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
  • C07K14/52—Cytokines; Lymphokines; Interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
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  • A61P15/18—Feminine contraceptives
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
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  • 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
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
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  • A61P31/14—Antivirals for RNA viruses
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
  • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5256—Virus expressing foreign proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention concerns the fields of molecular medicine and targeted delivery of therapeutic agents. More specifically, the present invention relates to compositions and methods for the in vitro identification of peptides that selectively target organs, tissues or cell types and the therapeutic or diagnostic use of such peptides.
• Therapeutic treatment of many disease states is limited by the systemic toxicity of the therapeutic agents used. Cancer therapeutic agents in particular exhibit a very low therapeutic index, with rapidly growing normal tissues such as skin and bone marrow affected at concentrations of agent that are not much higher than the concentrations used to kill tumor cells. Treatment of cancer and other organ, tissue or cell type confined disease states would be greatly facilitated by the development of compositions and methods for targeted delivery to a desired organ, tissue or cell type of a therapeutic agent.
• Phage display libraries expressing transgenic peptides on the surface of bacteriophage were initially developed to map epitope binding sites of immunoglobulins (Smith and Scott, 1986, 1993). Such libraries can be generated by inserting random oligonucleotides into cDNAs encoding a phage surface protein, generating collections of phage particles displaying unique peptides in as many as 10 9 permutations. (Pasqualini and Ruoslahti, 1996, Arap et al, 1998a; Arap et al 1998b).
• Attachment of therapeutic agents to targeting peptides has resulted in the selective delivery of the agent to a desired organ, tissue or cell type in the mouse model system.
• Targeted delivery of chemotherapeutic agents and proapoptotic peptides to receptors located in tumor angiogenic vasculature resulted in a marked increase in therapeutic efficacy and a decrease in systemic toxicity in tumor-bearing mouse models (Arap et al., 1998a, 1998b; Ellerby et al., 1999).
• Ad5 Adenovirus type 5 (Ad5)-based vectors have been commonly used for gene transfer studies (Weitzman et al, 1997; Zhang, 1999). The attachment of Ad5 to the target cell is mediated by the capsid's fiber knob region, which interacts with cell surface receptors, including the coxsackie adenovirus receptor (CAR) and possibly with MHC class I (Bergelson et al, 1997; Hong et al, 1997).
• CAR coxsackie adenovirus receptor
• the present invention solves a long-standing need in the art by providing compositions and in vitro methods for the identifying and using targeting peptides that are selective for specific organs, tissues or cell types.
• such methods and compositions may be used to identify one or more receptors for a targeting peptide.
• the compositions and methods may be used to identify naturally occurring ligands for known or newly identified receptors.
• the methods may comprise contacting a targeting peptide to an organ, tissue or cell containing a receptor of interest, allowing the peptide to bind to the receptor, and identifying the receptor by its binding to the peptide.
• the targeting peptide contains at least three contiguous amino acids selected from any of SEQ ID NO:5 through SEQ ID NO:65, SEQ ID NO:67 through SEQ ID NO: 165 and SEQ ID NO: 176 through SEQ ID NO:270.
• the targeting peptide comprises a portion of an antibody against the receptor.
• the targeting peptide may contain a random amino acid sequence.
• the contacting step can utilize intact organs, tissues or cells, or may alternatively utilize homogenates or detergent extracts of the organs, tissues or cells.
• the cells to be contacted may be genetically engineered to express a suspected receptor for the targeting peptide.
• the targeting peptide is modified with a reactive moiety that allows its covalent attachment to the receptor.
• the reactive moiety is a photoreactive group that becomes covalently attached to the receptor when activated by light.
• the peptide is attached to a solid support and the receptor is purified by affinity chromatography.
• the solid support comprises magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix.
• the targeting peptide inhibits the activity of the receptor upon binding to the receptor.
• receptor activity can be assayed by a variety of methods known in the art, including but not limited to catalytic activity and binding activity.
• the receptor is an endostatin receptor, a metalloprotease or an aminopeptidase.
• one or more ligands for a receptor of interest may be identified by the disclosed methods and compositions.
• One or more targeting peptides that mimic part or all of a naturally occurring ligand may be identified by phage display and biopanning.
• a naturally occurring ligand may be identified by homology with a single targeting peptide that binds to the receptor, or a consensus motif of sequences that bind to the receptor.
• an antibody may be prepared against one or more targeting peptides that bind to a receptor of interest. Such antibodies may be used for identification or immunoaffinity purification of the native ligand.
• the targeting peptides of the present invention are of use for the selective delivery of therapeutic agents, including but not limited to gene therapy vectors and fusion proteins, to specific organs, tissues or cell types in subjects.
• therapeutic agents including but not limited to gene therapy vectors and fusion proteins
• the skilled artisan will realize that the scope of the claimed methods of use include any disease state that can be treated by targeted delivery of a therapeutic agent to a desired organ, tissue or cell type in a subject.
• disease states include those where the diseased cells are confined to a specific organ, tissue or cell type, such as non- metastatic cancer, other disease states may be treated by an organ, tissue or cell type- targeting approach.
• One embodiment of the present invention concerns isolated peptides of 100 amino acids or less in size, comprising at least 3 contiguous amino acids of a targeting peptide sequence, selected from any of SEQ ID NO:5 through SEQ ID NO:65, SEQ ID NO:67 through SEQ ID NO: 165 and SEQ ID NO: 176 through SEQ ID NO:270.
• the isolated peptide is 50 amino acids or less, more preferably 30 amino acids or less, more preferably 20 amino acids or less, more preferably 10 amino acids or less, or even more preferably 5 amino acids or less in size.
• the isolated peptide of claim 1 comprises at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous amino acids of a targeting peptide sequence, selected from any of SEQ ID NO:5 through SEQ ID NO:65, SEQ ID NO:67 through SEQ ID NO:165 and SEQ ID NO:176 through SEQ ID NO:270.
• the isolated peptide is attached to a molecule.
• the attachment is a covalent attachment.
• the molecule is a drug, a chemotherapeutic agent, a radioisotope, a pro- apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, an imaging agent, a survival factor, an anti-apoptotic agent, a hormone antagonist, a nucleic acid or an antigen.
• Those molecules are representative only.
• Molecules within the scope of the present invention include virtually any molecule that may be attached to a targeting peptide and administered to a subject.
• the pro-aptoptosis agent is gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK) 2 (SEQ ID NO:l), (KLAKKLA) 2 (SEQ ID NO:2), (KAAKKAA) 2 (SEQ ID NO:3) or (KLGKKLG) 3 (SEQ ID NO:4).
• the anti-angiogenic agent is angiostatin5, pigment epithelium-drived factor, angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP- 10, Gro- ⁇ , thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, Docetaxel, polyamines, a proteasome inhibitor, a kinase inhibitor, a signaling peptide inhibitors,
• the cytokine is interleukin 1 (IL-1), IL-2, IL-5.
• IL-1 interleukin 1
• IL-2 IL-2
• IL-5 IL-5
• EL-10 interferon- ⁇
• IF- ⁇ interferon- ⁇
• IF- ⁇ IF- ⁇
• IF- ⁇ tumor necrosis factor-
• GM-CSF granulocyte macrophage colony stimulating factor
• the isolated peptide is attached to a macromolecular complex.
• the attachment is a covalent attachment.
• the macromolecular complex is a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic bead, a yeast cell, a mammalian cell, a cell or a microdevice. These are representative examples only. Macromolecular complexes within the scope of the present invention include virtually any macromolecular complex that may be attached to a targeting peptide and administered to a subject.
• the isolated peptide is attached to a eukaryotic expression vector, more preferably a gene therapy vector.
• the isolated peptide is attached to a solid support, preferably magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, " a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix.
• a solid support preferably magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, " a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix.
• Additional embodiments of the present invention concern fusion proteins comprising at least 3 contiguous amino acids of a sequence selected from any of SEQ ID NO:5 through SEQ ID NO:65, SEQ ID NO:67 through SEQ ID NO:165 and SEQ ID NO: 176 through SEQ ID NO:270.
• compositions comprising the claimed isolated peptides or fusion proteins in a pharmaceutically acceptable carrier.
• kits comprising the claimed isolated peptides or fusion proteins in one or more containers.
• a targeting peptide for a desired organ, tissue or cell type comprising selecting a targeting peptide for a desired organ, tissue or cell type, attaching said targeting peptide to a molecule, macromolecular complex or gene therapy vector, and providing said peptide attached to said molecule, complex or vector to a subject.
• the targeting peptide is selected to include at least 3 contiguous amino acids from any of SEQ ID NO:5 through SEQ ID NO:65, SEQ ID NO:67 through SEQ ID NO: 165 and SEQ ID NO: 176 through SEQ ID NO:270.
• the organ, tissue or cell type is bone marrow, lymph node, prostate cancer or prostate cancer that has metastasized to bone marrow.
• the molecule attached to the targeting peptide is a chemotherapeutic agent, an antigen or an imaging agent.
• chemotherapeutic agent an antigen or an imaging agent.
• any organ, tissue or cell type can be targeted for delivery, using targeting peptides attached to any molecule, macromolecular complex or gene therapy vector.
• nucleic acids of 300 nucleotides or less in size encoding a targeting peptide.
• the isolated nucleic acid is 250, 225, 200, 175, 150, 125, 100, 75, 50, 40, 30, 20 or even 10 nucleotides or less in size.
• the isolated nucleic acid is incorporated into a eukaryotic or a prokaryotic expression vector.
• the vector is a plasmid, a cosmid, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAG), a virus or a bacteriophage.
• the isolated nucleic acid is operatively linked to a leader sequence that localizes the expressed peptide to the extracellular surface of a host cell.
• Additional embodiments of the present invention concern methods of treating a disease state comprising selecting a targeting peptide that targets cells associated with the disease state, attaching one or more molecules effective to treat the disease state to the peptide, and administering the peptide to a subject with the disease state.
• the targeting peptide includes at least three contiguous amino acids selected from any of SEQ ID NO:5 through SEQ ID NO:65, SEQ ID NO:67 through SEQ ID NO: 165 and SEQ ID NO: 176 through SEQ ID NO:270.
• the disease state is diabetes mellitus, inflammatory disease, arthritis, atherosclerosis, cancer, autoimmune disease, bacterial infection and viral infection.
• the molecular adaptor is a targeting peptide that has been incorporated into a surface protein of a gene therapy vector.
• the gene therapy vector has been genetically engineered to contain AAN (adeno-associated virus) sequences.
• AAN adeno-associated virus
• Tumor targeting peptides identified by the methods disclosed in the instant application may be attached to therapeutic agents, including but not limited to molecules or macromolecular assemblages and administered to a patient with cancer, providing for increased efficacy and decreased systemic toxicity of the therapeutic agent.
• therapeutic agents within the scope of the present invention include but are not limited to chemotherapeutic agents, radioisotopes, pro-apoptosis agents, cytotoxic agents, cytostatic agents and gene therapy vectors.
• tumor targeting peptide is incorporated into the capsule of a phage gene therapy vector to target delivery of the phage to angiogenic endothelial cells in tumor blood vessels.
• Yet another embodiment of the present invention concerns methods of identifying targeting peptides against antibodies from an individual with a disease state, comprising obtaining a sample of serum from the individual, obtaining antibodies from the sample, adding a phage display library to the antibodies and collecting phage bound to the antibodies.
• the antibodies are attached to a solid support, more preferably attached to protein G attached to beads.
• a subtraction step is added where the phage display library is first screened against antibodies from an individual who does not have the disease state. Only phage that do not bind to these control antibodies are used to obtain phage binding to the diseased individual's antibodies.
• the antigen comprises one or more targeting peptides.
• the targeting peptides are prepared and immobilized on a solid support, serum containing antibodies is added and antibodies that bind to the targeting peptides are collected.
• a phage display library displaying the antigen binding portions of antibodies from a subject is prepared, the library is screened against one or more antigens and phage that bind to the antigens are collected.
• the antigen is a targeting peptide.
• FIG. 1 Selection of peptide library on immunoglobulins from the serum of metastatic prostate cancer patients. Each successive round of panning demonstrates an increase in selectivity as measured by the increase in total number of transducing units for cancer patients relative to the serum of control volunteers. Three metastatic androgen-independents (patients A, B, and D) serum samples and one metastatic androgen-dependent (patient C) serum sample were examined. Standard error of the mean (S.E.M.) from triplicate plating is shown.
• S.E.M. Standard error of the mean
• FIG. 2 Selection of peptide library on immunoglobulins from the serum of metastatic prostate cancer patients. A series of 100-fold dilutions (1:100-1:1200) was performed for each patient's serum to test specific binding of cancer antibodies to immobilized GST-fusion proteins by ELISA.
• FIG. 3 A Correlation between patients' survival outcome and peptides' reactivity according to the Kaplan-Meier method. The data represents all of the prostate cancer patients examined. A log-rank test was implemented to detect significant difference in survival time between peptide reacting group versus non-reacting group. No statistical significance was observed with peptide A (p- value of 0.75).
• FIG. 3B Correlation between patients' survival outcome and peptides' reactivity according to the Kaplan-Meier method. No statistical significance was observed with peptide B (p-value of 0.83).
• FIG. 4A-4F A homology search was run of the phage peptide sequences binding to endostatin versus known protein databases. Candidate proteins for endogenous receptors for endostatin are indicated, along with the degree of homology and, where available, the GenBank accession number.
• FIG. 5 Panning of phage peptide library on rhAngiostatin.
• the rhAngiostatin protein was immobilized onto microtiter wells and screened against a CX C library. Bound phage were recovered after infection with K91 bacteria. Results illustrate the average number of phage - transducing units x 10 2 recovered per well when coated with rhAngiostatin, rh-Endostatin and BSA after the first, second and third rounds of selection. (First round to left, second round in middle, and third round to right of FIG. 20).
• FIG. 6 Selectivity of rhAngiostatin-targeted phage to immobilized proteins. rhAngiostatin, rh-Endostatin and rmEndostatin and BSA were coated on microtiter wells at 1 ⁇ g/ml and used to bind phage expressing the selected angiostatin targetting peptides. The data represent the mean colony counts from triplicate wells, with standard error less than 10% of the mean.
• FIG. 7 CD13 and HGF bind to rhAngiostatin.
• the wells of a 96-well plate were coated with l ⁇ g/ml of protein (CD13, HGF, TSP-1, LN, COL IV, FN, VN or BSA), and incubated with rhAngiostatin.
• the amount of rhAngiostatin binding to each well was determined spectrophotometrically following the addition of anti-hAngiostatin polyclonal antibody, peroxidase conjugated anti-goat IgG and substrate. Error bars indicate standard error of the mean.
• FIG. 8 Selection of a peptide library on immunoglobulins from serum of a patient with Hodgkin's disease. Each successive round of panning show increased specificity since the number of IgG bound infective phage, as measured by the total number of transducing units, increases for the HD sera relative to the control sera.
• FIG. 9 Selected peptide motifs and homologous proteins. Targeting peptides against circulating antibodies from the sera of Hodgkin's disease patients were identified as described. Homologous proteins were identified by computer search of the Swiss Protein database.
• FIG. 10 Sequence homologies between HD targeting peptides and viral proteins from Macaca mulatta rhadinovirus, KSHN (Kaposi's sarcoma-associated virus) and EBN (Epstein Barr virus).
• FIG. 11 A Transduction of tumor cells by targeted phage is specific. Quantitative analysis of cell transduction by targeted and control phage. Tumor cells were incubated with targeted (HWGF- ⁇ -gal or RGD-4C- ⁇ -gal) or control insertless phage (fd-tet- ⁇ -gal). An anti- ⁇ -gal antibody was used for staining; gene expression was detected by immunofluorescence and results are expressed in % of ⁇ -gal positive cells. In each case, standard error of the mean (SEM) was calculated after counting 10 fields under the microscope in three independent experiments.
• SEM standard error of the mean
• FIG. 11B Transduction of tumor cells by targeted phage is specific. Inhibition of HWGF- ⁇ -gal phage transduction by the synthetic CTTHWGFTLC (SEQ ID NO: 167) peptide. Control peptides did not inhibit transduction of the tumor cells by the targeted phage. Non-specific transduction levels were determined by using the control insertless phage. Shown are mean ⁇ SEM obtained from duplicate wells.
• FIG. 11C Transduction of tumor cells by targeted phage is specific. Inhibition of RGD-4C- ⁇ -gal phage transduction by the synthetic RGD-4C peptide (SEQ ID NO: 166). Control peptides did not inhibit transduction of the tumor cells by the targeted phage. Non-specific transduction levels were determined by using the control insertless phage. Shown are mean ⁇ SEM obtained from duplicate wells.
• FIG. 12 Specific transduction in vivo by lung-targeting phage.
• Lung (targeted organ) and liver (control organ) were evaluated for ⁇ -gal expression after systemic administration of lung targeting GFE-phage or control phage into C57B1/6 immunocompetent mice.
• FIG. 13 Enhancement of transduction by genotoxic agents or genetic trans- complementation.
• Semi-confluent cells were infected with 10 5 TU of phage per cell for four hours. Next, the cells were incubated for 36 hours followed by addition of genotoxic drugs (topotecan, 10 ⁇ M; cisplatin, 10 ⁇ M) or application of physical agents such as ultraviolet radiation (UN; 15 J/m 2 ).
