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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
  • A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
  • A61K51/1018—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody 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
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

• This invention relates to compositions for treating central nervous system (CNS) disorders such as Alzheimer's disease (AD), and more particularly, to compositions that contain a ⁇ amyloid (A ⁇ ) polypeptide linked to a non-A ⁇ polypeptide.
• CNS central nervous system
• a ⁇ ⁇ amyloid
• the invention is based on the discovery that AP-immune complexes are transported across the BBB via a receptor-mediated process at a rate greater than that of antibody alone.
• transport of antibodies having specific binding affinity for A ⁇ across the BBB, or other polypeptides that have low permeability at the BBB can be enhanced when linked to an A ⁇ polypeptide.
• Polyamine modified antibodies having specific binding affinity for A ⁇ also have increased permeability at the BBB and can be used for passive immunization and treatment of AD.
• the invention features a composition that includes an A ⁇ polypeptide and a non-A ⁇ polypeptide, wherein the A ⁇ polypeptide and the non-A ⁇ polypeptide are linked (e.g., covalently).
• the composition further can include a pharmaceutically acceptable carrier or excipient.
• the non-A ⁇ polypeptide can be an antibody or a fragment thereof (e.g., a Fab fragment, a single chain Fv antibody fragment, or a F(ab) 2 fragment).
• the antibody can be labeled with a radioisotope or a contrast agent.
• the antibody can have specific binding affinity for amyloid.
• the non-A ⁇ polypeptide also can be an enzyme such as an antioxidant enzyme (e.g., catalase or superoxide dismutase), a cytokine such as an interferon, an interleukin, or a neurotrophic factor, or leptin.
• an antioxidant enzyme e.g., catalase or superoxide dismutase
• a cytokine such as an interferon, an interleukin, or a neurotrophic factor, or leptin.
• the A ⁇ polypeptide can include residues 1-40, 1-42, or 1-43 of SEQ ID NO:1.
• the invention also features a method of treating a patient diagnosed with AD.
• the method includes administering to the patient an amount of a composition effective to treat AD, wherein the composition includes an A ⁇ polypeptide and an antibody having specific binding affinity for the A ⁇ polypeptide.
• the antibody can be a Fab fragment, a single chain Fv antibody fragment, or a F(ab) 2 fragment.
• the invention features a method of treating a patient diagnosed with AD.
• the method includes administering to the patient an amount of an antibody effective to treat AD, wherein the antibody is polyamine modified and has specific binding affinity for an AP polypeptide.
• the invention features a method of diagnosing AD in a patient.
• the method includes administering a composition to the patient, wherein the composition includes an A ⁇ polypeptide and an antibody having specific binding affinity for amyloid, wherein the antibody is labeled, and detecting the presence or absence of the antibody bound to amyloid in the brain of the patient, wherein the patient is diagnosed with AD based on the presence of labeled amyloid (e.g., labeled amyloid deposits such as ⁇ -amyloid plaques).
• labeled amyloid e.g., labeled amyloid deposits such as ⁇ -amyloid plaques
• the detecting step can include diagnostic imaging (e.g., positron emission tomography, gamma-scintigraphy, single photon emission computerized tomography, magnetic resonance imaging, functional magnetic resonance imaging, or magnetoencephalography). Magnetic resonance imaging is particularly useful.
• diagnostic imaging e.g., positron emission tomography, gamma-scintigraphy, single photon emission computerized tomography, magnetic resonance imaging, functional magnetic resonance imaging, or magnetoencephalography.
• Magnetic resonance imaging is particularly useful.
• the antibody can be labeled with a contrast agent (e.g., gadolinium, dysprosium, or iron). Gadolinium is a particularly useful contrast agent.
• the invention features compositions containing A ⁇ polypeptides that can be used to enhance transport of non-A ⁇ polypeptides across the BBB.
• BBB permeability of a composition containing A ⁇ bound to a monoclonal antibody was significantly greater than that of the monoclonal antibody alone. Without being bound by a particular mechanism, A ⁇ itself may be responsible for transporting the antibody across the BBB.
