+

WO2008030251A1 - Anticorps deglycosylés anti-bêta amyloïde - Google Patents

Anticorps deglycosylés anti-bêta amyloïde Download PDF

Info

Publication number
WO2008030251A1
WO2008030251A1 PCT/US2006/040101 US2006040101W WO2008030251A1 WO 2008030251 A1 WO2008030251 A1 WO 2008030251A1 US 2006040101 W US2006040101 W US 2006040101W WO 2008030251 A1 WO2008030251 A1 WO 2008030251A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
abeta
antibody
antibodies
deglycosylated
Prior art date
Application number
PCT/US2006/040101
Other languages
English (en)
Inventor
Yasuji Matsuoka
Original Assignee
Georgetown University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Georgetown University filed Critical Georgetown University
Publication of WO2008030251A1 publication Critical patent/WO2008030251A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the invention relates to deglycosylated monoclonal antibodies to amyloid beta peptide.
  • AD Alzheimer's disease
  • Abeta Alzheimer's disease
  • AD Alzheimer's disease
  • Selkoe DJ 2002 Science 297:353-356 Active immunization with Abeta peptides reduced brain Abeta and improved cognitive performance in an AD mouse model
  • 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).
  • FcR Fc receptors
  • 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).
  • 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.
  • cytokine release in response to phagocytosis may occur with a passive immunization approach.
  • 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.
  • 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.
  • FIG. 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.
  • APP amyloid precursor protein
  • Figure 2 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.
  • FIG. 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).
  • 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 ⁇ m in Aa for Aa, 10 ⁇ m in Ab for Ab-Ad.
  • FIG. 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).
  • Abeta amyloid beta
  • 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.
  • Amyloid Precursor Protein and Proteolytic Fragments ⁇ -Amyloid protein is derived from amyloid precursor protein by sequential cleavages by proteases referred to as ⁇ -secretase and ⁇ -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.
  • 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.
  • amyloid precursor protein 751 that is in human platelets has been shown to inhibit factor XIa (a serine protease) in the clotting cascade.
  • amyloid precursor protein APP
  • 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 ⁇ -amyloid protein (A ⁇ ) 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 ⁇ -amyloid protein and TM regions is expanded.
  • the underlined residues represent the ⁇ - 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).
  • the first arrow indicates the site (after residue 687) of a cleavage by ⁇ -secretase that enables secretion of the large, soluble ectodomain of amyloid precursor protein (APP s - ⁇ ) 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 ⁇ -secretase at residue 711 or residue 713 to release the p3 peptides.
  • the fourth line depicts the alternative proteolytic cleavage after residue 671 by ⁇ -secretase that causes the secretion of the slightly truncated APP s - ⁇ molecule and the retention of a 99 residue C-terminal fragment (C99).
  • the C99 fragment can also undergo cleavage by ⁇ -secretase to release the ⁇ amyloid peptides.
  • Cleavage of both C83 and C99 by ⁇ -secretase releases the ⁇ amyloid precursor protein intracellular domain (AICD) into the cytoplasm.
  • AICD ⁇ amyloid precursor protein intracellular domain
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • An isolated nucleic acid may be substantially purified, but need not be.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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., ⁇ - 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.
  • the present invention derives, in part, from the isolation and characterization of monoclonal antibodies that selectively bind to Abeta.
  • 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.
  • the present invention provides a substantially full- length, anti-Abeta monoclonal antibody in isolated form and in pharmaceutical preparations.
  • 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.
  • 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.
  • an antibody molecule 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 are effectors of the complement cascade but are not involved in antigen binding.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment
  • 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.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDRl through CDR3 complementarity determining regions
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • chimeric antibodies including non-human sequences.
  • Some of the CDRs may be replaced as well.
  • 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.
  • chimeric antibodies bearing non-human mammalian Fc and FR sequences but incl ⁇ ding 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a preferred origin of replication is CoIEI found in pBR322 and a variety of other common plasmids.
  • 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.
  • 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).
  • 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 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.
  • 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.
  • eukaryotic cells such as CHO cells, mouse hybridomas, immortalized B-lymphoblastoid cells, and the like.
  • 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.
  • the present invention provides host cells, both prokaryotic and eukaryotic, transformed or transfected with, and therefore including, the vectors of the present invention.
  • 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 means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • 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.
  • conservative variation 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.
  • a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies having the substituted polypeptide also bind amyloid beta peptide.
  • 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.
  • kits 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.
  • container means such as vials, tubes, and the like
  • 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.
  • 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.
  • 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 memeposes 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.
  • 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.
  • the detectably labeled monoclonal antibody is given in a dose which is diagnostically effective.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetra-acetic acid
  • 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).
  • MRI magnetic resonance imaging
  • ESR electron spin resonance
  • any conventional method for visualizing diagnostic imaging can be utilized.
  • 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.
  • the monoclonal antibodies of the invention can be used in vitro and in vivo to monitor the course of therapies for Alzheimer's disease.
  • the monoclonal antibodies can also be used in prophylaxis and as therapy for Alzheimer's disease.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • MALDI-TOF matrix-assisted laser desorption/Ionization time-of-flight
  • 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
  • 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).
  • 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 form complexes that may interfere with Abeta ELISA quantification.
  • 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 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
  • Fig. 5 Deglycosylated and intact antibodies (50 ⁇ g 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
  • 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).
  • the intact and deglycosylated antibodies showed comparable short-term kinetics in plasma (Table 3).
  • 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).
  • 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.
  • 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).
  • 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
  • Nose M and Wigzell H 1983 Proc Natl Acad Sd USA 80:6632-6636 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.
  • 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.
  • therapeutic candidates should have favorable pharmacokinetics.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • deglycosylated antibody represents a simple Abeta binding agent that does not induce phagocytosis and cytokine production.
  • 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.
  • Preservative-free purified IgG was treated with peptide-N4-(acetyl-beta-glucosaminyl)-asparagine amidase, EC 3.5.1.52 (deglycosylation enzyme, 10 U/100 ⁇ g 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.
  • the cells were also incubated with rhodamine-conjugated phalloidin (0.2 ⁇ g/ml, Molecular Probes) and.Hoechst 33258 (6 ⁇ g/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).
  • 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
  • VH (SEQ ID NO: 1) EVKLVESGGGSVKPGGSLKVSCAASGFIFSNYGMSWVRQTPEKSLEWVASISRGGSTF