• genotoxic drugs topotecan, 10 ⁇ M; cisplatin, 10 ⁇ M
• a phage mixture of RGD-4C- ⁇ gal forward and reverse clones (molar ratio 1; termed For/Rev) at the same number of phage TU of RGD-4C- ⁇ gal phage was also tested.
• GFP normalized green fluorescent protein
• FIG. 14 Quantification of ⁇ -galactosidase expression in target cells after transduction with RGD-4C- ⁇ -gal phage particles.
• RGD4C- ⁇ -gal phage were incubated with MDA-MB-435 cells and HWGF- ⁇ -gal phage were incubated with KS1767 cells.
• the untargeted phage vector fd- ⁇ -gal was used as a control, ⁇ -galactosidase expression was assessed by immunofluorescence and counting positive cells.
• RFs Replicating forms
• a chimeric RGD4C-fMCSl phage vector was obtained by subcloning the 5.4 kb Bam Hi Sac ⁇ fragment of RF RGD4C into the Bam Hi Sac H sites of fMCSl.
• the fMCSl vector is a fd-tet derived phage vector not used for display, but which contains a multiple cloning site (MCS) including a Pstl site.
• MCS multiple cloning site
• the final RGD4C- ⁇ gal phage construct (14 kb) was obtained by subcloning a Pstl fragment containing a 4.5 kb CMV-driven eukaryotic ⁇ gal cassette into RGD4C- fMCSl in forward and reverse orientations.
• the resulting phage vectors were termed fRGD4C- ⁇ gal and rRGD4C- ⁇ Gal.
• FIG. 16 Gene expression of transgenic phage in eukaryotic cells. Uptake and expression of phage encoding a marker gene was examined in human cells.
• FIG. 18 Pancreatic islet targeting peptides and homologous proteins.
• FIG. 19 Pancreatic islet targeting peptides and homologous proteins.
• FIG. 20 Pancreatic islet targeting peptides and homologous proteins.
• Candidate endogenous proteins mimicked by the pancreatic islet targeting peptides CLASGMDAC (S ⁇ Q ID NO:243), CHD ⁇ RTGRC (S ⁇ Q ID NO:244), CAHHALM ⁇ C (SEQ ID NO:245) and CMQGAATSC (SEQ ID NO:246), identified by standard homology searches.
• FIG. 21 Pancreatic islet targeting peptides and homologous proteins.
• FIG. 22 Binding of phage containing the CVPELGHEC (SEQ ID NO:271) and CEELGFELGC (SEQ ID NO:272) targeting peptides to IgG's isolated from ovarian cancer patient #2 ascites, normal serum and BSA.
• FIG. 23 Binding of phage containing the CVPELGHEC (SEQ ID NO:271) targeting peptide to IgG's isolated from normal serum or from ascites of ovarian cancer patient #1 or #2. Control fd-tet phage contained no insert DNA.
• FIG. 24 Binding of phage containing the CVPELGHEC (SEQ ID NO:271) targeting peptide to IgG's isolated from serum of ovarian cancer patient #2, normal serum or BSA.
• FIG. 25 Homology between ovarian cancer targeting peptides ELGFELG (SEQ ID NO:250) and VPELGHE (SEQ ID NO:249) to matrix metalloproteinase proteins.
• a or “an” may mean one or more.
• the words “a” or “an” may mean one or more than one.
• another may mean at least a second or more of an item.
• a “targeting peptide” is a peptide comprising a contiguous sequence of amino acids, that is characterized by selective localization to an organ, tissue or cell type. Selective localization may be determined, for example, by methods disclosed below, wherein the putative targeting peptide sequence is incorporated into a protein that is displayed on the outer surface of a phage.
• a phage expressing a targeting peptide sequence is considered to be selectively locallized to a cell type, tissue or organ if it exhibits greater binding in that tissue or organ compared to a control tissue or organ.
• selective localization of a targeting peptide should result in a two-fold or higher enrichment of the phage in the target organ, tissue or cell type, compared to a control organ, tissue or cell type.
• An alternative method to determine selective localization is that phage expressing the putative target peptide preferably exhibit a two-fold, more preferably a three-fold or higher enrichment in the target organ compared to control phage that express a non-specific peptide or that have not been genetically engineered to express any putative target peptides.
• Another method to determine selective localization is that locallization to the target organ, tissue or cell type of phage expressing the target peptide is at least partially blocked by the co- administration of a synthetic peptide containing the target peptide sequence.
• Targeting peptide and “homing peptide” are used synonymously herein.
• a "phage display library” means a collection of phage that have been genetically engineered to express a set of putative targeting peptides on their outer surface.
• DNA sequences encoding the putative targeting peptides are inserted in frame into a gene encoding a phage capsule protein.
• the putative targeting peptide sequences are in part random mixtures of all twenty amino acids and in part non-random.
• the putative targeting peptides of the phage display library exhibit one or more cysteine residues at fixed locations within the targeting peptide sequence.
• a "macromolecular complex” refers to a collection of molecules that may be random, ordered or partially ordered in their arrangement.
• the term encompasses biological organisms such as bacteriophage, viruses, bacteria, unicellular pathogenic organisms, multicellular pathogenic organisms, and prokaryotic or eukaryotic cells.
• the term also encompasses non-living assemblages of molecules, such as liposomes, microcapsules, microparticles, magnetic beads and microdevices. The only requirement is that the complex contains more than one molecule.
• the molecules may be identical, or may differ from each other.
• a "receptor" for a targeting peptide includes but is not limited to any molecule or complex of molecules that binds to a targeting peptide.
• Non-limiting examples of receptors include peptides, proteins, glycoproteins, lipoproteins, epitopes, lipids, carbohydrates, multi-molecular structures, a specific conformation of one or more molecules and a morphoanatomic entity.
• a "receptor" is a naturally occurring molecule or complex of molecules that is present on the lumenal surface of cells forming blood vessels within a target organ, tissue or cell type.
• a “subject” refers generally to a mammal. In certain preferred embodiments, the subject is a mouse or rabbit. In even more preferred embodiments, the subject is a human.
• the methods described herein for identification of targeting peptides involve the in vitro administration of phage display libraries.
• Various methods of phage display and methods for producing diverse populations of peptides are well known in the art.
• the phage display technique involves genetically manipulating bacteriophage so that small peptides can be expressed on their surface (Smith et al, 1985, 1993).
• larger protein domains such as single-chain antibodies can also be displayed on the surface of phage particles (Arap et al, 1998a).
• Previous methods for identifying amino acid sequences for a targeting a given organ, tissue or cell type involved isolation by "biopanning" (Pasqualini and Ruoslahti, 1996; Pasqualini, 1999).
• a library of phage containing putative targeting peptides was administered to an animal model and samples of organs, tissues or cell types containing phage were collected.
• the phage may be recovered from a sample of organ, tissue or cell type that has been exposed to a phage library, hi alternative embodiments, phage may be recovered by infecting them into pilus-positive bacteria. The bacteria are not lysed by the phage but rather secrete multiple copies of phage that display a particular insert. Phage that bind to a target can be eluted from the organ, tissue or cell type and amplified by growing them in host bacteria. Alternatively, targeting peptide sequences may be amplified from the sample and inserted into fresh phage DNA, then infected into host bacteria. By either technique, targeting peptides may be recovered from the target and amplified.
• the amino acid sequence of the peptides may be determined by sequencing the DNA corresponding to the targeting peptide insert in the phage genome.
• the identified targeting peptide can then be produced , as a synthetic peptide by standard protein chemistry techniques (Arap et al, 1998a, Smith et al, 1985). This approach allows circulating targeting peptides to be detected in an unbiased functional assay, without any preconceived notions about the nature of their target.
• Once a candidate target is identified as the receptor of a targeting peptide, it can be isolated, purified and cloned by using standard biochemical methods (Pasqualini, 1999; Rajotte and Ruoslahti, 1999).
• Phage libraries displaying linear, cyclic, or double cyclic peptides may be used within the scope of the present invention. However, phage libraries displaying 3 to 10 random residues in a cyclic insert (CX 3 - ⁇ oC) are preferred, since single cyclic peptides tend to have a higher affinity for the target organ than linear peptides. Libraries displaying double-cyclic peptides (such as CX 3 C X 3 CX 3 C; Rojotte et al, 1998) have been successfully used. However, the production of the cognate synthetic peptides, although possible, can be complex due to the multiple conformers with different dissulfide bridge arrangements .
• a panel of peptide motifs that target the blood vessels of tumor xenografts in nude mice has been assembled (Arap et al, 1998a; reviewed in Pasqualini, 1999). These motifs include the sequences RGD-4C, NGR, and GSL.
• the RGD-4C peptide has previously been identified as selectively binding ⁇ v integrins and has been shown to home to the vasculature of tumor xenografts in nude mice (Arap et al, 1998a, 1998b; Pasqualini et al, 1997).
• the receptors for the tumor homing RGD4C targeting peptide has been identified as ⁇ v integrins (Pasqualini et al, 1997).
• the ⁇ v integrins play an important role in angiogenesis.
• the ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins are absent or expressed at low levels in normal endothelial cells but are induced in angiogenic vasculature of tumors (Brooks et al, 1994; Hammes et al, 1996).
• Aminopeptidase N/CD13 has recently been identified as an angiogenic receptor for the NGR motif (Burg et al, 1999). Aminopeptidase N/CD13 is strongly expressed in the angiogenic blood vessels of cancer and in other angiogenic tissues.
• Tumor-homing phage co-localize with their receptors in the angiogenic vasculature of tumors but not in non-angiogenic blood vessels in normal tissues (Arap et al, 1998b). Immunohistochemical evidence shows that vascular targeting phage bind to human tumor blood vessels in tissue sections (Pasqualini et al, 2000) but not to normal blood vessels. A negative control phage with no insert (fd phage) did not bind to normal or tumor tissue sections. The expression of the angiogenic receptors was evaluated in cell lines, in non-proliferating blood vessels and in activated blood vessels of tumors and other angiogenic tissues such as corpus luteum.
• Angiogenic neovasculature expresses markers that are either expressed at very low levels or not at all in non-proliferating endothelial cells (not shown).
• the markers of angiogenic endothelium include receptors for vascular growth factors, such as specific subtypes of VEGF and basic FGF receptors, and ⁇ v integrins, among many others (Mustonen and Alitalo, 1995).
• vascular growth factors such as specific subtypes of VEGF and basic FGF receptors, and ⁇ v integrins, among many others (Mustonen and Alitalo, 1995).
• tumor vascular markers are proteases and some of the markers also serve as viral receptors.
• Alpha v integrins are receptors for adenoviruses (Wickham et al., 1997c) and CD13 is a receptor for coronaviruses (Look et al, 1989).
• MMP-2 and MMP-9 are receptors for echo viruses (Koivunen et al, 1999).
• Aminopeptidase A also appears to be a viral receptor.
• Bacteriophage may use the same cellular receptors as eukaryotic viruses.
• separation of phage bound to the cells of a target organ, tissue or cell type from unbound phage is achieved using the BRASJL technique (Provisional Patent Application No. 60/231,266 filed September 8, 2000; U.S. Patent Application entitled, "Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL)" by Arap, Pasqualini and Giordano, filed concurrently herewith, incorporated herein by reference in its entirety).
• BRASIL Biopanning and Rapid Analysis of Soluble Interactive Ligands
• an organ, tissue or cell type is gently separated into cells or small clumps of cells that are suspended in an first, preferably aqueous phase.
• aqueous phase is layered over a second, preferably organic phase of appropriate density and centrifuged.
• Cells attached to bound phage are pelleted at the bottom of the centrifuge tube, while unbound phage remain in the aqueous phase. This allows a more efficient separation of bound from unbound phage, while maintaining the binding interaction between phage and cell.
• BRASIL may be performed in an in vitro protocol, in which organs, tissues or cell types are exposed to a phage display library in an aqueous phase in vitro before centrifugation.
• a subtraction protocol may be used with BRASIL or other screening protocols to further reduce background phage binding.
• the purpose of subtraction is to remove phage from the library that bind to cells other than the cell of interest, or that bind to inactivated cells.
• the phage library may be screened against a control cell line, tissue or organ sample that is not the targeted cell, tissue or organ. After subtraction the library may be screened against the cell, tissue or organ of interest.
• an unstimulated, quiescent cell line, tissue or organ may be screened against the library and binding phage removed. The cell line, tissue or organ is then activated, for example by administration of a hormone, growth factor, cytokine or chemokine and the activated cell line screened against the subtracted phage library.
• the present invention concerns novel compositions comprising at least one protein or peptide.
• a protein or peptide generally refers, but is not limited to, a protein of greater than about 200 amino acids, up to a full length sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids.
• proteins proteins
• polypeptide and “peptide are used interchangeably herein.
• the size of the at least one protein or peptide may comprise, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120, aboout 130, about 140, about 150, about 160, about 170, about 180, about
• amino acid residue refers to any naturally occuring amino acid, any amino acid derivitive or any amino acid mimic known in the art.
• the residues of the protein or peptide are sequential, without any non- amino acid interrupting the sequence of amino acid residues.
• the sequence may comprise one or more non-amino acid moieties.
• the sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties.
• protein or peptide encompasses amino acid sequences comprising at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid, including but not limited to those shown on Table 1 below.
• Proteins or peptides may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides.
• the nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art.
• One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http://www.ncbi.nlm.nih.gov/).
• the coding regions for known genes may be amplified and/or expressed using the techniques disclosed herein or as would be know to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.
• peptide mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al, "Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al, Eds., Chapman and Hall, New York (1993), incorporated herein by reference.
• the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.
• a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
• fusion proteins These molecules generally have all or a substantial portion of a targeting peptide, linked at the N- or C-terminus, to all or a portion of a second polypeptide or proteion.
• fusions may employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
• Another useful fusion includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification.
• fusion proteins include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.
• the fusion proteins of the instant invention comprise a targeting peptide linked to a therapeutic protein or peptide.
• proteins or peptides that may be incorporated into a fusion protein include cytostatic proteins, cytocidal proteins, pro-apoptosis agents, anti-angiogenic agents, hormones, cytokines, growth factors, peptide drugs, antibodies, Fab fragments antibodies, antigens, receptor proteins, enzymes, lectins, MHC proteins, cell adhesion proteins and binding proteins.
• fusion protein comprising a targeting peptide.
• Methods of generating fusion proteins are well known to those of skill in the art. Such proteins can be produced, for example, by chemical attachment using bifunctional cross-linking reagents, by de novo synthesis of the complete fusion protein, or by attachment of a DNA sequence encoding the targeting peptide to a DNA sequence encoding the second peptide or protein, followed by expression of the intact fusion protein.
• a protein or peptide may be isolated or purified.
• Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the homogenization and crude fractionation of the cells, tissue or organ to polypeptide and non-polypeptide fractions.
• the protein or polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity).
• Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing.
• An example of receptor protein purification by affinity chromatography is disclosed in U.S. Patent No. 5,206,347, the entire text of which is incorporated herein by reference.
• a particularly efficient method of purifying peptides is fast protein liquid chromatography (FPLC) or even HPLC.
• a purified protein or peptide is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
• An isolated or purified protein or peptide therefore, also refers to a protein or peptide free from the environment in which it may naturally occur.
• purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity.
• substantially purified this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins in the composition.
• Various methods for quantifying the degree of purification of the protein or peptide are known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
• a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity therein, assessed by a "-fold purification number.”
• the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification, and whether or not the expressed protein or peptide exhibits a detectable activity.
• Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
• Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule to which it can specifically bind to. This is a receptor-ligand type of interaction.
• the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (e.g., altered pH, ionic strength, temperature, etc.).
• the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
• the ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
• the targeting peptides of the invention can be synthesized in solution or on a solid support in accordance with conventional techniques.
• Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tarn et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Short peptide sequences, usually from about 6 up to about 35 to 50 amino acids, can be readily synthesized by such methods.
• recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell, and cultivated under conditions suitable for expression.
• the appropriate targeting peptide or receptor, or portions thereof may be coupled, bonded, bound, conjugated, or chemically-linked to one or more agents via linkers, polylinkers, or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions are familiar to those of skill in the art and should be suitable for administration to humans, i.e., pharmaceutically acceptable.
• Preferred agents are the carriers are keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).
• antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2 , single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
• cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, growth factors and traditional polypeptide hormones.
• cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-.alpha.
• growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone
• parathyroid hormone such as thyroxine
• insulin proinsulin
• relaxin prorelaxin
• glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH)
• FSH follicle stimulating hormone
• TSH thyroid stimulating hormone
• LH luteinizing hormone
• TGFs transforming growth factors
• CSFs colony stimulating factors
• M-CSF macrophage-CSF
• GM-CSF granulocyte- macrophage-CSF
• G-CSF granulocyte-CSF
• ILs ihterleukins
• Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine gene in combination with, for example, a cytokine gene, to enhance the recruitment of other immune system components to the site of treatment. Chemokines include, but are not limited to, RANTES, MCAF, MlPl-alpha, MDPl-Beta, and IP-10. The skilled artisan will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines. Imaging agents and radioisotopes
• the claimed peptides or proteins of the present invention may be attached to imaging agents of use for imaging and diagnosis of various diseased organs, tissues or cell types.