• compositions of the invention can be used in the diagnosis, treatment, and/or prevention of neurodegenerative disorders such as AD, Parkinson's disease, frontotemporal dementias (e.g., Pick's disease), and amyloidotic polyneuropathies, transmissible spongiform encephalopathies (i.e., prion diseases) such as Creutzfeldt-Jakob disease (CJD), Gerstmann-St Hurssler-Scheinker syndrome, and fatal familial insomnia, demyelinating diseases such as multiple sclerosis, and amyotropic lateral sclerosis.
• neurodegenerative disorders such as AD, Parkinson's disease, frontotemporal dementias (e.g., Pick's disease), and amyloidotic polyneuropathies, transmissible spongiform encephalopathies (i.e., prion diseases) such as Creutzfeldt-Jakob disease (CJD), Gerstmann-St syndromesler-Scheinker syndrome, and fatal familial insomnia,
• compositions of the invention include a purified A ⁇ polypeptide linked to a purified non-A ⁇ polypeptide.
• purified refers to a polypeptide that is separated from cellular components (e.g., other polypeptides, lipids, carbohydrates, and nucleic acids) that are naturally associated with the polypeptide.
• a purified polypeptide is any polypeptide that is removed from its natural environment and is at least 75% pure (e.g., at least about 80, 85, 90, 95, or 99% pure).
• a purified polypeptide will yield a single major band on a non-reducing polyacrylamide gel.
• a ⁇ polypeptide refers to 1) the naturally occurring human A ⁇ polypeptide (DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IAT, SEQ ID NO: 1) 2) polypeptides having one or more substitutions or insertions in the amino acid sequence of the naturally occurring human A ⁇ polypeptide that retain the ability to cross the BBB, and 3) fragments of 1) and 2) that retain the ability to cross the BBB. Permeability of an A ⁇ polypeptide at the BBB can be assessed according to the methods of Example 1. See also Poduslo et al., Proc. Natl. Acad.
• human A ⁇ polypeptide ranges in length from 39 to 43 amino acids (residues 1 to 39, 1 to 40, 1 to 41, 1 to 42, or 1 to 43 of SEQ ID NO:1), and is a proteolytic cleavage product of the amyloid precursor protein (APP).
• amino acid substitutions that can be introduced into human A ⁇ include substitutions at amino acid residues 5, 10, 13, 19, and 20 of SEQ ID NO:1, or combinations thereof.
• a glycine can be substituted for the arginine at residue 5
• a phenylalanine can be substituted for the tyrosine at residue 10
• an arginine can be substituted for the histidine at residue 13.
• substitutions do not alter the properties of human A ⁇ polypeptide. See Fraser et al., Biochemistry 31:10716-10723 (1992); and Hilbich et al., Eur. J. Biochem. 201:61-69 (1992).
• An isoleucine, leucine, threonine, serine, alanine, valine, or glycine can be substituted for the phenylalanine residues at positions 19 and 20.
• Suitable fragments of A ⁇ polypeptides are about 6 to 38 amino acid residues in length (e.g., 10 to 36, 10 to 34, 10 to 30, 12 to 28, 14 to 26, 16 to 24, or 18 to 22 amino acid residues in length) and retain the ability to cross the BBB.
• an A ⁇ polypeptide may contain residues 1 to 10, 1 to 15, 1 to 20, 5 to 15, 5 to 20, 5 to 25, 10 to 20, 10 to 25, 10 to 30, 15 to 25, 15 to 30, or 15 to 35 of SEQ ID NO:1.
• an A ⁇ polypeptide may include residues 20 to 30, 20 to 35, 20 to 40, 25 to 35, 25 to 40, 30 to 40, 25 to 42, or 30 to 42 of SEQ ID NO:1.
• a ⁇ polypeptides can be linked to non-A ⁇ polypeptides via covalent links.
• Covalent cross-linking techniques are known in the art. See, for example, “Chemistry of Protein Conjugation and Cross-Linking”, Shan S. Wong, CRC Press, Ann Arbor, 1991. Suitable cross-linking reagents do not interfere with the binding of the A ⁇ polypeptide to its cognate receptor and are chosen for appropriate reactivity, specificity, spacer arm length, membrane permeability, cleavability, and solubility characteristics. Similarly, suitable cross-linking reagents do not interfere with binding of a non-A ⁇ polypeptide to its binding partner (e.g., cognate receptor or epitope on a macromolecule). Cross-linking reagents are available commercially from many sources including Pierce Chemical Co., Rockford, Ill.
