deglycosylated antibodies, we developed a microplate assay. The assay system had a linear standard curve over the range of 10 pg to 10 ng IgG per well of a 96-well microplate (r ² >0.99). With both 82El and 6E10, the intact and deglycosylated antibodies showed comparable short-term kinetics in plasma (Table 3). Antibody, either the intact or deglycosylated, was intravenously injected (50 μg IgG/mouse) from the tail vein. Antibody levels in the plasma were determined at 24 and 48 hours after the injection. Data are presented as mean ± SD (n=5). We also determined the presence of antibodies in the brain at the terminal study point (48 hours after administration); the intact and deglycosylated antibodies showed similar brain entry, 0.06 and 0.10% in case of intact and deglycosylated 82El, and 0.05 and 0.08% in case of intact and deglycosylated 6E10, respectively. Table 3. Antibody levels in the plasma.

Antibody in the plasma [μg/ml]

A. 82El 24 hours 48 hours

Intact 22.8 ± 3.2 15.5 ± 2.5

Deglycosylated 28.8 ± 1.8 21.4 -t 1.6

B. 6E10

Intact 24.2 ± 0.5 20.1 ± 0.9

Deglycosylated 18.5 ± 3.1 16.4 ± 2.4

L

In contrast to intact antibody, deglycosylated antibody did not increase cytokine levels.

We determined the effects of intact and deglycosylated antibodies on cytokine levels using primary cultured microglia. Since 6E10 was prepared under conventional conditions and induces high levels of cytokine production by microglia due to pyrogen contamination, we used only 82El, which was prepared under non-pyrogenic condition, to investigate the effects of deglycosylation on cytokine production.

TNFalpha was detected in microglia culture medium even without Abeta stimulation. Treatment with the intact antibody significantly increased TNFalpha levels (Fig. 6A, ***P <0.001). However, deglycosylated antibody did not influence TNFalpha levels (significant difference compared to the intact antibody, JJJPO.001).

Treatment with Abeta increased TNFalpha levels (open columns in Fig. 6A vs 6B). The intact antibody further enhanced TNFalpha levels (Fig. 6B, ***P <0.001 compared to vehicle-treated controls). TNFalpha levels were significantly lower in deglycosylated antibody-treated microglia (JJJPO.001 compared to treatment with intact antibody, and similar to control). Discussion

Abeta is generated from a parental molecule, APP, by sequential proteolytic cleavage at the N and C termini of the Abeta domain by beta and gamma secretases, respectively. Therefore, beta and gamma secretase inhibitors are aggressively being pursued as therapeutic targets (Pollack SJ and Lewis H 2005 Curr Opin Investig Drugs 6:35-47). However, it is not certain yet that secretase inhibition will be sufficient to halt AD neurodegeneration, and combination therapy with other Abeta-lowering approaches may be required. Enhancement of Abeta metabolism and clearance represents an alternative approach to lower brain Abeta level. Several enzymes, such as neprilysin, insulin-degrading enzyme and plasmin, are known to degrade Abeta (Selkoe DJ 2001 Neuron 32:177-180). However, Abeta is not the sole substrate of these enzymes, and it may be difficult to enhance Abeta degradation specifically. Another plausible approach is the enhancement of Abeta clearance (Zlokovic BV 2004 J Neurochem 89:807-811). Antibodies present in the blood significantly enhanced Abeta transfer from the brain to the periphery (Abeta sequestration) (DeMattos RB et al. 2001 Proc Natl Acad Sd USA 98:8850-8855; Lemere CA et al. 2003 Neurobiol Dis 14:10-18). In addition, simple Abeta

binding agents reduced brain Abeta load (Matsuoka Y et al. 2003 J Neurosci 23:29-33; Bergamaschini L et al. 2004 J Neurosci 24:4181-4186).

The ideal molecular properties for therapeutic agents based on Abeta sequestration are not yet known, but Abeta binding affinity is presumably important. Good antibodies achieve sub nM affinity, but it is very challenging to develop low molecular weight drug candidates that interact with the primary structure of the peptide at similarly high affinity. Currently available Abeta binding agents, such as Congo red, thioflavin T and their derivatives, have limited affinity to non-aggregated Abeta, which is the target of Abeta sequestration; their potency to induce Abeta sequestration is limited (Matsuoka Y et al. 2005 Curr Alzheimer Res 2:265-268). Intact antibody has high affinity, but evokes immune responses that may be undesirable. The glycan portion of IgG is critically involved in binding to immune response effectors including FcR (Heyman B 2000 Annu Rev Immunol 18:709-737; Radaev S and Sun PD 2001 J Biol Chem 276:16478-16483), complement CIq (Winkelhake JL et al. 1980 J Biol Chem 255:2822-2828), and others (Nose M and Wigzell H 1983 Proc Natl Acad Sd USA 80:6632-6636). Here, we confirmed that deglycosylation of anti-amyloid antibodies did not affect their affinity. Deglycosylated antibodies showed limited ability to activate microglia in vitro (Rebe S and Solomon B 2005 Am J Alzheimers Dis Other Demen 20:303-313). In addition, chronic treatment with deglycosylated antibody, in comparison to intact antibody, is associated with reduced CD45- immunopositive activated microglia surrounding plaques in an AD model mouse in vivo (Carty NC et al. 2006 J Neuroinflammation 3:11; Wilcock DM et al. 2006 J Neurosci 26:5340-5346). These previous studies examined effects of deglycosylation on microglial activation, but effects on microglial phagocytosis and cytokine generation have not previously been studied. In the present disclosure, we found that deglycosylated antibody did not increase Abeta phagocytosis or cytokine release above the control level. Brain immune responses are not involved in amyloid reduction by Abeta sequestration, and immune inactive deglycosylated antibody therefore is envisioned as representing the basis for a safe and effective therapeutic compound. hi addition to potent pharmacological effects, therapeutic candidates should have favorable pharmacokinetics. Although brain penetration is a major issue for CNS-acting drugs, Abeta sequestering agents act in the periphery. Since antibody needs to be administered by intravenous infusion, a long half-life is desired to minimize the dosing frequency. Intact antibodies typically have a half-life of several weeks, allowing once per