• imaging agents are known in the art, as are methods for their attachment to proteins or peptides (see, e.g., U.S. patents 5,021,236 and 4,472,509, both incorporated herein by reference).
• Certain attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a DTPA attached to the protein or peptide (U.S. Patent 4,472,509).
• Proteins or peptides also may be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
• Non-limiting examples of paramagnetic ions of potential use as imaging agents include chromium (UI), manganese (II), iron (DI), iron (II), cobalt (II), nickel (IT), copper (II), neodymium (DI), samarium (HI), ytterbium (HI), gadolinium (HI), vanadium (H), terbium (HI), dysprosium (HI), holmium (HI) and erbium (HI), with gadolinium being particularly prefened.
• Ions useful in other contexts, such as X-ray imaging include but are not limited to lanthanum (HI), gold (HI), lead (II), and especially bismuth (HI).
• Radioisotopes of potential use as imaging or therapeutic agents include astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 cobalt, copper 67 , 152 Eu, gallium 67 , 3 hydrogen, iodine 123 , iodine 125 , iodine 131 , indium 111 , 59 iron, 32 phosphorus, rhenium 186 , rhenium 188 , 75 selenium, 35 sulphur, technicium 99 " 1 and yttrium 90 .
• 125 I is often being prefened for use in certain embodiments, and technicium 99 " 1 and indium 111 are also often prefened due to their low energy and suitability for long range detection.
• Radioactively labeled proteins or peptides of the present invention may be produced according to well-known methods in the art. For instance, they can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
• a chemical oxidizing agent such as sodium hypochlorite
• an enzymatic oxidizing agent such as lactoperoxidase.
• Proteins or peptides according to the invention may be labeled with technetium- 99 " 1 by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the peptide to this column or by direct labeling techniques, e.g., by incubating pertechnate, a reducing agent such as SNC1 2 , a buffer solution such as sodium-potassium phthalate solution, and the peptide.
• Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to peptides are diethylenetriaminepentaacetic acid (DTP A) and ethylene diaminetetracetic acid (EDTA).
• fluorescent labels including rhodamine, fluorescein isothiocyanate and renographin.
• the claimed proteins or peptides may be linked to a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate.
• suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase.
• Prefened secondary binding ligands are biotin and avidin or streptavidin compounds. The use of such labels is well known to those of skill in the art in light and is described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.
• Bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies.
• Homobifunctional reagents that carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different macromolecules or subunits of a macromolecule, and linking of polypeptide ligands to their specific binding sites.
• Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially.
• the bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino, sulfhydryl, guanidino, indole, carboxyl specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied.
• a majority of heterobifunctional cross-linking reagents contains a primary amine-reactive group and a thiol-reactive group.
• ligands can be covalently bound to liposomal surfaces through the cross-linking of amine residues.
• Liposomes in particular, multilamellar vesicles (MLV) or unilamellar vesicles such as microemulsified liposomes (MEL) and large unilamellar liposomes (LUNET), each containing phosphatidylethanolamine (PE), have been prepared by established procedures.
• MLV multilamellar vesicles
• MEL microemulsified liposomes
• LUNET large unilamellar liposomes
• PE in the liposome provides an active functional residue, a primary amine, on the liposomal surface for cross-linking purposes.
• Ligands such as epidermal growth factor (EGF) have been successfully linked with PE-liposomes. Ligands are bound covalently to discrete sites on the liposome surfaces. The number and surface density of these sites are dictated by the liposome formulation and the liposome type. The liposomal surfaces may also have sites for non-covalent association.
• cross-linking reagents have been studied for effectiveness and biocompatibility.
• Cross-linking reagents include glutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), and a water soluble carbodiimide, preferably l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
• GAD glutaraldehyde
• OXR bifunctional oxirane
• EGDE ethylene glycol diglycidyl ether
• EDC water soluble carbodiimide
• heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described (U.S. Patent 5,889,155, specifically incorporated herein by reference in its entirety).
• the cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling in one example, of aldehydes to free thiols.
• the cross-linking reagent can be modified to cross-link various functional groups.
• Nucleic acids according to the present invention may encode a targeting peptide, a receptor protein or a fusion protein.
• the nucleic acid may be derived from genomic DNA, complementary DNA (cDNA) or synthetic DNA. Where incorporation into an expression vector is desired, the nucleic acid may also comprise a natural intron or an intron derived from another gene. Such engineered molecules are sometime refened to as "mini-genes.”
• nucleic acid as used herein includes single-stranded and double-stranded molecules, as well as DNA, RNA, chemically modified nucleic acids and nucleic acid analogs. It is contemplated that a nucleic acid within the scope of the present invention may be of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, " 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94
• targeting peptides, fusion proteins and receptors may be encoded by any nucleic acid sequence that encodes the appropriate amino acid sequence.
• the design and production of nucleic acids encoding a desired amino acid sequence is well known to those of skill in the art, using standardized codon tables (see Table 2 below).
• the codons selected for encoding each amino acid may be modified to optimize expression of the nucleic acid in the host cell of interest. Codon preferences for various species of host cell are well known in the art.
• the present invention encompasses complementary nucleic acids that hybridize under high stringency conditions with such coding nucleic acid sequences.
• High stringency conditions for nucleic acid hybridization are well known in the art.
• conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M ⁇ aCl at temperatures of about 50°C to about 70°C.
• the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleotide content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.
• expression vectors are employed to express the targeting peptide or fusion protein, which can then be purified and used.
• the expression vectors are used in gene therapy. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger R ⁇ A stability and translatability in host cells also are known.
• expression construct or "expression vector” are meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid coding sequence is capable of being transcribed, i prefened embodiments, the nucleic acid encoding a gene product is under transcriptional control of a promoter.
• a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
• under transcriptional control means that the promoter is in the conect location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
• the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.
• a human cell it is preferable to position the nucleic acid coding region adjacent and under the control of a promoter that is capable of being expressed in a human cell.
• a promoter might include either a human or viral promoter.
• the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
• CMV cytomegalovirus
• the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
• a cDNA insert typically one will typically include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
• the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed, such as human growth hormone and SV40 polyadenylation signals.
• a terminator also contemplated as an element of the expression construct. These elements can serve to enhance message levels and to minimize read through from the construct into other sequences.
• the cells containing nucleic acid constructs of the present invention may be identified in vitro or in vivo by including a marker in the expression construct.
• markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
• a drug selection marker aids in cloning and in the selection of transformants.
• genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers.
• enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed.
• Immunologic markers also can be employed.
• the selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
• the expression construct comprises a virus or engineered construct derived from a viral genome.
• Prefened gene therapy vectors are generally viral vectors.
• viruses that can accept foreign genetic material are limited in the number of nucleotides they can accommodate and in the range of cells they infect, these viruses have been demonstrated to successfully effect gene expression.
• adenoviruses do not integrate their genetic material into the host genome and therefore do not require host replication for gene expression making them ideally suited for rapid, efficient, heterologous gene expression. Techniques for preeparing replication infective viruses are well known in the art.
• a prefened means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.
• DNA viruses used as gene vectors include the papovaviruses (e.g., simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).
• papovaviruses e.g., simian virus 40, bovine papilloma virus, and polyoma
• adenoviruses Rosgeway, 1988; Baichwal and Sugden, 1986.
• filamentous bacteriophage expressing targeting peptides in a surface protein may be genetically engineered to contain elements of viral sequences, such as AAV sequences, and used as targeted expression vectors.
• adenovirus expression vector is meant to include, but is not limited to, constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense or a sense polynucleotide that has been cloned therein.
• the expression vector comprises a genetically engineered form of adenovirus.
• retroviral infection the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
• adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
• Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
• ITRs inverted repeats
• the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
• the El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication.
• MLP major late promoter
• TPL 5'- tripartite leader
• recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
• adenovirus vectors which are replication deficient depend on a unique helper cell line, designated 293, which is transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the cunent adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the E3, or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra kb of DNA.
• the maximum capacity of the cunent adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector- borne cytotoxicity. Also, the replication deficiency of the El-deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the cunently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).
• MOI multiplicities of infection
• Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
• the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells.
• the prefened helper cell line is 293.
• Racher et al, (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.
• natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue.
• Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) are employed as follows.
• a cell innoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h.
• the medium is then replaced with 50 ml of fresh medium and shaking is initiated.
• cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking is commenced for another 72 hr.
• the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
• Adenovirus type 5 of subgroup C is the prefened starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
• a typical vector applicable to practicing the present invention is replication defective and will not have an adenovirus El region.
• the position of insertion of the construct within the adenovirus sequences is not critical.
• the polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al, (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.
• Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 9 -10 ⁇ plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
• Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Animal studies have suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al, 1990; Rich et al, 1993).
• retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription (Coffin, 1990).
• the resulting DNA then stably integrates into cellular chromosomes as a pro virus and directs synthesis of viral proteins.
• the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
• the retroviral genome contains three genes, gag, pol, and env. that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
• a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
• Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences, and also are required for integration in the host cell genome (Coffin, 1990).
• a nucleic acid encoding protein of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
• Retroviral vectors are capable of infecting a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).
• retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus et al, 1981).
• Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This may result from recombination events in which the intact sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome.
• new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al, 1988; Hersdorffer et Z., 1990).
• viral vectors may be employed as expression constructs.
• Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988), adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984), and herpes viruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).
• Non- viral methods for the transfer of expression constructs into cultured mammalian cells include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990), DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
• the expression construct may be entrapped in a liposome.
• Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reanangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. Also contemplated are lipofectamine-DNA complexes.
• Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
• Wong et al, ' (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells.
• Nicolau et al, (1987) accomplished successful liposome- mediated gene transfer in rats after intravenous injection.
• a number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively.
• anti- metabolite resistance can be used as the basis of selection for dhfr: that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; and hygro, that confers resistance to hygromycin.
• compositions - expression vectors, virus stocks, proteins, antibodies and drugs - it may be necessary to prepare pharmaceutical compositions - expression vectors, virus stocks, proteins, antibodies and drugs - in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of impurities that could be harmful to humans or animals.
• Aqueous compositions of the present invention comprise an effective amount of the protein or peptide, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are refened to as innocula.
• pharmaceutically or pharmacologically acceptable refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
• the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the proteins or peptides of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
• compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention may be accomplished via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial or intravenous injection. Such compositions normally would be administered as pharmaceutically acceptable compositions, described supra.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• a coating such as lecithin
• surfactants for example, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the prefened methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• chemotherapeutic agents may be attached to a targeting peptide or fusion protein for selective delivery to a tumor.
• Agents or factors suitable for use may include any chemical compound that induces DNA damage when applied to a cell.
• Chemotherapeutic agents include, but are not limited to, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide (VP16), farnesyl-protein transferase inhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum
• chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous agents, and any analog or derivative variant thereof.
• Chemotherapeutic agents and methods of administration, dosages, etc. are well known to those of skill in the art (see for example, the “Physicians Desk Reference”, Goodman & Gilman's “The Pharmacological Basis of Therapeutics” and in “Remington's Pharmaceutical Sciences", incorporated herein by reference in relevant parts), and may be combined with the invention in light of the disclosures herein. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Examples of specific chemotherapeutic agents and dose regimes are also described herein.
• Alkylating agents are drugs that directly interact with genomic DNA to prevent the cancer cell from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific.
• An alkylating agent may include, but is not limited to, a nitrogen mustard, an ethylenimene, a methylmelamine, an alkyl sulfonate, a nitrosourea or a triazines. They include but are not limited to: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan.
• Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, they specifically influence the cell cycle during S phase. Antimetabolites can be differentiated into various categories, such as folic acid analogs, pyrimidine analogs and purine analogs and related inhibitory compounds. Antimetabolites include but are not limited to, 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.
• 5-FU 5-fluorouracil
• Ara-C cytarabine
• fludarabine gemcitabine
• gemcitabine gemcitabine
• methotrexate methotrexate
• Natural products generally refer to compounds originally isolated from a natural source, and identified has having a pharmacological activity. Such compounds, analogs and derivatives thereof may be, isolated from a natural source, chemically synthesized or recombinantly produced by any technique known to those of skill in the art. Natural products include such categories as mitotic inhibitors, antitumor antibiotics, enzymes and biological response modifiers.
• Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors include, for example, docetaxel, etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine.
• Mitotic inhibitors include, for example, docetaxel, etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine.
• Taxoids are a class of related compounds isolated from the bark of the ash tree, Taxus brevifolia. Taxoids include but are not limited to compounds such as docetaxel and paclitaxel. Paclitaxel binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules. Vinca alkaloids are a type of plant alkaloid identified to have pharmaceutical activity. They include such compounds as vinblastine (VLB) and vincristine.
• Antitumor antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific so they work in all phases of the cell cycle. Examples of antitumor antibiotics include, but are not limited to, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin (mithramycin) and idarubicin.
• Corticosteroid hormones are considered chemotherapy drugs when they are implemented to kill or slow the growth of cancer cells. Corticosteroid hormones can increase the effectiveness of other chemotherapy agents, and consequently, they are frequently used in combination treatments. Prednisone and dexamethasone are examples of corticosteroid hormones.
• Progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate have been used in cancers of the endometrium and breast.
• Estrogens such as diethylstilbestrol and ethinyl estradiol have been used in cancers such as breast and prostate.
• Antiestrogens such as tamoxifen have been used in cancers such as breast.
• Androgens such as testosterone propionate and fluoxymesterone have also been used in treating breast cancer.
• Antiandrogens such as flutamide have been used in the treatment of prostate cancer.
• Gonadotropin-releasing hormone analogs such as leuprolide have been used in treating prostate cancer.
• Some chemotherapy agents do not fall into the previous categories based on their activities. They include, but are not limited to, platinum coordination complexes, anthracenedione, substituted urea, methyl hydrazine derivative, adrenalcortical suppressant, amsacrine, L-asparaginase, and tretinoin. It is contemplated that they may be used within the compositions and methods of the present invention.
• Platinum coordination complexes include such compounds as carboplatin and cisplatin ( s-DDP).
• An anthracenedione such as mitoxantrone has been used for treating acute granulocytic leukemia and breast cancer.
• a substituted urea such as hydroxyurea has been used in treating chronic granulocytic leukemia, polycythemia vera, essental thrombocytosis and malignant melanoma.
• a methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH) has been used in the treatment of Hodgkin's disease.
• An adrenocortical suppressant such as mitotane has been used to treat adrenal cortex cancer, while aminoglutethimide has been used to treat Hodgkin's disease.
• Apoptosis or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Ken et al., 1972).
• the Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems.
• the Bcl-2 protein plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985; Geary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986).
• the evolutionarily conserved Bcl-2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists.
• Bcl-2 acts to suppress cell death triggered by a variety of stimuli. Also, it now is apparent that there is a family of Bcl-2 cell death regulatory proteins which share in common structural and sequence homologies. These different family members have been shown to either possess similar functions to Bcl-2 (e.g., BCI XL , Bclw, Bcl s , Mcl-l, Al, Bfl-1) or counteract Bcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
• Non-limiting examples of pro-apoptosis agents contemplated within the scope of the present invention include gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK) 2 (SEQ ID NO:l), (KLAKKLA) 2 (SEQ ID NO:2), (KAAKKAA) 2 (SEQ HD NO:3) or (KLGKKLG) 3 (SEQ ID NO:4).
• the present invention may concern administration of targeting peptides attached to anti-angiogenic agents, such as angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP- 10, Gro- ⁇ , thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon- alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, plate
• Bone manow is the prefened site of metastasis in the large majority of patients with prostate cancer (Fidler, 1999). This striking selectivity has been viewed as an example of site-specific interactions that were essential to cancer progression (Rak, 1995; Zetter, 1998). Despite the clinical relevance, little is known about the mechanisms that control prostate cancer spread to bone. In addition, there were no effective strategies for targeting therapeutic agents for the treatment of metastatic prostate cancer (Brodt et. al, 1996).
• a subset of peptides capable of selective homing to bone manow through the circulation is likely to simulate the behavior of prostate cancer cells during bone metastasis formation.
• the vascular markers targeted by using phage display might also be utilized by tumor cells to metastasize. This concept has already been proven to be true for lung-homing peptides. Peptides that home to lung blood vessels inhibit experimental metastasis. These results fit a "modified seed and soil" model, in which the basis for site-specific metastasis is the presence of homing receptors in blood vessels of certain tissues to which metastasis preferentially occurs.
• Such selective vascular markers are exposed to tumor cells during adhesion, the first step of the metastastic cascade.
• Isolation of bone manow-homing peptides is of utility for identifying those vascular markers that mediate prostate cancer cell homing during the metastatic process, and for potential therapeutic intervention in preventing metastases to bone, or in selectively imaging and/or treating cancer that has already metastasized to bone.
• compositions and methods disclosed herein were of use to develop new anti-prostate cancer therapeutic strategies that focus on the prevention and treatment of bone metastasis.
• the phage library remained in contact with the bone manow surface for 1-2 hours, at 4°C.
• the tissue was then washed gently about 5-10 times with 1 ml of DMEM/BSA-PI.
• K91 E. coli were infected and phage recovery was performed at room temperature for 1-2 hours.