• An A ⁇ polypeptide and a non-A ⁇ polypeptide can be covalently cross-linked using, for example, glutaraldehyde, a homobifunctional cross-linker, or a heterobifunctional cross-linker.
• Glutaraldehyde cross-links polypeptides via their amino moieties.
• Homobifunctional cross-linkers e.g., a homobifunctional imidoester, a homobifunctional N-hydroxysuccinimidyl (NHS) ester, or a homobifunctional sulfhydryl reactive cross-linker
• a homobifunctional imidoester e.g., a homobifunctional N-hydroxysuccinimidyl (NHS) ester, or a homobifunctional sulfhydryl reactive cross-linker
• Homobifunctional NHS esters and imido esters cross-link amine containing polypeptides. In a mild alkaline pH, imido esters react only with primary amines to form imidoamides, and overall charge of the cross-linked polypeptides is not affected.
• Homobifunctional sulfhydryl reactive cross-linkers include bismaleimidhexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-di-(3′,2′-pyridyldithio) propionamido butane (DPDPB).
• BMH bismaleimidhexane
• DFDNB 1,5-difluoro-2,4-dinitrobenzene
• DPDPB 1,4-di-(3′,2′-pyridyldithio) propionamido butane
• Heterobifunctional cross-linkers have two or more different reactive moieties (e.g., an amine reactive moiety and a sulfhydryl-reactive moiety) and are cross-linked with one of the polypeptides via the amine or sulfhydryl reactive moiety, then reacted with the other polypeptide via the non-reacted moiety.
• Multiple heterobifunctional haloacetyl cross-linkers are available, as are pyridyl disulfide cross-linkers.
• Carbodiimides are a classic example of heterobifunctional cross-linking reagents for coupling carboxyls to amines, which results in an amide bond.
• an A ⁇ polypeptide can be linked to a non-A ⁇ polypeptide such as an antibody via the specific binding affinity of the antibody for the A ⁇ polypeptide.
• Purified A ⁇ polypeptide and antibody can be incubated together at 37° C. in an appropriate buffer (e.g., phosphate buffered saline) to form an immune complex.
• an appropriate buffer e.g., phosphate buffered saline
• Such an immune complex constitutes a composition of the invention.
• a ⁇ polypeptides can be linked to any non-A ⁇ polypeptide, and in particular, to any polypeptide that is useful for diagnosing or treating a disorder of the CNS.
• Non-A ⁇ polypeptides are at least six amino acid residues in length.
• an A ⁇ polypeptide can be linked to an enzyme such as an antioxidant enzyme, which can protect cells against reactive oxygen species.
• antioxidant enzymes include catalase (E.C. 1.11.1.6), superoxide dismutase (E.C. 1.15.1.1), glutathione peroxidase (E.C. 1.6.4.2), and glutathione reductase (E.C. 1.11.1.9).
• a ⁇ polypeptides also can be linked to cytokines such as an interferon (e.g., interferon ⁇ , ⁇ , or ⁇ ), interleukin (IL) (e.g., IL-1a or b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, or IL-12), neurotrophic factors such as neurotrophins (e.g., nerve growth factor or brain-derived neurotrophic factor), neuropoietic factors such as cholinergic differentiation factor, ciliary neurotrophic factor, oncostatin M, growth-promoting factor, and sweat gland factor, and growth factor peptides such as glial-cell line-derived neurotrophic factor, or a hormone such as leptin.
• IL interleukin
• neurotrophic factors such as neurotrophins (e.g., nerve growth factor or brain-derived neurotrophic factor), neuropoietic factors such as cho
• a ⁇ polypeptides can be linked to an antibody.
• an A ⁇ polypeptide can be linked to an antibody having specific binding affinity for amyloid deposits of A ⁇ or of a prion protein (PrP).