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Neurosurgery (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Hospice & Palliative Care (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Psychiatry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des anticorps monoclonaux déglycosylés qui se lient à la bêta-amyloïde (Abeta). L'invention concerne de tels anticorps et des compositions pharmaceutiques qui comprennent de tels anticorps. L'invention concerne en outre des acides nucléiques isolés qui codent pour les anticorps de l'invention ainsi que les cellules hôtes transformées avec ceux-ci. De plus, l'invention concerne des procédés prophylactiques, thérapeutiques et diagnostiques qui utilisent les anticorps dé-glycosylés de l'invention.
PCT/US2006/040101 2006-09-08 2006-10-13 Anticorps deglycosylés anti-bêta amyloïde WO2008030251A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US84346006P 2006-09-08 2006-09-08
US60/843,460 2006-09-08

Publications (1)

Publication Number Publication Date
WO2008030251A1 true WO2008030251A1 (fr) 2008-03-13

Family

ID=37912518

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/040101 WO2008030251A1 (fr) 2006-09-08 2006-10-13 Anticorps deglycosylés anti-bêta amyloïde

Country Status (1)

Country Link
WO (1) WO2008030251A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8497072B2 (en) 2005-11-30 2013-07-30 Abbott Laboratories Amyloid-beta globulomer antibodies
KR20130136968A (ko) * 2010-07-30 2013-12-13 에이씨 이뮨 에스.에이. 안전하고 기능적인 인간화 항 베타-아밀로이드 항체
US8691224B2 (en) 2005-11-30 2014-04-08 Abbvie Inc. Anti-Aβ globulomer 5F7 antibodies
US8877190B2 (en) 2006-11-30 2014-11-04 Abbvie Inc. Aβ conformer selective anti-Aβ globulomer monoclonal antibodies
US8895004B2 (en) 2007-02-27 2014-11-25 AbbVie Deutschland GmbH & Co. KG Method for the treatment of amyloidoses
US8987419B2 (en) 2010-04-15 2015-03-24 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US9062101B2 (en) 2010-08-14 2015-06-23 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US9146244B2 (en) 2007-06-12 2015-09-29 Ac Immune S.A. Polynucleotides encoding an anti-beta-amyloid monoclonal antibody
US9176150B2 (en) 2003-01-31 2015-11-03 AbbVie Deutschland GmbH & Co. KG Amyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof
US9175094B2 (en) 2007-06-12 2015-11-03 Ac Immune S.A. Monoclonal antibody
US9403902B2 (en) 2007-10-05 2016-08-02 Ac Immune S.A. Methods of treating ocular disease associated with amyloid-beta-related pathology using an anti-amyloid-beta antibody
US11434284B2 (en) 2020-07-23 2022-09-06 Othair Prothena Limited Anti-Abeta antibodies