• the bone sample was removed after infection and aliquots of the K91 culture were plated in serial dilutions. After the antibiotic concentrations were adjusted the culture was grown overnight. These were the bulk recovered phage. It was necessary to grow 200 individual colonies after each round for RH, and also sequence phage from every round. The inputs were titrated retrospectively after each round. Triplicate platings were done for statistical significance. For sequencing, it was important to obtain colonies that were well spread and distant from each other.
• the cultures were processed for successive rounds of panning as follows: K91 E. coli cultures were spun at 8,000, and PEG precipitated. Phage formed a white pellet that was usually visible and has to be re-suspended in 100-200 ⁇ l of PBS. Usually half (50 ⁇ l) of the material could be used for the next round of panning. The process was repeated for at least three rounds. To evaluate if there was selectivity during the screenings, control phage containing no peptide insert (Fd tet) were tested side-by-side at similar concentrations. The number of phage recovered using bulk vs. single colony preps also showed if there were differences based on selectivity.
• Human ribs were obtained from the Department of Pathology at the MD Anderson Cancer Center. These materials were generated during kidney and lung cancer surgeries and removed to provide access to the tumor site. The ribs were sectioned in half to expose the manow surface. No significant damage was inflicted on the tissue and morphology was preserved during these procedures. This material was suitable for the isolation of specific peptides that bind to human bone marrow, listed below. Bone-binding assays were performed in vitro to confirm that a number of these peptides bind to the rib surfaces. Targeting peptide sequences that homed to human bone manow were shown in Table 3. Table 3. Phage recovered from human bone marrow surfaces in multiple rounds of selection
• VLGPRAM (SEQ HD NO:9)
• a system has been designed to analyze the data resulting from peptide library screenings, adapted from the SAS package. The system is available upon request from the M.D. Anderson Cancer Center. Table 4 summarizes the data compiled after 400 phage clones selected ex-vivo were entered in the program. A peptide library pool was used in these experiments. Shown are peptide motifs that appeared more than 3 times.
• bone manow targeting peptide sequences identified herein will be of use for numerous applications within the scope of the present invention, including but not limited to targeted delivery of therapeutic agents or gene therapy, in vivo imaging of normal or diseased organs, tissues or cell types, identification of receptors and receptor ligands in organs, tissues or cell types, and therapeutic treatment of a number of human diseases, particularly metastatic prostate cancer.
• Example 2 Fingerprinting the circulating pool of immunoglobulins elicited against prostate cancer in human patients provides a novel prostate cancer marker that is prognostic for disease progression
• phage libraries are screened against the pool of immunoglobulins from an individual with a disease state or other characteristic.
• the antibody pool provides a structural sampling of ligands targeted to naturally occuring receptors, some of which may constitute novel disease markers. Biopanning against an antibody pool may be used to identify disease markers and to further characterize the molecular events underlying the disease state.
• the present example shows the feasibility of this approach by identifying a novel marker for prostate cancer.
• the results further show that this marker has prognostic value for predicting which individuals with prostate cancer are likely to have an unfavorable clinical outcome, resulting in death of the patient.
• this marker has prognostic value for predicting which individuals with prostate cancer are likely to have an unfavorable clinical outcome, resulting in death of the patient.
• there is a great need in the field of prostate cancer for a reliable method to separate those individuals whose prostate cancer will prove lethal (and therefore are candidates for more agressive therapeutic intervention) from individuals who will not die from prostate cancer.
• the present example represents a significant advance in prostate cancer prognosis and illustrates the utility of the claimed methods and compositions.
• the present example deals with prostate cancer
• the methods and compositions disclosed are suitable for use with any disease state or condition in which the host immune system is likely to produce antibodies against a molecular marker associated with the disease or condition.
• the repertoire of circulating antibodies from the serum of prostate cancer patients with advanced disease was used to screen a phage display library. Certain peptides binding to those antibodies conespond to tumor antigens expressed in bone metastasis of prostate cancer.
• a panel of prostate cancer serum samples from patients with recorded clinical outcome was screened by an ELISA assay against those peptides. The results show that reactivity against one particular peptide ("peptide C”) can be used to identify patients with metastatic androgen-independent prostate cancer.
• patients with detectable levels of circulating antibodies against peptide C exhibited decreased survival compared to individuals without such antibodies.
• Sera was selected from patients diagnosed with androgen-dependent and androgen-independent prostate cancer.
• a CX6C peptide library was screened against this pool of IgGs in a two-step procedure. First, the peptide library was pre-cleared against a pool of purified IgGs from normal serum samples using Protein G. This step removed peptides from the phage display library that bound to immunoglobulins from patients without prostate cancer. Next, the pre-cleared peptide library was screened against the pool of purified IgGs from the serum of prostate cancer patients. This step selected peptides binding specifically to IgGs elicited against prostate cancer.
• Human plasma samples were prospectively collected from 91 patients with locally advanced, metastatic androgen-dependent and metastatic androgen-independent adenocarcinoma of the prostate. The following criteria were applied to the locally advanced group: no evidence of regional and distant metastasis, clinical stage Tic or T2 with high grade disease (Gleason score 8-10) on initial biopsy or clinical stage T2b-T2c with Gleason score 7 and PSA > 10 or clinical stage T3, negative bone scan and CT of the abdomen and pelvis. Patients in the metastatic androgen-dependent group were responsive to androgen ablation (either combined or LHRH agonist alone) at the time blood samples were collected. A castrate serum testosterone level ⁇ 50 ng/dl and a rising PSA for two consecutive measurements at least two weeks apart defined androgen independence.
• Plasma from 34 healthy individual donors was obtained from the Blood Bank at the University of Texas M. D. Anderson Cancer Center (UTMDACC).
• Archived tissue paraffin blocks were obtained from the Department of Pathology at UTMDACC. The blood samples were initially allowed to clot at room temperature and then centrifuged to separate the cellular component from the supernatant. Aliquots of supernatant were promptly frozen and stored at -80°C until assayed. Biopanning.
• a 6-mer cyclic peptide library (CX6Q, constructed as described above, was used for the biopanning.
• CX6Q 6-mer cyclic peptide library
• a pre-clearing stage was employed to remove non-specific peptides by pre- absorbing the peptide library onto purified IgGs from pooled normal serum (five healthy male individuals). The pre-cleared peptide library was screened onto the
• a 20 ⁇ g/ml solution of purified GST or GST-fusion proteins in 0.1M NaHCO3 was used to coat maxisorp multi-well plates (Nalge Nunc International Corporation) and incubated overnight at 4°C. The plates were blocked in a blocking buffer (4% milk, 2% casein, and 0.05% Tween-20) for 3-4 hours.
• a series of 100-fold dilutions (1:100-1:1200) of sera from prostate cancer patients or healthy individuals was added and incubated for 1.5 hours and then washed five times with washing buffer (1% milk, 0.5% casein, and 0.025% Tween-20), followed by incubation at 4°C with anti-human alkaline phosphatase-conjugated antibodies (Gibco). The plates were then washed six times in washing buffer and developed using p-NPP (Sigma) as a substrate.
• An automatic ELISA plate reader (BIO-TEK instrument) recorded the results at OD405 nm.
• GST-fusion proteins containing the peptide sequence from patient C were coated on multi-well plates. After incubating the plates with the patient's serum, the plates were washed. The bound IgG antibodies were eluted with 50 ⁇ l of 0.1 M glycine buffer, pH 2.2, neutralized by addition of 10 ⁇ l 1 M Tris pH 9.0, and dialyzed in PBS overnight followed by concentration of the antibody using Centricon-30 (Millipore) filters. The purified antibody (500 ⁇ g) was coupled to biotin according to the manufacturer's instructions (Vector). The biotinylated antibody was analyzed by SDS- gel electrophoresis.
• Paraffin sections from patient C were stained with purified biotinylated antibodies and peptide antibodies by immunoperoxidase detection using the Dako antigen retrieval kit and DAB as a substrate. All of the sections (4 ⁇ m) were counter- stained with hematoxylin. The biotinylated immunopurified antibodies were used at a dilution of 1:100. Peptide C antibodies and purified pre-immune antibodies were used at 0.01 ⁇ g/ ⁇ l. For the inhibition staining, peptide C antibodies were pre-incubated for 30 minutes at room temperature with the GST-peptide C (500 ⁇ g) prior to staining. Peptide antibodies were generated in rabbits and purified using a T-gel immunoglobulin purification kit and protein G column (Pierce).
• Probabilities of survival for each group were estimated using the Kaplan-Meier method. A log-rank test was implemented in order to detect significant differences between the groups. Reactivity was considered to be detected if the ratio between GST- peptide and GST alone was greater or equal to two by the ELISA data.
• the Cox proportional hazards model was applied to analyze the effect of single and multiple risk factors in association with survival. Martingale residual plots were used to assess the proportional hazard assumption. P-values less than 0.05 were considered significant. All analyses were performed using SPLUS statistical software.
• the reactivity profile of 91 sera obtained from clinically annotated prostate cancer patients was examined. Sera from 34 healthy individuals were also tested as a control for specificity. ELISA was performed to evaluate each serum using the three selected peptides (A, B, and C).
• the sera obtained from patients with prostate cancer were divided into three groups: 35 originated from patients with localized adenocarcinomas, 27 from patients with metastatic androgen-dependent disease, and 29 originated from patients with metastatic androgen-independent disease.
• Antibodies against peptide C were examined by immunohistochemistry to determine whether they would recognize tumor-associated antigens, using tissue sections from bone manow metastasis (derived from surgical specimens from patient C). Strong tumor staining was observed using immunopurified antibodies from patient C's serum (not shown). Specific immunostaining was also observed using a rabbit polyclonal antibody against the synthetic form of peptide C (not shown). However, no staining was observed with pre-immune antibodies (not shown). The staining was completely inhibited by the GST-fusion protein containing peptide C (not shown). Normal prostate tissue was completely negative when tested for reactivity with the anti- peptide C polyclonal antibody (not shown).
• peptide sequences are of value for a variety of applications, including but not limited to prostate cancer detection, diagnosis and prognosis, therapeutic vaccine development, rapid immunodiagnostic screening, and the identification of the natural antigen.
• disease-specific antigens identified using this approach could be employed to define common or unique features in the immune response of individuals to the same disease, i.e., immunofingerprinting the immune response against a given antigen.
• the aligned sequences were also used to search unique epitopes by cross- referencing the peptide sequences in Table 6 using the Mac Vector software package (Oxford Molecular Group). To confirm these findings, a short peptide database and analysis software were developed in collaboration with the Department of Biostatistics at UTMDACC. This biostatistical program determines the cumulative frequency by which any combination of 3 or more amino acid residues occurs. It was found that the conserved regions identified from the LAIIGN program matched frequently occurring residue sequences identified from the biostatistical analysis.
• Table 6 presents a few of the selected targeting peptides, including peptide C.
• Peptide motifs with the consensus sequences CNXSDKSC (SEQ ID NO:61) or CNXTDKSC (SEQ ID NO:62) were identified. Table 6. Peptides isolated by biopanning on immunoglobulins from a prostate cancer patient.
• peptide C is apparently a mimeotope of an endogenous protein against which circulating antibodies are present in a high percentage of individuals with metastatic prostate cancer.
• the following methods were used to identify the endogenous peptide C antigen.
• the endogenous antigen is important as a diagnostic and prognostic marker for prostate cancer, a potential therapeutic target for treatment of prostate cancer and a potential antigen for vaccine development against prostate cancer.
• the endogenous antigen may also be of significance in understanding the biochemical mechanisms underlying prostate cancer metastasis.
• the DU145 prostate cell line was used as the source material for purification of the peptide C antigen.
• Ten plates of cells were grown to confluence before harvesting. The cells were rinsed 3X in PBS before adding 750 ul of TM buffer (0.01M tris-CL, 0.002M MgC12, 1% tritonXlOO) per plate. Cells from all plates were combined in a 50 ml tube by scraping the cells off the plates. The cells were sheared by passing 3X through a 22-gauge needle to separate the nuclei from rest of the cell. Aliquouts of 10 ul were visualized under a microscope to check for complete separation.
• the homogenate was centrifuged at 800 rpm for 10 min and the supernatant (cytosolic/membrane fraction) transfened to new tubes (500 ul/tube).
• the remaining pellet containing the cell nuclear fraction was resuspended with 800 ul of nuclear lysis buffer (0.1% SDS, 0.5% Triton X100, 50 mM Tris-Cl, 10 mM NaCl).
• Antibodies against peptide C were prepared by standard techniques. Briefly, synthetic peptide C was conjugated to KLH by ANASPEC and purified by HPLC. The conjugated peptide (100 ⁇ g) was injected into rabbits, who were subsequently boosted with the same peptide. Pre-immune serum was collected prior to the initial injection.
• the 80 kDa band was excised for protein sequencing and further analysis.
• the gel slices containing excised protein were crushed in 3 ml of running buffer. The supernatant was recovered and concentrated using a Centricon-30 filter. About 40 ul of partially purified protein (excised from SDS-PAGE) was loaded onto an 8% SDS gel and Western blot analysis was performed. Pre-immune serum was used a control. The partially purified protein showed a somewhat diffuse band of about 80 kDa. Additional gel slices were removed from the SDS gel and analyzed for amino acid sequence by mass spectrometry.
• peptide sequences were obtained from the protein excised from SDS gels. All five peptides matched portions of the 78 kDa glucose regulated protein (Table 7, GRP78, SEQ ID NO:66, GenBank Accession Numbers CAB71335 and XM 044202). The locations of the five sequenced peptides within GRP78 are indicated in Table 7 in bold font. A commercial antibody against GRP78 reacted on Western blot with the purified peptide C antigen from DU145 cells showed positive reactivity (not shown).
• the original peptide C sequence (SEQ ID NO:60) is not found within the GRP78 sequence (SEQ ID NO:66), indicating that the epitope recognized in vivo by anti- peptide C antibodies is formed from discontiguous regions of the GRP78 protein.
• GRP78 is the endogenous antigen against which circulating antibodies are present in a high percentage of metastatic prostate cancer patients. These results are consistent with the reported characteristics of GRP78.
• the GRP78 protein is a chaperone that is normally present in endoplasmic reticulum (Triantafilou et al., 2001). However, it has recently been reported to also exist as a cell surface protein, where it associates with MHC class I (Triantafilou et al, 2001). GRP78 is about 60% homologous to the hsp70 heat shock protein (U.S. Patent No. 5,188,964). GRP78 is normally overexpressed in response to glucose starvation (U.S. Patent No. 5,188,964).
• GRP78 induction of GRP78 in response to thapsigargin, a pro-apoptotic agent, has been reported in human rabdomiosarcoma cells and rat brain tumor cells (Delpino et al, 1998; Chen et al, 2000). Furuya et al. (1994) reported that glucose regulated protein was induced in rat and human prostate cancer cells treated with thapsigargin. Androgen independent prostatic cancer cells treated with thapsigargin underwent apoptosis within several days (Furuya et al, 1994). Circulating antibodies against GRP78 have been reported in ovarian cancer patients (Chinni et al, 1997).
• the present disclosure is the first to report that antibodies reactive with GRP78 are present in a high percentage of individuals with metastatic prostate cancer, and that the presence of such antibodies in sera of prostate cancer patients is significantly associated with a substantial decrease in patient survival.
• Examples 2 and 3 above illustrated the use of phage display screening against circulating antibodies from prostate cancer patients to identify a novel prostate cancer marker that is prognostic for patient survival.
• the present example illustrates a further embodiment of the methods, using phage display library screening to examine the progression in circulating antibodies accompanying disease progression.
• Example 2 The methods used were similar to those described in Example 2.
• a subtraction protocol was used, in which IgG from a normal individual was coupled to protein G chromatography beads.
• Antibodies from patient M prostate cancer patient
• the phage display library that had been pre-exposed to normal IgG's was exposed to the IgG pool from patient M. After thorough washing of the column, the phage that bound to the prostate cancer IgG (but did not bind to normal IgG) was eluted and amplified. This procedure was followed for three rounds of screening and targeting peptides against patient M's antibodies were obtained.
• Serum samples from the same patient were obtained from archival specimens and used to obtain targeting peptides.
• Patient M's serum from 1994 (early stage cancer), 1998 (intermediate stage) and 2000 (late stage) were used to obtain antibody targeting peptides as described above. These peptides were shown in Table 8.
• the numbers in parentheses indicate the number of phage exhibiting the sequence out of the total number of clones obtained.
• CTFAGSSC SEQ ID NO:67
• Phage display libraries may be screened against cancer patient samples to identify targeting peptides that bind to antibodies against tumor specific or tumor associated antigens.
• the identified targeting peptides may be used, for example, to purify antitumor antibodies using affinity chromatograpy or other well-known techniques.
• the purified anti-tumor antibodies can be used in diagnostic kits to identify individuals with cancer.
• chemotherapeutic agents such as chemotherapeutic agents, radioisotopes, anti-angiogenic agents or pro-apoptosis agents and used for cancer therapy.
• the targeting peptides against anti-tumor antibodies may also be used to identify novel tumor specific or tumor-associated antigens, of diagnostic or therapeutic use.
• Phage display antibody libraries may also be constructed and screened against tumor targeting peptides. By this method, it is possible to isolate and purify large quantities of antibodies specific for tumor antigens.