month administration. In this disclosure, we found, preliminarily, that the deglycosylated antibodies have comparable kinetics to intact antibodies. Further study is required, but this result indicates that deglycosylated antibodies are likely to be effective in sequestering Abeta using dosing regimens similar to those of intact antibodies. In Abeta sequestration, there is enhanced Abeta efflux from the brain to the periphery, where the sequestered Abeta forms complexes with the administered agent. Unbound Abeta (not complexed with an Abeta binding agent) is cleared from the body within 10 minutes (Kandimalla KK et al. 2005 J Pharmacol Exp Ther 313:1370-1378). Sequestration agents including anti-Abeta antibodies apparently stabilize Abeta in the blood by prolonging the clearance process. No adverse effects of high plasma Abeta have been reported, but this could be a potential issue with antibody-mediated sequestration. Abeta binding agents that are rapidly cleared after capturing Abeta would be a safe and efficient method for Abeta sequestration. hi this disclosure, we compared two anti-Abeta antibodies, clones 82El and 6E10. Both intact and deglycosylated 82El showed more significant Abeta elevation compared to clone 6E10. Clone 82El is specific for Abeta and not cross-reactive with uncleaved APP (Horikoshi Y et al. 2004 Biochem Biophys Res Commun 319:733-737), while clone 6E10 is fully cross reactive. APP is more abundant than cleaved Abeta, and Abeta-specific (non- APP cross reactive) antibodies are more efficient in Abeta sequestration. Although the active immunization clinical trial was terminated early, follow up studies indicate that patients who received the Abeta immunization showed apparent reduction of Abeta plaque load (Nicoll JA et al. 2003 Nat Med 9:448-452), and subjects who had an immune response to the immunization showed slowing of cognitive decline (Hock C et al. 2003 Neuron 38:547-554). Because most generated antibodies remain in the periphery, presumably the therapeutic benefit was derived at least partially through Abeta sequestration, hi this disclosure, we showed that deglycosylated antibody represents a simple Abeta binding agent that does not induce phagocytosis and cytokine production. Recently, chronic treatment with another deglycosylated anti-Abeta C terminus antibody reduced brain Abeta load and improved cognitive function (Carty NC et al. 2006 J Neuroinflammation 3:11; Wilcock DM et al. 2006 JNeurosci 26:5340-5346) (The antibody was raised against aa33-40 of human Abeta; the antibody binds Abeta terminating at position 40 preferentially over peptides ending at position 42). Microhemorrhage, another critical adverse event associated with active immunization, was significantly reduced.

Taken together, the evidence supports the role envisioned for deglycosylated antibody as being the basis for a potent and safe therapeutic agent for AD.

Example 1

Preparation of deglycosylated antibodies We used two mouse monoclonal anti-Abeta antibodies: clones 82El (Immuno-

Biological Laboratories, Takasaki, Gunma, Japan) (Horikoshi Y et al. 2004 Biochem Biophys Res Commun 319:733-737) and 6E10 (Signet Laboratories, Dedham, MA) (Kim KS et al. 1988 Neurosci Res Commun 2:121-130). The subclass of these antibodies is IgGl and the epitopes are amino acids 1-5 and 3-8 of Abeta, respectively. Preservative-free purified IgG was treated with peptide-N4-(acetyl-beta-glucosaminyl)-asparagine amidase, EC 3.5.1.52 (deglycosylation enzyme, 10 U/100 μg IgG, Prozyme, San Leandro, CA) in phosphate buffered saline (PBS), pH 7.4, for 18 hours at 37°C. Deglycosylation was validated using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Affinity of the intact and deglycosylated antibodies to Abeta was determined using surface plasmon resonance, BIAcore.

