[go: up one dir, main page]

WO1997004794A1 - Modulateurs d'expression et de fonction de la proteine associee au recepteur de la lipoproteine basse densite (lrp) dans la maladie d'alzheimer - Google Patents

Modulateurs d'expression et de fonction de la proteine associee au recepteur de la lipoproteine basse densite (lrp) dans la maladie d'alzheimer Download PDF

Info

Publication number
WO1997004794A1
WO1997004794A1 PCT/US1996/012686 US9612686W WO9704794A1 WO 1997004794 A1 WO1997004794 A1 WO 1997004794A1 US 9612686 W US9612686 W US 9612686W WO 9704794 A1 WO9704794 A1 WO 9704794A1
Authority
WO
WIPO (PCT)
Prior art keywords
app
lrp
ofthe
receptor
agent
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US1996/012686
Other languages
English (en)
Inventor
Dudley K. Strickland
Bradley T. Hyman
Maria Z. Kounnas
Robert D. Moir
Rudolph E. Tanzi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
American National Red Cross
General Hospital Corp
Original Assignee
American National Red Cross
General Hospital Corp
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 American National Red Cross, General Hospital Corp filed Critical American National Red Cross
Publication of WO1997004794A1 publication Critical patent/WO1997004794A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention broadly relates to the treatment, diagnosis, and prophylactic prevention of Alzheimer's disease. More specifically, the present invention relates to methods and compositions for preventing the endocytosis and cellular internalization of integral membrane amyloid ⁇ -precursor protein (APP) and its subsequent catabolism by blocking or interfering with the association or binding of APP with members of the low density lipoprotein receptor family.
  • APP integral membrane amyloid ⁇ -precursor protein
  • AD Alzheimer's disease
  • a ⁇ peptide is derived from an integral membrane protein termed amyloid ⁇ -protein precursor protein (APP) (Tanzi et al., Science 255:880 (1987); Kang et al., Nature 525:733-736 (1987)).
  • APP amyloid ⁇ -protein precursor protein
  • APP The function and metabolism of APP have been the subject of intensive study due to the fact that mutations in APP are associated with an autosomal dominant form of AD, (Goate et al., Nature 349:704-707 (1991)) and over-production of APP is the presumptive cause of AD in trisomy 21 (Tanzi et al., Science 255:880 (1987); Hyman et al., Proc. Natl. Acad. Sci. USA P2-.3586-3590 (1995)).
  • Multiple APP isoforms can be generated by alternatively splicing of mRNAs.
  • the major isoforms in brain are APP695,
  • These isoforms are transmembranous proteins having large extracellular regions, with hydrophobic membrane spanning domains and short cytoplasmic segments.
  • APP is also a member of an evolutionary conserved family of proteins which include the APP-like proteins, APLP1 and APLP2 (Wasco et al., Proc. Natl. Acad. Sci. USA 89: 10758- 10762 (1992); Wasco et al., Nature Genet. 5:95-100 (1993); Slunt et ai, J. Biol. Chem. 269:2637-2644 (1994)).
  • APP/PN-2 binds with high affinity to cultured fibroblasts (Johnson- Wood et al., Biochem. Biophys. Res. Commun. 200:1685-1692 (1994)), and APP/PN-2:proteinase complexes are internalized and degraded by cultured cells (Knauer and Cunningham, Proc. Natl. Acad. Sci.
  • LRP low density lipoprotein receptor-related protein
  • LRP is a large multiligand receptor (Krieger and Herz, Annu. Rev. Biochem. 63:60 -637 (1994)) that is a member ofthe LDL receptor family, which also includes the LDL receptor (Yamamoto et al., Cell 59:27-38 (1984)), the
  • VLDL receptor (Takahashi et al, Proc. Natl. Acad. Sci. USA S9:9252-9256 (1992)), and glycoprotein 330 (Saito et al, Proc. Natl Acad. Sci. USA 97:9725- 9729 (1994)).
  • RAP receptor associated protein
  • a 39 kDa protein termed the receptor associated protein (RAP) (Strickland et al, J. Biol. Chem. 255:13364-13369 (1991)) binds to members of the LDL receptor family (Williams et al, J. Biol Chem. 257:9035-9040 (1992); Kounnas et al , J. Biol. Chem. 267:21162-21166 (1992); Battey et al. , J. Biol.
  • RAP receptor associated protein
  • LRP mediates the cellular uptake and subsequent degradation of proteinases, such as tissue-type plasminogen activator (Bu et al, Proc. Natl. Acad. Sci. USA 89:7427- 7431 (1992)) and urokinase-type plasminogen activator (Kounnas et al, J. Biol. Chem. 268:21862-21867 (1993)), proteinase-inhibitor complexes, such as ⁇ 2 - macroglobulin-proteinase complexes (Ashcom et al, J. Cell Biol.
  • LRP is expressed in many tissues and is a major apoE receptor in the central nervous system (Rebeck et al, Neuron 77:575-580 (1993)). Genetic data implicate inheritance ofthe ⁇ 4 allele of apoE as a risk factor in AD (Strittmatter et al, Proc. Natl Acad. Sci. USA 90:1977-1981 (1993); Rebeck et al, Neuron 77:575-580 (1993); Poirier et al, Lancet 342:697-699 (1993); Saunders et al,
  • LRP LRP-like protein
  • LRP is capable of binding and mediating the internalization and degradation of APP s 770as well as its complexes with proteinases.
  • a first embodiment ofthe present invention therefore relates to agents which bind to the APP-binding site on the LRP particle (Group I agents) and agents which bind to the LRP-binding site found on APP (Group II agents).
  • An additional embodiment ofthe present invention relates to DNA molecules which encode peptides and antibodies that are Group I agents and/or Group II agents and to host organisms that have been transformed with the DNA molecules ofthe present invention.
  • the present invention also relates to processes for preparing DNA molecules which encode a functional derivative or a fragment of LRP, RAP or
  • a further embodiment ofthe present invention relates to methods for preparing polypeptides that are functional derivatives of LRP which comprise taking the polypeptide from the native receptor molecule by enzymatic, such as proteolytic, or chemical, such as reductive, treatment.
  • An additional embodiment ofthe invention relates to a process for preparing Group I agents which are a functional derivative of APP or RAP which comprise expressing a recombinant DNA molecule according to the invention.
  • the present invention further relates to Group II agents which are antibodies, or an antibody fragment containing the antigen binding domain, that bind to the LRP binding site found on APP.
  • Another embodiment ofthe present invention relates to processes for preparing Group II agents which are a functional derivative of LRP which comprise expressing a recombinant DNA molecule according to the invention.
  • the present invention additionally relates to hybrid cell lines that secrete monoclonal antibodies against the LDL-receptor protein which interfere with APP attachment to the LRP receptor.
  • An additional embodiment ofthe present invention includes the use of
  • Group I agents and/or Group II agents for qualitatively and/or quantitatively determining or purifying the presence of LRP which is found in a sample.
  • a further embodiment ofthe present invention includes a test kit for determining whether a polypeptide is a Group I agent and/or a Group II agent, this kit comprising a carrier means having in close confinement therein one or more container means at least one of which contains an antibody that binds to the LRP binding site found on APP.
  • Another embodiment ofthe present invention relates to processes for preparing antibodies that bind to the LRP binding site found on APP, in which a host animal is immunized with one or more polypeptides of Group I and/or Group
  • An additional embodiment ofthe present invention relates to the use of Group I agents and/or Group II agents, or the native receptor molecules ofthe LDL-receptor family or pharmaceutically suitable salts thereof, for the therapeutic or prophylactic treatment ofthe human body.