• CTFAGSSC SEQ ID NO:67
• CTFAGSSC SEQ ID NO:67
• the peptide may also be used to prepare monoclonal or polyclonal antibodies that are of use for tumor diagnosis, imaging or therapy.
• Example 5 Identification of Receptor/Ligand Pairs: Endostatin receptors revealed by phage display
• Endostatin is a recently characterized cell protein with reported anti-angiogenic properties (U.S. Patent Serial No. 6,174,861). It apparently acts at least in part by inhibiting endothelial cell proliferation, thus blocking the growth of new blood vessels (U.S. Patent Serial No. 6,174,861). Administration of endostatin is reported to inhibit tumor growth in model systems (U.S. Patent Serial No. 6,174,861). Despite its clinical significance, the mechanisms by which endostatin exerts these effects remain unknown. Elucidating the function of endostatin would be facilitated by identification of targeting peptides that bind to endostatin and potentially act as mimeotopes of endogenous endostatin ligands. Such peptides may also be of potential use as novel anti-angiogenic or anti-tumor agents.
• CX6C and CX7C phage libraries were screened. using recombinant His-tag fusion proteins that contained endostatin coated onto microtiter wells. An immobilized His-tag control protein was used as a negative control for enrichment during the panning. Phage were sequenced from randomly selected clones after three rounds of panning as described (Koivunen et al, 1994, 1995; Pasqualini et al, 1995). Successful isolation of distinct sequences that interacted specifically with endostatin is reported in Table 9.
• CAMGSPEC (SEQ HD NO:90) CEAGRGGC (SEQ HD NO:91) CKLSGTRC (SEQ HD NO:92) CNGIVQVC (SEQ HD NO:93) CASSHAVC (SEQ HD NO:94) CWQGSVSC (SEQ HD NO:95) CMVGYIVC (SEQ HD NO:96) CWNRGSTC (SEQ HD NO:97) CPERGTRC (SEQ HD NO:98) CVNKYIPC (SEQ HD NO:99) CGTAEGVC (SEQ HD NO: 100) CASPNLAC (SEQ HD NO: 101) CDNGNASC (SEQ HD NO: 102) CSQLKLGC (SEQ HD NO: 103) CMGTKSSC (SEQ HD NO: 104) CHDTSELC (SEQ HD NO: 105) CGRVPQMC (SEQ HD NO: 106) CAGFSSPC (SEQ HD NO: 107) CSRSSFLC (SEQ HD NO: 108) CIRPNDHC (SEQ HD NO: 109)
• a homology search provided numerous examples of protein candidates for the endogenous endostatin receptors. These are shown in FIG. 4A-4F.
• the skilled artisan will realize that the targeting peptide sequences identified herein are of potential use for the development of novel agents that may be either anti-angiogenic or pro-angiogenic, depending upon their interactions with endostatin, the endogenous endostatin receptor(s) and the binding interaction between endostatin and its receptor(s). Further, the putative endostatin receptor proteins identified by homology to the endostatin targeting peptides are potential targets for therapeutic treatment directed towards anti- angiogenesis.
• angiostatin a proteolytic fragment of collagen XVHI.
• endostatin the molecular mechanisms by which angiostatin induces these effects is unknown.
• the present example identifies angiostatin targeting peptides by phage display. Potential receptors for angiostatin are determined by homology with angiostatin targeting peptide sequences.
• Anti-human angiostatin and anti-mouse endostatin (R&D systems: AF226, AF570), CD13 (Sigma St Louis, MO: L-9776), thrombospondin and hepatocyte growth factor (Calbiochem, San Diego, CA: 605225, 375228) were purchased from commercial sources.
• rh-Angiostatin and rh-Endostatin were produced by EntreMed, Inc. (Rockville, MD).
• APN/CD13 enzyme and L-Alanine-p-nitroanilide hydrochloride substrate were purchased from Sigma, St Louis, MO (# L-9776; # A9325).
• a phage library displaying random cyclic peptides with the structure CX 7 C (C, cysteine; X, any residue) was prepared.
• An aliquot of the library containing 3 x 10 10 transducing units (TU) was screened with rhAngiostatin protein coated on microtiter wells as described (Koivunen et al 1993).
• the amount of protein used was 10 ⁇ g/well.
• the wells were coated with a decreased concentration of rhAngiostatin protein (100 ng/well) in the third panning. Phage remained bound after extensive washing. Bound phage were recovered by infection into F-pilus positive K91 bacteria.
• rhAngiostatin binding To determine the specificity of rhAngiostatin binding, selected phage were assayed as described but using rh- Endostatin and BSA proteins to coat the wells. In all assays, insertless phage (fdtet) was used as a control.
• Phage were sequenced from randomly selected clones after three rounds of panning (Koivunen et al., 1994, 1995; Pasqualini et al., 1995). A number of distinct angiostatin-targeting sequences were identified (Table 10). Randomly selected clones from rounds H and HI were sequenced. Amino acid sequences of the phagemid encoded peptides were deduced from nucleotide sequences. A consensus sequence appeared on the third round in 61% of the sequences examined (26 out of 42) - CWSLEXXKC (SEQ HD NO: 141). Including the closely related sequence CWSAEWTKC (SEQ ID NO: 142) the consensus motif accounted for 71% of Angiostatin binding peptides. The ratios in the last two columns of Table 10 were calculated by dividing the number of colonies recovered from rhAngiostatin-coated wells by those recovered from rh- Endostatin or BSA wells.
• Phage binding assays with rhAngiostatin selected clones.
• HGF hepatocyte growth factor
• CD 13 Aminopeptidase N
• TSP-1 Thrombospondin 1
• angiostatin binds to HGF, CD 13 and TSP-1, but not to other ECM proteins such as COL TV (collagen IV), LN (laminin), FN (fibronectin), or VN (vitronectin) (FIG. 7).
• Binding of angiostatin to CD 13 was competitive with the NGR peptide (Burg et al, 1999) in a dose-dependent manner (not shown). Control experiments were run with the CARAC peptide (SEQ ID NO: 169) (not shown). Binding of angiostatin to CD13 was inhibited by more than 50% by 0.5 ⁇ g of NGR peptide, while binding was unaffected by up to 500 ⁇ g of CARAC peptide (SEQ HD NO: 169) (not shown).
• the targeting peptide sequences identified herein are of potential use for the development of novel agents of potential use as anti- angiogenic or pro-angiogenic activity.
• the putative angiostatin receptor proteins identified by homology to the angiostatin targeting peptides are potential targets for therapeutic treatment directed towards anti-angiogenesis or pro- angiogenesis.
• Anti-angiogenic agents of therapeutic use for tumor treatment are also within the scope of the present invention.
• HD Hodgkin's disease
• EBN Epstein-Ban virus
• the incidence of EBN in HD patients ranges from 40-50% in developed countries and up to 94% in developing countries (Chang and Weiss, 1996).
• the incidence of HD with EBN seropositivity also varies with histological subtype, being about 80-90% in mixed cellularity (HDMC) and 40- 50% in nodular sclerosis (HD ⁇ S) (Lyons and Liebowitz, 1998).
• EBN D ⁇ A is localized to nearly all RS (Reed Sternberg) cells (Brousset et al., 1991; Herbst et al., 1991, 1992; Pallesen et al., 1991; Weiss et al., 1991).
• RS Raster Sternberg
• Phage display has been used to identify peptide epitopes from random peptide libraries in viral diseases such as hepatitis C (Pereboeva et al., 1998, Prezzi et al., 1996) and measles (Owens et al., 2000). Identification of viral and tumor cell surface epitopes has the potential to be clinically useful for developing neutralizing antibodies that may protect against viral infections and for developing serodiagnostic testing (Pereboeva et al., 2000).
• IgGs were isolated from the serum of HD patients by batch binding to Protein G agarose, using Pierce ImmunoPure Immobilized Protein G binding buffer. The bound IgGs were eluted with a Protein G elution buffer (Pierce) and immediately neutralized with 0.1 volume of 1 M Tris-Cl, pH 9. After identification of targeting peptides as described below, purified IgGs were incubated with the glutathione Sepharose 4B- bound GST-fusion proteins to affinity purify the specific IgGs which recognized the conesponding targeting peptide epitopes. The glutathione Sepharose 4B resin was pelleted by centrifugation, and rinsed to remove non-specific binding IgGs. IgGs were eluted with acidic elution buffer and immediately neutralized with 0.1 volume 1 M Tris-Cl, pH 9.
• Peptide coding sequences from selected phage were amplified by PCR using forward and reverse primers containing BamHI and EcoRI sites, respectively.
• the amplified sequences were cloned into the BamHI-EcoRI site of the GST vector, pGEX- 2TK (Amersham/Pharmacia), and the presence of the inserted sequences was verified by sequence analysis.
• GST-fusion proteins were affinity purified from bacterial lysates by affinity chromatography to immobilized glutathione using established protocols (Smith and Johnson, 1988). Briefly, the GST-fusion proteins were batch-bound to glutathione Sepharose 4B beads, and the resin extensively rinsed to remove non-specific proteins. GST-fusion proteins were eluted by incubating the resin with an excess of reduced glutathione, followed by extensive dialysis of the eluted protein against phosphate buffered saline, pH 7.4 (PBS) to remove the glutathione. To purify epitope-specific HD IgGs, the GST-fusion proteins were not eluted from the solid support as described below.
• Cryostat samples were processed using published methods (Bielenberg et al., 1999). Briefly, samples were fixed with cold acetone, acetone: chloroform (1:1) at RT, and acetone at RT for 5 minutes each, rinsed 3x with PBS, blocked for 20 minutes at RT with PBS supplemented with 5% normal horse serum, and incubated with affinity purified primary human IgG overnight at 4°C. Fixed tissues were rinsed 3x with PBS, blocked in 5% horse serum/PBS for 10 minutes, and incubated in mouse anti-human IgG secondary antibody conjugated to flurochrome dyes such as Cyanine 3 or Cyanine 5 (Amersham/Pharmacia) for 1 hour at RT, and rinsed in PBS.
• flurochrome dyes such as Cyanine 3 or Cyanine 5 (Amersham/Pharmacia) for 1 hour at RT, and rinsed in PBS.
• the affinity GST-fusion proteins were used to screen a collection of HD patient sera by enzyme linked immunosorbent assay (ELISA).
• the purified GST-fusion proteins were used to coat a 96-well plate at 100 ng/well at room temperature (RT) or at 4°C overnight. Following coating, the wells were emptied, rinsed, and non-specific sites were blocked with 200 ⁇ l 3% BSA/PBS at RT for 1-2 hours. HD sera were applied to each coated and blocked well at 1:100 dilution and then incubated at RT for 1 hour.
• the wells were rinsed 3x with 3% BSA/PBS containing 0.01% Tween 20, and then incubated for 1 hour with 50 ⁇ l each of anti-human alkaline phosphatase conjugated antibody at 1:2000 dilution. Signals were detected in the presence of p- nitrophenyl phosphate by measuring OD 4 o 5 at specific intervals to follow the course of color development.
• a positive control was the HD sera the peptide was identified from, and a negative control was EBV negative normal sera or BSA.
• a CX C phage display library in the fUSE5 vector was generated as described above. Bulk amplification was used between each selection round to elute phage.
• IgGs were isolated from the serum of HD patients using established methods.
• a phage display random peptide library was screened on this pool of IgGs (Smith, 1985) in a two-step procedure.
• the peptide library was pre-cleared on a pool of IgGs from control normal serum. This step removed nonspecific peptide interactions.
• the pre-cleared peptide library was screened on the pool of IgGs from the serum of HD patients. This step selected specific interactions between peptides and HD IgGs.
• HD IgGs bound to Protein G agarose were incubated with a CX C phage peptide library that had been pre-cleared on IgGs isolated from non-HD sera.
• the resultant HD IgG-bound phage were eluted from the solid support, neutralized immediately, and used to infect E. coli strain K91.
• the phage were amplified and precipitated for a subsequent round of panning (Koivunen et al., 1999). Phage infected K91 were plated onto tetracycline LB agar plates and individual clones were subjected to colony PCR and sequence analysis.
• FIG. 8 A representative experiment after four rounds of selection on HD IgG is shown (FIG. 8). The figure shows that the selectivity of the targeting phage improves with each round of selection, compared to control phage without insert.
• FIG. 9 shows selected viral motifs that were identified.
• a number of similar proteins from a variety of viruses were aligned using the LALIGN program from the ExPASY website to identify conserved regions.
• the aligned sequences were also used to search unique epitopes by cross-referencing the peptide sequences in FIG. 9, using the Mac Vector software package (Oxford Molecular Group). This analysis confirmed that similar viral capsid proteins contain conserved regions within their primary structure.
• the targeting peptide sequences identified were CSLLPASSC (SEQ HD NO:157), CIGKGTSLC (SEQ HD NO: 158), CYVNVQVSC (SEQ HD NO: 159), CLGDIVERC (SEQ HD NO: 160), CMLVKRKNC (SEQ HD NO: 161) CAHFHNSC (SEQ HD NO: 162), CYYPGEKSC (SEQ HD NO: 163), CFSSFFRCC (SEQ HD NO: 164) and CGIRGPNKC (SEQ HD NO: 165). Screening the same peptide library on IgGs from other human solid tumors did not yield peptides sequences that shared sequence identity to known viral proteins (data not shown). Moreover, the same peptides were selected in different HD patients but not in controls.
• Viruses such as the human papilloma virus 16, hepatitis B and C, Kaposi's sarcoma-associated virus (KSHV), and the Epstein Ba virus (EBV) are recognized to cause a variety of human cancers. Although tumors are known to elicit an immune response against mutated, altered, or overexpressed antigens, a limited number of immunogenic tumor antigens have been thus far identified.
• the work here illustrates how phage display technology can be used to screen immunoglobulins (IgGs) from Hodgkin's disease (HD) patients to search for novel viral etiological agents.
• IgGs immunoglobulins
• phage display can be applied to distinguish unique epitopes from IgGs isolated from HD sera in an unbiased fashion.
• the peptides identified from these studies may be of use for identifying novel viral agents in EBV- negative HD patients.
• the clinical applications of this work range from the development of vaccines and/or anti-idiotype antibodies for immunotherapy, increased accuracy for diagnostic/prognostic testing, and directed specificity in tumor targeting.
• Example 8 A new generation of targeted phage-based vectors for systemic gene delivery in humans
• compositions and methods disclosed herein are of use for targeted delivery of therapeutic agents to selected organs, tissues or cell types, including cancer cells.
• the targeted therapeutic agent is an expression vector.
• the present example discloses a non-limiting embodiment illustrating the use of targeting peptides for delivery of novel expression vectors encoding therapeutic proteins or peptides.
• Heterologous ligands also have been incorporated into the envelopes of retroviruses or the capsids of adenoviruses and adeno-associated viruses, thereby targeting these vectors to integrins (Dmitriev et al., 1998; Vigne et al., 1993; Girod et al., 1999), T-cell receptors (Engelstadter et al., 2000) or melanoma- associated antigens (Martin et al., 1999).
• integrins Dmitriev et al., 1998; Vigne et al., 1993; Girod et al., 1999
• T-cell receptors Engelstadter et al., 2000
• melanoma- associated antigens Martin et al., 1999
• tumor vasculature is a suitable target for intervention because the vascular endothelium is composed of non-malignant cells that are genetically stable but epigenetically diverse.
• In vivo phage display has been used to isolate probes that home selectively to different vascular beds and target receptors expressed only on certain blood vessels.
• vascular receptors are attractive targets for systemic delivery of gene therapy, in particular because such receptors are readily accessible through the circulation and often can mediate internalization of ligands by cells. While incorporation of vascular homing peptides derived from in vivo phage display screenings into viral vectors has been attempted, this strategy has proven quite challenging because the structure of the capsid and the targeting properties of the peptides can be adversely affected (Wickham, 2000).
• phage vectors have several advantages over mammalian viruses conventionally used for gene therapy. Receptors for prokaryotic viruses such as untargeted (wild-type) phage are not expressed on mammalian cells (Id.). Receptor-mediated internalization by mammalian cells can occur if re-targeted phage vectors display certain peptide ligands (Larocca et al, 1999).
• the parental tumor-homing phage used here are known to target receptors expressed in the activated blood vessels of multiple types of human and murine tumors, including carcinomas, melanomas, and sarcomas in mouse models (Pasqualini et al, 1997; Arap et al, 1998; Koivunen et al, 1999).
• the lung-homing phage and its conesponding receptor expressed in the lung vasculature have also been characterized in mice (Rajotte et al, 1998; Rajotte and Ruoslahti, 1999).
• targeted systemic gene delivery to the vascular endothelium may be feasible using phage particles homing to cell surface receptors on blood vessels to provide selective tissue expression and adequate vector uptake into the targeted tissue.
• the present example demonstrates the feasibility of this approach.
• a new generation of phage-based gene delivery vectors is provided that targets the molecular diversity of the vascular endothelium in vivo.
• the present example shows that targeted phage vectors can promote gene expression in mammalian cells in vitro following specific receptor- mediated internalization.
• Systemic targeted tissue-specific transduction of the lungs in immunocompetent mice and angiogenesis-related transduction of tumors in immunodeficient mice bearing human tumor xenografts is demonstrated.