Abeta phagocytosis assay using primary cultured microglia

Primary cultured microglia (over 97% pure) were prepared from newborn rats as previously described (Kitamura Y et al. 2001 J Neurosci Res 64:553-563). Human Abeta 1-42 (Anaspec, San Jose, CA), 1 μM, was pre-incubated with the intact or deglycosylated antibody (5 μg IgG/ml) in culture medium for 1 hour prior to the treatment. Subsequently, the culture medium in the well was replaced. Twelve hours after the treatment, cells were subjected to analysis.

For cytochemical examination, cells were rinsed with PBS and fixed with 4% paraformaldehyde in 100 mM PBS for 30 minutes. Cells were incubated with a rabbit polyclonal anti-Abeta antibody. (1 :2,000, Chemicon International, Temecula, CA) in PBS containing 0.3% Triton X-100, and visualized by fluorescein-labeled anti-rabbit IgG antibody (4 μg/ml, Molecular Probes, Eugene, OR). The cells were also incubated with rhodamine-conjugated phalloidin (0.2 μg/ml, Molecular Probes) and.Hoechst 33258 (6 μg/ml, Molecular Probes) which are markers for actin filaments and nuclei, respectively, to visualize the cellular structure. Fluorescence was detected using a laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

Levels of Abeta phagocytosis and cytokine production were determined by ELISAs. Culture medium was collected and snap frozen for cytokine assay. Cells were rinsed with

PBS and then collected in PBS containing 0.1% Triton X-100. Abeta levels in the cell lysate were determined by ELISA using antibodies against the middle region and the C terminus, clones 12B2 and 1C3 (epitope: Abeta 11-28 and 38-42, respectively) as previously described (Horikoshi Y et al. 2004 Biochem Biophys Res Commun 319:733- 737). Levels of tumor necrosis factor-alpha (TNFalpha) were determined using a kit (Biosource International, Camarillo, CA). Plasma Abeta elevation in vivo

Triple transgenic mice expressing mutant amyloid precursor protein (APP), presenilin-1 and tau (Oddo S et al. 2003 Neuron 39:409-421) at 13 ± 1 weeks of age were used (n=5 in each group). Blood was collected from the tail vein, mixed with EDTA, and plasma was prepared after brief centrifugation. Blood was collected 1 day prior to the injection, and used to determine the baseline level of Abeta. Deglycosylated and intact antibodies (50 μg IgG/mouse) were injected intravenously into the tail vein, and blood was collected at 24 and 48 hours after the injection. Plasma Abeta levels were determined using the ELISA as described above.

Determination of antibody levels in the brain and plasma

A 96-well Maxisorp plate (Nunc, Rochester, NY) was coated with 500 ng Abeta/well, and non-specific binding was blocked with Block Ace (Serotec, Oxford, UK). The brain homogenate and plasma were incubated overnight. Known amounts of intact and deglycosylated anti-Abeta antibody (25 pg-25 ng IgG/well), mixed in non-transgenic mouse brain homogenate or plasma, and used to draw the standard curve. The captured anti-Abeta antibody was detected by HRP-coupled anti-mouse IgG, and visualized using TMB as a substrate (Pierce, Rockford, IL). Statistical analysis Statistical significance of differences was determined by analysis of variance

(ANOVA) followed by Bonferroni/Dunn post-hoc tests (StatView, Abacus Concepts,

Berkeley, CA).

***

While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. AU figures, tables, and appendices, as well as patents, applications, and publications, referred to above, are hereby incorporated by reference.

Appendix 1

82El

VH (SEQ ID NO: 1) EVKLVESGGGSVKPGGSLKVSCAASGFIFSNYGMSWVRQTPEKSLEWVASISRGGSTF

YSDRVKGRFTISRENGRNILYLQMNSLRSEDTAIYYCVRYDYDEGATDYWGQGTTLT

VSS

FRL(SEQ ID NO: 2) EVKLVESGGGSVKPGGSLKVSCAASGFIFS

CDRl (SEO ID NO: 3)

NYGMS FR2_(SEQ ID NO: 4)

WVRQTPEKSLEWVA

CDR2 (SEO ID NO: 5)

SISRGGSTFYSDRVKG

FR3_(SEQ ID NO: 6) RFTISRENGRNILYLQMNSLRSEDTAIYYCVR

CDR3 (SEO ID NO: 7) YDYDEGATDY

FR4 (SEO ID NO: 8)

WGQGTTLTVSS YL(SEQ ID NO: 9)

DWMTQTPLNLSVTIGQPASISCKSSQSLLDRDGKTYLNWLFQRPGQSPKRL IYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFG GGTKLEIK FRL(SEQ ID NO: 10)

DWMTQTPLNLSVTIGQPASISC

CDRl (SEO ID NO: 11)

KSSQSLLDRDGKTYLN

FR2 (SEO ID NO: 12)

^• WLFQRPGQSPKRLIY

CDR2 (SEO ID NO: 13) LVSKLDS

FR3_(SEQ ID NO: 14) GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC

CDR3 (SEO ID NO: 15)

WQGTHFPR

FRl(SEQ ID NO: 16) TFGGGTKLEIK