  • the invention also relates to methods for reducing the rate of onset or the severity of Alzheimer's disease, comprising administering to an animal, such as a human, one or more Group I agents and/or one or more Group II agents in an amount effective to reduce the rate of APP attachment to its receptor.
  • a further embodiment of the present invention relates to pharmaceutical compositions for therapeutic treatment of Alzheimer's disease, comprising one or more of Group I agents and/or one or more Group II agents and/or the native receptor molecule ofthe LDL-receptor family and a pharmaceutically acceptable carrier.
  • An additional embodiment of the invention relates to the use of LRP for inhibiting the binding of natural ligands to a member ofthe LDL-receptor family of proteins.
  • An alternative embodiment ofthe present invention relates to methods for identifying substances which inhibit the binding ofa ligand (RAP) or APP to a protein derived from the LDL-receptor family, comprising the steps of: a) incubating the receptor, or a soluble form ofthe receptor, with RAP or APP in the presence of a potential inhibitor substance; and b) determining the extent of binding of RAP or APP to the receptor or receptor fragment.
  • RAP ligand
  • Another embodiment ofthe present invention relates to methods for detecting receptors ofthe LDL-receptor family, comprising the steps of: a) incubating a substance derived from a fragment of RAP or APP which contains a binding activity for the receptor with a sample; and b) determining the extent of binding of the RAP or APP material to the sample.
  • a further embodiment ofthe present invention relates to methods for supplying a therapeutically active substance into a carrying cell, characterized in that a) a fragment of RAP or APP with a binding activity on the LDL- receptor is coupled with the therapeutic substance; and b) the said material is added to the corresponding cell material, bound to the receptor and in this way the therapeutically active substance is introduced into the cell.
  • Figure 1 shows the catabolism of APP by fibroblasts containing LRP (panel A) compared to the absence of catabolism of APP by fibroblasts deficient in LRP (panel B) and the absence of catabolism of APP lacking the KPI domain by either LRP-containing or LRP-deficient fibroblasts (panels C and D). Note the inhibition of catabolism of APP by the addition of RAP in fibroblasts containing
  • Figure 2 shows the internalization and degradation of LDL by both LRP- containing and LRP-deficient fibroblasts (panels A and B) and the internalization and degradation of ⁇ 2 -macroglobulin by LRP-containing fibroblasts and the lack of both internalization and degradation of ⁇ 2 -macroglobulin by LDL-deficient fibroblasts (panels C and D).
  • FIG. 3 shows the internalization and degradation of APP by LRP- containing fibroblasts (panels A and B) and the substantially lower level of internalization and degradation of APP by LRP-deficient fibroblasts (panels C and D). Note the inhibition of internalization of APP by the addition of heparin
  • Figure 4 shows the inhibition of APP degradation by LRP-binding ligands in LRP-containing fibroblasts (panel A) and the lesser effect ofthese ligands on APP degradation by LRP-deficient fibroblasts (panel B).
  • Figure 5 shows the direct ability of APP to bind LRP as measured by enzyme-linked immunosorbant assay (ELISA) using anti-APP (panel A) as compared to the amount of LRP available for binding (panel B). Note the inhibition of APP binding by RAP, an LRP ligand (panel C).
  • ELISA enzyme-linked immunosorbant assay
  • Figure 6 shows the enhancement of degradation of APP in LRP- containing fibroblasts by coupling with coagulation factor IXa (panel A) as compared to the degradation of APP in LRP-deficient fibroblasts (panel B). Note the inability of this complexing to avoid inhibition of degradation by RAP (panel A).
  • Figure 7 shows a slight inhibition of APP degradation in LRP-containing fibroblasts by competition with apolipoprotein E3/ ⁇ very low density lipoprotein or apolipoprotein E4/ ⁇ very low density lipoprotein (panel A) as compared to the same competition in LRP-deficient fibroblasts.
  • the LDL-receptor family is defined as those proteins which are recognized in the art as being formed from four structurally-related cell surface receptors and which mediate the endocytosis of lipoproteins and other plasma proteins (Brown et al, Curr. Opin. Lipidology 2:65-72 (1991)). These receptors share the following common features: cysteine-rich repeats which are responsible for ligand binding; cysteine-rich repeats ofthe epidermal growth factor-type (EGF-type); tyrosine-tryptophan-threonine-aspartate repeats; a single region spanning the membrane; and at least one internalizing signal (Willnow et al, J. Biol. Chem. 257:26172-21180 (1992)).
  • an agent is said to reduce the amount or rate of binding if the amount or rate of binding is less in the presence ofthe agent than when the agent is absent. Under conditions when the amount or rate of reduction is nearly complete, there will be an actual inhibition or total blocking of binding.
  • the agents ofthe present invention i.e. the Group I agents and the Group II agents, may be any composition of matter provided that it has the ability to bind to the APP-binding site on LRP (Group I) and/or the ability to bind to the LRP-binding site on APP (Group II).
  • Suitable agents exhibiting these properties include, but are not limited to, peptides, antibodies, carbohydrates, nucleic acids, vitamins, pharmaceutical agents, and the like, including derivatives thereof.
  • the agents ofthe present invention may be identified and/or prepared according to any ofthe methods and techniques known to those skilled in the art. These agents, particularly peptide agents and antibody agents, may occur or be produced as monomer, dimers, trimers, tetrameres or multimers. Such multimers can be prepared using enzymatic or chemical treatment ofthe native receptor molecules or be prepared using recombinant techniques. Preferably, the agents ofthe present invention are selected and screened at random or rationally selected or designed using protein modeling techniques.
  • candidate agents are selected at random and assayed for their ability to reduce the amount or rate of binding ofthe amyloid ⁇ - precursor protein (APP) to the LDL receptor-related protein (LRP). Any ofthe suitable methods and techniques known to those skilled in the art may be employed to assay candidate agents.
  • APP amyloid ⁇ - precursor protein
  • LRP LDL receptor-related protein
  • the agent is selected based on the configuration ofthe LRP binding site found on APP or the APP binding site found on the LRP. Any ofthe suitable methods and techniques known to those skilled in the art may be employed for rational selection or design.
  • one skilled in the art can readily adapt currently available procedures to generate antibodies, peptides, pharmaceutical agents and the like capable of binding to a specific peptide sequence of LRP or APP. Illustrative examples of such available procedures are described, for example, in Hurby et al, "Application of Synthetic Peptides: Antisense Peptides," in Synthetic Peptides, A User's Guide, W.H. Freeman, NY, pp. 289-307 (1992); Kaspczak et al, Biochemistry 25:9230 (1989); and Harlow, Antibodies, Cold Spring Harbor Press, NY (1990).
  • agents ofthe present invention can alternatively be identified using modification of methods known in the art.
  • suitable peptide agents may be identified using the filter binding assay described by Mischak et al. (Mischak et al. , J. Gen. Virol. 59:2653-2656 (1988) and Mischak et al , Virology
  • peptide is applied to a suitable membrane, such as nitrocellulose, and the membrane is saturated with a detergent mixture in order to block any non-specific binding.