• AAV adeno-associated virus
• AAP adeno-associated phage
• AAP vectors appear to combine favorable biological features of both prokaryotic viruses (peptide display system for receptor targeting and high production yield in host bacteria) and mammalian viruses (long-term transduction stability) and may be of use for systemic gene therapy targeting applications in vivo.
• KS1775 Kaposi's sarcoma
• ATCC Manassas, VA
• MDA-MB-435 breast carcinoma The following human cell lines were used: Kaposi's sarcoma (termed KS1767), 293 embryonic kidney (ATCC; Manassas, VA), and MDA-MB-435 breast carcinoma.
• Cell lines were maintained in minimal essential medium (MEM; Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal calf serum (FCS; Gibco-BRL, Rockville, MD) plus sodium pyruvate, L-glutamine, and penicillin/streptomycin (Gibco-BRL).
• a fUSE5-based filamentous phage display vector was modified to transduce mammalian cells with the ⁇ -galactosidase reporter gene under the control of the cytomegalovirus (CMV) immediate early gene promoter.
• CMV cytomegalovirus
• the sequences for the targeting peptides were inserted into the Sfi I sites of the fUSE5 phage display cloning vector of the gene IH coat protein (pHI).
• the RGD-4C targeting peptide (CDCRGDCFC, SEQ HD NO: 166) was chosen for its ability to bind to and be internalized by ⁇ v3/5 integrin expressing cells in tumor vasculature.
• a second tumor homing targeting peptide the MMP-2/9 binding HWGF peptide (CTTHWGFTLC, SEQ HD NO: 167) was also selected.
• CTTHWGFTLC MMP-2/9 binding HWGF peptide
• an fd-tet phage derived construct that carries the CMV- ⁇ -gal cassette but does not display a targeting peptide was used.
• Targeted RGD4C- ⁇ -gal phage vector was engineered in a two-step process that included the generation of an intermediate construct (termed RGD-4C-fMCSl) and subsequent production of RGD-4C- ⁇ -gal.
• RGD-4C-fMCSl contained the oligonucleotide insert encoding the RGD-4C targeting peptide (SEQ HD NO: 166) and a fragment of the fMCSl plasmid that had a multicloning site (MCS) for insertion of transgenes.
• RGD-4C phage-derived fUSE5 DNA (Koivunen et al, 1995) and fd-tet phage-derived fMCSl DNA were purified from lysates of host bacteria (E. coli MC1061).
• the intermediate RGD-4C-fMCSl vector was prepared by ligating a 5.4-kb Bam ⁇ fflSacll fragment of the RGD-4C plasmid to the 4.1 kb BamHUSac ⁇ . fragment of the fMCSl plasmid.
• a 14 kb RGD-4C- ⁇ -gal phage plasmid was obtained by insertion of a 4.5 kb Pstl CMV- ⁇ -gal fragment derived from pCMV ⁇ (Clontech, Palo Alto, CA) into the Pstl site of RGD-4C-fMCSl. This allowed cloning of the CMV- ⁇ -gal cassette in either forward or reverse orientation.
• Orientations of resulting vectors were differentiated by EcoRV restriction analysis and by DNA sequencing.
• Targeted phage vectors were designated fRGD4C- ⁇ -gal (forward) and rRGD4C- ⁇ -gal (reverse).
• Other targeting (HWGF- ⁇ -gal, GF ⁇ - ⁇ -gal) phage and insertless control (fd- ⁇ -gal) phage were constructed using the same methods.
• GF ⁇ phage contained the lung targeting peptide GF ⁇ -1 (CGFECVRQCPERC, SEQ ID NO: 168).
• a targeted phage/ AAV chimeric vector was produced by cloning a 2.8 kb fragment of pAAV-eGFP (enhanced GFP; Stratagene) from ITR to LTR into the Pstl site of RGD-fMSC. Briefly, pAAV was digested with P d to release a 2.8 kb fragment, which was blunted with DNA polymerase and cloned into the blunted Pstl site of RGD-fMSC (thus destroying, the Pstl restriction site). In each of the constructs, conect orientation of insert was verified by restriction analysis. Single clones in each orientation were sequenced. Unless otherwise stated, the forward vectors were used in this example.
• the double-stranded DNAs of the replicative forms of targeted (RGD4C- ⁇ -gal, HWGF- ⁇ -gal, GFE- ⁇ -gal) and insertless control (fd- ⁇ -gal) constructs were prepared using a Plasmid Maxi kit (Qiagen).
• the single-stranded DNAs of the infective forms of the phage vectors were extracted from the phage capsid proteins using Strataclean resin (Stratagene), followed by two ethanol precipitations. DNA was quantified by spectrophotometry with 1.0 A 26 o equal to 40 ⁇ g/ml for single-stranded DNA or 50 ⁇ g/ml for double-stranded DNA.
• the 293 recipient cells were transfected with 5 ⁇ g of either double-stranded or single-stranded phage DNA into 5 xlO 5 cells, using the SuperFect ® reagent (Qiagen) according to the manufacture's protocol. Both the gene expression and enzyme activity of ⁇ -gal were evaluated at least 48 hours post- transfection. Cells were incubated with the X-gal substrate for 3 hours at 37°C and enzyme activity was visualized by using an in situ ⁇ -galactosidase staining kit (Stratagene) according to the manufacturer's instructions.
• Phage vectors were isolated and purified from the culture supernatant as disclosed (Pasqualini et al, 2000b; Smith and Scott, 1993). Phage were re-suspended in Tris-buffered saline (pH 7.4) and re-centrifuged to remove residual bacteria and debris. The resulting supernatant containing the phage in suspension was filtered through a 0.45 ⁇ m filter and titered according to standard protocols (Id.).
• MDA-MB-435 and KS1767 cells were cultured on 8-well chamber glass slides.
• the culture media was replaced by 200 ⁇ l of MEM with 2% FCS and 5 X10 10 TU of RGD-4C- ⁇ -gal, HWGF- ⁇ -gal, or fd- ⁇ -gal phage vectors (at 10 5 transducing units/cell in each case).
• Phage were incubated with cells for 3 hr at 37°C, followed by a medium change to MEM plus 10% FCS. The cells were incubated for 72 hr at 37°C to allow for ⁇ -gal gene expression.
• MDA-MB-435 cells were cultured on 12- well plates and then incubated with 10 ⁇ g of RGD-4C peptide (SEQ HD NO: 166) or control peptides (CARAC, SEQ HD NO: 169 or CKDRFERC, SEQ HD NO:59) in normal growth media for 30 minutes.
• KS1767 cells were grown on 12-well plates and then incubated with 40 ⁇ g CTTHWGFTLC (SEQ HD NO: 167) or control peptides in normal growth media for 30 minutes.
• the transduced cells were washed with PBS and permeabilized with 0.2% Triton X-100 for five minutes on ice, followed by blocking with 1% BSA in PBS.
• An anti- ⁇ -gal antibody (Sigma) diluted to 1:2,000 in blocking solution was then incubated with the cells overnight.
• a Texas Red-conjugated secondary antibody (Caltag, Burlingame, CA) diluted to 1:600 in PBS was incubated with the cells for 1 hour.
• the degree of ⁇ -gal gene expression was determined by counting fluorescent cells in at least ten fields under an inverted microscope (Nikon, Japan).
• Quantification of the ⁇ -gal activity in cell culture was measured as relative light units (RLU) in a luminometer and then normalized to the amount of protein in micrograms, as determined by the Lowry method in a protein assay kit (Bio-Rad Protein Assay ® ; Hercules, CA). Subsequently, blue cells were counted under an inverted microscope (Nikon).
• RLU relative light units
• ⁇ -gal activity in cell lysates was detected by the Galacto-Star ® chemiluminescent reporter gene system (Tropix, Bedford, MA) according to the manufacturer's protocol.
• 293 cells were plated at 3 x 10 5 cells/well and incubated with either 1 mg/ml of RGD-4C peptide (SEQ HD NO: 166) or inelevant control peptides (CARAC, SEQ HD NO: 169 or CKDRFERC, SEQ HD NO:59). After 30 minutes, cells were washed and 10 5 TU of phage per cell were added for 4 hours in serum free media. After the 4 hours, 10% FCS supplemented medium was added.
• FACS fluorescence activated cell sorting
• mice Female 4-month old nude mice and female 4-month old immu ⁇ ocompetent C57B1/6 mice (Harlan Sprague Dawley, San Diego, CA) were used. Avertin (0.015 ml/g) was used as an anesthetic.
• Tumor xenografts derived from human Kaposi's sarcoma KS1767 cells were established by injecting tumor cells (10 6 cells per mouse in 200 ⁇ l of serum-free MEM) into the mammary fat pad of nude mice. Tumor-bearing mice with matched tumor sizes were used for systemic gene transfer experiments 20 to 40 days afterwards when tumors reached 0.5 to 1.5 cm in diameter.
• RGD-4C- ⁇ -gal, HWGF- ⁇ -gal, and fd- ⁇ -gal phage (10 9 TU/mouse) were injected intravenously (tail vein) into female nude mice carrying subcutaneous tumor xenografts.
• GFE- ⁇ -gal phage and fd- ⁇ -gal control phage (10 9 TU/mouse) were injected intravenously into female C57B1/6 mice. Lungs and livers were harvested two weeks after vector administration.
• ⁇ -gal activity in the lung and control tissues were detected by a chemiluminescent assay system (Tropix).
• the fUSE5 -based filamentous phage display vector was modified by inserting a ⁇ -galactosidase ( ⁇ -gal)-encoding gene under the control of a CMV promoter into an intergenic region of the phage genome to construct a fUSE5- ⁇ -gal backbone vector.
• ⁇ -gal ⁇ -galactosidase
• DNA olignonucleotide sequences encoding the targeting peptides CDCRGDCFC (SEQ HD NO.-166, referred to as RGD-4C) (Pasqualini et al, 1997; Arap et al, 1998), CTTHWGFTLC (SEQ HD NO: 167, refened to as HWGF) (Koivunen et al, 1999), and CGFECVRQCPERC (SEQ HD NO: 168, refened to as GFE) (Rajotte et al, 1998; Rajotte and Ruoslahti, 1999) were inserted into the Sfi I site of the gene HI minor coat protein (pIH) of the fUSE5 phage.
• RGD-4C Pasqualini et al, 1997; Arap et al, 1998)
• CTTHWGFTLC SEQ HD NO: 167, refened to as HWGF
• CGFECVRQCPERC SEQ HD NO: 168, refened to
• the resulting viral constructs (RGD-4C- ⁇ -gal, HWGF- ⁇ -gal, and GFE- ⁇ -gal) were used for production of targeted phage particles that displayed the targeting peptides on their outer surface and carried a CMV- ⁇ -gal reporter gene.
• RGD-4C- ⁇ -gal and HWGF- ⁇ -gal were designed to target ⁇ v integrins and matrix metalloproteinases (MMP-2 and MMP-9), respectively. Both receptors are expressed in angiogenic vasculature.
• MMP-2 and MMP-9 matrix metalloproteinases
• Both receptors are expressed in angiogenic vasculature.
• the GFE- ⁇ -gal phage was designed to target membrane dipeptidase (MDP) expressed in lung vasculature. The same strategy was used to construct the other targeting and control vectors.
• embryonic human kidney cells were transfected with the infective forms of the phage DNA constructed to contain the reporter transgene in either forward or reverse orientation.
• a CMV-driven mammalian expression vector was used as a positive control and an empty vector as a negative control for ⁇ -gal expression.
• Transfer of the modified single- stranded DNA of the phage infective form promoted transgene expression in mammalian cells (not shown).
• the orientation of the transgene cassette did not significantly influence the level of gene expression (not shown). Therefore all subsequent experiments used the vector with the ⁇ -gal expression cassette in the forward orientation.
• Receptor-mediated internalization and specific transduction of recipient cells by targeted phage vectors in vitro Receptor-mediated internalization and specific transduction of recipient cells by targeted phage vectors in vitro.
• transgene constructs were functional, the transduction of human cell lines expressing receptors targeted by the RGD-4C- ⁇ -gal and HWGF- ⁇ -gal phage vectors was examined.
• the untargeted fUSE5-derived control phage vector (termed fd- ⁇ -gal) was used as a negative control.
• RGD-4C- ⁇ -gal phage and HWGF- ⁇ - gal phage were incubated with breast cancer and Kaposi's sarcoma cells (MDA-MB- 435 and KS1767 lines, respectively).
• Both cell lines express high levels of the RGD- 4C-receptors ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins (Pasqualini et al, 1996) and of the HWGF receptors MMP-2 and MMP-9 (Koivunen et al, 1999).
• each vector was intravenously injected into nude mice bearing human KS1767 Kaposi's sarcoma xenografts.
• KS1767 cells are suitable because they form well-vascularized tumors and the receptor expression profiles in tumor cells and tumor-associated blood vessels has been characterized, ⁇ v integrins and gelatinases (MMP-2 and -9) are highly expressed on the KS1767-derived tumor xenografts and their angiogenic vasculature.
• phage displaying RGD-4C and HWGF peptides target KS1767 tumors efficiently and specifically in vivo (Pasqualini et al, 1997; Arap et al, 1998; Koivunen et al, 1999).
• Tumors and control organs liver and brain
• the RGD-4C- ⁇ -gal, HWGF- ⁇ -gal and control fd- ⁇ -gal vectors were analyzed. Strong ⁇ -gal immunostaining was observed within tumors, while negligible immunostaining was seen in control organs (not shown).
• tissues recovered from mice that received untargeted negative control fd- ⁇ -gal phage vector did not show detectable ⁇ - gal expression in either the tumor or the control organs, including liver and brain (not shown).
• ⁇ -gal reactivity matched the conesponding immunostaining pattern of phage targeting to the vascular endothelium of blood vessels in tumors vivo (Pasqualini et al, 1997; Arap et al, 1998; Koivunen et al, 1999).
• Targeted gene delivery was evaluated in vivo by using GFE- ⁇ -gal, a phage vector targeted to MDP in the vascular endothelium of lung blood vessels.
• the lung- homing GFE- ⁇ -gal vector was injected intravenously into immunocompetent C57B1/6 mice.
• Substantial ⁇ -gal activity was seen in the lungs of mice injected with GFE- ⁇ -gal phage but not in the lungs of mice injected with fd- ⁇ -gal control (FIG. 12).
• the ⁇ -gal activity in the liver of mice injected with the GFE- ⁇ -gal phage was similar to that of background ⁇ -gal activity from mice injected with control phage (FIG. 12).
• Phage/ AAV chimeric vectors markedly improve gene transduction stability
• This examples provides the first demonstration that systemic gene delivery can be achieved by genetically adapting targeted phage clones selected from screenings of phage display random peptide libraries.
• targeting peptides can be integrated into conventional gene therapy vectors and used for organ, tissue or cell type selective delivery. These strategies have been technically challenging but not necessarily efficient.
• the present example resolves issues of specificity and efficiency by taking advantage of peptide ligands selected from phage libraries in vitro and in vivo.
• the example demonstrates that the null-tropism of wild-type phage towards mammalian cells can be modified to target and deliver genes to receptors expressed on the vascular endothelium of normal organs (such as the lung) and tumors.
• the- phage vectors disclosed herein have a number of potential advantages. Their targeting to selective vascular beds is based on receptor expression patterns that are known and characterized. The receptors are accessible to circulating probes. These ligand-receptor pairs provide internalization of the vector into targeted cells.
• AAP adeno-associated phage
• AAP adeno-associated phage
• the biological features of AAP are distinct from either targeted phage or AAV. While the enhanced duration of gene transduction by AAP is similar to the long-term expression patterns associated with AAV transduction, the receptor-mediated targeting is characteristic of phage clones selected in in vivo screenings. Thus, AAP are endowed with several advantages as a gene therapy vector. AAP are easy to produce in high titers in host bacteria. No helper viruses or trans-acting factors are needed. The native tropism of AAV for human cells is eliminated because there is no AAV capsid formation.
• the AAP vectors are targeted because they incorporate peptides that have been isolated in vivo and are defined by their ability to home to selective vascular beds. Targeted gene delivery specific to the ligand-receptor pair to which the phage is directed was possible, and gene expression was maintained for over two months.
• the present example describes a new generation of targeted phage-based vectors that enable systemic gene delivery and robust long-term transgene expression.
• Novel chimeric phage-based vector containing genetic elements from adeno-associated virus (AAV) have been designed and tested. These vectors (i) specifically home to receptors that have been well characterized for selective expression on the vascular endothelium, (ii) can deliver genes to angiogenic or tissue-specific blood vessels, and (iii) markedly increase transduction stability and duration of gene expression.
• AAV adeno-associated virus
• Example 9 In vitro results with targeted phage delivery
• Phage particles were isolated from the E. coli host strain XLl-Blue MR (Stratagene, San Diego.CA). The phage particles were purified from the culture supernatant by two precipitations in 0.15 volume polyethylene glycol 8000 (Sigma, St. Louis, MO). The phage particles were resuspended in Tris-buffered saline (pH 7.4) and centrifuged to remove any residual bacteria and contaminating debris. The resulting supernatant containing the phage suspension was filtered through a 0.45 ⁇ m filter and titered following standard protocols (Smith & Scott, 1993).