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003086310A2 (fr) * 2002-04-12 2003-10-23 Ramot At Tel Aviv University Ltd. Prevention de l'inflammation du cerveau en tant que resultat d'une reponse auto-immune induite
WO2006036291A2 (fr) * 2004-07-30 2006-04-06 Rinat Neuroscience Corp. Anticorps anti peptide amyloide beta, et leurs procedes d'utilisation
WO2006066171A1 (fr) * 2004-12-15 2006-06-22 Neuralab Limited Anticorps amyloide ???? utilises afin d'ameliorer la cognition

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003086310A2 (fr) * 2002-04-12 2003-10-23 Ramot At Tel Aviv University Ltd. Prevention de l'inflammation du cerveau en tant que resultat d'une reponse auto-immune induite
WO2006036291A2 (fr) * 2004-07-30 2006-04-06 Rinat Neuroscience Corp. Anticorps anti peptide amyloide beta, et leurs procedes d'utilisation
WO2006066171A1 (fr) * 2004-12-15 2006-06-22 Neuralab Limited Anticorps amyloide ???? utilises afin d'ameliorer la cognition

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
D. WILCOCK ET AL.: "Deglycosylated anti-amyloid beta antibodies eliminate cognitive deficits and reduce prenchymal amyloid with minimal vascular consequences in aged amyloid precursor protein transgenic mice.", THE JOURNAL OF NEUROSCIENCE, vol. 26, no. 20, 17 May 2006 (2006-05-17), U.S.A., pages 5340 - 5346, XP002430460 *
N. CARTY ET AL.: "Intracranial administration of deglycosylated C-terminal-specific anti-Abeta antibody efficiently clears amyloid plaques without activating microglia in amyloid-depositing transgenic mice.", JOURNAL OF NEUROINFLAMMATION, vol. 3, no. 1, 10 May 2006 (2006-05-10), London, GB, pages 1 - 11, XP021019066 *
S. REBE & B. SOLOMON: "Deglycosylation of anti-beta amyloid antibodies inhibits microglia activation in BV-2 cellular model.", AMERICAN JOURNAL OF ALZHEIMER'S DISEASE AND OTHER DEMENTIAS, vol. 20, no. 5, September 2005 (2005-09-01), U.S.A., pages 303 - 313, XP008078061 *
Y. HORIKOSHI ET AL.: "Abeta N-terminal-end specific antibody reduced beta-amyloid in Alzheimer-model mice.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 325, no. 2, 10 December 2004 (2004-12-10), Orlando, FL, U.S.A., pages 384 - 387, XP004626804 *
Y. HORIKOSHI ET AL.: "Development of Abeta terminal end-specific antibodies and sensitive ELISA for Abeta variant.", BIOCHEMICAL AND BIOPHYSICAL REASEARCH COMMUNICATIONS, vol. 319, no. 3, 2 July 2004 (2004-07-02), Orlando, FL, USA, pages 733 - 737, XP002430459 *