  • a suitable membrane such as nitrocellulose
  • the treated membrane is then incubated with labeled rhinovirus, e.g. with HRN2 labeled with 35 S-methionine, in order to check the specific binding. After washing and drying ofthe membrane, specific binding can be visualized by autoradiography.
  • the Group I agents ofthe present invention include those agents which bind directly to the APP binding site found on the LRP. Additionally, the Group I agents ofthe present invention bind to the LRP in interfering proximity with the APP binding site or bind to the LRP in such a manner so as to conformationally alter the APP binding site. Suitable Group I agents can therefore be first identified by their ability to bind the LRP and then by their ability to reduce the amount or rate at which APP binds to LRP.
  • Group I agents ofthe present invention include, but are not limited to: soluble fragments of APP contaimng the KPI domain; anti-LRP antibodies; soluble fragments of receptor associated protein (RAP); ⁇ 2 - macroglobulin:proteinase complexes; pregnancy zone protein (PZP):proteinase complexes; tissue-type plasminogen activator; pro-urokinase-type plasminogen activator; tissue factor pathway inhibitor; apolipoprotein E-enriched lipoproteins; lipoprotein lipase; hepatic lipase; thrombospondin; and lactoferrin.
  • Preferred Group I agents are based on and derived from the amino acid sequence ofthe receptor associated protein (RAP).
  • RAP receptor associated protein
  • An especially preferred type of Group I agent is isolated RAP or a fragment thereof, such as a soluble fragment of RAP which contains the LRP binding site.
  • Such agents act as competitive inhibitors of APP binding to its receptor in vitro as well as in vivo.
  • the preferred fragments of RAP are soluble under physiological conditions.
  • the C-terminus ofthese polypeptides can be shortened as desired, provided that the binding capacity for the LRP particle remains intact.
  • the preferred amino acid sequence of RAP corresponds to the human protein. Suitable RAP sequences can also be derived from the amino acid sequence of RAP isolated from other mammals or amphibia.
  • RAP may be produced using any ofthe methods and techniques known to those skilled in the art.
  • RAP can be purified from a source which naturally expresses the protein, can be isolated from a recombinant host which has been altered to express RAP or fragment thereof, or can be synthesized using protein synthesis techniques known in the art.
  • the skilled artisan can readily adapt a variety of techniques in order to obtain Group I peptide agents which contain the LRP binding site found on RAP.
  • isolated native protein may be converted by enzymatic and/or chemical cleavage to generate fragments ofthe whole protein, for example by reacting cell lines which express an RAP with an enzyme such as papain or trypsin or a chemical such as cyanogen bromide.
  • an enzyme such as papain or trypsin or a chemical such as cyanogen bromide.
  • Proteolytically active enzymes or chemicals are preferably selected in order to release the extracellular receptor region. Fragments which contain the LRP binding site, especially fragments which are soluble under physiological conditions, can then be isolated using known methods.
  • RAP or a fragment of RAP may be expressed in a recombinant bacteria, as described, for example, in Williams et al, J. Biol. Chem.
  • the Group II agents ofthe present invention include compositions which bind to the LRP binding site found on APP. Additionally, the Group II agents of the present invention include compositions that bind to APP in interfering proximity to the LRP binding site. Suitable Group II agents can therefore be first identified by their ability to bind to APP and then by their ability to reduce the amount or rate at which APP binds to LRP. Illustrative examples ofthe Group II agents ofthe present invention include, but are not limited to, antibodies which bind to the LRP binding site found on APP and soluble fragments of LRP. Preferred Group II agents include antibodies and antibody fragments which are capable of binding to a residue found on APP and consequently act as a competitive inhibitor for LRP binding.
  • the most preferred antibodies or antibody fragments ofthe present invention bind to an LRP-specific epitope in APP.
  • Antibodies, or other Group II agents such as anti-sense peptides, which bind to epitopes within this sequence reduce the amount or rate of APP binding to LRP.
  • the antibodies ofthe present invention include polyclonal and monoclonal antibodies, as well as antibody fragments and derivatives that contain the relevant antigen binding domain ofthe antibodies. Such antibodies or antibody fragments are preferably used in the diagnostic and therapeutic embodiments of the present invention.
  • Suitable monoclonal and polyclonal antibodies may be prepared by any ofthe methods and techniques well known in the art, such as described in, for example, A.M. Campbell, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers,
  • an antibody capable of binding to a domain of APP can be generated by immunizing an animal with a polypeptide whose sequence is encoded by that domain.
  • Any animal which is known to produce antibodies can be utilized to produce antibodies with the desired specificity and suitable methods for immunization ofthese animals are well known in the art, including, for example, subcutaneous or intraperitoneal injection ofthe polypeptide.
  • One skilled in the art will recognize that the amount of polypeptide used for immunization will vary based on a number of factors, including the animal which is immunized, the antigenicity ofthe polypeptide selected, and the site of injection.
  • polypeptides used as an immunogen may be modified as appropriate or administered in an adjuvant in order to increase the peptide antigenicity.
  • Suitable methods increasing antigenicity are well known in the art, and include, for example, coupling the antigen with a heterologous protein (such as globulin or ⁇ -galactosidase) or through the inclusion of an adjuvant during immunization.
  • a preferred method of generating monoclonal antibodies comprises removing spleen cells from the immunized animals, fusing these cells with myeloma cells, such as SP2/0-Agl4 myeloma cells, and allowing them to become monoclonal antibody-producing hybridoma cells.
  • any one of a number of methods well known in the art may be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al, Exp. Cell Res. 775:109-124 (1988); Kishimoto et al, Proc. Natl. Acad. Sci. USA 57:2244-2248 (1990)).
  • Hybridomas secreting the desired antibodies are cloned and the class and subclass ofthe secreted antibodies are determined using procedures known in the art (Campbell, A.M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984)).
  • antibody-containing antisera is preferably isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one ofthe above-described procedures.
  • the present invention further provides hybrid cell lines which secrete monoclonal antibodies selective for the peptide agents of Group I and/or Group II. These monoclonal antibodies are capable of wholly or partially neutralizing the activity ofthe polypeptides or specifically binding to one ofthe said polypeptides.
  • the present invention therefore also includes test systems which contain the monoclonal antibodies herein described.
  • Antibodies may be used as an isolated whole antibody, or can be used as a source for generating antibody fragments which contain the antigen binding site ofthe antibody. Examples of such antibody fragments include, but are not limited to the F v , the F(ab), the F(ab) 2 , fragment, as well as single chain antibodies. Various methods known in the art can be used to generate such fragments without undue experimentation.