• the replicative form of forward and reverse RGD4C- ⁇ -gal and HWGF- ⁇ -gal plasmids were prepared using the Plasmid Maxi kit (Qiagen).
• the non-replicative ssDNA phage genome was extracted from the phage capsid proteins using Strataclean resin (Stratagene), followed by ethanol precipitation.
• Human embryonic kidney 293 cells (American Type Culture Collection), MDA- MB-435 human breast carcinoma cells, and KS1767 human Kaposi's sarcoma cells (Herndier et al., 1996) were grown in minimal essential media (Irvine Scientific, Santa Ana, CA), supplemented with 10% fetal calf serum (FCS) (Tissue Culture Biologicals, Tulare, CA).
• FCS fetal calf serum
• the MO7e leukemia cell line was grown in RPMI 1640 (Irvine Scientific) supplemented with 10% FCS.
• 5 X 10 5 293 cells were transfected using 5 ⁇ g of DNA with the SuperFect transfection reagent (Qiagen) following the manufacturer's recommendations. The incubation time allowed for reporter gene expression was 48 h for dsDNA transfection and 72 h for ssDNA transfection.
• MDA-MB-435 cells were grown on 8-well chamber plastic slides (Nunc/Nalgene, Houston, TX) to 80% confluency. The cells were then incubated with 10 10 phage particles/well for 8 h at 37°C. The cells were washed six times with phosphate-buffered saline (PBS) and treated with glycine buffer (50 mM glycine, pH 2.8; 500 mM NaCl) three times for 10 min to elute externally bound phage particles. The cells were neutralized with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature.
• PBS phosphate-buffered saline
• glycine buffer 50 mM glycine, pH 2.8; 500 mM NaCl
• MDA-MB-435 and KS1767 cells were grown on 8-well chamber glass slides to 60-80% confluency.
• the growth medium was replaced by 200 ⁇ l MEM containing 2% FCS and 10 11 transducing units of either RGD-4C ⁇ -gal, HWGF- ⁇ -gal, or fd- ⁇ -gal phage (100,000 phage units/cell).
• Phage vectors were incubated on cells for 3 h at 37°C, followed by medium change to MEM plus 10% FCS. The cells were incubated for 72 h at 37°C to allow for ⁇ -galactosidase gene expression.
• reporter gene expression was analyzed by immunofluorescence.
• the cells were washed with PBS and permeabilized with 0.2% Triton X-100 for 5 min on ice, followed by blocking with 1% BSA in PBS.
• the monoclonal anti- ⁇ -galactosidase antibody (Sigma) diluted 1:2000 in blocking solution was incubated with the cells overnight.
• a Texas Red-conjugated secondary antibody diluted 1:600 in PBS was incubated with the cells for 1 h. The degree of ⁇ -gal expression was determined by counting the fluorescent cells seen under the inverted Nikon microscope.
• ⁇ -galactosidase enzyme activity was visualized using the In Situ ⁇ -galactosidase Staining Kit (Stratagene) following manufacturer's instructions. Cells were incubated with the X-gal substrate for 3 h at 37°C. Blue cells were counted in at least ten fields under the inverted Nikon microscope.
• Tumor homing phage were constructed that displayed the RGD-4C peptide (Pasqualini et al., 1997) or the HWGT peptide (Koivunen et al., 1999) and contained a ⁇ -galactosidase reporter gene under the control of a CMV promoter.
• These vectors, RGD-4C- ⁇ -gal and HWGF- ⁇ -gal specifically transduced two tumor cell lines, MDA- MB-435 and KS1767, that express the RGD-4C receptor, ⁇ v ⁇ 3/ ⁇ v ⁇ 5 integrins, or the HWGF receptor, MMP-2/-9, respectively.
• a control phage with the same transgene cassette but without targeting peptide did not transduce these cell lines.
• the double- stranded (ds) phage DNA (replicative form) and the single stranded (ss) phage DNA (infective form) were transfected into 293 human embryonic kidney cells and ⁇ - galactosidase transgene expression was determined histochemically. Plasmids with both orientations of the transgene cassette, forward and reverse, were evaluated. While transfection of 293 cells with ds RGD-4C- ⁇ -gal DNA resulted in ⁇ -gal activity in approximately 40% of the cells, transfection with ssDNA produced ⁇ -gal activity in 1- 3% of the cells (not shown).
• the orientation of the transgene cassette did not significantly influence the level of ⁇ -galactosidase expression (not shown). Therefore, DNA was used with the transgene cassette in the forward orientation in all remaining studies.
• the transfection data confirmed the functionality of phage-derived hybrid DNA constructs in achieving transgene expression in mammalian cells. Targeted phage vectors are internalized by tumor cells.
• Immunofluorescence identified phage in the cells that were incubated with the targeted phage vectors, while staining of cells incubated with fd- ⁇ -gal was close to the level of background (not shown). Omitting the permeabilization step prior to staining in cells that were incubated with RGD4C- ⁇ -gal phage vectors almost completely abolished immunofluorescence, confirming the presence of phage particles exclusively inside the targeted cells (not shown).
• Targeted phage vectors transduce cells expressing suitable receptors.
• RGD-4C- ⁇ -gal and HGWF- ⁇ -gal could deliver a reporter gene to mammalian cells
• those phage vectors were incubated with MDA-MB-435 cells, expressing high levels of RGD-4C-receptors ( ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins), or KS1767 cells, expressing high levels of HWGF-receptors (MMP-2 and MMP-9).
• Anti- ⁇ -gal immunofluorescence detected transgene expression in cells treated with the targeted phage vectors, while cells treated with the fd- ⁇ -gal vector showed minimal immunofluorescence (FIG. 14).
• RGD-4C- ⁇ -gal phage vector Specific uptake of the RGD-4C- ⁇ -gal phage vector was observed in ⁇ v ⁇ 3 and ⁇ v ⁇ 5-positive MDA-MB-435 cells, while MO7e cells showed no ⁇ -gal expression. This is consistent with previous reports that RGD displaying phage particles are internalized by integrin-expressing cells (Hart et al., 1994). Although MMP-2 and MMP-9 have been disclosed as targets for cancer therapy, their potential as a receptor for targeted gene delivery has not been studied. The present results show that the HWGF- ⁇ -gal phage is internalized and confers ⁇ -galactosidase expression in MMP-2 and MMP-9 positive KS1767 cells. The skilled artisan will realize that RGD-4C and HWGF-displaying phage may be of use as vectors for targeted gene delivery.
• the present example confirms and extends the results of Example 8, demonstrating the feasibility of using targeting peptide modified phage as gene therapy vectors for in vivo or in vitro delivery of therapeutic genes to human cells, tissues and organs.
• the replicative form of the phage genome is dsDNA
• the infective form of the phage is ssDNA.
• a LacZ reporter gene embedded in an ss phage genome was tested for expression in human cells. After single-stranded IFs were transfened into human cells, LacZ expression confirmed that the gene was converted from ss DNA to the ds DNA form (FIG. 16).
• Phage containing a CMV- ⁇ gal cassette were transfected into the human 293 cell line. Both the ds phage genome (RF) and the ss phage genome (IF) were transfected.
• the extraction of the ss DNA genome from the phage capsid proteins was performed with Strataclean resin (Stratagene), followed by ethanol precipitation.
• the DNA was quantified by spectrophotometry with 1.0 A 26 o equal to 40 ⁇ g/ml for ss DNA or 50 ⁇ g/ml for ds DNA. Both ss and ds DNA forms of the phage genome were expressed, as shown by beta-galactosidase activity.
• a phage internalization assay was used to show that RGD4C- ⁇ Gal exhibits receptor-mediated cell uptake in human cancer cells.
• Human KREB sarcoma cells or angiogenic KS1767 Kaposi sarcoma cells were used to determine whether RGD4C- ⁇ gal phage can deliver a reporter gene to mammalian cells for expression. After cells were grown to 70% confluency, RGD4C- ⁇ gal phage or a control phage with no insert were added at 10 10 T.UVwell and cells were incubated for 8 hr at 37°C. Cells were washed 6 times with PBS and treated with glycine buffer 3 times to elute externally bound phage.
• Cells were fixed with 4% paraformaldehyde for 15 minutes at RT, permeabilized with 0.05% saponin/TBS or treagted with TBS alone, stained with an anti-M13 antibody, and counterstained with hematoxylin. Cells were treated with RGD4C- ⁇ gal phage with or without permeabilization or were treated with control phage with no insert with or without permeabilization.
• Intravenous protein administration is possible and appears to be safe in the case of angiogenesis inhibitors such as angiostatin and endostatin.
• angiogenesis inhibitors such as angiostatin and endostatin.
• these inhibitors must be injected at very high doses on a weekly basis to produce long-lasting antitumor effects. Therapy cannot be interrupted without the recunence of the tumor, unless the proteins are given over a period of many months (Boehm et al., 1997).
• Genetic therapy based on the inhibition of angiogenesis appears to be feasible since, in the case of endostatin, the protein has been shown to be biologically active when secreted from gene-transduced cells (Cao et al., 1998; Griscelli et al., 1998; Tanaka et al., 1998).
• vector systems suitable for this kind of treatment would have to promote high and long-lasting expression.
• the AAP vectors described in Example 8 may have advantages for anti- angiogenic gene therapy because they are known to generate high, stable levels of gene expression. Recent data from a number of groups indicates that AAP-type vectors may be particularly useful in skeletal muscle, where reporter expression has been demonstrated over one year following a single injection (Muzyczka et al., 1994).
• In vivo screenings were performed to isolate a panel of muscle-homing phage. When injected intravenously, the phage accumulated specifically within the vasculature, and at later time points, in muscle tissue. These results may be of use for targeted AAP transduction of skeletal muscle as a depot for sustained secretion of endostatin.
• AAP vectors combined with muscle homing peptides may be used to increase the level and duration of gene expression in skeletal muscle.
• PALM Pulsitioning and Ablation with Laser Microbeams
• the PALM Robot-MicroBeam uses a precise, computer-guided laser for microablation.
• a pulsed ultra-violet (UV) laser is interfaced into a microscope and focused through an objective to a beam spot size of less than 1 micrometer in diameter.
• the principle of laser cutting is a locally restricted ablative photodecomposition process without heating (Hendrix, 1999). The effective laser energy is concentrated on the minute focal spot only and most biological objects are transparent for the applied laser wavelength.
• Tissue samples may be retrieved by circumcising a selected zone or a single cell after phage administration to the subject. A clear-cut gap between selected and non-selected area is typically obtained.
• the isolated tissue specimen can be ejected from the object plane and catapulted directly into the cap of a common microfuge tube in an entirely non-contact manner.
• LPC Laser Pressure Catapulting
• PALM was used in the present example to select targeting phage for mouse pancreatic tissue, as described below.
• a CX C peptide phage library (10 9 TU) was pre-screened by injected into the tail vein of a C57BL/6 male mouse, and the pancreas was harvested to recover the phage by bacterial infection. Phage from 246 colonies were grown separately in 5 mis LB/kanamycin (100 ⁇ g/ml)/tetracycline (40 ⁇ g/ml) at 37°C in the dark with agitation. Overnight cultures were pooled and the phage purified by NaCl/PEG precipitation for another round of in vivo bio-panning. Three hundred colonies were picked from the second round of panning, and the phage were recovered by precipitation.
• Phage from the second bio-panning round was then used for another round of in vivo panning and also was incubated with thawed frozen murine pancreatic sections for one in vitro panning round.
• 10 9 TU phage from the second round were injected into a third mouse and allowed to circulate for six minutes, followed by an intravenous injection of 50 ⁇ l of FTTC-Iectin (Vector Laboratories, Inc.). After a two-minute circulation, the mouse was perfused through the left ventricle with 3 mis MEM Earle salts. The pancreas was harvested, frozen at " 80 °C in Tissue Tek (Sakura), and sectioned onto prepared slides.
• Phage were recovered from cryo-preserved FITC-lectin stained mouse pancreatic islets and sunounding acinar cells that were microdissected from 14 ⁇ m sections using the PALM (Positioning and Ablation with Laser Microbeams) cold laser pressure catapulting system. Pancreatic islet and control sections were catapulted into 1 mM EDTA, pH 8, and frozen at -20 °C until enough material was collected for PCR amplification.
• PALM Positioning and Ablation with Laser Microbeams
• Phage DNA was amplified with fUSE5 primers: forward primer 5' TAA TAC GAC TCA CTA TAG GGC AAG CTG ATA AAC CGA TAG AATT 3' (SEQ HD NO: 170), reverse primer 5' CCC TCA TAG TTA GCG TAA CGA TCT 3' (SEQ HD NO: 171).
• the PCR products were subjected to another round of PCR using a nested set of primers. The 3' end of the second primer set was tailed with the M13 reverse primer for sequencing purposes.
• the nested primer set used was: forward nested primer 5' CCTTTCTATTCTCACTCGGCCG 3' (SEQ HD NO: 172), reverse nested primer 5' CAGGAAACAGCTATGACCGCTAAACAACTTTCAACAGTTTCGGC 3' (SEQ HD NO: 173).
• forward library primer 5' CACTCGGCCGACGGGGC 3' SEQ HD NO: 174
• reverse primer 5' CAGTTTCGGCCCCAGCGGCCC 3' SEQ HD NO: 175.
• PCR products generated from the nested primers were gel purified (Qiagen), and confirmed for the presence of a CX 7 C peptide insert sequence using the Ml 3 reverse primer by automated sequencing.
• PCR products generated from the library primers were gel purified (Qiagen), ligated into CsCl 2 purified fUSE5/SfiI, electroporated into electrocompetent MC1061 cells, and plated onto LB/streptomycin (100 ⁇ g/ml)/tetracycline (40 ⁇ g/ml) agar plates. Single colonies were subjected to colony PCR using the fUSE5 primers to verify the presence of a CX 7 C insert sequence by gel electrophoresis. Positive clones were sequenced using BigDye terminators (Perkin Elmer)
• Pancreatic islet and control sections were catapulted into 1 mM AEBSF, 20 ⁇ g/ml aprotinin, 10 ⁇ g/ml leupeptin, 1 mM elastase inhibitor I, 0.1 mM TPCK, 1 nM pepstatin A in PBS, pH 7.4, and frozen for 48 hours or less until enough material was collected.
• Each culture was transfened to 1.2 mis LB/Kan/Tet (0.2 ⁇ g/ml) and incubated in the dark at RT for 40 minutes.
• the tetracychne concentration was increased to 40 ⁇ g/ml for each culture, and the cultures were incubated overnight at 37 °C with agitation.
• Each culture was plated out the following day onto LB/Kan/Tet agar plates and incubated for 14 hours at 37 °C in the dark. Positive clones were picked for colony PCR and automated sequencing. Results
• phage were either bulk amplified or else single colonies of phage from pancreas, kidney, lung and adrenal glands were amplified and subjected to additional rounds of in vivo screening. Both bulk amplified and colony amplified phage from mouse pancreas showed successive enrichment with increasing rounds of selection (not shown). After three rounds of selection, the colony amplified phage showed almost an order of magnitude higher enrichment than bulk amplified phage (not shown).
• Table 12 lists selected targeting sequences and consensus motifs identified by pancreatic screening.
• VVG CEGVVGIVC SEQ HD NO:206
• VGG CVGGARALC SEQ HD NO:209 (SEQ HD NO: 181) CVGGVRGGC (SEQ HD NO: 199) CLAHRVGGC (SEQ HD NO:210)
• ALV CALVNVHLC (SEQ HD NO:224)
• VSG CMVSGVLLC (SEQ HD NO.226)
• FIG. 17 shows a general protocol for recovery of phage insert sequences from PALM selected thin section materials.
• phage may be recovered by direct infection of E. coli host bacteria, after protease digestion of the thin section sample.
• phage inserts may be recovered by PCR amplification and cloned into new vector DNA, then electroporated or otherwise transformed into host bacteria for cloning.
• Pancreatic sequences recovered by direct bacterial infection included CVPRRWDVC (SEQ HD NO:233), CQHTSGRGC (SEQ HD NO.234), CRARGWLLC (SEQ HD NO.235), CVSNPRWKC (SEQ HD NO.236), CGGVHALRC (SEQ ID NO:220), CFNRTWIGC (SEQ HD NO:237) and CSRGPAWGC (SEQ HD NO:238).
• Pancreatic targeting sequences recovered by amplification of phage inserts and cloning into phage include CWSRGQGGC (SEQ HD NO:239), CHVLWSTRC (SEQ HD NO:240), CLGLLMAGC (SEQ HD NO:241), CMSSPGVAC (SEQ HD NO:242), CLASGMDAC (SEQ HD NO:243), CHDERTGRC (SEQ HD NO:244), CAHHALMEC (SEQ HD NO:245), CMQGAATSC (SEQ HD NO:246), CMQGARTSC (SEQ HD NO:247) and CVRDLLTGC (SEQ HD NO:248).
• CWSRGQGGC SEQ HD NO:239)
• CHVLWSTRC SEQ HD NO:240
• CLGLLMAGC SEQ HD NO:241
• CMSSPGVAC SEQ HD NO:242
• CLASGMDAC SEQ HD NO:243
• CHDERTGRC SEQ HD NO:244
• CAHHALMEC
• FIG. 18 through FIG. 21 show sequence homologies identified for selected pancreatic targeting sequences.