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9176150B2 (en) 2003-01-31 2015-11-03 AbbVie Deutschland GmbH & Co. KG Amyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof
US10464976B2 (en) 2003-01-31 2019-11-05 AbbVie Deutschland GmbH & Co. KG Amyloid β(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof
US10323084B2 (en) 2005-11-30 2019-06-18 Abbvie Inc. Monoclonal antibodies against amyloid beta protein and uses thereof
US10538581B2 (en) 2005-11-30 2020-01-21 Abbvie Inc. Anti-Aβ globulomer 4D10 antibodies
US8497072B2 (en) 2005-11-30 2013-07-30 Abbott Laboratories Amyloid-beta globulomer antibodies
US8691224B2 (en) 2005-11-30 2014-04-08 Abbvie Inc. Anti-Aβ globulomer 5F7 antibodies
US9540432B2 (en) 2005-11-30 2017-01-10 AbbVie Deutschland GmbH & Co. KG Anti-Aβ globulomer 7C6 antibodies
US10208109B2 (en) 2005-11-30 2019-02-19 Abbvie Inc. Monoclonal antibodies against amyloid beta protein and uses thereof
US8877190B2 (en) 2006-11-30 2014-11-04 Abbvie Inc. Aβ conformer selective anti-Aβ globulomer monoclonal antibodies
US9951125B2 (en) 2006-11-30 2018-04-24 Abbvie Inc. Aβ conformer selective anti-Aβ globulomer monoclonal antibodies
US9359430B2 (en) 2006-11-30 2016-06-07 Abbvie Inc. Abeta conformer selective anti-Abeta globulomer monoclonal antibodies
US9394360B2 (en) 2006-11-30 2016-07-19 Abbvie Inc. Aβ conformer selective anti-Aβ globulomer monoclonal antibodies
US8895004B2 (en) 2007-02-27 2014-11-25 AbbVie Deutschland GmbH & Co. KG Method for the treatment of amyloidoses
US9175094B2 (en) 2007-06-12 2015-11-03 Ac Immune S.A. Monoclonal antibody
US9146244B2 (en) 2007-06-12 2015-09-29 Ac Immune S.A. Polynucleotides encoding an anti-beta-amyloid monoclonal antibody
US9585956B2 (en) 2007-06-12 2017-03-07 Ac Immune S.A. Polynucleotides encoding anti-amyloid beta monoclonal antibodies
US9403902B2 (en) 2007-10-05 2016-08-02 Ac Immune S.A. Methods of treating ocular disease associated with amyloid-beta-related pathology using an anti-amyloid-beta antibody
US8987419B2 (en) 2010-04-15 2015-03-24 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US9822171B2 (en) 2010-04-15 2017-11-21 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
KR101713365B1 (ko) 2010-07-30 2017-03-08 에이씨 이뮨 에스.에이. 안전하고 기능적인 인간화 항 베타-아밀로이드 항체
KR20130136968A (ko) * 2010-07-30 2013-12-13 에이씨 이뮨 에스.에이. 안전하고 기능적인 인간화 항 베타-아밀로이드 항체
US9221900B2 (en) 2010-07-30 2015-12-29 Ac Immune S.A. Methods for identifying safe and functional humanized antibodies
US10047121B2 (en) 2010-08-14 2018-08-14 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US9062101B2 (en) 2010-08-14 2015-06-23 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US11434284B2 (en) 2020-07-23 2022-09-06 Othair Prothena Limited Anti-Abeta antibodies
US11434283B2 (en) 2020-07-23 2022-09-06 Othair Prothena Limited Anti-abeta antibodies
US11434285B2 (en) 2020-07-23 2022-09-06 Othair Prothena Limited Anti-Abeta antibodies
US11440953B2 (en) 2020-07-23 2022-09-13 Othair Prothena Limited Anti-abeta antibodies

Similar Documents

Publication Publication Date Title
WO2008030251A1 (fr) Anticorps deglycosylés anti-bêta amyloïde
US10208111B2 (en) Alpha-synuclein antibodies and uses thereof
EP2074145B1 (fr) Anticorps humanise contre amyloide-beta
EP2046833B9 (fr) Anticorps humanisé contre l'amyloid beta
EP2764022B9 (fr) Anticorps phosphospécifiques reconnaissant tau
EP2625198B1 (fr) Anticorps dirigés contre tau phosphorylée
CN102936287B (zh) 能够特异性结合Aβ寡聚体的抗体及其应用
CN103380145B (zh) 人类抗-sod1抗体
DK2593475T3 (en) Anti-ADDL monoclonal antibody and uses thereof
KR102781727B1 (ko) 항-알파 시누클레인 항체
EP2343318A2 (fr) Anticorps se liant à un fragment C-terminal de l'apolipoprotein E
KR20080090408A (ko) 항-Ａβ 글로불로머 항체, 이의 항원-결합 잔기, 상응하는하이브리도마, 핵산, 벡터, 숙주 세포, 당해 항체의 제조방법, 당해 항체를 포함하는 조성물, 당해 항체의 용도 및당해 항체의 사용 방법
EP3502248B1 (fr) Anticorps dirigés contre hmgb1 et composition les comprenant pour le traitement ou la prévention de la maladie d'alzheimer
SG185316A1 (en) ANTIBODY CAPABLE OF SPECIFICALLY BINDING TO Aβ OLIGOMER, AND USE THEREOF
TR201812636T4 (tr) İnsan anti-tau antikorları.
WO2011045945A1 (fr) ANTICORPS RECONNAISSANT UNE STRUCTURE EN SPIRE D'AMYLOÏDE β
EP3309172B1 (fr) Anticorps humanisés contre le peptide amyloïde-bêta
EP3239295A1 (fr) Anticorps reconnaissant de façon très spécifique une structure en coude aux positions 22-23 dans l'amyloïde bêta
JP2022502369A (ja) α−シヌクレインの立体配座特異的エピトープ、それに対する抗体、およびそれに関連する方法
EP3988566A1 (fr) Anticorps humanisés contre le peptide amyloïde-bêta
EA039569B1 (ru) Антитела, специфичные к гиперфосфорилированному тау-белку, и способы их применения
AU2013202799A1 (en) Humanized antibody against amyloid beta

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 06825922

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06825922

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载