  • Recombinant techniques are preferred for generating large quantities of antibodies, antibody fragments and single chain antibodies, as described, for example, in Pluckthum, Bio/Technology 70:163-167 (1992); Carter et al, Bio/Technology 70:167-170 (1992); and Mullinax et al, Biotechniques 72:864-869 (1992).
  • recombinant techniques may be used to generate heterobifunctional antibodies.
  • recombinant production of antibodies, antibody fragments or derivatives thereof uses mRNA encoding an antibody which is isolated from hybridoma cells that produce the desired antibody. This mRNA is then used as a source for generating a cDNA molecule which encodes the antibody, or a fragment thereof. Once obtained, the cDNA may be amplified and expressed according to known methods in a variety of eukaryotic and prokaryotic hosts.
  • the present invention further includes derivatives of antibodies (antibody derivatives).
  • an "antibody derivatives” contain an antibody ofthe present invention, or a fragment thereof, as well as an additional moiety which is not normally a part ofthe antibody. Such moieties may improve the solubility, absorption, biological half-life, etc., ofthe antibody, decrease the toxicity ofthe antibody, eliminate or attenuate any undesirable side effect ofthe antibody, or serve as a detectable marker ofthe presence ofthe antibody. Moieties capable of mediating such effects are well known in the art.
  • Detectably labeled antibodies constitute a special class ofthe antibody derivatives ofthe present invention.
  • An antibody is said to be "detectably labeled” ifthe antibody, or fragment thereof, is attached to a molecule which is capable of identification, visualization, or localization using known methods.
  • Suitable detectable labels include radioisotopic labels, enzyme labels, non-radioactive isotopic labels, fluorescent labels, toxin labels, affinity labels, chemiluminescent labels and nuclear magnetic resonance contrast agents.
  • suitable enzyme labels include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase, and acetylcholine esterase.
  • radioisotopic labels include, but are not limited to, 3 H, , ⁇ In, 125 1, 131 1, 32 P, 35 S, 14 C, 5, Cr, "To, 58 Co, 59 Fe, 75 Se, 152 Eu, °°Y, 67 Cu, 2,7 Ci, 21 'At, 212 Pb, 7 Sc, ,09 Pd, etc.
  • '"In is a preferred isotope where in vivo imaging is used since its avoids the problem of dehalogenation ofthe I25 I or 13 'I-labeled monoclonal antibody by the liver.
  • this radionucleotide has a more favorable gamma emission energy for imaging (Perkins et al, Eur. J. Nucl Med.
  • non-radioactive isotopic labels include, but are not limited to, ,57 Gd, 55 Mn, ,62 Dy, 52 Tr, and 56 Fe.
  • fluorescent labels include, but are not limited to, an 15 Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o- phthaldehyde label, and a fluorescamine label.
  • Illustrative examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.
  • Illustrative examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, and an aequorin label.
  • nuclear magnetic resonance contrasting agents include paramagnetic heavy metal nuclei such as Gd, Mn, and Fe.
  • Typical techniques for binding the above-described labels to antibodies are provided by Kennedy et al. (Clin. Chim. Acta 70:1-31 (1976)), and Schurs et al. (Clin. Chim. Acta 57:1-40 (1977)), which are both herein incorporated in their entirety by reference. Coupling techniques mentioned in the latter reference include the glutaraldehyde method, the periodate method, the dimaleimide method, and the m-maleimidobenzyl-N-hydroxy-succinimide ester method.
  • the present invention additionally includes humanized forms ofthese antibodies.
  • Humanized forms ofthe antibodies may be produced, for example, by replacing an immunogenic portion of an antibody with a corresponding, non- immunogenic portion (chimeric antibodies) (Better, et al, Science 240:1041-1043 (1988)).
  • suitable "humanized” antibodies can be produced by CDR or CEA grafting/substitution as described, for example, in Jones, et al, Nature 321:552-525 (1986); Nerhoeyan et al, Science 239:1534 (1988); and Beidler, et al, J. Immunol. 141:4053-4060 (1988).
  • Antisense-peptide sequences are short peptides which are specifically designed to bind to a particular amino acid sequence.
  • antisense peptide agents may be generated using methods known in the art, such as those described, for example, in Hurby et al, "Application of Synthetic Peptides: Antisense Peptides,” in Synthetic Peptides, A User's Guide, W.H. Freeman, ⁇ Y, pp. 289-307 (1992) and Kaspczak et al, Biochemistry 25:9230-8 (1989).
  • an additional class ofthe Group II agents ofthe invention are natural ligands of APP.
  • a natural ligand of APP is defined as any substance which binds to APP, such as soluble fragments of LRP containing the APP binding site.
  • Such soluble fragments may be prepared by any suitable method known to those skilled in the art, such as the method of Davis et al, Nature 525:760-765 (1987), which involves deletion ofthe entire EGF domain.
  • soluble forms ofthe receptor may be formed by inserting a stop codon in front ofthe region of D ⁇ A encoding the cytoplasmic or transmembrane domain (Yokade et al, J. Cell Biol 117:39 (1992)).
  • the agents ofthe present invention may be used in vitro and/or in vivo to study LRP attachment and to reduce the rate of onset and/or ameliorate the duration and severity of Alzheimer's disease.
  • the agents ofthe present invention may be used in qualitative, quantitative and preparative assays and purification procedures to isolate, identify and facilitate the purification of APP.
  • the agents ofthe present invention may be provided to a patient as a means of reducing the amount or rate of APP binding to LRP (Hayden, et al, Antiviral Res. 9:233-247 (1988)).
  • compositions comprising a Group I agent and or a Group II agent.
  • These pharmaceutical compositions may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
  • pharmaceutically acceptable carrier is intended to mean a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • a particular mode of administration can be made empirically based upon considerations such as the particular disease state being treated; the type and degree ofthe response to be achieved; the specific agent or composition employed; the age, body weight, general health, sex and diet ofthe patient; the time of administration and rate of excretion ofthe agent or composition; the duration ofthe treatment; drugs (such as a chemotherapeutic agent) used in combination or coincidental with the specific composition; and like factors well known in the medical arts.
  • compositions ofthe present invention for parenteral injection may comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include, but are not limited to, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylceuulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance ofthe required particle size in the case of dispersions, and by the use of surfactants.
  • compositions ofthe present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorgamsms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged abso ⁇ tion ofthe injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the absorption from subcutaneous or intramuscular injection in order to prolong the effect ofthe therapeutic agent or inhibitor, it is desirable to slow the absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature ofthe particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
  • Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules.
  • the active compounds are preferably mixed with at least one pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and
  • the dosage form may also comprise buffering agents as appropriate.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part ofthe intestinal tract, optionally, in a delayed manner.