• Several proteins known to be present in pancreatic tissues were identified. The results of this example show that the PALM method may be used for selecting cell types from tissue thin sections and recovering targeting phage sequences. The skilled artisan will realize that this method could be used with virtually any tissue to obtain targeting sequences directed to specific types of cells in heterologous organs, tissues or cell types.
• Ovarian cancer is the fifth most common cancer among American women, with 23,000 new cases diagnosed annually.
• the five-year survival rates for ovarian cancer by stage are: Stage I (93%), Stage H (70%), Stage HI (37%), and Stage IV (25%).
• Stage I 93%
• Stage H 70%
• Stage HI 37%
• Stage IV 25%
• delayed detection results in a drastic reduction in survival rate.
• Approximately two thirds of patients are cunently diagnosed with advanced stage disease. Most patients are asymptomatic or have only vague symptoms such as abdominal or pelvic fullness before metastasis occurs.
• the majority of women who have been successfully treated for ovarian cancer and in whom tumor control is achieved will eventually develop recunent disease. More women die of ovarian cancer than from all other gynecologic malignancies combined.
• CA125 is the most extensively studied. It has a well-defined and validated role as a reliable indicator of response or progression. However, it is a poor predictor of long term prognosis (Maggino and Gadduci, 2000; Mayer and Rustin, 2000).
• Ovarian malignancy may result in the accumulation of ascitic fluid in the peritoneal cavity.
• This exudate often contains tumor cells as well as tumor-related compounds such as carcinoembryonic antigen (Booth et al, 1977; Breborowicz et al, 1977), ⁇ fetoproteins (Khoo and Mackay, 1977), glycoproteins (Booth et al, 1977) and many tumor associated immunoglobulins (Dorsett et al, 1975). It has also been shown that immunoglobulins isolated from ascitic fluid react with the serum as well as tumor tissue from the patient from which the ascitic fluid was isolated (Hill et al, 1978).
• in vitro phage display may be used to screen immunoglobulins from the ascitic fluid of ovarian cancer patients and identify markers for the disease.
• IgGs from normal donor serum and ovarian cancer cell free ascitic fluid were bound to Protein agarose (Pierce) in the Pierce acetate, pH5 binding buffer.
• a two step biopanning procedure was employed in which the first step involved pre-clearing the phage peptide library by incubating it with IgGs isolated from normal donor serum in order to remove common antigens. This was followed by a second step where the pre- cleared library was used to screen cancer specific IgGs. Approximately 10 8 transforming units were added to the IgGs for the panning procedure.
• the resultant IgG-bound phage were recovered by eluting the phage with 0.1M glycine buffer, pH2.2, neutralizing the phage with 0.1 volume 1 M Tris-Cl, pH 9, and using the phage to infect stationary phase Escherichia coli strain K91. Serial dilutions of phage infected-K91 were plated onto tetracychne (40 ⁇ g/ml) LB agar plates and grown overnight at 37°C. Individual clones were picked, amplified, and precipitated for subsequent rounds of panning. A total of three rounds of selection were performed. Phage clones from the second and third rounds were subject to PCR followed by sequence analysis to evaluate enrichment of the most consistently binding peptide sequences.
• Peptide specific sequences to ovarian cancer cells may also be selected by utilizing BRASIL.
• BRASIL is based on the fast separation of phage bound to cells from an aqueous medium into an oil phase. Cells mixed with a phage display library are layered on an oil phase and centrifuged. Because intact cells are denser than the oil, they pellet at the bottom of the tube. Only the phage bound to the cell surface can pass through the oil. Unbound, water-soluble phage are left in the aqueous phase at the top of the tube.
• Ovarian cancer cells isolated from ascitic fluid may be harvested with PBS and 1% EDTA (5 minutes), washed with PBS, resuspended in MEM containing 1% BSA at 10 6 cells/ml and incubated with phage on ice. After 4 h, 100 ⁇ l of the cell suspension is transfened to a 400 ⁇ l Eppendorf tube containing 200 ⁇ l of a dibutyl phthalate yclohexane mixture (9:1) and centrifuged at lO.OOOg for 10 minutes. The tubes are snap frozen in liquid N 2 , the bottom of the tubes were cut off, and the pellets transfened to a new tube. The phage bound to cancer cells are rescued by infection with 200 ⁇ l of Escherichia coli strain K91kan cells in log phase. Preferably, three rounds of selection are performed. Subcloning, Expression, and Purification of GST fusion Proteins
• Peptide coding sequences of interest obtained from selection were amplified by colony PCR and cloned into the GST vector pGEX-2TK (Amersham Pharmacia) at the BamHI-EcoRI sites. Automated sequencing was used for verification of positive clones. Positive clones were transformed into the bacterial expression host strain, BL21 (DE3) pLys (Strategene), by electroporation. GST fusion proteins were affinity purified from bacterial lysates by affinity chromotography using glutathione Sepharose 4B resin (Amersham/Pharmacia) in 0.02 M Tris-Cl, pH 8.0, 0.1 M NaCl, 1 mM EDTA, 20% NP-40.
• Affinity purified GST fusion proteins were used to screen banked ascitic fluid and serum from ovarian cancer patients by ELISA.
• a solution of either GST or GST fusion proteins in 0.1 M NaHCO 3 were used to coat maxisorp multi-well plates (Nalge Nunc International Corporation) at 1 ⁇ g/well at 4°C overnight. Following coating, the plates were rinsed and subsequently blocked with a blocking buffer composed of 4% milk, 2% casein, and 0.05% Tween-20 for approximately 3-4 hours. Ascitic fluid or serum was applied to the coated and blocked wells at varying dilutions.
• the plates were washed with a washing buffer (1% milk, 0.5% casein, and .025%Tween-20) and anti-human alkaline phosphatase (Sigma) was added to each well.
• the colorimetric signal was developed using p-nitrophenyl phosphate (Sigma) and measuring OD 4 o 5 . Background signals were determined with GST alone and normal donor serum.
• GST fusion proteins made from inserting recombinant peptide sequences of interest in an expression vector were coated on maxisorp multi-well plates (Nalge Nunc International Corporation). The plates are incubated with the ascitic fluid from which the peptide was originally isolated. Following a washing procedure to remove unbound IgGs, bound IgGs were eluted with 0.1 M glycine buffer, pH2.2, neutralized with 1 M Tris-Cl, pH9.0, and dialyzed in PBS overnight. To concentrate the IgG, centricon-30 columns (Millipore) were used.
• the present example demonstrates that circulating antibodies against ovarian cancer cell surface markers are present in ascites fluid samples.
• Peptides that are mimeotopes of the endogenous cancer markers may be identified by phage display panning against ascites immunoglobulins.
• Peptide motifs identified by panning ascitic IgG are listed in Table 13.
• VPELGHE SEQ HD NO:249
• QRLVHP SEQ HD NO:260
• FIG. 22 shows that phage bearing the sequence CVPELGHEC (SEQ HD NO:271) bind with very high selectivity to IgG from the ovarian cancer patient, but not to IgG from a normal patient or to BSA.
• the phage binding was also selective for ascites from the ovarian cancer patient from which the phage sequence was originally selected (patient #2).
• the circulating antibody that the CVPELGHEC (SEQ HD NO:271) sequence bound to was present in the blood serum of patient #2.
• Peptide motifs identified by panning against patient #2 ascites showed homology to the catalytic domain of matrix metalloproteinases (MMPs), as shown in FIG. 25.
• MMPs matrix metalloproteinases
• Homology searches identified several other candidate protein homologs for the ovarian cancer targeting peptides or motifs identified by ascites screening, including an unnamed protein product from HUVEC cells (FTRWRYL, SEQ HD NO:267); coxsackie and adenovirus receptor protein (SLGGMSG, SEQ HD NO:263); estrogen receptor (GLPSGL, SEQ HD NO:268), TSH, FSH, LH and ⁇ -hCG receptor (CVPELGHEC, SEQ HD NO.-271, CELGFELGC, SEQ HD NO:272); endothelin-converting enzyme (ELGFELG, SEQ HD NO:250), and fibronectin leucine rich transmembrane protein 1 (FFLRDWF, SEQ HD NO:253).
• FTRWRYL SEQ HD NO:267
• SSGGMSG coxsackie and adenovirus receptor protein
• GLPSGL coxsackie and adenovirus receptor protein
• the targeting peptides identified herein may be used to purify antibodies against ovarian cancer markers or as antigens to produce monoclonal or polyclonal antibodies against ovarian cancer markers.
• the peptides and antibodies are also of use for identifying the endogenous ovarian cancer antigenic proteins against which the circulating IgG's are induced.
• Such antibodies may be of use for ovarian cancer diagnosis and/or prognosis, for imaging ovarian cancer, for anti-cancer therapy and for targeted delivery of anti-cancer agents.
• the methods disclosed may be applied to markers for any type of cancer or any other disease state against which circulating antibodies may be found in blood, ascites, lymphatic fluid or any other sample from an individual suspected of exhibiting cancer or another disease.
• MAb 13C03 anti-human CD13, IgGl was from Neomarkers, LabVision Corporation (Fremont, CA); mAb WM15 (anti-human CD13, IgGl) was from Pharmingen (San Diego, CA); Human recombinant TNF and NGR-TNF (consisting of human TNF M57 fused with the C-te ⁇ ninus of CNGRCG, SEQ HD NO:271) were prepared by recombinant DNA technology and purified from E.coli cell extracts, according to Curnis et al. (2000). mAb 78 (IgGl) was obtained from Dr E. Barbanti (Pharmacia-Upjohn, Milan, Italy). MAb 78 is an anti-human TNF antibody able to form stable complexes with soluble TNF Q£__ 3.2 x 10 "10 M) and to neutralize its interaction with membrane receptors (Barbanti et al, 1993).
• Surgical specimens of human tissues (Bouin-fixed for 4-6 h, paraffin-embedded specimens 5-6 ⁇ m thick) were adsorbed on polylysine-coated slides. Antigens were detected using the avidin-biotin complex method.
• Tissue sections were rehydrated using xylenes and a graded alcohol series, according to standard procedures. Tissue sections were placed in a vessel containing 1 mM EDTA and boiled for 7 min using a microwave oven (1000 W). The vessel was then refilled with 1 mM EDTA and boiled again for 5 min. The tissue sections were left to cool and incubated in PBS containing 0.3% hydrogen peroxide for 15 min to quench endogenous peroxidase. The samples were then rinsed with PBS and incubated with 100-200 ⁇ l of PBS-BSA (1 h at room temperature) followed by the primary antibody or NGR-TNF/78 complex in PBS-BSA (overnight at 4°C).
• NGR-TNF/78 Complexes of human NGR-TNF and anti-TNF mAb 78 (termed NGR-TNF/78) were prepared by incubating a mixture of 1 ⁇ g/ml NGR-TNF and 1 ⁇ g/ml mAb 78, both in PBS containing 2% BSA (PBS-BSA) for 20 min (20°C).
• a mixture of TNF and mAb 78 (termed TNF/78) was prepared in the same way using human TNF instead of NGR-TNF.
• the slides were then washed 3 times (3 min each) with PBS and incubated with PBS-BSA containing 2% normal horse serum (PBS-BSA-NHS) (Vector Laboratories, Burlingame, CA) for 5 min.
• PBS-BSA-NHS normal horse serum
• a tablet of 3,3'-diamino- benzidine-tetrahydrochloride (Merck, Darmstadt, Germany) was dissolved in 10 ml of deionized water containing 0.03% hydrogen peroxide, filtered through a 0.2 ⁇ m membrane and overlaid on tissue sections for 5-10 min. The slides were washed as above and counterstained with Harris' hematoxylin.
• a CD13 isoform associated with tumor vessels is a receptor for NGR-TNF in renal cell carcinoma
• NGR-TNF/78 anti-human TNF mAb 78
• Controls with TNF/78 complexes or mAb 78 alone were also included. These complexes offer the advantage that they can be used as a single reagent in parallel with other antibodies.
• the staining patterns obtained with NGR-TNF/78 were very similar to those of WM15 and distinct from those of 13C03 (not shown). Like WM15, NGR-TNF/78 interacted with tumor associated vessels but not with the brush border of renal proximal tubule epithelial cells (not shown). No binding was observed with controls, such as TNF/78 or mAb 78 alone (not shown).
• tissue thin sections for immunohistochemistry and detection of receptors demonstrated in Example 13, was confirmed for angiostatin receptors.
• Angiostatin was incubated with tissue sections of metastitic human bone manow. After washing, the tissue sections were developed with an anti-angiostatin Ab, followed by the conesponding secondary Ab conjugated to peroxidase. Tissue sections exhibited staining with a "vessel-like" structure. The staining was specific since only this structure and not other cells were stained. This result suggests that the angiostatin receptor is localized in the vasculature.
• An ti -human Angiostatin were purchased (R&D Systems: AF226). rhAngiostatin was produced by EntreMed, Inc. (Rockville, MD). Surgical specimens of human metastases (Bouin-fixed 4-6 h, paraffin-embedded sections, 5-6 ⁇ m thick) were adsorbed on polylysine-coated slides. Tissue slides were incubated with 100 ⁇ g/ml of rh-angiostatin. The slides were washed 3 times and incubated with anti- angiostatin antibody. The slides were washed again 3 times and bound antibodies were detected using an anti-goat peroxidase-conjugate antibody. Results
• Tissue sections stained with angiostatin binding exhibited a stained with "vessel-like" structure (not shown). The staining was specific since only this structure and not others were stained. Staining did not represent in situ angiostatin, since anti- angiostatin antibody by itself showed no staining of the samples (not shown).
• angiostatin receptors are present in angiogenic tissues. They also confirm that targeting peptides or their endogenous analogs can be used in immunohistological staining to detect the presence of receptors for targeting peptide sequences in tissue thin sections.
• Zetter B Inhibition of angiogenesis by tissue inhibitor of metallopeinase-3. Invest.
• Integrin ⁇ v ⁇ 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 1157-1164, 1994b
• MIKHEEVA G., BELOUSOVA, N., and CUR1EL, D.T. (1998).
• An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackie virus and adenovirus receptor-independent cell entry mechanism. J. Virol. 72; 9706-9713.
• Thalidomide a phase E study in advanced melanoma, renal cell, ovarian and breast cancer. Br J Cancer 82, 812-817, 2000.
• GOLDMAN C.K., ROGERS, B.E., DOUGLAS, J.T., SOSNOWSKI, B.A., Y1NG, W., SH ⁇ GAL, G.P., BAIRD, A., CAMPAIN, J.A., and CURIEL, D.T. (1997).
• aminopeptidase-N is a marker for antigen presenting cells and apears to be co-
• HONG S.S., GALAUP, A., PEYTAVI, R., CHAZAL, N., and BOULANGER, P.A. (1999). Enhancement of adenovirus-mediated gene delivery by use of an oligopeptide with dual binding specificity. Hum. Gene Ther. 10; 2577-2586. HONG, S.S., KARYAN, L., TOURNHER, J., CUREL, D.T., and BOULANGER, P.A. (1997). Adenovirus type 5 fiber knob binds to MHC class I alpha-2 domain at the surface of human epithelial and B lymphoblastoid cells. EMBO J. 16; 2294-2306.
• Joliot, A.H. Triller, A., Volovitch, M. Pernelle, C, and Prochiantz, A. alpha-2,8- Polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide. New Biol.3:1121-1131, 1991a.
• Human myeloid plasma membrane glycoprotein CD13 (gpl50) is identical to aminopeptidase N. J. Clin. Invest.
• MICHAEL S.I., HONG, J.S., CURIEL, D.T., and ENGLER, J.A. (1995). Addition of a short peptide ligand to the adenovirus fiber protein. Gene Ther. 2; 660-668.
• AAV vectors Muzyczka N. Adeno-associated virus (AAV) vectors: will they work? J. Clin. Invest. 94:1351, 1994
• Nicolas and Rubinstein In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988.
• Vascular Endothelial Growth factor B a novel growth factor for endothelial cells. Proc Natl Acad Sci USA, 93, 2576-2581.
• a peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins. J. Cell Biol. 130:1189- 1196, 1995.
• Pelleymounter et al Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269: 540-543, 1994. Pereboeva, L. A., A. V. Pereboev, and G. E. Morris. 1998. Identification of antigenic sites on three hepatitis C virus proteins using phage-displayed peptide libraries. J Med Virol 56:105-11.
• mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants. Embo J 17, 3521-3533 (1998).
• ROELV1NK P.W., LEE, G.M., EINFELD, D.A., KOVESDI, I., and WICKHAM, TJ. (1999). Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing adenoviridae. Science 286; 1568-1571.
• ROMANCZUK H., GALER, C.E., ZABNER, J., BARSOMIAN, G., WADSWORTH, S.C., and O'RIORDAN, CR. (1999). Modification of an adenoviral vector with biologically selected peptides: a novel strategy for gene delivery to cells of choice. Hum. Gene Ther. 10; 2615-2626.
• MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93, 411-422, 1998.
• WATKINS S.J., MESYANZHINOV, V.V., KUROCHKINA, L.P., and HAWKINS, R.E. (1997).
• Watson CA Camera-Benson L, Palmer-Croker R and Pober JS.Nariability among human umbilical vein endothelial cell cultures. Science 268: 447-448, 1995.

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