• PrP prion protein
• antibodies includes polyclonal or monoclonal antibodies, humanized or chimeric antibodies, and antibody fragments such as single chain Fv antibody fragments, Fab fragments, and F(ab) 2 fragments. Monoclonal antibodies are particularly useful.
• a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Chimeric antibodies can be produced through standard techniques.
• Antibody fragments can be generated by known techniques. For example, F(ab′) 2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of F(ab′) 2 fragments. Alternatively, Fab expression libraries can be constructed. See, for example, Huse et al., Science, 246:1275 (1989). Single chain Fv antibody fragments are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge (e.g., 15 to 18 amino acids), resulting in a single chain polypeptide. See, for example, U.S. Pat. No. 4,946,778.
• amino acid bridge e.g. 15 to 18 amino acids
• the A ⁇ polypeptide and/or the non-A ⁇ polypeptide are labeled to facilitate diagnosis of a CNS disorder.
• Typical labels that are useful include radioisotopes and contrast agents used for imaging procedures in humans.
• Non-limiting examples of labels include radioisotope such as 123 I(iodine), 18 F (fluorine), 99m Tc (technetium), 111 In (indium), and 67 Ga (gallium), and contrast agents such as gadolinium (Gd), dysprosium, and iron.
• Radioactive Gd isotopes ( 153 Gd) also are available and suitable for imaging procedures in non-human mammals.
• Polypeptides can be labeled through standard techniques.
• polypeptides can be iodinated using chloramine T or 1,3,4,6-tetrachloro-3 ⁇ ,6 ⁇ -diphenylglycouril.
• fluorination polypeptides are synthesized and fluorine is added during the synthesis by a fluoride ion displacement reaction. See, Muller-Gartner, H., TIB Tech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst., 16(2):209-244 (1999) for a review of synthesis of proteins with such radioisotopes.
• Polypeptides also can be labeled with a contrast agent through standard techniques.
• polypeptides can be labeled with Gd by conjugating low molecular Gd chelates such as Gd diethylene triamine pentaacetic acid (GdDTPA) or Gd tetraazacyclododecanetetraacetic (GdDOTA) to the polypeptide.
• GdDTPA Gd diethylene triamine pentaacetic acid
• GdDOTA Gd tetraazacyclododecanetetraacetic
• Antibodies can be labeled with Gd by, for example, conjugating polylysine-Gd chelates to the antibody.
• antibodies can be labeled with Gd by incubating paramagnetic polymerized liposomes that include Gd chelator lipid with avidin and biotinylated antibody. See, for example, Sipkins et al. Nature Med., 4 623-626 (1998).
• Isolated nucleic acid molecules encoding A ⁇ and non-A ⁇ polypeptides of the invention can be produced by standard techniques.
• isolated refers to a sequence corresponding to part or all of a gene encoding an A ⁇ or non-A ⁇ polypeptide, but free of sequences that normally flank one or both sides of the wild-type gene in a mammalian genome.
• An isolated nucleic acid can be, for example, a recombinant DNA molecule, provided one or both of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent.
• isolated nucleic acids include, without limitation, a DNA that exists as a separate molecule (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote.
• an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid.
• a nucleic acid existing among hundreds to millions of other nucleic acids within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.
• Isolated nucleic acid molecules are at least about 18 nucleotides in length.
• the nucleic acid molecule can be about 18 to 20, 20-50, 50-100, or greater than 150 nucleotides in length.
• Nucleic acid molecules can be DNA or RNA, linear or circular, and in sense or antisense orientation.
• Specific point changes can be introduced into the nucleic acid sequence encoding the naturally-occurring human A ⁇ polypeptide by, for example, oligonucleotide-directed mutagenesis.
• a desired change is incorporated into an oligonucleotide, which then is hybridized to the wild-type nucleic acid.
• the oligonucleotide is extended with a DNA polymerase, creating a heteroduplex that contains a mismatch at the introduced point change, and a single-stranded nick at the 5′ end, which is sealed by a DNA ligase. The mismatch is repaired upon transformation of E.
• kits for introducing site-directed mutations can be purchased commercially.
• Muta-Gene® in-vitro mutagenesis kits can be purchased from Bio-Rad Laboratories, Inc. (Hercules, Calif.).