  • opacifying agents such as enteric coatings and other coatings well known in the pharmaceutical formulating art.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active agents of Group I and/or Group II can also be in micro ⁇ encapsulated form, if appropriate, with one or more ofthe above-mentioned excipients.
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetiahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solub
  • the oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to the agent or inhibitor, stabilizers, preservatives, excipients, and the like.
  • Preferred lipids are phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art.
  • agents ofthe present invention can be formulated according to known methods to prepare pharmaceutically acceptable compositions, whereby these materials, or their functional derivatives, are combined in a mixture with a pharmaceutically acceptable carrier vehicle.
  • Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are well known in the art.
  • compositions will contain an effective amount of one or more agents ofthe present invention.
  • Control release preparations may be achieved through the use of polymers to complex or absorb the therapeutic agents ofthe invention.
  • the controlled delivery may be exercised by selecting appropriate macromolecules (such as polyesters, polyamino acids, polyvinyl, pyrroiidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and methods of incorporation in order to control release.
  • Another possible method to control the duration of action by controlled release preparations is to incorporate antibodies into particles of a polymeric material such as polyesters, polyamino acids, hydro ⁇ gels, poly(lactic acid) or ethylene vinyl acetate copolymers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine- microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal dmg delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • the pharmaceutical formulations ofthe present invention are prepared, for example, by admixing the active agent with solvents and/or carriers, optionally using emulsifiers and/or dispersants, whilst if water is used as the diluent, organic solvents may be used as solubilizing agents or auxiliary solvents.
  • the excipients used include, for example, water, pharmaceutically acceptable organic solvents such as paraffins, vegetable oils, mono- or polyfunctional alcohols, carriers such as natural mineral powders, synthetic mineral powders, sugars, emulsifiers and lubricants.
  • inventive therapeutic agents can be determined empirically and may be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt, ester or prodmg form.
  • the agonist or antagonist my be administered in compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage ofthe agents and compositions ofthe present invention will be decided by the attending physician within the scope of sound medical judgement.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the type and degree ofthe response to be achieved; the specific agent or composition employed; the age, body weight, general health, sex and diet ofthe patient; the time of administration, route of administration, and rate of excretion ofthe agent or composition; the duration ofthe treatment; dmgs (such as a chemotherapeutic agent) used in combination or coincidental with the specific composition; and like factors well known in the medical arts.
  • a dosage of antibody or peptide agent in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient).
  • the therapeutically effective dose can be lowered if the agent ofthe present invention is additionally administered with another compound.
  • one compound is said to be additionally administered with a second compound when the administration ofthe two compounds is in such proximity of time that both compounds can be detected at the same time in the patient's serum.
  • satisfactory results are obtained by oral administration of therapeutic dosages on the order of from 0.05 to 10 mg/kg/day, preferably 0J to 7.5 mg/kg/day, more preferably 0J to 2 mg/kg/day, administered once or, in divided doses, 2 to 4 times per day.
  • dosages on the order of from 0.01 to 5 mg/kg/day, preferably 0.05 to 1.0 mg/kg/day and more preferably 0J to 1.0 mg/kg/day can be used.
  • Suitable daily dosages for patients are thus on the order of from 2.5 to 500 mg p.o., preferably 5 to 250 mg p.o., more preferably 5 to 100 mg p.o., or on the order of from 0.5 to 250 mg i.v., preferably 2.5 to 125 mg i.v. and more preferably 2.5 to 50 mg i.v.
  • Dosaging may also be arranged in a patient specific manner to provide a predetermined concentration of an agent in the blood, as determined by the RIA technique. Thus patient dosaging may be adjusted to achieve regular on-going trough blood levels, as measured by RIA, on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.
  • the agents ofthe present invention are intended to be provided to a patient in an amount sufficient to reduce the amount or rate of binding of human
  • An amount is said to be sufficient to "reduce the amount or rate of APP binding" ifthe dosage, route of administration, etc. ofthe agent is sufficient to reduce the amount or rate of APP attachment to the LRP receptor.
  • Such an effect can be assayed, for example, by examining the onset of Alzheimer's disease symptoms occurring in vivo, or by correlating in vitro blocking studies with predicted in vivo efficacy.
  • the administration ofthe agents ofthe present invention may be for either prophylactic or therapeutic purpose.
  • the agent is provided in advance of any Alzheimer's disease symptoms.
  • the prophylactic administration ofthe agent serves to prevent or reduce the rate of onset of symptoms.
  • the agent is provided at (or shortly after) the onset ofthe appearance of symptoms of actual disease.
  • the therapeutic administration ofthe agent serves to reduce the severity and duration of Alzheimer's disease.
  • the present invention further includes the use ofthe agents ofthe present invention in diagnostic applications.
  • the Group I agents ofthe present invention can be used to detect the presence of LRP in a test sample.
  • the Group II agents of the present invention can be used to detect the presence of APP in a test sample. Conditions for incubating an agent with a test sample vary.
  • Incubation conditions will depend on factors such as the type of agent, format, and detection system employed for the assay, as well as the nature ofthe test sample used in the assay. For example, condition will vary slightly when a whole antibody, a single chain antibody, a F(ab) fragment, or a peptide agent is used.
  • immunological assay formats such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based ouchterlony, or rocket immunofluorescent assays
  • examples of such assays can be found in T.
  • the agent ofthe present invention is immobilized on a solid support for use in the diagnostic assay.
  • solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads.
  • plastics such as polycarbonate
  • complex carbohydrates such as agarose and sepharose
  • acrylic resins such as polyacrylamide and latex beads.
  • Techniques for coupling agents such as antibodies, peptides and the like to such solid supports are well known in the art, as described, for example, in D.M. Weir et al. , Handbook of
  • one or more ofthe agents ofthe present invention which is used in one ofthe above-described methods can be detectably labelled prior to use, for example, through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horse radish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labelling are well- known in the art (L.A. Stemberger et al, J. Histochem. Cytochem. 75:315 (1970);
  • the present invention provides a compartmentalized kit to receive in close confinement, one or more containers which comprises: a) a first container comprising an agent capable of binding to the LRP binding site; and b) one or more other containers comprising one or more ofthe following: wash reagents and reagents capable of detecting the presence of bound agents from the first container.
  • a compartmentalized kit includes any kit in which reagents are contained in separate containers.
  • Illustrative examples of such containers include, but are not limited to, small glass containers, plastic containers or strips of plastic or paper.
  • Particularly preferred types of containers allow the skilled worker to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers include, but are not be limited to, a container which will accept the test sample, a container which contains one or more ofthe agents ofthe present invention used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound agent.
  • wash reagents such as phosphate buffered saline, Tris-buffers, etc.
  • detection reagents which can be used in the above described kits include, but are not limited to, labelled secondary agents, or in the altemative, ifthe primary agent is labelled, enzymatic or agent binding reagents which are capable of reacting with the labelled agent.
  • agents of the present invention can be readily incorporated into one ofthe established kit formats which are well known in the art.
  • a further aspect ofthe present invention concerns DNA molecules which encode for the polypeptide and antibody agents of Group I and/or Group II.
  • the starting nucleotide molecules can be obtained by the person skilled in the art using known methods.
  • the DNA molecules, where the amino acid sequence is known may be produced synthetically (e.g. according to Edge et al, Nature 292:756-762 (1981)) or by methods such as PCR (Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
  • DNA sequences ofthe present invention include not only the actual nucleotide sequence used by the organism from which the receptor protein is derived but also includes all degenerate forms which encode a peptide with the desired sequence.
  • the invention includes DNA sequences which have been modified utilizing methods known in the art, such as those generated by mutation, deletion, transposition or addition.
  • the preferred mutations will introduce stop codons within the RAP sequence so that a truncated protein will be generated.
  • the present invention further includes DNA vectors which contain the DNA sequences described above and below.
  • these may be vectors in which the DNA molecules described are functionally linked to control sequences which allows expression ofthe corresponding polypeptides.
  • These are preferably plasmids which can be replicated and/or expressed in prokaryotes such as E. coli and/or in eukaryotic systems such as yeasts or mammalian cell lines.
  • These vectors may also be mammalian viral vectors which can be replicated and/or expressed in eukaryotes such as mammalian cell lines and in the human patient, as
  • host for integration into the cellular genome ofthe patient and expression as genetic therapy systems.
  • the invention also includes host organisms transformed with the above vectors. Expression in prokaryotes and eukaryotes may be carried out using techniques known in the art.
  • the DNA sequences according to the invention may be expressed as fusion polypeptides or as intact, native polypeptides. Fusion proteins may advantageously be produced in large quantities. They are generally more stable than the native polypeptide and are easy to purify.
  • the expression of these fusion proteins can be controlled by normal host DNA sequences.
  • the DNA sequences according to the invention can be cloned and expressed as lacZ fusion genes in E. coli.
  • a person skilled in the art has a variety of vector systems available for this purpose, e.g. the pUR- vector series (U. R ⁇ ther and B.
  • the bacteriophage promoter ⁇ p R may also be used, in the form ofthe vectors pEX-1 to -3, for expressing large amounts of Cro- ⁇ -galactosidase fusion protein (K.K.
  • tac promoter which can be induced with IPTG can also be used, for example in the form ofthe pROK- vector series (CLONTECH Laboratories).
  • the prerequisite for producing intact native polypeptides using E. coli is the use of a strong, regulatable promoter and an effective ribosome binding site.
  • Promoters which may be used for this purpose include the temperature sensitive bacteriophage ⁇ p L -promoter, the tac-promoter inducible with IPTG or the T7- promoter.
  • plasmids with suitable promoter stmctures and efficient ribosome binding sites have been described, such as for example pKC30 ( ⁇ p L ; Shimatake and Rosenberg, Nature 292:128 (1981), pKK173-3 (tac, Amann and Brosius, Gene 40: 183 (1985)) or pET-3 (T7-promoter (Studier and Moffat, J. Mol.
  • E. coli strains which are specifically tailored to a particular expression vector are known to those skilled in the art (Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).
  • the experimental performance ofthe cloning experiments, the expression ofthe polypeptides in E. coli and the working up and purification of the polypeptides are known and are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989).
  • eukaryotic microorganisms such as yeast may also be used.
  • yeast the plasmid YRp7 (Stinchcomb et al. Nature
  • the plasmid YRp7 contains the TRP 1 -gene which provides a selection marker for a yeast mutant (e.g. ATCC No. 44076) which is incapable of growing in tryptophan-free medium.
  • the presence ofthe TRP1 defect as a characteristic ofthe yeast strain used then constitutes an effective aid to detecting transformation when cultivation is carried out without tryptophan.
  • the same is tme with the plasmid YEpl3, which contains the yeast gene LEU-2, which can be used to complete a LEU-2-minus mutant.
  • yeast hybrid vectors also contain a replication start and a marker gene for a bacterial host, particularly E. coli, so that the constmction and cloning ofthe hybrid vectors and their precursors can be carried out in a bacterial host.
  • Other expression control sequences suitable for expression in yeast include, for example, those of PHO3- or PHO5-gene.
  • suitable promoter sequences for yeast vectors contain the 5 '- flanking region ofthe genes of ADH I (Ammerer, Methods of Enzymology 707:192-210 (1983)), 3-phosphoglycerate kinase (Hitzeman et al, J. Biol.
  • enolase glycerinaldehyde-3-phosphate- dehydrogenase, hexokinase, pyruvate-decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglucose-isomerase and glucokinase.
  • enolase glycerinaldehyde-3-phosphate- dehydrogenase
  • hexokinase hexokinase
  • pyruvate-decarboxylase phosphofructokinase
  • glucose-6-phosphate isomerase phosphoglucose-isomerase
  • glucokinase glucokinase
  • promoters are the promoter regions ofthe genes for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase and enzymes which are responsible for the metabolism of maltose and galactose.
  • Promoters which are regulated by the yeast mating type locus such as promoters ofthe genes BARI, MF ⁇ l, STE2, STE3, STE5 can be inserted in temperature regulated systems by the use of temperature-dependent sir mutations. (Rhine, Ph.D. Thesis, University of Oregon, Eugene, Oregon (1979); Herskowitz and Oshima, The Molecular Biology ofthe Yeast Saccharomyces, Cold Spring Harbor Laboratory Press, part I, pp. 181-209 (1981)). Generally, however, any vector which contains a yeast- compatible promoter and origin replication and termination sequences is suitable.
  • hybrid vectors which contain sequences homologous to the yeast 2 ⁇ plasmid DNA may also be used. Such hybrid vectors are incorporated by recombination within the cells of existing 2 ⁇ -plasmids or replicate autonomously.