• PCR Polymerase chain reaction
• PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA.
• Primers are typically 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length.
• General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, Ed. by Dieffenbach, C. and Dveksler, G., Cold Spring Harbor Laboratory Press, 1995.
• Nucleic acids encoding A ⁇ and non-A ⁇ polypeptides also can be produced by chemical synthesis, either as a single nucleic acid molecule or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector.
• oligonucleotides e.g., >100 nucleotides
• DNA polymerase is used to extend the oligonucleotides, resulting in a double-
• Purified A ⁇ and non-A ⁇ polypeptides of the invention can be obtained from commercial sources, or alternatively, can be obtained by extraction from a natural source (e.g., liver tissue), chemical synthesis, or by recombinant production in a host cell.
• recombinant polypeptides are produced by introducing an expression vector that contains a nucleic acid encoding the polypeptide of interest operably linked to regulatory elements necessary for expression of the polypeptide into a bacterial or eukaryotic host cell (e.g., insect, yeast, or mammalian cells). Regulatory elements do not typically encode a gene product, but instead affect the expression of the nucleic acid sequence.
• a strain of Escherichia coli such as BL-21 can be used.
• Suitable E. coli vectors include the pGEX series of vectors that produce fusion proteins with glutathione S-transferase (GST). Transformed E. coli are typically grown exponentially then stimulated with isopropylthiogalactopyranoside (IPTG) prior to harvesting. Such fusion proteins typically are soluble and can be purified easily from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
• the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
• a nucleic acid encoding a polypeptide of the invention can be cloned into, for example, a baculoviral vector such as pBlueBac (Invitrogen, San Diego, Calif.) and then used to co-transfect insect cells such as Spodoptera frugiperda (Sf9) cells with wild type DNA from Autographa californica multinuclear polyhedrosis virus (AcMNPV). Recombinant viruses producing polypeptides of the invention can be identified by standard methodology.
• a nucleic acid encoding a polypeptide of the invention can be introduced into a SV40, retroviral, or vaccinia based viral vector and used to infect suitable host cells.
• Mammalian cell lines that stably express a polypeptide of interest can be produced using an expression vector that contains a selectable marker and standard techniques.
• the eukaryotic expression vector pCR3.1 (Invitrogen, San Diego, Calif.) can be used to express polypeptides of interest in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC).
• a nucleic acid encoding the polypeptide of interest can be ligated into a mammalian expression vector such as pcDNA3 (Invitrogen, San Diego, Calif.) then transcribed and translated in vitro using wheat germ extract or rabbit reticulocyte lysate.
• pcDNA3 Invitrogen, San Diego, Calif.
• Polypeptides of interest can be purified by known chromatographic methods including DEAE ion exchange, gel filtration, and hydroxylapatite chromatography
• Polypeptides can be “engineered” to contain an amino acid sequence that allows the polypeptide to be captured onto an affinity matrix.
• a tag such as c-myc, hemagglutinin, polyhistidine, or FlagTM tag (Kodak) can be used to aid polypeptide purification.
• tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino termini.
• Other fusions that could be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase.
• Immunoaffinity chromatography also can be used to purify polypeptides of interest.
• polyamine modification of an antibody having specific binding affinity for A ⁇ enhances permeability of the modified antibody at the BBB.
• polyamine-modified monoclonal antibody against A ⁇ has a PS product that is 36 fold higher in the cortex compared to unmodified antibody and may provide a better approach to passive immunization for AD.
• Antibodies having specific binding affinity for A ⁇ can be modified with polyamines that are either naturally occurring or synthetic. See, for example, U.S. Pat. No. 5,670,477.
• Useful naturally occurring polyamines include putrescine, spermidine, spermine, 1,3-diaminopropane, norspermidine, syn-homospermidine, thermine, thermospermine, caldopentamine, homocaldopentamine, and canavalmine. Putrescine, spermidine, and spermine are particularly useful.
• Synthetic polyamines are composed of the empirical formula C x H y N z , and can be cyclic or acyclic, branched or unbranched, hydrocarbyl chains of 3-12 carbon atoms that further include 1-6 NR or N(R) 2 moieties, wherein R is H, (C 1 -C 4 ) alkyl, phenyl, or benzyl. Polyamines can be linked to an antibody using the cross-linking techniques described above.
• compositions of the invention can be formulated with a pharmaceutically acceptable carrier and administered to a mammal.
• a composition of the invention can be administered to a non-human animal (e.g., a transgenic mouse model of Alzheimer's disease) or to a human to aid in the diagnosis of a CNS disorder such as Alzheimer's disease or for treating a human patient that has been diagnosed with a CNS disorder.
• treatment refers to administering a composition of the invention to a patient, regardless of whether the patient responds to the treatment, with the proviso that when the same composition is administered to a population of patients, a statistically significant number of patients within the population exhibit a clinically recognized improvement or stabilization of one or more clinical features of the disorder.
• compositions of the invention are administered intravenously (i.v.), although other parenteral routes of administration, including subcutaneous, intramuscular, intra-arterial, intranasal, intracarotid, and intrathecal also can be used.
• parenteral routes of administration including subcutaneous, intramuscular, intra-arterial, intranasal, intracarotid, and intrathecal also can be used.
• Formulations for parenteral administration may contain pharmaceutically acceptable carriers such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, and the like.
• the dosage of the composition to be administered can be determined by the attending physician taking into account various factors known to modify the action of drugs. These include health status, body weight, sex, diet, time and route of administration, other medications, and any other relevant clinical factors. Typically, the dosage is about 1-3000 ⁇ g/kg body weight (e.g., from about 10-1000 ⁇ g/kg body weight or 50-500 ⁇ g/kg body weight). Therapeutically effective dosages may be determined by either in vitro or in vivo methods.
• Treatment of a CNS disorder can be assessed by determining if one or more clinical features of the disorder (e.g., cognitive function, memory, behavior, language skills, motor skills, or rigidity of the patient) improve or are stabilized in the patient.
• one or more clinical features of the disorder e.g., cognitive function, memory, behavior, language skills, motor skills, or rigidity of the patient
• the composition that is administered to the patient contains at least one polypeptide that is labeled as described above. Presence or absence of the labeled polypeptide (e.g., labeled antibody or labeled A ⁇ polypeptide) is detected in the CNS in vivo (e.g., in the brain of the patient) using, for example, imaging techniques such as positron emission tomography (PET), gamma-scintigraphy, magnetic resonance imaging (MRI), functional magnetic resonance imaging (FMRI), magnetoencephalography (MEG), and single photon emission computerized tomography (SPECT).
• PET positron emission tomography
• MRI magnetic resonance imaging
• FMRI functional magnetic resonance imaging
• MEG magnetoencephalography
• SPECT single photon emission computerized tomography
• MRI is particularly useful as the spatial resolution and signal-to-noise ratio provided by MRI (30 microns) is suitable for detecting amyloid deposits, which can reach up to 200 microns in size.
• the CNS disorder can be diagnosed based on the presence, for example, of labeled amyloid (e.g., labeled amyloid deposits).
• a ⁇ Proteins Human A ⁇ 1-42 was synthesized by using f-moc chemistry in a Perkin-Elmer peptide synthesizer in the Mayo Protein Core Facility. The amino acid sequence of human A ⁇ is provided in SEQ ID NO:1. Purity of the peptide was evaluated by peptide sequencing and laser desorption mass spectrometry.
• B-cell hybridomas were generated following the procedure of St. Groths and Scheidegger ( J. Immunol. Methods 35:1 (1980)) in the Mayo Monoclonal Core Facility. Human A ⁇ 1-42 that was aggregated and fibrilized by incubating at 37° C. for 24 hours was used as antigen. Positive subclones were isotyped and cryopreserved and further characterized by ELISA and immunohistochemistry labeling of AD transgenic mouse brain sections. A non-specific, monoclonal antibody was obtained from ATCC HB96 L227 (anti-human Ia).
• PS/V p Measurements at the BBB for Radioiodinated Monoclonal IgG (MoIgG): Aliquots of the proteins (MoIgG or A ⁇ ) were labeled with 125 I or 131 I using the chloramine T procedure described by Poduslo et al., Proc. Natl. Acad. Sci. USA 9:5705-5709 (1994). PS/V p measurements were performed as described by Poduslo et al., Neurobiol. Disease, 8:555-567 (2001) and Poduslo et al., Proc. Natl. Acad. Sci. USA 89:2218-2222 (1992).
• the procedure for quantifying BBB permeability of proteins was adapted from the rat to the mouse and included catheterizing the femoral artery and vein of the mouse instead of the brachial artery and vein as for the rat. Because of the smaller blood volume in the mouse, serial sampling of 20 ⁇ l of blood from the femoral artery was performed and directly TCA precipitated to generate a whole blood washout curve for the intact protein. Briefly, an I.V. bolus injection of phosphate-buffered saline (PBS) containing 125 I-MoIgG (100 ⁇ C) was rapidly injected into the femoral vein in pentobarbital-anesthetized mice.
• PBS phosphate-buffered saline
• I-MoIgG 100 ⁇ C
• V p and PS measurements were calculated as described by Poduslo et al., Neurobiol. Disease, 8:555-567 (2001) and Poduslo et al., Proc. Natl. Acad. Sci. USA 89:2218-2222 (1992). All procedures were performed using humane and ethical protocols approved by the Mayo Clinic Institutional Animal Care and Use Committee, in accordance with the National Institute of Health's Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize both the suffering and the number of animals used.
• the BBB permeability of a non-specific monoclonal antibody (anti-human Ia; L227; IgG 1 ⁇ ), monoclonal antibody against human A ⁇ 1-42 (PC2; IgG 1 ⁇ ), and the immune complex [(human A ⁇ 42)-L227 or (human A ⁇ 42)-PC2)] at various mole ratios was determined in the normal adult mouse (B6SJL) as described in Example 1 by quantifying the permeability coefficient x surface area (PS) product for each protein after correction for the residual plasma volume (V p ) occupied by the protein in blood vessels in different brain regions following an I.V. bolus injection.
• PS permeability coefficient x surface area
• the V p value was determined with a second aliquot of the same protein radioiodinated with a different isotope of iodine ( 125 I vs. 131 I) given 30-60 seconds before the end of the experiment.
• a different isotope of iodine 125 I vs. 131 I
• a dual isotope approach allows for the determination of the vascular space in each individual animal. The PS product at the BBB for different radioiodinated proteins is corrected, therefore, for the V p with a second tracer of the same protein.
• the PS product for the non-specific monoclonal antibody ranged from 0.5-1.1 ⁇ 10 ⁇ 6 ml/g/sec in six different brain regions (Table 1).
• the PS values for the monoclonal antibody to human A ⁇ 1-42 ranged from 0.6-1.4 ⁇ 10 ⁇ 6 ml/g/sec in the same brain regions and were not significantly different.
• V p values ranged from 12.8-28.4 ⁇ l/g for L227 and from 11.8-28.0 ⁇ l/g for PC2 and were not significantly different (Table 1).
• the PS values for both monoclonal antibodies are low and less than that observed for albumin. Both IgG and albumin are considered to be transported at the BBB by passive diffusion or fluid phase endocytosis.
• insulin has very high PS values in mice (27.7-43.0 ⁇ 10 ⁇ 6 ml/g/sec) and is transported at the BBB by a receptor-mediated transport.
• Insulin has a PS product at the BBB that is approximately 28.3-49.9 fold greater than that of the monoclonal antibody to human A ⁇ 42 (PC2).
• the V p values for insulin and the monoclonal antibody to human A ⁇ 42 (PC2) are similar.
• PS values ranging from 21.5-33.0 ⁇ 10 ⁇ 6 ml/g/sec in six different brain regions were observed for a polyamine modified monoclonal antibody to human A ⁇ (PC2).
• These PS values for PUT-PC2 were highly significant (P ⁇ 0.0001) and ranged from 22.8-37.9 fold higher than the antibody (PC2) alone.
• Polyamine modification of the monoclonal antibody may allow for better delivery across the BBB. This approach is not dependant upon circulating A ⁇ levels and may allow for a more dramatic reduction in amyloid burden in the Alzheimer brain following passive immunization.

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