  • yeasts other eukaryotic systems may, of course, be used to express the polypeptides according to the invention. Since post-translational modifications such as disulphide bridge formation, glycosylation, phosphorylation and/or oligomerization are frequently necessary for the expression of biologically active eukaryotic proteins by means of recombinant DNA, it may be desirable to express the DNA according to the invention not only in mammalian cell lines but also insect cell lines.
  • Functional prerequisites ofthe corresponding vector systems comprise, in particular, suitable promoter, termination and polyadenylation signals as well as elements which make it possible to carry out replication and selection in mammalian cell lines.
  • suitable promoter, termination and polyadenylation signals as well as elements which make it possible to carry out replication and selection in mammalian cell lines.
  • the DNA molecules according to the invention it is particularly desirable to use vectors which are replicable both in mammalian cells and also in prokaryotes such as E. coli.
  • Vectors derived from viral systems such as SV40, Epstein-Barr-virus, etc., include, for example, pTK2, pSV2-dhfv, pRSV-neo, pKO-neo, pHyg, p205, pHEBo, etc. (Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y. (1989)).
  • suitable host cells e.g. CHO-cells
  • corresponding transformed cells may be obtained with the aid of selectable markers (thymidine- kinase, dihydrofolate-reductase etc.) and the corresponding polypeptides are isolated after expression.
  • the host cells suitable for the vectors are known, as are the techniques for transformation (micro-injection, electroporation, calcium phosphate method, etc.) as described, for example, in Sambrook et al, Molecular
  • the selected vector may cut, for example, with a restriction endonuclease and, optionally after modification ofthe linearized vector thus formed, an expression control sequence equipped with corresponding restriction ends is inserted.
  • the expression control sequence contains the recognition sequence of a restriction endonuclease, so that the vector already containing the expression control sequence is digested with the said restriction enzyme and the DNA molecule according to the invention, provided with ends which fit, can be inserted.
  • the invention also relates to processes for preparing the vectors described herein, particularly expression vectors.
  • vectors are characterized in that a DNA provided with corresponding ends and coding for a functional derivative or a fragment ofthe LRP receptor is inserted into a vector DNA cut with restriction endonucleases and containing the expression control sequences described by way of example, in such a way that the expression control sequences regulate the expression ofthe DNA inserted.
  • the peptides and antibody agents ofthe present invention which are obtained by the expression of recombinant DNA or from the native receptor molecule may, of course, also be derivatized by chemical or enzymatic processes. The present invention is described in further detail in the following non ⁇ limiting examples.
  • Example 1 m I-labeled APP 70 Degradation by LRP-expressing Fibroblasts
  • APPs770 (Panels A and B) or 125I-APPs695 (Panels C and D)for 10 h at 37 °C in the absence of competitor (•) or in the presence of either 1 ⁇ M RAP (O), or 100 ⁇ g ml heparin ( ⁇ ).
  • the cellular degradation of ,25 I-labeled APP S 770 by LRP- expressing MEF cells is shown in panel A and by LRP-deficient PEA 13 cells in panel B.
  • the cellular degradation of 125 I-APP S 695 by LRP-expressing cells is depicted in panel C and by LRP-deficient cells in panel D. Plotted values represent means of duplicate determinations.
  • the migration position of molecular mass standards are indicated on the right in kDa.
  • Example 2 LRP-deficient cells internalize and degrade 12S I-LDL, but not 12S I-
  • Wells containing 2 x IO 5 LRP-expressing (•) or LRP-deficient (O) mouse fibroblasts were incubated with 125 I-LDL (8 ⁇ g/ml) or 125 I- ⁇ 2 M* (1.4 nM) for selected time intervals. Specific internalization and degradation was determined by incubation of 125 I-ligand in the presence of a 200-fold excess unlabeled ligand. Shown are the amounts of 125 I-LDL internalized (panel A) and degraded (panel B), 125 I- ⁇ 2 M* internalized (panel C) and degraded (panel D). The plotted values represent means of duplicate determinations.
  • Mouse fibroblasts (2 x 10 5 cells per well) were incubated for selected times with l25 I- APP S 770 (1 nM) in the absence of competitor (•) or in the presence of either 1 ⁇ M RAP (O), or 100 ⁇ g/ml heparin (Q). The amount of radioactivity internalized and degraded was determined at the indicated time intervals. Shown is the amount of internalization and degradation of 125 I- APP S 770 by LRP-expressing MEF cells ⁇ panels A and B, respectively) and the internalization and degradation of 1 5 I-APP S 770 by LRP-deficient PEA13 cells (panels C andD, respectively). The plotted values are means of duplicate determinations.
  • Example 4 LRP-antibodies inhibit the degradation of 12S I-APP s 770 by mouse fibroblasts
  • Mouse fibroblasts (2 x 10 5 cells/well) were incubated for 18 h at 37 °C with ,25 I-APP S 770 (10 nM) in the absence of competitor or in the presence of either heparin (100 ⁇ g/ml), chloroquine (0J mM), RAP (1 ⁇ M), affinity purified anti-LRP IgG (100 ⁇ g/ml), or control IgG against the cytoplasmic domain of LRP (100 ⁇ g/ml).
  • Panels show the amount of ,25 I-APP S 770 degraded by mouse fibroblasts expressing LRP (A) or ones deficient in LRP expression (75). The data shown are means of duplicate determinations ⁇ S. E.
  • Example 5 LRP binds directly to APP 70 in ELISA
  • Example 6 12S I-APP s 770:Factor LXa complex is degraded by LRP-expressing cells but not by LRP-deficient cells Wells containing 2 x 10 5 LRP-expressing (MEF) or LRP-deficient (PEA13) cells were incubated with ,25 I-APP S 770 (5 nM) or ,25 I-APP s 770:FlXa (3 nM) for 18 h at 37 °C in the absence of competitor or in the presence of 1 ⁇ M RAP.
  • MEF LRP-expressing
  • PEA13 LRP-deficient
  • Example 7 Effect ofapoE isoforms on the degradation of I25 I-APP s 770 by mouse fibroblasts
  • Mouse fibroblasts (2 x IO 5 ) were incubated for 18 h at 37°C with 125 1- APP/PN-2 (8.8 nM) in the absence of competitor or in the presence of either RAP (1 ⁇ M)), 10 ⁇ g/ml apoE3/ ⁇ VLDL, 10 ⁇ g/ml apoE4/ ⁇ VLDL , or 10 ⁇ g/ml ⁇ VLDL.
  • the concentration of apoE-enriched ⁇ VLDL represent the ⁇ VLDL protein before enrichment with apoE.
  • Panels show the amount of 125 I-APP S 770 degradation by mouse fibroblasts expressing LRP (A) or deficient in LRP expression (B). The data shown are means of duplicate deterrninations ⁇ S.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Neurology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Neurosurgery (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention se rapporte généralement au traitement, au diagnostic et la prophylaxie de la maladie d'Alzheimer. Plus particulièrement, l'invention se rapporte à des procédés et à des compositions pour la prévention de l'endocytose et de l'internalisation cellulaire de la protéine membranaire intégrale dite protéine précurseur β amyloïde (APP) et de son catabolisme ultérieur en bloquant ou en interférant avec l'association ou la liaison de l'APP avec des membres de la famille de récepteurs de la lipoprotéine basse densité.
PCT/US1996/012686 1995-07-27 1996-07-29 Modulateurs d'expression et de fonction de la proteine associee au recepteur de la lipoproteine basse densite (lrp) dans la maladie d'alzheimer Ceased WO1997004794A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US160095P 1995-07-27 1995-07-27
US60/001,600 1995-07-27
US165395P 1995-07-28 1995-07-28
US60/001,653 1995-07-28

Publications (1)

Publication Number Publication Date
WO1997004794A1 true WO1997004794A1 (fr) 1997-02-13

Family

ID=26669253

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/012686 Ceased WO1997004794A1 (fr) 1995-07-27 1996-07-29 Modulateurs d'expression et de fonction de la proteine associee au recepteur de la lipoproteine basse densite (lrp) dans la maladie d'alzheimer

Country Status (2)

Country Link
CA (1) CA2246561A1 (fr)
WO (1) WO1997004794A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001015729A1 (fr) * 1999-08-27 2001-03-08 Fibrogen, Inc. Recepteur du facteur de croissance du tissu conjonctif, agonistes et antagonistes dudit recepteur, et leurs utilisations therapeutiques et diagnostiques
WO2001090758A3 (fr) * 2000-05-23 2002-08-29 Univ Southern California Role du systeme vasculaire cerebral et du transport a travers la barriere hemato-encephalique dans la clairance du peptide beta-amyloide d'alzheimer dans le systeme nerveux central
US6472140B1 (en) 1997-09-05 2002-10-29 The General Hospital Corporation α-2- macroglobulin therapies and drug screening methods for Alzheimer's disease.
US6638711B1 (en) 1999-04-29 2003-10-28 The General Hospital Corporation Methods for identifying an agent that inhibits oxygen-dependent hydrogen peroxide formation activity but does not inhibit superoxide-dependent hydrogen peroxide formation
US7045531B1 (en) 1997-03-11 2006-05-16 The General Hospital Corporation Composition comprising a metal chelator and a method of treating amyloidosis by administering the metal chelator
US7179462B2 (en) 2000-06-02 2007-02-20 University Of Connecticut Health Center α (2) macroglobulin receptor as a heat shock protein receptor and uses thereof
US7186515B1 (en) 2000-06-02 2007-03-06 University Of Connecticut Health Center Alpha(2) macroglobulin receptor as a heat shock protein receptor and uses thereof
US7449557B2 (en) 2000-06-02 2008-11-11 University Of Connecticut Health Center Complexes of alpha (2) macroglobulin and antigenic molecules for immunotherapy
EP1718324A4 (fr) * 2003-06-11 2009-07-15 Socratech L L C Derive soluble de la proteine liee au recepteur de lipoproteine de basse densite de liaison directe au peptide amyloide beta d'alzheimer
US8877204B2 (en) 2003-02-20 2014-11-04 University Of Connecticut Health Center Methods and compositions for the treatment of cancer and infectious disease using alpha (2) macroglobulin-antigenic molecule complexes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ANNALS OF NEUROLOGY, Volume 37, Number 2, issued February 1995, REBECK et al., "Multiple, Diverse Senile Plaque-Associated Proteins are Ligands of an Apolipoprotein E Receptor, the alpha-2-Macroglobulin Receptor/Low-Density-Lipoprotein Receptor-Related Protein", pages 211-217. *
CELL, Volume 6, issued 1995, KOUNNAS et al., "LDL Receptor-related Protein (LRP) Binds Secreted beta-Amyloid Precursor Protein and Mediates its Degradation", page 327A, Abstract 1901. *
CELL, Volume 82, No. 2, issued 28 July 1995, KOUNNAS et al., "LDL Receptor-Related Protein, a Multifunctional ApoE Receptor, Binds Secreted beta-Amyloid Precursor Protein and Mediates Its Degradation", pages 331-340. *
FIBRINOLYSIS, Volume 10, issued June 1996, HORN et al., "Recombinant Fragments of the Second Cluster of Complement-type Repeats of LRP Mediate High Affinity Binding of Different Ligands as Determined by Surface Plasmon Resonance", Abstract 61. *
JOURNAL OF LIPID RESEARCH, Volume 36, Number 9, issued 1995, RAFFAI et al., "Molecular Characterization of Two Monoclonal-Antibodies Specific for the LDL Receptor-Binding Site of Human Apolipoprotein E", pages 1905-1918. *
NEUROSCIENCE, Volume 65, Number 4, issued 1995, LORENT et al., "Expression in Mouse Embryos and in Adult Mouse Brain of Three Members of the Amyloid Precursor Protein Family, of the Alpha-2-Macroglobulin Receptor/Low Density Lipoprotein Receptor-Related Protein and of its Ligands Apolipoprotein E, Lipoprotein Lipase, alpha-2-Macroglobulin and the 40,000 Molecular Weight Receptor-Associated Protein", pages 1009-1025. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7045531B1 (en) 1997-03-11 2006-05-16 The General Hospital Corporation Composition comprising a metal chelator and a method of treating amyloidosis by administering the metal chelator
US6472140B1 (en) 1997-09-05 2002-10-29 The General Hospital Corporation α-2- macroglobulin therapies and drug screening methods for Alzheimer's disease.
US6638711B1 (en) 1999-04-29 2003-10-28 The General Hospital Corporation Methods for identifying an agent that inhibits oxygen-dependent hydrogen peroxide formation activity but does not inhibit superoxide-dependent hydrogen peroxide formation
US6555322B1 (en) 1999-08-27 2003-04-29 Fibrogen, Inc. α2-macroglobulin receptor as a receptor for CTGF
WO2001015729A1 (fr) * 1999-08-27 2001-03-08 Fibrogen, Inc. Recepteur du facteur de croissance du tissu conjonctif, agonistes et antagonistes dudit recepteur, et leurs utilisations therapeutiques et diagnostiques
US7695903B2 (en) 2000-05-23 2010-04-13 The University Of Southern California Low-density lipoprotein receptor related protein-1 (LRP-1)in clearance of alzheimer's amyloid-beta peptide from the central nervous system
WO2001090758A3 (fr) * 2000-05-23 2002-08-29 Univ Southern California Role du systeme vasculaire cerebral et du transport a travers la barriere hemato-encephalique dans la clairance du peptide beta-amyloide d'alzheimer dans le systeme nerveux central
US7179462B2 (en) 2000-06-02 2007-02-20 University Of Connecticut Health Center α (2) macroglobulin receptor as a heat shock protein receptor and uses thereof
US7449557B2 (en) 2000-06-02 2008-11-11 University Of Connecticut Health Center Complexes of alpha (2) macroglobulin and antigenic molecules for immunotherapy
US7186515B1 (en) 2000-06-02 2007-03-06 University Of Connecticut Health Center Alpha(2) macroglobulin receptor as a heat shock protein receptor and uses thereof
US8877204B2 (en) 2003-02-20 2014-11-04 University Of Connecticut Health Center Methods and compositions for the treatment of cancer and infectious disease using alpha (2) macroglobulin-antigenic molecule complexes
US9566348B2 (en) 2003-02-20 2017-02-14 University Of Connecticut Health Center Methods and compositions for the treatment of cancer and infectious disease using alpha(2) macroglobulin-antigenic molecule complexes
EP1718324A4 (fr) * 2003-06-11 2009-07-15 Socratech L L C Derive soluble de la proteine liee au recepteur de lipoproteine de basse densite de liaison directe au peptide amyloide beta d'alzheimer
US7608586B2 (en) 2003-06-11 2009-10-27 The University Of Rochester Soluble low-density lipoprotein receptor related protein binds directly to Alzheimer's amyloid-beta peptide

Also Published As

Publication number Publication date
CA2246561A1 (fr) 1997-02-13

Similar Documents

Publication Publication Date Title
US6156311A (en) Modulators of expression and function of LRP in alzheimer's disease
CA2212887C (fr) Anticorps monoclonal specifique du peptide .beta.a4
US7541438B2 (en) Monoclonal antibody against connective tissue growth factor and medicinal uses thereof
US20030026803A1 (en) Compositions for inhibiting macrophage activity
EA016193B1 (ru) Антитела, направленные против бета-амилоидного пептида, и способы их использования
US20060134121A1 (en) DII4 antagonists, assays, and therapeutic methods thereof
JPH10504458A (ja) 単鎖Ｆｖ分子の製造方法
KR100496335B1 (ko) 항-Fas 리간드항체 및 이 항체를 이용한 측정법
EP2325203A2 (fr) Eléments et procédés permettant la production de cadherines
JPWO1996029350A1 (ja) Ｆａｓリガンドに特異的に反応するモノクローナル抗体及びその製造方法
WO1997004794A1 (fr) Modulateurs d'expression et de fonction de la proteine associee au recepteur de la lipoproteine basse densite (lrp) dans la maladie d'alzheimer
CA2245956A1 (fr) Anticorps monoclonaux specifiques des cadherines de cellules endotheliales et leurs utilisations
WO1999032520A1 (fr) Dosage de ldl denaturees
WO1999015652A1 (fr) Proteine megsine
US20060088528A1 (en) Methods and compositions for treating alzheimer's disease
JP4537507B2 (ja) 結合組織増殖因子に対するモノクローナル抗体及びその医薬用途
EP1367123A1 (fr) Neurotonine et utilisation
EP0956508A1 (fr) Icam-4 et ses utilisations diagnostiques
WO2002024929A2 (fr) MONOCHAINE DE MICROGLOBULINE BETA 2 SOLUBLE (β2M)/HFE UTILE POUR DES APPLICATIONS BIOTECHNOLOGIQUES ET THERAPEUTIQUES
MXPA97006189A (en) Specific monoclonal antibody for beta peptide
HK1114105B (en) Monoclonal antibody specific for beta a4 peptide
WO1999018441A1 (fr) Icam-4 et ses utilisations diagnostiques

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP MX

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

CFP Corrected version of a pamphlet front page
CR1 Correction of entry in section i
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2246561

Country of ref document: CA

Ref country code: CA

Ref document number: 2246561

Kind code of ref document: A

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase