US20150374853A1 - Brain tumor targeting peptides and methods - Google Patents

Brain tumor targeting peptides and methods Download PDF

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US20150374853A1
US20150374853A1 US14/789,549 US201514789549A US2015374853A1 US 20150374853 A1 US20150374853 A1 US 20150374853A1 US 201514789549 A US201514789549 A US 201514789549A US 2015374853 A1 US2015374853 A1 US 2015374853A1
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peptide
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Stephen Mark Robbins
Jennifer Rahn
Donna Lorraine Senger
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Arch Cancer Therapeutics Inc
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Arch Cancer Therapeutics Inc
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    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
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    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A61K47/48246—
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1866—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
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    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A—HUMAN NECESSITIES
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0085—Brain, e.g. brain implants; Spinal cord
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/007—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests for contrast media
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
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    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/48—NMR imaging systems
    • G01R33/54—Signal processing systems, e.g. using pulse sequences ; Generation or control of pulse sequences; Operator console
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    • G01R33/5601—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution involving use of a contrast agent for contrast manipulation, e.g. a paramagnetic, super-paramagnetic, ferromagnetic or hyperpolarised contrast agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
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    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to the field of peptides capable of targeting malignant glioma cells, and in particular, a brain tumor initiating cell (BTIC) subtype of human glioblastoma multiforme (GBM) cells and highly invasive glioma cell (HIGC) subtype of human GBM cells, and to methods employing the peptides.
  • BTIC brain tumor initiating cell
  • GBM human glioblastoma multiforme
  • HIGC highly invasive glioma cell
  • Glioblastoma multiforme is a complex and heterogeneous disease, with prevalent short-term relapse and a median survival time of about 1 year when treated with surgery, radiotherapy and temozolomide [1-3].
  • Categorization by transcriptional clustering into proneural (better patient survival profile), indeterminant (“neural”), mesenchymal (associated with NF-1 loss) and proliferative (“classical”; associated with EGFR mutation or amplification) subtypes (reviewed in [1]) has underscored the usefulness of individualized patient profiles in determining prognosis as well as rational selection of targeted therapeutics, however a practical approach to targeting these subtypes clinically needs to be developed.
  • invasive glioma has been characterized by recent clinical and in vitro studies which have shown that genetically and phenotypically distinct cells can form long tendrils which extend several centimeters away from the main tumor mass, or form diffusely spread, invasive subpopulations of tumor that are resistant to chemo- and radiotherapy, by virtue of their remote localization from the main tumor site [4-6], as well as expression of drug resistance genes and enhanced DNA repair capabilities [7-10]. These cells are referred to herein as highly invasive glioma cells, or HIGC's.
  • the second putative reservoir is based on concept of Cancer Stem Cells (CSCs), which arose because mechanisms of self-renewal without terminal differentiation were similar between stem cells and cancer cells [11].
  • CSCs Cancer Stem Cells
  • the cancer stem cell hypothesis proposes that a rare population of transformed stem cells, or progenitor cells with acquired self-renewal properties are the source of tumor cell renewal.
  • Evidence for the existence of cancer stem cells has been suggested for a number of hematological malignancies [12-14] and more recently for a number of solid tumors [14-18].
  • BTICs brain tumor-initiating cells
  • BTICs be defined as patient-derived cells with the ability to self-renew, differentiate into multiple lineages and form tumors in vivo [28].
  • the invention includes, in one aspect, a peptide composition for targeting one of (i) a highly invasive glioma cell (HIGC) subtype of human glioblastoma multiforme (GBM) cells characterized by their ability to migrate from one brain hemisphere into which the cells are injected into the contralateral hemisphere, and (ii) a brain tumor initiating cell (BTIC) subtype of human GBM cells characterized by their stem-cell like properties of being able to self renew, generate spheres without the addition of exogenous mitogens and growth factors, and induce tumor formation in vivo when placed in the brains of immunocompromised mice.
  • the composition includes an isolated peptide of between 12-20 amino acids and containing a sequence selected from the group consisting of SEQ ID NOS: 1-10, 13, and 17 for targeting HIGCs, and SEQ ID NOS: 11-16, for targeting BTICs.
  • the composition may include multiple GMB-binding peptides, for example, at least one peptide that binds preferentially to HIGC's and at least one peptide that binds preferentially to BTIC's.
  • the peptide of the composition may be composed of L-amino acids, D-amino acids, a mixture of L- and D-amino acids, or a retro-inverso peptide formed of D-amino acids arranged in reverse order.
  • the composition may include a contrast agent coupled to the peptide.
  • the composition may further include an anti-tumor agent coupled to the peptide.
  • the peptide may contain a sequence selected from the group consisting of SEQ ID NOS: 7 (H10), 13 (E10) and 17 (modified H10), and preferably SEQ ID NOS: 17 (modified H10).
  • the peptide may contain a sequence selected from the group consisting of SEQ ID NOS: 11 (7A), 14 (3F), 16 (3B), and 13 (E10), and preferably SEQ ID NOS: 11 (7A).
  • the isolated peptide may be coupled to a carrier peptide having the sequence identified by SEQ ID NOS: 18 or 19, and in another embodiment, may be encapsulated within a nanoparticle formed of poly(lactide-co-glycolide) copolymer, a cyclodextrin, or cetyl alcohol/polysorbate.
  • compositions for detecting the presence of HIGC or BTIC subtypes of cells in a patient with a human GBM tumor where the composition includes a detectable contrast agent coupled to the peptide.
  • the peptide in an exemplary composition for detecting the presence of an HIGC subtype of cells contains the sequence SEQ ID NO: 7 or 17, and may additionally include the peptide identified by SEQ ID NO: 18 or 19.
  • the peptide in an exemplary composition for detecting the presence of a BTIC subtype class of cells contains the sequence SEQ ID NO: 11, and may additionally include the peptide identified by SEQ ID NO: 18 or 19.
  • the above peptide composition for inhibiting or killing HIGC or BTIC subtypes of cells in a patient with a human glioblastoma multiform (GBM) tumor, where the composition includes an anti-tumor agent coupled to the peptide.
  • the peptide in one exemplary composition for inhibiting or killing an HIGC subtype of cells contains the sequence SEQ ID NO: 7 or 17.
  • the peptide in an exemplary composition for inhibiting or killing a BTIC subtype of cells contains the sequence SEQ ID NO: 11.
  • the invention includes a method of characterizing a glioblastoma multiforme (GBM) tumor in a patient, by the steps of:
  • a peptide composition containing (i) a first peptide having between 12-20 amino acids in length that has been selected for its preferential binding to a highly invasive glioma cell (HIGC) subtype of human GBM cells characterized by their ability to migrate from one brain hemisphere into which the cells are injected into the contralateral hemisphere, and coupled to first peptide, a first contrast agent that allows the first peptide, when bound to cells in the patient tumor region, to be imaged in vivo;
  • HIGC highly invasive glioma cell
  • Steps (a) and (b) may be carried out by one of:
  • the contrast agent is selected from the group consisting of a gadolinium-based contrast agent, an iron oxide contrast agent, and a manganese contrast agent;
  • positron emission tomography PET or scintigraphy
  • the contrast agent is selected from the group consisting of 64 Cu diacetyl-bis(N 4 -methylthiosemicarbazone), 18 F-fluorodeoxyglucose, 18 F-fluoride, 3′-deoxy-3′-[ 18 F]fluorothymidine (FLT), 18 F-fluoromisonidazole, gallium, Technetium-99m, and thallium; and
  • contrast agent is selected from the group consisting of barium, gastrografin, and iodine contrast agents.
  • the peptide compositions may be administered intravenously.
  • steps (a) and (b) are carried out together, and the first and second contrast agents allow the distributions of bound first and second peptides to be independently determined.
  • steps (a) and (b) are carried sequentially.
  • the first peptide administered may contain a sequence selected from the group consisting of SEQ ID NOS: 7 (H10), 13 (E10) and 17 (modified H10).
  • the second peptide administered may contain a sequence selected from the group consisting SEQ ID NOS: 11 (7A), 13 (E10), 14 (3F) and 16 (3B).
  • the invention includes a method of characterizing the gene expression profiles of cellular phenotypes in a human glioblastoma multiforme (GBM) tumor in a patient, by the steps of:
  • identifying cells in the tumor corresponding to one of (i) a differentiated GBM tumor cell, (ii) a highly invasive glioma cell (HIGC) subtype of human glioblastoma multiforme (GBM) cells characterized by their ability to migrate from one brain hemisphere into which the cells are injected into the contralateral hemisphere, and (iii) a brain tumor initiating cell (BTIC) subtype of human GBM cells characterized by their stem-cell like properties of being able to self renew, generate spheres without the addition of exogenous mitogens and growth factors, and induce tumor formation in vivo when placed in the brains of immuno-compromised mice, and
  • FIG. 1A is a schematic paradigm for the selection of phage that bound preferentially to the highly invasive glioma cell population developed by in vivo serial passage referred to as U87R.
  • Peptide selection was performed in a two-step process using a series of biopanning steps where the PhD-12 M13 combinatorial phage display library was first subtracted for phage that bound to non-target cells, the non-invasive U87T cells, to remove any phage common between the cell types. Next, a positive selection was performed for phage that bound preferentially to the target cells, U87R. Any non-bound phage was discarded and the remaining phage were amplified in a series of steps that enriched for the target specific phage referred to as the 12R library.
  • FIG. 1B shows a whole cell ELISA assay that detects phage that is bound to the surface of the cells.
  • the 12R phage library was incubated with the invasive U87R or non-invasive U87T cells, any non-bound phage were removed and an HRP-anti-M13 antibody and TMB substrate (blue) was used to detect bound phage.
  • the 12R library preferentially bound to the highly invasive U87R cells.
  • FIG. 2A illustrates the inhibitory ability of the 12R library subclone H10 to selectively inhibit glioma invasion.
  • Plating serial dilutions of phage with host bacteria on agarose plates was used to isolate phage subclones. Individual clones were isolated, amplified and tested for their inhibitory ability in in vitro invasion assays.
  • the phage designated as H10 specifically and significantly blocked the migration of the invasive U87R cells through brain-like matrix coated transwell membranes (3 ⁇ 10 10 pfu; 4 hrs at 37° C.). Assessing the effects of LPS or random phage controlled for potential contamination by the host bacterial culture;
  • FIG. 2B shows that in addition to the H10 phage, the H10 synthetic peptide (50 uM) effectively and selectively inhibits migration of the highly invasive U87R cells, as well as the U87MG cells transfected with p75 NTR , a protein shown to mediate glioma invasion;
  • FIG. 2C demonstrates the clinical relevance of the H10 peptide, as the migration of 3/5 (60%) brain tumor initiating cell (BTIC) isolates from glioblastoma patients were inhibited by the H10 peptide. Asterisks indicate a statistically significant difference from untreated control; Dunnett's test (p ⁇ 0.05);
  • FIG. 3A is a bar graph showing that cholesterol oxidase inhibits cell migration.
  • the invasive U87R cells were plated on matrix-coated transwell membranes and treated with cholesterol oxidase (1.8 U/mL) for 4 hours. Cells that migrated to the underside of the transwell membrane were stained with crystal violet and counted by light microscopy;
  • FIG. 3B shows that cholesterol oxidase prevents internalization of a lipid raft marker GM1.
  • U87R cells were plated onto matrix-coated transwell membranes and treated with cholesterol oxidase and Alexa 555-cholera toxin B subunit for 120 minutes to assess differences in the uptake and/or turn over of its receptor, GM1.
  • the white arrow indicates a membranous accumulation of GM1;
  • FIG. 4A shows accumulation of GM1 in the presence of H10.
  • Invasive U87R cells were plated onto matrix-coated transwell membranes and incubated with H10 phage in the presence of Alexa 555-cholera toxin B subunit for 30 minutes to assess differences in the uptake and/or turn over of the lipid raft marker GM1.
  • Cells treated with H10 showed a generalized accumulation of GM1 staining, characterized by globular internal structures and membranous localization (white arrows);
  • FIG. 4B are western blots showing that treatment of invasive glioma cells with H10 or cholesterol oxidase results in the accumulation of higher molecular weight complexes of p75 NTR , a membrane protein found in lipid rafts and known to promote cell migration.
  • Density gradient fractions were prepared from U87R cells plated onto collagen and treated with PBS (control), H10 phage, F2 phage, or cholesterol oxidase for 2 hours, and analyzed by Western Blot for p75 NTR .
  • White arrows indicate higher molecular weight p75 NTR -containing complexes. Lines underneath the figure indicate the fractions in which common organelles are generally found;
  • FIG. 4C shows that treatment of glioma cells with H10 results in accumulation of p75 NTR at the plasma membrane.
  • BTIC 25 cells no GFP
  • the p75 NTR receptor which is normally cleaved during p75 NTR signaling, is greatly increased at the cell membrane after H10 treatment.
  • p75 NTR does not appear to accumulate in the H10-affected GM1 compartment, suggesting H10 may act on more than one class of membrane structure;
  • FIG. 5 shows single Z-stack projections from confocal micrographs of BTIC isolates stained with biotinylated 7A peptide. Binding specificity of the 7A peptide was assessed with non-target (U87MG; NSC) and target (BTIC) cells using a biotinylated 7A peptide (red) followed by confocal imaging.
  • FIG. 6A illustrates the in vivo homing capability of H10.
  • Invasive U87R or non-invasive U87T cells were grown in the right brain hemisphere of SCID mice. Once tumors were established, mice were anaesthetized and perfused with H10 phage for 10 minutes. Unbound phage was flushed from the system with PBS. Brains were cryosectioned and immunohistochemically stained for M13 phage or human nuclear antigen (hNA). In comparison to the tumors established using the U87T cells, the H10 phage homed more efficiently to the U87R tumor cells as demonstrated by increased staining on the U87R cell bodies and along the edge of the xenograft mass.
  • hNA human nuclear antigen
  • FIG. 6B shows that 7A homes in vivo to BTIC12 and BTIC25 xenografts.
  • Cells were injected into the right brain hemisphere of SCID mice and allowed to establish for three months. Mice were then perfused with 7A or Al (random control) phage for 10 minutes. The distribution of phage binding was unique between the two BTIC xenografts and again reveals heterogeneity within the samples. 7A homing was not observed in mice bearing tumors generated from the U251N cell line. Perfusion of a BTIC 25 xenograft with a randomly selected phage, Al, showed minimal staining;
  • FIG. 7 demonstrates that the 7A peptide can distinguish subpopulations within a BTIC isolate with defined cellular behavior.
  • BTIC25 was subcloned by limiting dilution and screened for binding to biotinylated 7A peptide (red).
  • Subpopulations that bound to 7A had a higher propensity to form neurospheres in culture, were not highly proliferative as a xenograft in SCID mice, and were found preferentially near the ventricles.
  • Human xenografts were implanted in the right brain hemisphere of mice and visualized by an antibody to a human nuclear antigen (brown). Sections were counterstained with Toluidine blue to visualize all cell nuclei.
  • FIG. 8 is a table showing binding specificities of BTIC-selective peptides on a representative selection of BTIC isolates
  • FIG. 9A are confocal images of p75 NTR transfected glioma cells (U87p75) imaged for uptake of FITC-transferrin.
  • U87p75 NTR grown in the presence of H10 and cholesterol oxidase were assessed for FITC-transferrin uptake 2 hours after cells were plated on collagen-coated transwell membranes. H10 and cholesterol oxidase had no effect on distribution of transferrin or transferrin receptor, markers of clathrin-coated vesicles;
  • FIG. 9B shows cellular fractionation of invasive U87R cells using iodixanol gradient separation.
  • U87R cells were treated with H10, F2, cholesterol oxidase or control PBS for two hours. Cells were lysed, fractioned using an iodixanol gradient, and Western Blot analysis for the transferrin receptor was performed;
  • FIG. 10A shows an anti-p75 NTR Western blot and full-length p75 NTR , the C-terminal fragment (a-secretase) and intracellular domain ( ⁇ -secretase) cleavage products were visualized after treatment with H10, MbCD or H10+MbCD;
  • FIG. 10B shows Western Blot analysis of full-length p75 NTR after exposure to H10 for 4 hours and 1 week;
  • FIG. 10C shows in vitro invasion assays performed in the presence of cholesterol oxidase and methyl- ⁇ -cyclodextran. Both cholesterol oxidase and methyl- ⁇ -cyclodextran significantly inhibited cell migration of U87p75 glioma cells;
  • FIG. 11 shows confocal images of a panel of patient-derived BTIC isolates (rows) that have been screened for binding specificity to a number of biotinylated synthetic peptides (red; columns).
  • FIGS. 12A and 12B show in vitro transwell migration assays in the presence of different extracellular matrices.
  • the presence of brain matrix was essential for H10-mediated abrogation of cell migration in vitro ( FIG. 12A ), with collagen I mediating the majority of the effect ( FIG. 12B ).
  • FIGS. 13A and 13B are high magnification images of thin sections from five mouse brains that were bisected close to a tumor-implantation site ( 13 A), and stained with biotinylated 7A peptide, and thin sections from five mouse brains injected with non-7A binding subclones ( 13 B);
  • FIG. 14A shows the ability of fluorescently labeled 7A peptide to home in a live mouse to a large implanted tumor grown from BT025 cells ( 14 A).
  • FIGS. 14B and 14C are lower and higher magnifications, respectively, of thin sections taken from the brain in FIG. 14A after it was bisected close to the tumor implantation site and stained for human nuclear antigen (green) and total DNA (blue) to outline the positioning of the cells. The red peptide signal is visible on cells immediately adjacent to the main tumor mass as well as spread diffusely in the surrounding tissue.
  • FIG. 15A shows an intact mouse brain carrying a BT025-implanted tumor, after the mouse was injected via the carotid artery with fluorescently tagged 3B peptide;
  • FIG. 15B is a fluorescently stained thin section of the brain in 15 A, showing that the red peptide signal is both within and outside of the main tumor mass;
  • FIG. 16A shows am intact mouse brain bisected at the tumor injection site and cut sides turned upwards
  • FIGS. 16B and 16C are lower and higher magnification photomicrograghs, respectively, of brain sections from animals who received intracarotid injection of the 3F peptide, showing that 3F binds primarily to cells that are diffusely spread outside the main tumor mass;
  • FIGS. 17A and 17B show that similar results to those seen in FIGS. 16B and 16C can be when the peptide is administered by tail vein injection rather than intracarotid injection;
  • FIG. 18A shows a whole mouse brain and the site to tumor injection
  • FIGS. 18B and 18C are photomicrographs of brain sections from animals who received intracarotid injections of the H10 peptide, showing that H10 binds primarily to cells that are diffusely spread outside of the main tumor mass
  • FIG. 18D shows a similar result when the peptide is administered by tail vein;
  • FIG. 19A shows a whole mouse brain and the site to tumor injection
  • FIGS. 19B and 19C are photomicrographs of brain sections from animals who received intracarotid injections of the E10 peptide, showing that E10 binds to a portion of cells within the tumor mass ( 19 B), and to cells outside the main tumor mass ( 19 C); Similar results were observed when the E10 peptide was injected via the tail vein ( FIG. 19D ); and
  • FIG. 20 shows gene clustering of microarray data generated from mRNA isolated from three independently isolated 7A binding cell subclones and three independently isolated 7A non-binding subclones.
  • GBM Human glioblastoma multiforme
  • HIGCs Highly invasive glioma cells
  • GBM cells characterized by an ability to migrate from one brain hemisphere into which the cells are injected into the contralateral hemisphere.
  • An example of an HIGC is the U87R subtype of the U87MG human glioblastoma cells.
  • Brain-tumor initiating cells are a subtype (subpopulation) of human GBM cells characterized by their stem-cell like properties of being able to self renew, generate spheres without the addition of exogenous mitogens and growth factors, and induce tumor formation in vivo when placed in the brains of immuno-compromised mice.
  • Peptide-displaying phage refers to bacteriophage that have been engineered to express a library, typically a combinatorial library, of exogenous peptides on their surfaces, allowing phage selection based on the presence on the phage surface of an exogenous library peptide.
  • HIGC-specific peptides refers to peptides, typically associated with peptide-displaying phage, that bind preferentially to HIGCs under the conditions of phage-display panning described in Section VIIIC below.
  • BTIC-specific peptides are peptides, typically associated with peptide-displaying phage, that bind preferentially to BTICs under the conditions of phage-display panning described in Section VIIID below.
  • Peptide-displaying phage “bind preferentially to HIGCs or BTICs” if the phage remain bound to immobilized HIGC or BTIC target cells under the phage-panning wash conditions described in Section VIIIC and VIIID, respectively, below.
  • Amino acid residues are indicated herein by their standard one-letter code (see, for example, www.mun.ca/biochem/courses/3107/aasymbols.html).
  • a “contrast agent” refers to an imaging agent used in connection with an imaging technique, such as magnetic resonance imaging (MRI), positron emission tomography (PET), scintigraphy, computed tomography (CT), and x-ray imagining, to enhance the information available from the image, particularly as it relates to the binding of the imaging agent to target anatomical structures or cells.
  • an imaging technique such as magnetic resonance imaging (MRI), positron emission tomography (PET), scintigraphy, computed tomography (CT), and x-ray imagining
  • Exemplary contrast agents include (i) gadolinium-based contrast agents, iron oxide contrast agents, and manganese contrast agents used in MRI; 64 Cu diacetyl-bis(N 4 -methylthiosemicarbazone), 18 F-fluorodeoxyglucose, 18 F-fluoride, 3′-deoxy-3′-[ 18 F]fluorothymidine (FLT), 18 F-fluoromisonidazole, gallium, Technetium-99m, and thallium, used in PET or scintigraphy; and barium, gastrografin, and iodine contrast agents used in x-ray imaging.
  • agent is “coupled” to a peptide if it is attached directly or indirectly to the peptide in a manner that allows the agent to be carried with the peptide, e.g., in the bloodstream, as a stable two-component composition.
  • the agent may be covalently coupled to the peptide, or carried in a structure, such as a chelate, cavitand, nanoparticle, or lipid particle, which is itself covalently coupled to the peptide, or may be carried with the peptide within a stable structure, such as a nanoparticle or lipid structure.
  • the invasive glioma and BTIC compartments have been newly modeled, allowing detailed examination of potential targetable components.
  • the invasive glioma model described herein was developed by in vivo serial passaging of GFP neo-transfected U87MG human glioblastoma cells through mouse brains to isolate the invasive subpopulation (U87R, remote from primary tumor) from brain hemispheres that were contralateral to the injection sites [5, 6].
  • U87R remote from primary tumor
  • the BTIC model consists of a series of primary cell cultures derived from freshly resected human brain tumor specimens. Originating tumors are tested against a routine panel of antibodies to confirm a diagnosis of GBM, then subpopulations are selected via their ability to form self-renewing neurospheres in culture, as well as differentiate into astrocytic, oligodendrocytic or neuronal lineages. Upon injection of as few as ten cells into mouse brains, these brain tumor initiating cells (BTICs) tend to recapitulate the primary tumor as well as robustly invade, similar to what is seen with clinical GBM specimens [28]. Although it is unclear if BTICs are the best representation of brain cancer stem cells, they remain an excellent model for delineating the behavior of the human disease.
  • peptides useful in targeting, characterizing and manipulating cells from disease reservoirs implicated in relapse of post-treatment GBM have been identified.
  • the peptide selection was accomplished through phage display techniques already proven successful in other studies. For example, biopanning a phage display library against target cells or purified molecules has led to the characterization of cell surface proteins unique to defined cell subpopulations (e.g. the vascular address system [30, 31]), the identification of motifs important for specific protein function (e.g. the RGD motif necessary for integrin engagement [32-34]), as well as the isolation of peptide reagents with functional utility [35-38].
  • the present inventors have earlier described an in vivo selection for a population of human glioma cells that were highly invasive (U87R) [6].
  • U87R highly invasive
  • a biopanning experiment using a combinatorial phage display library was employed. Initially subtractive biopanning was performed with the U87 cells that were non-invasive (U87T) followed by selective biopanning with the U87R cells to isolate a library of phage ( FIG. 1A ) that were confirmed to preferentially interact with U87R cells by whole-cell ELISA ( FIG. 1B ).
  • phage were subcloned by plating serial dilutions of phage with host bacteria on agarose plates, and the sequences of the peptide inserts were determined, and listed in Table 1, which shows, from left-to-right, (i) the peptide identifier, (ii) peptide sequence e, (iii) SEQ ID NO, and (iv) number cell-binding phages containing that sequence.
  • phage were tested for the ability to functionally affect the behavior of the U87R cells.
  • the H10 phage-displayed sequence (SEQ ID NO:7) was found to abrogate the migration of the U87R cells through membranes coated with a brain-like extracellular matrix ( FIG. 2A ).
  • Other phage peptides e.g., M32 and M24, also showed a similar inhibitory trend as H10, although not as strong an effect.
  • the possibility that bacterial components from the host culture could be causing the inhibition was ruled out by also testing the effects of LPS and a 20 ⁇ volume of randomly selected phage.
  • a synthetic version of the H10 peptide was also able to inhibit the migration of U87R cells, as well as U87MG cells transfected with p75 NTR ( FIG. 2B ), a molecule shown to be sufficient to confer a migratory phenotype in brain tumor xenografts [6]. These results confirmed the inhibitory property of this peptide in the absence of the phage particle.
  • H10 peptide The global utility of the H10 peptide was tested in the transwell assay by using BTIC lines established from individual patient specimens. As these were isolated directly from resected tumors, it was felt they would give an indication of the true clinical potential of H10. The migration of 60% (3/5) of the BTIC lines tested was significantly inhibited, although all five showed an inhibitory trend ( FIG. 2C ). In experiments where H10 had no significant effect, the cells were not highly invasive to begin with.
  • the p75 neurotrophin receptor can be detected differentially in lipid rafts isolated with specific detergents [49].
  • intracellular vesicles can deliver molecules required for a particular function to the polar extremes of a cell, for example, transport of digestive enzymes or structural molecules such as integrins to the leading edge during migration [50-52].
  • the p75 NTR can be detected differentially in lipid rafts isolated with specific detergents [49], and must be proteolytically processed to trigger cell migration in glioma cells [5]. Thus, it is probable that p75 NTR is also cleaved within a lipid raft after endocytosis [53]. To determine if H10 may be having an impact on endosomally located p75 NTR , cells were hypertonically lysed (to keep vesicles and organelles at least partially intact) and separated on a 10-40% sucrose/iodixanol gradient from which fractions were individually collected.
  • H10 and cholesterol oxidase have no effect on the intracellular localization or gradient fractionation of transferrin or its receptor, which are markers of clathrin-coated vesicles ( FIGS. 9A and 9B ), and while H10 does not show an obvious effect on the processing of over-expressed transfected p75 NTR , it may cause a slight accumulation of endogenously expressed full length protein ( FIGS. 10A-10C ).
  • a BTIC-specific phage library was generated as described for the 12R library, using U87MG cells, U251N cells, and human normal fetal astrocytes as subtraction cells to deplete the library of background phage, and a mixture of BTIC isolates (BTIC 12, 25, 42, 50) from different patients for the selection.
  • the most abundant phage that was isolated was called 7A (25/50) and encoded the 12-mer sequence N-PSPHRQRQHILR-C (SEQ ID NO: 11).
  • 5 other independent phage (10C, E10, 3F, C1 and 3B) were isolated more than once and comprised 50% (25/50) of the subclones screened, shown in Table 3.
  • FIG. 5B shows binding specificities of BTIC-specific peptide hits on a representative selection of BTICs.
  • E10 binds to both U87MG and BTIC's, and has further been found to bind to both U87R (HIGC) and U87T (main tumor) cells.
  • HIGC U87R
  • U87T main tumor
  • the ability of the selected phage or their corresponding peptides was examined for their ability to ‘home’ to their respective U87R or BTIC glioma cell targets in vivo, according to their ability to detect glioma cells in an orthotopic xenograft model.
  • SCID mice carrying U87R or U87T xenografts were injected with the test phage for ten minutes, which was subsequently perfused with PBS to remove any unbound particles.
  • Serial sections of the brains were immunohistochemically stained for M13 phage, demonstrating that H10 homed specifically to the U87R, but not the U87T tumors ( FIG. 6A ).
  • the studies reported above indicate that 7A phage/peptide recognizes only a subset of cells within an individual BTIC line ( FIG. 5 , FIG. 6B ). It was therefore of interest to determine whether there were unique properties of these cells of which 7A binding may be an indicator.
  • the BTIC 25 cells were subcloned by limiting dilution and screened for peptide binding. The population of cells enriched for 7A binding was distinctly different from the non-binding counterparts, forming neurospheres in culture as opposed to adherent colonies ( FIG. 7 ).
  • the 7A binding subpopulation did not proliferate well in orthotopic xenografts (3 months growth time), and could only be detected in small numbers within the subventricular zone of the brain, a region in the brain known to host a neural stem cell niche.
  • the 7A non-binding subpopulation were very tumorigenic and formed very large tumor masses with limited invasion into surrounding tissue. Therefore, the 7A peptide distinguishes a population of cells associated with neurosphere formation, low proliferative activity in vivo, and a preference to locate within the subventricular zone. The fact that both populations were derived from a BTIC culture indicates that 7A is able to track the progression of cells within this reservoir from hidden precursor to recurred tumor.
  • the invention includes a peptide composition for targeting either (i) a highly invasive glioma cells (HIGC) subtype of human GBM cells, e.g., the U87R subtype of U87GM cells, or (ii) a brain tumor initiating cell (BTIC) subtype of human GBM cells, as characterized above.
  • HIGC highly invasive glioma cells
  • BTIC brain tumor initiating cell
  • the peptide in the composition is 12-20 amino acid residues in length and contains one of the sequences identified as SEQ ID NOS-1-17, or a sequence that is at least 90% homologous with the given sequence. That is, the peptide has the same sequence as one of SEQ ID NOS: 1-17, or a sequence that differs from the given sequence by at most one amino acid residue.
  • the peptide may contain only the given sequence of amino acids, or may contain additional N- and/or C-terminal residues up to a total of 20 residues.
  • the peptide corresponding to SEQ ID NO: 7, TNSIWYTAPYMF (H10) may have this exact sequence, the same sequence but with a single amino acid substitution, addition or deletion at any of the 12 residue positions, or a peptide having a total of up to eight additional residues at one or both of the N- or C-terminals.
  • SEQ ID NO:17 contains the 12 residues of SEQ ID NO: 7 (H10), plus three N-terminal residues (KKG) which make the H10 peptide substantially more water soluble by virtue of the two additional lysine residues.
  • the peptide of the invention is formed by conventional solid-phase or recombinant DNA synthetic methods, using amino acids having the natural L-isomer form, the D-amino acid form, or a mixture of the two.
  • Peptides having an all D-form or partial D-form composition are expected to be more resistant to proteolytic breakdown under biological conditions, e.g., when administered to a patient.
  • Solid-phase peptide synthesis methods for preparing peptides composed of all L-amino acids, all D-amino acids or a mixture of D- and L-amino acids utilizing activated D- or L-form amino acid reagents are described, for example, in Guichard, G., et al., Proc. Nat. Acad. Sci.
  • the peptides may be composed of D-amino acids synthesized in a reverse-sequence direction, that is, in a carboxy to amine end direction, to produce a so-called retro-inverso (RI) pilin peptide.
  • RI retro-inverso
  • Methods for synthesizing RI-form peptides are detailed, for example, in Fletcher, M. D. and Campbell, M. M., Partially Modified Retro-Inverso Peptides: Development, Synthesis, and Conformational Behavior, Chem Rev, 1998, 98:763-795, which is incorporated herein by reference.
  • the peptide For use in targeting HIGCs, the peptide contains a sequence selected from the group consisting of SEQ ID NOS: 1-10, 13, and 17, including SEQ ID NOS: 2 (M32), 7 (H10), and 9 (M24), of which SEQ ID NO: 7 (H10) is exemplary, 13 binds to both BTIC's, HIGC's and a population of differentiated GMB tumor cells, and 17 is modified for enhanced solubility.
  • the peptide For use in targeting BTICs, the peptide contains a sequence selected from the group consisting of SEQ ID NOS: 11-16, preferably SEQ ID NOS: 11(7A), 14 (3F) and 16 (3B), or which SEQ ID NO: 11 is exemplary.
  • the peptide may be coupled to a contrast agent, such as fluorescent moiety, for use in imaging GBM cell types in vitro, or a contrast agent, for use in imaging GBM cell types in vivo.
  • a contrast agent such as fluorescent moiety
  • Major imaging technologies that presently utilize contrast agents for in vivo imaging include x-ray/Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Positron Emission
  • contrast agents are (i) gadolinium-based contrast agents, iron oxide contrast agents, and manganese contrast agents used in MRI; 64 Cu diacetyl-bis(N 4 -methylthiosemicarbazone), 18 F-fluorodeoxyglucose, 18 F-fluoride, 3′-deoxy-3′-[ 18 F]fluorothymidine (FLT), 18 F-fluoromisonidazole, gallium, Technetium-99m, and thallium, used in PET or scintigraphy; and barium, gastrografin, and iodine contrast agents used in x-Ray imaging.
  • the contrast agent or coordination complex carrying the agent e.g., a radio-isotopic metal, may be covalently attached to the peptide according to well known methods, e.g., through a covalently attached ligand coordination complex.
  • the peptide may be coupled to one or a variety of anti-tumor agents, including, for example, alkylating agents, anti-metabolites, plant alkaloids and terpenoids, e.g. taxol, topoisomerases, proeosome inhibitors, and monoclonal antibodies.
  • anti-tumor agents may be covalently attached to the peptide according to well-known methods.
  • the peptides are attached to the surfaces of particles, e.g., liposomes, that carry the anti-tumor agent in encapsulated form.
  • the peptide of the invention is formed as a fusion peptide with a peptide carrier, such as AngioPep-2 or AngioPep-7 (SEQ ID NOS: 18 and 19, respectively) that is capable of facilitating passage of the peptide across the blood brain barrier (BBB) (62).
  • a peptide carrier such as AngioPep-2 or AngioPep-7 (SEQ ID NOS: 18 and 19, respectively) that is capable of facilitating passage of the peptide across the blood brain barrier (BBB) (62).
  • composition of the invention may include (i) the peptide alone, (ii) the peptide in a suitable carrier, e.g., sterile physiological saline, in liquid or dried form, (iii) the peptide in coupled form as described above, or (iv) the peptide formulated in a suitable peptide-delivery nanoparticle, such as encapsulated within nanoparticles of poly(lactide-co-glycolide) copolymer, cyclodextrin nanoparticles, or cetyl alcohol/polysorbate.
  • Nanoparticles useful for delivering drugs across the BBB barrier are well known in the art (e.g., 68-71).
  • the nanoparticles are preferably in the 30-100 nm size range and may be coated with polyethyleneglycol for enhanced circulation time in the bloodstream.
  • the particles may be suspended in an injectable medium, e.g., sterile physiological saline, or supplied in dehydrated form.
  • the peptide composition may contain multiple GBM binding peptides, and/or peptide conjugates, for example, at least one peptide or peptide conjugate that binds preferentially to BTIC's and at least one peptide or peptide conjugate that binds preferentially to HIGC's.
  • the agent may include any pharmaceutical agent used, or potentially useful, in treating a neurological condition, such as depression, anxiety, schizophrenia, hyperactivity, Alzheimer's disease, or Parkinson's disease, or any anti-tumor compound, such as from the classes named above, that would be effective against cancers of the brain or spine if the blood brain barrier could be breached.
  • a neurological condition such as depression, anxiety, schizophrenia, hyperactivity, Alzheimer's disease, or Parkinson's disease
  • any anti-tumor compound such as from the classes named above, that would be effective against cancers of the brain or spine if the blood brain barrier could be breached.
  • the agent is coupled to the peptide according to known methods, such those involving direct attachment of the compound to an activated hydroxyl, sulfide, amine or carboxyl groups on the peptide or attachment of the peptide to a corresponding activated group on the compound, or by the use of a bifunctional coupling reagent.
  • the invention provides methods for diagnosis and/or treating patients with human GBM tumors, and in particular, provides an improvement in the treatment of GBM tumors in human patients, by characterizing and/or treating subpopulations of tumor cells that are likely causes of tumor recurrence.
  • the patient may have been previously treated with one or more known treatment modalities, such as chemotherapy, x-radiation therapy and/or surgical resection.
  • the initial treatment may cause a significant reduction in tumor size or growth, but the tumor may likely recur due to the presence of a HIGC subpopulation of cells that may reside outside the treatment region, or through a BTIC subpopulation of cells capable of reseeding the tumor with actively dividing GBM cells
  • the invention provides a method of characterizing a glioblastoma multiforme (GBM) tumor in a patient according to the density and distribution of different cell phenotypes within the tumor, including the HIGC and BTIC phenotypes, associated with tumor reservoir that may escape current treatment methods.
  • GBM glioblastoma multiforme
  • the method involves generating one or more images of the patient's brain tumor that indicates the density and distribution of both HIGC and BTIC subtypes within the tumor region, and optionally, the main differentiated tumor cells making up the majority of the tumor.
  • the patient is administered a cocktail (multiple-peptide composition) of two or more single-peptide compositions, one containing a peptide that preferentially targets HIGC cells and is coupled to a first contrast agent, and the second containing a peptide that targets BTIC cells and is coupled to a second contrast agent, where the two contrast agents allow visualization of binding to each peptide to its target cell type.
  • an individual image is formed after administration of each peptide composition.
  • the same contrast agent may be used for each image.
  • the separate images can be integrated into a single image by known image processing techniques.
  • the techniques that may be used include magnetic resonance imaging (MRI), positron emission tomography (PET), scintigraphy, computed tomography (CT), and x-ray.
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • CT computed tomography
  • x-ray a variety of suitable contrast agents are available, including (i) gadolinium-based contrast agents, iron oxide contrast agents, and manganese contrast agents used in MRI; 64 Cu diacetyl-bis(N 4 -methylthiosemicarbazone), 18 F-fluorodeoxyglucose, 18 F-fluoride, 3′-deoxy-3′-[ 18 F]fluorothymidine (FLT), 18 F-fluoromisonidazole, gallium, Technetium-99m, and thallium, used in PET or scintigraphy; and barium, gastrografin, and iodine contrast agents used in x-ray imaging.
  • the contrast agents may be coupled to the associated
  • the physician can readily assess the density and distribution, e.g., localization of the first and second peptides and their associated contrast agent within the tumor and at the margins of the tumor.
  • FIGS. 13-19 demonstrate in vivo targeting to BTIC- and HIGC-specific peptides to specific cells of a human GBM tumor implanted in a mouse brain.
  • brain tumors were implanted in mice with a patient-derived BTIC cell line BT025 identified as above by its binding to the 7A peptide. After tumor growth from the implanted cells, the tumors were removed, sectioned and stained for microscopic visualization.
  • a fluorescent peptide was administered intravenously, either by intracorotid injection or via tail vein into mice having an implanted GBM tumor from the BT025 cell line ( FIGS.
  • FIG. 13A shows high magnification images of thin sections from five mouse brains that were previously injected with the BT025 cell line, and stained with antibodies specific to an antigen present only in human nuclei, and a dark brown detection reagent. The figure confirms the diffusion of 7A binding cells through the brain tissue.
  • FIG. 13B shows thin sections from five separate mouse brains injected with the BT025 subclones.
  • the toluidine blue staining clearly shows massive tumor growth and disruption of normal tissue architecture.
  • FIG. 14A shows the ability of fluorescently labeled 7A peptide (SEQ ID NO: 11, preferential for BTIC binding) to home in a live mouse to a large implanted tumor grown from BT025 cells.
  • the peptide was administered to the mouse via intracarotid injection and allowed to circulate for approximately half an hour to allow clearance of background signal before the mouse was sacrificed and the brain removed for imaging using an Olympus OV100 whole mouse imaging system. By non-magnified inspection, the large outgrowth of the tumor is obvious.
  • FIG. 14A The fluorescent signal appears strongly in part of the tumor, but clearly does not detect all tumor cells present.
  • FIGS. 14B and 14C are lower and higher magnification photomicrographs of a thin section taken from the brain in FIG. 14A after it was bisected close to the tumor implantation site and stained for human nuclear antigen (green) and total DNA (blue) to outline the positioning of the cells.
  • the red peptide signal is visible on cells immediately adjacent to the main tumor mass as well as spread diffusely in the surrounding tissue.
  • FIG. 14C in particular, shows that the 7A peptide binds primarily to cells that are diffusely spread outside the main tumor mass.
  • FIGS. 15A and 15B show similar sections views, where the animal was injected with a fluorescently labeled 3B peptide (SEQ ID NO: 16, also preferential for BTIC cell binding).
  • FIG. 15B shows that 3B binds to cells that are both within and outside the main tumor mass. This indicates that 3B can detect both proliferative and diffusely migratory tumor cell populations.
  • FIGS. 16A and 16B show similar views, where the animal was injected with a fluorescently labeled 3F peptide (SEQ ID NO: 14, also preferential for BTIC cell binding).
  • FIG. 16B shows that when thin sectioned, most of the red signal appears outside of the main tumor mass, but close to the green signal for human nuclear antigen on cells that have diffusely migrated in the surrounding tissue.
  • FIGS. 17A and 17B shows that similar results can be obtained when the peptide is administered by tail vein injection rather than intracarotid.
  • FIGS. 18 A and 18 B- 18 D show similar views, where the animal was injected with a fluorescently labeled modified H10 peptide (SEQ ID NO: 17, preferential for HIGC binding), in animals carrying a tumor initiated with the U87MG human glioblastoma cell line.
  • a fluorescently labeled modified H10 peptide SEQ ID NO: 17, preferential for HIGC binding
  • the tumors were not grown as large, and a far-red fluorescent signal is not obvious from inspection without sectioning ( FIG. 18A ).
  • FIG. 18B-18C show that the modified H10 peptide binds primarily to cells that are diffusely spread outside the main tumor mass, as would be expected of HIGC's.
  • FIG. 18D shows that the modified H10 peptide binds primarily to cells that are diffusely spread outside the main tumor mass, as would be expected of HIGC's.
  • FIG. 18D shows that the modified H10 peptide binds primarily to cells that are diffusely spread outside the
  • FIGS. 19A and 19B and 19 D show similar views, where the animal was injected with a fluorescently labeled modified E10 peptide (SEQ ID NO: 13, which binds to both BTIC's and HIGC's), in animals carrying a tumor initiated with the U87MG human glioblastoma cell line.
  • a fluorescently labeled modified E10 peptide SEQ ID NO: 13, which binds to both BTIC's and HIGC's
  • FIG. 19A shows that E10 binds to a portion of cells within the main tumor mass
  • FIG. 19C shows that E10 also binds to cells outside of the main tumor mass. Similar results were obtained when the peptide was administered via the tail vein.
  • both the main tumor and the tumor margin that may contain reservoir cells, particularly HIGC's but also BTIC's.
  • This information will allow the surgeon to better define the margins of the tumor that need to be removed in order to reduce the risk of relapse, and will allow the radiation oncologist to more accurately define the brain volume and coordinates that need to be included in the radiation treatment.
  • the method can also be used to monitor the effectiveness of treatment in eliminating or reducing reservoir cells.
  • the brain may be imaged in accordance with the method at an initial time zero, then reimaged at one or more times over the course of the treatment, to track the effect of the tumor on reservoir cells.
  • the treatment method may be used to tailoring a chemotherapy for GBM tumor therapy, using the different expression profiles of different tumor-cell phenotypes within the tumor.
  • FIG. 20 show gene clustering of microarray data generated from mRNA isolated from three independently isolated 7A binding cell subclones and three independently isolated 7A non-binding subclones. The gene expression profiles of all the 7A binding subclones clustered together (demonstrating similar gene expression), as did the 7A non-binding subclones. This strengthens the notion that 7A distinguishes between BTIC subtypes that are transcriptionally unique, and whose behavior in vivo can be predicted.
  • the gene expression data may allow the oncologist to examine the different gene expression profiles of cells in identifying chemotherapeutic agents that are most effective against particular gene expression profiles.
  • the discovery of peptides that bind preferentially to particular peptides to GBM cell subtypes that in turn have different gene expression profiles provides a novel method for characterizing a tumor in terms of its component cells. According to this method, the oncologist would correlate the identified cell type with the known gene expression pattern of that cell type, then determine the presence and/or distribution and/or number of cells of each particular type with the peptides of the invention, as a basis for tailoring the therapy to a known identified gene expression profile.
  • the peptide composition of the invention are useful for targeting therapeutic agents to particular tumor cell types for tumor treatment.
  • the oncologist might adopt one known therapy, e.g., surgery, radiation, or chemotherapy or a combination of these, for treating the tumor, and secondarily use the peptide composition of the invention to target therapeutic agents to the reservoir cells of the tumor, to minimize the reservoir effect in relapse after the initial treatment.
  • a number of delivery system are available for delivering the peptide composition to the brain (62-71), including intravenous administration.
  • the H10 peptide is itself able to pass the blood brain barrier, and so may be administered by any systemic route including intra-arterial, IV, IM, subQ, nasal, mucosal or through the lungs, either as a peptide alone or peptide coupled to an imaging or an anti-tumor agent.
  • the studies conducted on mice tumor further shows that all of the peptides studied were able to target brain tissue following intravenous administration.
  • Neurosurgical-based strategies include intraventricular drug infusion, intracerebral implants, and BBB disruption.
  • Pharmacologic-based strategies include the use of nanoparticle carriers (described, for example, in 68-71).
  • Physiologic-based strategies take advantage of the normal, endogenous pathways of either carrier-mediated transport of nutrients or receptor-mediated transport of peptides.
  • ANG1005 consists of three paclitaxel molecules linked to a novel peptide, Angio-Pep-2, that allows it to be transported across the blood-brain barrier via the low-density lipoprotein receptor-related protein 1 receptor (62).
  • Angio-pep-2 is 19-amino acid peptide (SEQ ID NO: 17) that binds to low density lipoprotein receptor-related protein (LRP) receptors at the BBB and has the potential to deliver drugs to brain by receptor-mediated transport.
  • LRP low density lipoprotein receptor-related protein
  • the BTIC- or HIGC-specific peptide with or without associated radio-imaging or anti-tumor agent, is coupled to the Angio-pep peptide, and delivered by a systemic route.
  • the composition of the invention can be administered either directly in wafers or by installing a convection enhanced delivery pump to deliver the peptides on a routine schedule.
  • Convection-enhanced delivery uses an infusion catheter whose tip is placed close to the target site.
  • a cannula is inserted directly into the area of the brain to be treated, and the therapeutic agent is delivered through the cannula via bulk flow, circumventing the BBB.
  • the peptide composition could alternatively be administered after brief disruption of the BBB using mannitol or other BBB disruption agent. This approach has recently been used to deliver Avastin to the brain tumors of patients (67).
  • Still another delivery method uses nasal transport routes via the olfactory nerve pathway (axonal transport) and the olfactory epithelial pathway.
  • the diagnostic method is carried out to image reservoirs of BTICs and/or HIGCs, typically after an initial treatment of the GBM tumor, e.g., by surgery, radiation therapy or chemotherapy.
  • the dose of diagnostic composition administered to the patient, or the route of administration may be varied until a meaningful diagnostic result is obtained.
  • the patient may be treated with the peptide in therapeutic form, to knock out or reduce populations of the reservoir cells.
  • progress in reducing HIGC or BTIC populations can be checked periodically with the diagnostic method, and the therapeutic dose of the composition may be varied, if necessary, to increase the extent of inhibition or destruction of the targeted HIGCs or BTICs.
  • Treatment is continued, e.g., by twice weekly or weekly administration of the composition, until a desired endpoint is observed.
  • the human glioma cell line U87 was obtained from the American Type Culture Collection, transfected with GFP and separated into the U87T and U87R subpopulations as previously described [6].
  • the human glioma cell line U251N and human fetal astrocytes were a kind gift from V. W. Yong (University of Calgary, Calgary, Alberta, Canada).
  • DMEM Dulbecco's modified eagle's medium
  • FBS heat-inactivated fetal bovine serum
  • 0.1 mM nonessential amino acids 2 mM L-glutamine, 1 mM sodium pyruvate
  • transfected cells 400 ⁇ g/ml of G418 (Invitrogen) at 37° C. in a humidified 5% CO 2 incubator.
  • G418 Invitrogen
  • BTICs Brain tumour initiating cells
  • phage libraries were amplified as follows: Overnight cultures of the bacterial host strain ER2738 were diluted 1/100 in LB broth with 20 ⁇ g/mL tetracycline and inoculated with 10 ⁇ L of phage ( ⁇ 10 8 -10 10 pfu/ ⁇ L), then cultured with shaking at 37° C. overnight. Bacteria were pelleted and the phage were precipitated from the supernatant by adding 1/6 volume of 20% w/v PEG-8000, 2.5M NaCl and storing at 4° C. overnight, followed by centrifugation for 15 minutes at 10000 ⁇ g. The pellet contained phage and was reconstituted in PBS. Purified phage were put through a 0.22 ⁇ m filter if intended for mouse work.
  • Phage titers were determined by adding 10 ⁇ L of serially diluted phage and 200 ⁇ L of ER2738 to 3 mL of 7% agarose in LB warmed to 50° C., and poured over a warmed plate of LB agar with 20 ⁇ g/mL tetracycline 40 ⁇ g/mL XGal and 50 ⁇ g/mL IPTG. After overnight culture at 37° C., blue plaques were counted for calculation of pfu/ ⁇ L.
  • Plaques could be subcloned by removing an isolated plaque from the agar plate using a Pasteur pipette and extracting the phage in 100 ⁇ L of 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 6 mM MgSO 4 at 4° C. overnight. Amplification cultures were inoculated with 20 ⁇ L of the extraction buffer, as described above.
  • Biopanning was performed as described in [61].
  • Subtraction cells e.g. U87T
  • 1 ⁇ 10 7 cells were resuspended in 1% BSA in PBS with 1.5 ⁇ 10 11 pfu of Ph.D.-12 M13 Phage Library (New England Biolabs).
  • the cells and phage were incubated in an eppendorf tube for 1 hour at room temperature with gentle shaking, before pelleting the cells and retaining the supernatant, which was transferred to a fresh tube with another aliquot of subtraction cells.
  • the incubation and transfer of supernatant was performed three times, with the supernatant being transferred to an empty tube on the last round.
  • Selection cells were released from tissue culture plastic with Puck's EDTA (e.g. U87R cells) and washed in PBS. 5 ⁇ 10 6 cells were resuspended in the 3 ⁇ subtracted supernatant and incubated for 4 hours at 4° C. with slow shaking The cell pellet was washed 5 ⁇ in cold 1% BSA/0.1% Tween 20 in PBS five times, changing the tube after each wash. Bound phage were eluted by rocking the cells gently for 10 minutes at 4° C. in 1 mL of 0.2M Glycine-HCl pH 2.2 with 1 mg/mL BSA, and the supernatant was immediately neutralized with 150 ⁇ L of 1M Tris-HCl pH 9.1. The final library was amplified as per manufacturer's protocol.
  • Puck's EDTA e.g. U87R cells
  • a BTIC-specific phage library was generated as described for the U87R library, this time using U87MG cells, U251N cells, and human normal fetal astrocytes to deplete the library of background phage, and a mixture of BTIC cell lines from different patients were used for the selection. Cells were released with EDTA if adherent, or if grown as neurospheres (e.g. BTIC cells) were separated into single cells by repeated pipetting through a small bore pipette tip. Otherwise, the subtractive and selective biopanning were performed as described for the U87R library.
  • test cells 1 ⁇ 10 4 test cells were plated into a well of a 24-well dish, in 0.5 mL of media and allowed to equilibrate overnight under normal culture conditions, then the media was replaced with HEPES-buffered culture media. 5 ⁇ 10 9 pfu phage were added to the wells with gentle shaking for 1 hour at room temperature followed by three washes with PBS. Bound phage were detected with anti-M13-HRP antibody (GE Healthcare) and insoluble TMB substrate (Sigma).
  • the membranes of 8 ⁇ m pore sized transwell inserts were coated with brain-like matrix (720 ⁇ L of 3 mg/mL collagen I (PureCol); 1804, of 10 ⁇ DMEM (Invitrogen); 9 uL of 1 mg/mL human plasma fibronectin (Sigma); 9 uL of 1 mg/mL chondroitin sulfate proteoglycans (Chemicon); 16 ⁇ L of 0.15 mg/mL laminin (Chemicon)) on both sides, allowed to dry, then plated with 5 ⁇ 10 4 cells in 100 ⁇ L media in the upper chamber and 500 ⁇ L media in the lower chamber. Inclusion of collagen was essential to see the inhibitory effect of H10 (Fig.
  • test phage 3 ⁇ 10 10 pfu of test phage were added to the upper chambers containing the cells, which were incubated for 4 hours under normal culture conditions. At the end of the incubation, the media was aspirated and the cells were fixed and stained in 1% crystal violet in 95% ethanol for one minute. Transwells were rinsed in PBS, then cells on the upper membrane surfaces were removed with a cotton swab. Cells which had migrated to the undersurface of the membranes were counted by light microscopy, and the sum of cells in five microscopic fields was recorded for each membrane.
  • Adherent cells 13 mm coverslips were coated with neutralized 3 mg/mL Collagen I (PureCol), allowed to dry, then plated with test cells at 1 ⁇ 10 4 /mL, 0.5 mL volume and allowed to equilibrate 20 minutes under normal culture conditions. Coverslips were fixed for 10 minutes in 3% formaldehyde in PBS, washed, then incubated in biotinylated peptide (3 mM stock) or primary antibody at a 1:100 dilution in 2% BSA/0.02% Tween 20 in PBS for 1 hour at room temperature. After washing in PBS, secondary antibody or streptavidin-Alexa 568 was applied at a dilution of 1:250 for 1 hour before coverslips were washed and mounted with DAKO mounting media with antifade.
  • Suspended neurospheres 504, of densely suspended neurospheres were stained as described above, except in suspension in an eppendorf tube. The stained neurospheres were mounted in a drop of DAKO mounting media under a coverslip.
  • 3 ⁇ 10 4 U87T or 3 ⁇ 10 5 U87R cells were injected in a 34 volume into one brain hemisphere of a SCID mouse and allowed to grow for 4 or 6 weeks respectively.
  • unlabeled primary antibodies FITC-transferrin or A1exa555-cholera toxin B subunit (Invitrogen) were diluted 1:100 in 2% BSA/0.02% Tween 20 in PBS and used in place of biotinylated peptides.
  • species-specific fluorescently labeled secondary antibodies (Molecular Probes, Invitrogen) were diluted 1:500.
  • Cells were scraped into 1 mL of 0.25M sucrose, 140 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, and dounce homogenized until cells were no longer visible by microscopy. Debris was pelleted for 5 min, 800 ⁇ g and the supernatant was loaded on top of a 10 mL continuous 10-40% gradient, prepared by the dilution of OptiPrep (Sigma, 60% iodixanol) with 0.25M sucrose, 140 mM NaCl, 3 mM EDTA, 60 mM Tris-HCl, pH8.0.
  • OptiPrep Sigma, 60% iodixanol
  • 3 ⁇ 10 4 U87T cells, 3 ⁇ 10 5 U87R cells or 5 ⁇ 10 4 BTICs were injected in a 34 volume into one brain hemisphere of a SCID mouse and allowed to grow for 4, 6 or 12 weeks respectively.
  • the mice were anaesthetized, then treated with 150 ⁇ L of 20% (w/v) mannitol for 15 minutes before injecting 5 ⁇ 10 9 pfu into the brain via the carotid. After allowing the phage to circulate for 10 minutes, unbound phage were flushed out of the circulatory system with 15 mL of PBS injected into the left ventricle after the right atrium was clipped.
  • the brains were harvested, immediately frozen on dry ice and embedded in OCT for sectioning.
  • SVSVGMKPSPRP (SEQ ID NO: 1) (A2) GISLSSYLQSTQ (SEQ ID NO: 2) (M32) EHMALTYPFRPP (SEQ ID NO: 3) (C12) HWAPSMYDYVSW (SEQ ID NO: 4) (M5) RTVPDYTAHVRT (SEQ ID NO: 5) (M43) SGHQLLLNKMPN (SEQ ID NO: 6) (M19) TNSIWYTAPYMF (SEQ ID NO: 7) (H10) GMSLSRQMLWSL (SEQ ID NO: 8) (F2) HLFPQSNYGGHS (SEQ ID NO: 9) (M24) CIQLANPPRLXG (SEQ ID NO: 10) (M23) PSPHRQRQHILR (SEQ ID NO: 11) (7A) QTIRIIIRRSRT (SEQ ID NO: 12) (10C) SLHMRHKRKPRR (SEQ ID NO: 13) (E10) SSRSM

Abstract

A method of diagnosing and treating a human glioblastoma multiforme (GBM) brain tumor in a subject is disclosed. The method includes administering to the subject, an effective amount of composition having a peptide 12-20 amino acid residues in length and selected for its ability to bind preferentially to a subtype of human GBM cells identified as brain tumor initiating cells (BTICs) or highly invasive glioma cells (HIGCs).

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation-in-part of U.S. application Ser. No. 12/950,855, filed Nov. 19, 2010, which claims the benefit of U.S. Provisional Application No. 61/264,064, filed Nov. 24, 2009, both of which are incorporated herein by reference in their entirety.
    • FIELD OF THE INVENTION
  • The present invention relates to the field of peptides capable of targeting malignant glioma cells, and in particular, a brain tumor initiating cell (BTIC) subtype of human glioblastoma multiforme (GBM) cells and highly invasive glioma cell (HIGC) subtype of human GBM cells, and to methods employing the peptides.
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    BACKGROUND OF THE INVENTION
  • Glioblastoma multiforme (GBM) is a complex and heterogeneous disease, with prevalent short-term relapse and a median survival time of about 1 year when treated with surgery, radiotherapy and temozolomide [1-3]. Categorization by transcriptional clustering into proneural (better patient survival profile), indeterminant (“neural”), mesenchymal (associated with NF-1 loss) and proliferative (“classical”; associated with EGFR mutation or amplification) subtypes (reviewed in [1]) has underscored the usefulness of individualized patient profiles in determining prognosis as well as rational selection of targeted therapeutics, however a practical approach to targeting these subtypes clinically needs to be developed.
  • Despite intensive radio- and chemotherapy, tumor regrowth is virtually inevitable and typically occurs within a few centimeters of the resection margin [4]. There are two potential disease reservoirs that may contribute to treatment failure. First, invasive glioma has been characterized by recent clinical and in vitro studies which have shown that genetically and phenotypically distinct cells can form long tendrils which extend several centimeters away from the main tumor mass, or form diffusely spread, invasive subpopulations of tumor that are resistant to chemo- and radiotherapy, by virtue of their remote localization from the main tumor site [4-6], as well as expression of drug resistance genes and enhanced DNA repair capabilities [7-10]. These cells are referred to herein as highly invasive glioma cells, or HIGC's.
  • The second putative reservoir is based on concept of Cancer Stem Cells (CSCs), which arose because mechanisms of self-renewal without terminal differentiation were similar between stem cells and cancer cells [11]. The cancer stem cell hypothesis proposes that a rare population of transformed stem cells, or progenitor cells with acquired self-renewal properties are the source of tumor cell renewal. Evidence for the existence of cancer stem cells has been suggested for a number of hematological malignancies [12-14] and more recently for a number of solid tumors [14-18].
  • There is now accumulating in vitro and in vivo data supporting the involvement of CSCs in glioblastoma [19-25]. The concept of brain tumor stem cells, or as they are referred to herein, as brain tumor-initiating cells or BTICs, is potentially important since they would define a tumor's behavior including proliferation, progression and response to therapy. One of the most important features of BTICs is that they closely resemble the human disease and therefore may be the best system for understanding brain tumor biology and developing therapeutics [23]. In addition, BTICs have fewer cytogenetic and molecular abnormalities [21, 23], which should make identifying causal events (i.e. instead of changes which occur as a consequence of transformation) in brain tumor formation easier. It should be clearly stated that the presence of a BTIC and the exact operational definition and use of terminology is a topic of great debate. As CD133 is controversial as an indicator of “sternness” [26-28], it is proposed herein that BTICs be defined as patient-derived cells with the ability to self-renew, differentiate into multiple lineages and form tumors in vivo [28].
    • SUMMARY OF THE INVENTION
  • The invention includes, in one aspect, a peptide composition for targeting one of (i) a highly invasive glioma cell (HIGC) subtype of human glioblastoma multiforme (GBM) cells characterized by their ability to migrate from one brain hemisphere into which the cells are injected into the contralateral hemisphere, and (ii) a brain tumor initiating cell (BTIC) subtype of human GBM cells characterized by their stem-cell like properties of being able to self renew, generate spheres without the addition of exogenous mitogens and growth factors, and induce tumor formation in vivo when placed in the brains of immunocompromised mice. The composition includes an isolated peptide of between 12-20 amino acids and containing a sequence selected from the group consisting of SEQ ID NOS: 1-10, 13, and 17 for targeting HIGCs, and SEQ ID NOS: 11-16, for targeting BTICs.
  • The composition may include multiple GMB-binding peptides, for example, at least one peptide that binds preferentially to HIGC's and at least one peptide that binds preferentially to BTIC's.
  • The peptide of the composition may be composed of L-amino acids, D-amino acids, a mixture of L- and D-amino acids, or a retro-inverso peptide formed of D-amino acids arranged in reverse order.
  • For use in localizing of HIGCs or BTICs in a subject with a human GBM tumor, the composition may include a contrast agent coupled to the peptide.
  • For use in inhibiting or killing HIGCs or BTICs in a subject with a human GBM tumor, the composition may further include an anti-tumor agent coupled to the peptide.
  • For use in targeting HIGCs, the peptide may contain a sequence selected from the group consisting of SEQ ID NOS: 7 (H10), 13 (E10) and 17 (modified H10), and preferably SEQ ID NOS: 17 (modified H10).
  • For use in targeting BTICs, the peptide may contain a sequence selected from the group consisting of SEQ ID NOS: 11 (7A), 14 (3F), 16 (3B), and 13 (E10), and preferably SEQ ID NOS: 11 (7A).
  • For use in delivery to a patient human GBM tumor across the blood-brain barrier, the isolated peptide may be coupled to a carrier peptide having the sequence identified by SEQ ID NOS: 18 or 19, and in another embodiment, may be encapsulated within a nanoparticle formed of poly(lactide-co-glycolide) copolymer, a cyclodextrin, or cetyl alcohol/polysorbate.
  • Also disclosed is the use of the above peptide composition for detecting the presence of HIGC or BTIC subtypes of cells in a patient with a human GBM tumor, where the composition includes a detectable contrast agent coupled to the peptide. The peptide in an exemplary composition for detecting the presence of an HIGC subtype of cells contains the sequence SEQ ID NO: 7 or 17, and may additionally include the peptide identified by SEQ ID NO: 18 or 19. The peptide in an exemplary composition for detecting the presence of a BTIC subtype class of cells contains the sequence SEQ ID NO: 11, and may additionally include the peptide identified by SEQ ID NO: 18 or 19.
  • Further disclosed is the use of the above peptide composition, for inhibiting or killing HIGC or BTIC subtypes of cells in a patient with a human glioblastoma multiform (GBM) tumor, where the composition includes an anti-tumor agent coupled to the peptide. The peptide in one exemplary composition for inhibiting or killing an HIGC subtype of cells contains the sequence SEQ ID NO: 7 or 17. The peptide in an exemplary composition for inhibiting or killing a BTIC subtype of cells contains the sequence SEQ ID NO: 11.
  • In another aspect, the invention includes a method of characterizing a glioblastoma multiforme (GBM) tumor in a patient, by the steps of:
  • (a) generating an image of the patient's brain tumor after administering to the patient a peptide composition containing (i) a first peptide having between 12-20 amino acids in length that has been selected for its preferential binding to a highly invasive glioma cell (HIGC) subtype of human GBM cells characterized by their ability to migrate from one brain hemisphere into which the cells are injected into the contralateral hemisphere, and coupled to first peptide, a first contrast agent that allows the first peptide, when bound to cells in the patient tumor region, to be imaged in vivo;
  • (b) generating an image of the patient's brain tumor after administering to the patient a peptide composition containing (i) second peptide having between 12-20 amino acids in length that has been selected for its preferential binding to a brain tumor initiating cell (BTIC) subtype of human GBM cells characterized by their stem-cell like properties of being able to self renew, generate spheres without the addition of exogenous mitogens and growth factors, and induce tumor formation in vivo when placed in the brains of immunocompromised mice, and coupled to the second peptide, a second contrast agent that allows the second peptide, when bound to cells in the patient tumor region, to be imagined in vivo; and
  • (c) determining, from the distribution of the first and second peptides and their associated contrast observed in the image(s) generated in steps (a) and (b), at least one of:
  • (ci) the boundaries of the tumor, for purposes of surgical resection of the tumor,
  • (cii) the boundaries of the tumor for purposes of radiation therapy of the tumor,
  • (ciii) the expression profiles of different tumor-cell phenotypes within the tumor, for purposes of tailoring a chemotherapeutic regimen for treating the tumor; and
  • (civ) the change in the distribution of the first and second peptides and their associated contrast agents over a given time course in which steps (a) and (b) are repeated over time.
  • Steps (a) and (b) may be carried out by one of:
  • (i) MRI, wherein the contrast agent is selected from the group consisting of a gadolinium-based contrast agent, an iron oxide contrast agent, and a manganese contrast agent;
  • (ii) positron emission tomography (PET) or scintigraphy, wherein the contrast agent is selected from the group consisting of 64Cu diacetyl-bis(N4-methylthiosemicarbazone), 18F-fluorodeoxyglucose, 18F-fluoride, 3′-deoxy-3′-[18F]fluorothymidine (FLT), 18F-fluoromisonidazole, gallium, Technetium-99m, and thallium; and
  • (iii) x-ray imaging, where the contrast agent is selected from the group consisting of barium, gastrografin, and iodine contrast agents.
  • The peptide compositions may be administered intravenously. In one embodiment, steps (a) and (b) are carried out together, and the first and second contrast agents allow the distributions of bound first and second peptides to be independently determined. In another embodiment, (a) and (b) are carried sequentially.
  • The first peptide administered may contain a sequence selected from the group consisting of SEQ ID NOS: 7 (H10), 13 (E10) and 17 (modified H10). The second peptide administered may contain a sequence selected from the group consisting SEQ ID NOS: 11 (7A), 13 (E10), 14 (3F) and 16 (3B).
  • In yet another aspect, the invention includes a method of characterizing the gene expression profiles of cellular phenotypes in a human glioblastoma multiforme (GBM) tumor in a patient, by the steps of:
  • (a) identifying cells in the tumor corresponding to one of (i) a differentiated GBM tumor cell, (ii) a highly invasive glioma cell (HIGC) subtype of human glioblastoma multiforme (GBM) cells characterized by their ability to migrate from one brain hemisphere into which the cells are injected into the contralateral hemisphere, and (iii) a brain tumor initiating cell (BTIC) subtype of human GBM cells characterized by their stem-cell like properties of being able to self renew, generate spheres without the addition of exogenous mitogens and growth factors, and induce tumor formation in vivo when placed in the brains of immuno-compromised mice, and
  • (b) correlating the identified cell type with the known gene expression pattern of that cell type.
  • These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying figures.
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1A is a schematic paradigm for the selection of phage that bound preferentially to the highly invasive glioma cell population developed by in vivo serial passage referred to as U87R. Peptide selection was performed in a two-step process using a series of biopanning steps where the PhD-12 M13 combinatorial phage display library was first subtracted for phage that bound to non-target cells, the non-invasive U87T cells, to remove any phage common between the cell types. Next, a positive selection was performed for phage that bound preferentially to the target cells, U87R. Any non-bound phage was discarded and the remaining phage were amplified in a series of steps that enriched for the target specific phage referred to as the 12R library.
  • FIG. 1B shows a whole cell ELISA assay that detects phage that is bound to the surface of the cells. The 12R phage library was incubated with the invasive U87R or non-invasive U87T cells, any non-bound phage were removed and an HRP-anti-M13 antibody and TMB substrate (blue) was used to detect bound phage. The 12R library preferentially bound to the highly invasive U87R cells.
  • FIG. 2A illustrates the inhibitory ability of the 12R library subclone H10 to selectively inhibit glioma invasion. Plating serial dilutions of phage with host bacteria on agarose plates was used to isolate phage subclones. Individual clones were isolated, amplified and tested for their inhibitory ability in in vitro invasion assays. The phage designated as H10 specifically and significantly blocked the migration of the invasive U87R cells through brain-like matrix coated transwell membranes (3×1010 pfu; 4 hrs at 37° C.). Assessing the effects of LPS or random phage controlled for potential contamination by the host bacterial culture;
  • FIG. 2B shows that in addition to the H10 phage, the H10 synthetic peptide (50 uM) effectively and selectively inhibits migration of the highly invasive U87R cells, as well as the U87MG cells transfected with p75NTR, a protein shown to mediate glioma invasion;
  • FIG. 2C demonstrates the clinical relevance of the H10 peptide, as the migration of 3/5 (60%) brain tumor initiating cell (BTIC) isolates from glioblastoma patients were inhibited by the H10 peptide. Asterisks indicate a statistically significant difference from untreated control; Dunnett's test (p<0.05);
  • FIG. 3A is a bar graph showing that cholesterol oxidase inhibits cell migration. The invasive U87R cells were plated on matrix-coated transwell membranes and treated with cholesterol oxidase (1.8 U/mL) for 4 hours. Cells that migrated to the underside of the transwell membrane were stained with crystal violet and counted by light microscopy;
  • FIG. 3B shows that cholesterol oxidase prevents internalization of a lipid raft marker GM1. U87R cells were plated onto matrix-coated transwell membranes and treated with cholesterol oxidase and Alexa 555-cholera toxin B subunit for 120 minutes to assess differences in the uptake and/or turn over of its receptor, GM1. The white arrow indicates a membranous accumulation of GM1;
  • FIG. 4A shows accumulation of GM1 in the presence of H10. Invasive U87R cells were plated onto matrix-coated transwell membranes and incubated with H10 phage in the presence of Alexa 555-cholera toxin B subunit for 30 minutes to assess differences in the uptake and/or turn over of the lipid raft marker GM1. Cells treated with H10 showed a generalized accumulation of GM1 staining, characterized by globular internal structures and membranous localization (white arrows);
  • FIG. 4B are western blots showing that treatment of invasive glioma cells with H10 or cholesterol oxidase results in the accumulation of higher molecular weight complexes of p75NTR, a membrane protein found in lipid rafts and known to promote cell migration. Density gradient fractions were prepared from U87R cells plated onto collagen and treated with PBS (control), H10 phage, F2 phage, or cholesterol oxidase for 2 hours, and analyzed by Western Blot for p75NTR. White arrows indicate higher molecular weight p75NTR-containing complexes. Lines underneath the figure indicate the fractions in which common organelles are generally found;
  • FIG. 4C shows that treatment of glioma cells with H10 results in accumulation of p75NTR at the plasma membrane. BTIC 25 cells (no GFP) were plated on matrix-coated transwell membranes and treated with phage for 2 hours. The p75NTR receptor, which is normally cleaved during p75NTR signaling, is greatly increased at the cell membrane after H10 treatment. p75NTR does not appear to accumulate in the H10-affected GM1 compartment, suggesting H10 may act on more than one class of membrane structure;
  • FIG. 5 shows single Z-stack projections from confocal micrographs of BTIC isolates stained with biotinylated 7A peptide. Binding specificity of the 7A peptide was assessed with non-target (U87MG; NSC) and target (BTIC) cells using a biotinylated 7A peptide (red) followed by confocal imaging.
  • FIG. 6A illustrates the in vivo homing capability of H10. Invasive U87R or non-invasive U87T cells were grown in the right brain hemisphere of SCID mice. Once tumors were established, mice were anaesthetized and perfused with H10 phage for 10 minutes. Unbound phage was flushed from the system with PBS. Brains were cryosectioned and immunohistochemically stained for M13 phage or human nuclear antigen (hNA). In comparison to the tumors established using the U87T cells, the H10 phage homed more efficiently to the U87R tumor cells as demonstrated by increased staining on the U87R cell bodies and along the edge of the xenograft mass.
  • FIG. 6B shows that 7A homes in vivo to BTIC12 and BTIC25 xenografts. Cells were injected into the right brain hemisphere of SCID mice and allowed to establish for three months. Mice were then perfused with 7A or Al (random control) phage for 10 minutes. The distribution of phage binding was unique between the two BTIC xenografts and again reveals heterogeneity within the samples. 7A homing was not observed in mice bearing tumors generated from the U251N cell line. Perfusion of a BTIC 25 xenograft with a randomly selected phage, Al, showed minimal staining;
  • FIG. 7 demonstrates that the 7A peptide can distinguish subpopulations within a BTIC isolate with defined cellular behavior. BTIC25 was subcloned by limiting dilution and screened for binding to biotinylated 7A peptide (red). Subpopulations that bound to 7A had a higher propensity to form neurospheres in culture, were not highly proliferative as a xenograft in SCID mice, and were found preferentially near the ventricles. Human xenografts were implanted in the right brain hemisphere of mice and visualized by an antibody to a human nuclear antigen (brown). Sections were counterstained with Toluidine blue to visualize all cell nuclei.
  • FIG. 8 is a table showing binding specificities of BTIC-selective peptides on a representative selection of BTIC isolates;
  • FIG. 9A are confocal images of p75NTR transfected glioma cells (U87p75) imaged for uptake of FITC-transferrin. U87p75NTR grown in the presence of H10 and cholesterol oxidase were assessed for FITC-transferrin uptake 2 hours after cells were plated on collagen-coated transwell membranes. H10 and cholesterol oxidase had no effect on distribution of transferrin or transferrin receptor, markers of clathrin-coated vesicles;
  • FIG. 9B shows cellular fractionation of invasive U87R cells using iodixanol gradient separation. U87R cells were treated with H10, F2, cholesterol oxidase or control PBS for two hours. Cells were lysed, fractioned using an iodixanol gradient, and Western Blot analysis for the transferrin receptor was performed;
  • FIG. 10A shows an anti-p75NTR Western blot and full-length p75NTR, the C-terminal fragment (a-secretase) and intracellular domain (γ-secretase) cleavage products were visualized after treatment with H10, MbCD or H10+MbCD;
  • FIG. 10B shows Western Blot analysis of full-length p75NTR after exposure to H10 for 4 hours and 1 week;
  • FIG. 10C shows in vitro invasion assays performed in the presence of cholesterol oxidase and methyl-β-cyclodextran. Both cholesterol oxidase and methyl-β-cyclodextran significantly inhibited cell migration of U87p75 glioma cells;
  • FIG. 11 shows confocal images of a panel of patient-derived BTIC isolates (rows) that have been screened for binding specificity to a number of biotinylated synthetic peptides (red; columns).
  • FIGS. 12A and 12B show in vitro transwell migration assays in the presence of different extracellular matrices. The presence of brain matrix was essential for H10-mediated abrogation of cell migration in vitro (FIG. 12A), with collagen I mediating the majority of the effect (FIG. 12B).
  • FIGS. 13A and 13B are high magnification images of thin sections from five mouse brains that were bisected close to a tumor-implantation site (13A), and stained with biotinylated 7A peptide, and thin sections from five mouse brains injected with non-7A binding subclones (13B);
  • FIG. 14A shows the ability of fluorescently labeled 7A peptide to home in a live mouse to a large implanted tumor grown from BT025 cells (14A). FIGS. 14B and 14C are lower and higher magnifications, respectively, of thin sections taken from the brain in FIG. 14A after it was bisected close to the tumor implantation site and stained for human nuclear antigen (green) and total DNA (blue) to outline the positioning of the cells. The red peptide signal is visible on cells immediately adjacent to the main tumor mass as well as spread diffusely in the surrounding tissue.
  • FIG. 15A shows an intact mouse brain carrying a BT025-implanted tumor, after the mouse was injected via the carotid artery with fluorescently tagged 3B peptide; FIG. 15B is a fluorescently stained thin section of the brain in 15A, showing that the red peptide signal is both within and outside of the main tumor mass;
  • FIG. 16A shows am intact mouse brain bisected at the tumor injection site and cut sides turned upwards; FIGS. 16B and 16C are lower and higher magnification photomicrograghs, respectively, of brain sections from animals who received intracarotid injection of the 3F peptide, showing that 3F binds primarily to cells that are diffusely spread outside the main tumor mass;
  • FIGS. 17A and 17B show that similar results to those seen in FIGS. 16B and 16C can be when the peptide is administered by tail vein injection rather than intracarotid injection;
  • FIG. 18A shows a whole mouse brain and the site to tumor injection; FIGS. 18B and 18C are photomicrographs of brain sections from animals who received intracarotid injections of the H10 peptide, showing that H10 binds primarily to cells that are diffusely spread outside of the main tumor mass; FIG. 18D shows a similar result when the peptide is administered by tail vein;
  • FIG. 19A shows a whole mouse brain and the site to tumor injection; FIGS. 19B and 19C are photomicrographs of brain sections from animals who received intracarotid injections of the E10 peptide, showing that E10 binds to a portion of cells within the tumor mass (19B), and to cells outside the main tumor mass (19C); Similar results were observed when the E10 peptide was injected via the tail vein (FIG. 19D); and
  • FIG. 20 shows gene clustering of microarray data generated from mRNA isolated from three independently isolated 7A binding cell subclones and three independently isolated 7A non-binding subclones.
    •  DETAILED DESCRIPTION OF THE INVENTION I. Definitions
  • “Human glioblastoma multiforme (GBM)” refers to the most common and aggressive type of primary brain tumor in humans. GBM tumors are characterized by the presence of small areas of necrotizing tissue that is surrounded by anaplastic cells (pseudopalisading necrosis). This characteristic, as well as the presence of hyperplastic blood vessels, differentiates the tumor from Grade 3 astrocytomas, which do not have these features.
  • “Highly invasive glioma cells,” or “HIGCs,” are a subtype (subpopulation) of human GBM cells characterized by an ability to migrate from one brain hemisphere into which the cells are injected into the contralateral hemisphere. An example of an HIGC is the U87R subtype of the U87MG human glioblastoma cells.
  • “Brain-tumor initiating cells,” or “BTICs,” are a subtype (subpopulation) of human GBM cells characterized by their stem-cell like properties of being able to self renew, generate spheres without the addition of exogenous mitogens and growth factors, and induce tumor formation in vivo when placed in the brains of immuno-compromised mice.
  • “Peptide-displaying phage” refers to bacteriophage that have been engineered to express a library, typically a combinatorial library, of exogenous peptides on their surfaces, allowing phage selection based on the presence on the phage surface of an exogenous library peptide.
  • “HIGC-specific peptides” refers to peptides, typically associated with peptide-displaying phage, that bind preferentially to HIGCs under the conditions of phage-display panning described in Section VIIIC below.
  • “BTIC-specific peptides” are peptides, typically associated with peptide-displaying phage, that bind preferentially to BTICs under the conditions of phage-display panning described in Section VIIID below.
  • Peptide-displaying phage “bind preferentially to HIGCs or BTICs” if the phage remain bound to immobilized HIGC or BTIC target cells under the phage-panning wash conditions described in Section VIIIC and VIIID, respectively, below.
  • Amino acid residues are indicated herein by their standard one-letter code (see, for example, www.mun.ca/biochem/courses/3107/aasymbols.html).
  • A “contrast agent” refers to an imaging agent used in connection with an imaging technique, such as magnetic resonance imaging (MRI), positron emission tomography (PET), scintigraphy, computed tomography (CT), and x-ray imagining, to enhance the information available from the image, particularly as it relates to the binding of the imaging agent to target anatomical structures or cells. Exemplary contrast agents include (i) gadolinium-based contrast agents, iron oxide contrast agents, and manganese contrast agents used in MRI; 64Cu diacetyl-bis(N4-methylthiosemicarbazone), 18F-fluorodeoxyglucose, 18F-fluoride, 3′-deoxy-3′-[18F]fluorothymidine (FLT), 18F-fluoromisonidazole, gallium, Technetium-99m, and thallium, used in PET or scintigraphy; and barium, gastrografin, and iodine contrast agents used in x-ray imaging.
  • An agent is “coupled” to a peptide if it is attached directly or indirectly to the peptide in a manner that allows the agent to be carried with the peptide, e.g., in the bloodstream, as a stable two-component composition. The agent may be covalently coupled to the peptide, or carried in a structure, such as a chelate, cavitand, nanoparticle, or lipid particle, which is itself covalently coupled to the peptide, or may be carried with the peptide within a stable structure, such as a nanoparticle or lipid structure.
    • II. U87R-Cell Specific Peptides
  • In accordance with the present invention, the invasive glioma and BTIC compartments have been newly modeled, allowing detailed examination of potential targetable components. The invasive glioma model described herein was developed by in vivo serial passaging of GFP neo-transfected U87MG human glioblastoma cells through mouse brains to isolate the invasive subpopulation (U87R, remote from primary tumor) from brain hemispheres that were contralateral to the injection sites [5, 6]. When cultured in vitro and compared to their non-invasive counterparts (U87T, tumor forming), these cells retained a higher propensity to invade both in vitro and upon reinjection into mouse brains. Microarray analyses showed that many genes were either down- or up-regulated in the U87R cells when compared to the U87T cells, including increased expression of p75NTR. Using a combination of functional, biochemical, and clinical studies, it was found that p75NTR dramatically enhanced migration and invasion of genetically distinct glioma cells and frequently exhibited robust expression in highly invasive glioblastoma patient specimens [6]. These observations suggest that the U87R subpopulation is an appropriate model cell line for the subsequent peptide screening. The U87R subtype is an example of a highly invasive glioma cell (HIGC).
  • The BTIC model consists of a series of primary cell cultures derived from freshly resected human brain tumor specimens. Originating tumors are tested against a routine panel of antibodies to confirm a diagnosis of GBM, then subpopulations are selected via their ability to form self-renewing neurospheres in culture, as well as differentiate into astrocytic, oligodendrocytic or neuronal lineages. Upon injection of as few as ten cells into mouse brains, these brain tumor initiating cells (BTICs) tend to recapitulate the primary tumor as well as robustly invade, similar to what is seen with clinical GBM specimens [28]. Although it is unclear if BTICs are the best representation of brain cancer stem cells, they remain an excellent model for delineating the behavior of the human disease.
  • In accordance with the invention, peptides useful in targeting, characterizing and manipulating cells from disease reservoirs implicated in relapse of post-treatment GBM have been identified. The peptide selection was accomplished through phage display techniques already proven successful in other studies. For example, biopanning a phage display library against target cells or purified molecules has led to the characterization of cell surface proteins unique to defined cell subpopulations (e.g. the vascular address system [30, 31]), the identification of motifs important for specific protein function (e.g. the RGD motif necessary for integrin engagement [32-34]), as well as the isolation of peptide reagents with functional utility [35-38]. In practical terms, selection of unique phage displayed peptide sequences useful in diagnosis or therapy of patient glioblastomas can only be done ex vivo, necessitating the development of our unique, clinically relevant model systems, which recapitulate particular characteristics of GBM that cause therapeutic difficulties, allowing the causative cell types to be studied in isolation.
  • The final panels of selected peptides described herein have shown to be useful in demonstrating the molecular heterogeneity between cell populations and within a given cell population, and may lead to further refinement in the characterization of glioblastoma subtypes which will allow matching of tumor phenotypes with patient outcome, or with a particular therapeutic regimen, essential components of personalized medicine in cancer therapy. Alternately, because of the in vivo utility demonstrated, these peptides can be used as clinical imaging tools, or reagents for targeting chemotherapeutics to a particular tumor subtype. There is also inherent therapeutic potential of the unmodified peptides, as one of these dramatically inhibits the migration and invasive abilities of U87R cells as well as patient isolates that exhibit invasive properties.
  • A. Selective Biopanning of the Phage Display Library Resulted in the Isolation of a U87R-Specific Peptide Sequence that Inhibits Glioma Cell Migration
  • The present inventors have earlier described an in vivo selection for a population of human glioma cells that were highly invasive (U87R) [6]. In order to isolate peptides that could bind specifically to these highly invasive glioma cells, a biopanning experiment using a combinatorial phage display library was employed. Initially subtractive biopanning was performed with the U87 cells that were non-invasive (U87T) followed by selective biopanning with the U87R cells to isolate a library of phage (FIG. 1A) that were confirmed to preferentially interact with U87R cells by whole-cell ELISA (FIG. 1B). These phage were subcloned by plating serial dilutions of phage with host bacteria on agarose plates, and the sequences of the peptide inserts were determined, and listed in Table 1, which shows, from left-to-right, (i) the peptide identifier, (ii) peptide sequence e, (iii) SEQ ID NO, and (iv) number cell-binding phages containing that sequence.
  • To select a phage subclone for further biochemical and in vivo evaluation, phage were tested for the ability to functionally affect the behavior of the U87R cells. The H10 phage-displayed sequence (SEQ ID NO:7) was found to abrogate the migration of the U87R cells through membranes coated with a brain-like extracellular matrix (FIG. 2A). Other phage peptides, e.g., M32 and M24, also showed a similar inhibitory trend as H10, although not as strong an effect. The possibility that bacterial components from the host culture could be causing the inhibition was ruled out by also testing the effects of LPS and a 20× volume of randomly selected phage. A synthetic version of the H10 peptide was also able to inhibit the migration of U87R cells, as well as U87MG cells transfected with p75NTR (FIG. 2B), a molecule shown to be sufficient to confer a migratory phenotype in brain tumor xenografts [6]. These results confirmed the inhibitory property of this peptide in the absence of the phage particle.
  • TABLE 1
    U87R-binding peptides
    A2 SVSVGMKPSPRP (SEQ ID NO: 1) 21/100
    M32 GISLSSYLQSTQ (SEQ ID NO: 2) 20/100
    C12 EHMALTYPFRPP (SEQ ID NO: 3) 13/100
    M5 HWAPSMYDYVSW (SEQ ID NO: 4)  7/100
    M43 RTVPDYTAHVRT (SEQ ID NO: 5)  5/100
    M19 SGHQLLLNKMPN (SEQ ID NO: 6)  4/100
    H10 TNSIWYTAPYMF (SEQ ID NO: 7)  3/100
    F2 GMSLSRQMLWSL (SEQ ID NO: 8)  2/100
    M24 HLFPQSNYGGHS (SEQ ID NO: 9)  2/100
    M23 CIQLANPPRLXG (SEQ ID NO: 10)  2/100
  • The global utility of the H10 peptide was tested in the transwell assay by using BTIC lines established from individual patient specimens. As these were isolated directly from resected tumors, it was felt they would give an indication of the true clinical potential of H10. The migration of 60% (3/5) of the BTIC lines tested was significantly inhibited, although all five showed an inhibitory trend (FIG. 2C). In experiments where H10 had no significant effect, the cells were not highly invasive to begin with.
  • B. H10 Affects the Turn Over of GM1-Containing Structures and Other Lipid Raft Components
  • To determine the molecular mechanism by which the H10 peptide is inhibiting glioma invasion, a biotinylated version of the peptide was used to pull down potential binding partners which were then analyzed by mass spectrometry. Many of the proteins detected had some role or association with endosomal or vesicular transport (Table 2).
  • As lipid rafts are involved in some endocytotic processes [39-44], the reactions of the U87R cells to treatment with cholesterol oxidase, a known lipid raft disruptor [45, 46], was examined. Cell migration was abrogated in the transwell assay (FIG. 3A), and GM1, a component of both non-caveolar lipid rafts and caveolae [47, 48], was seen to accumulate within the cells, particularly at the plasma membrane (FIG. 3B). Similar effects were seen when cells were treated with H10, in comparison to a control phage, in three independent cell lines (FIG. 4A).
  • TABLE 2
    Mass Spec Hits of Interest
    LIM
    Focal Domain Endosomes/
    Adhesions/CSK Containing Vesicle Plasminogen
    Reorganization: Proteins: Transport: Receptors:
    ESP-2 Zyxin RAB1B Alpha-enolase
    Zyxin ESP-2 RAB1A Annexin A2
    Gamma- TRIP6 MEL
    filamin
    Transgelin 2 Lasp-1 REB35
    IQGAP1 ABP-278 Rab-15
    TRIP6 TIP47
    Lasp-1 Clathrin
    ABP-278 Transferrin
    Receptor Protein
    1
    Talin 1 Transmembrane
    protein
    33
    Glyceraldehyde-3-
    phosphate
    dehydrogenase
    SEC13
    Coatomer protein
    complex
  • The p75 neurotrophin receptor (NTR) can be detected differentially in lipid rafts isolated with specific detergents [49]. Alternately, intracellular vesicles can deliver molecules required for a particular function to the polar extremes of a cell, for example, transport of digestive enzymes or structural molecules such as integrins to the leading edge during migration [50-52].
  • The p75NTR can be detected differentially in lipid rafts isolated with specific detergents [49], and must be proteolytically processed to trigger cell migration in glioma cells [5]. Thus, it is probable that p75NTR is also cleaved within a lipid raft after endocytosis [53]. To determine if H10 may be having an impact on endosomally located p75NTR, cells were hypertonically lysed (to keep vesicles and organelles at least partially intact) and separated on a 10-40% sucrose/iodixanol gradient from which fractions were individually collected. Both H10 and cholesterol oxidase resulted in a shift of higher molecular weight forms of p75NTR into the denser gradient fractions (FIG. 4B). While the majority of p75NTR does not appear to be associated with GM1-containing lipid rafts, incubation of the cells with H10 did cause increased co-localisation of the p75NTR intracellular and extracellular domains (FIG. 4C). Taken together, the data suggests that (i) H10 can inhibit the turn over of both GM1 and p75NTR-containing lipid rafts and (ii) H10 appears to be preventing the release of at least a portion of the p75NTR extracellular domain produced within a cell. In contrast, H10 and cholesterol oxidase have no effect on the intracellular localization or gradient fractionation of transferrin or its receptor, which are markers of clathrin-coated vesicles (FIGS. 9A and 9B), and while H10 does not show an obvious effect on the processing of over-expressed transfected p75NTR, it may cause a slight accumulation of endogenously expressed full length protein (FIGS. 10A-10C).
    • III. BTIC-Cell Specific Peptides
  • A BTIC-specific phage library was generated as described for the 12R library, using U87MG cells, U251N cells, and human normal fetal astrocytes as subtraction cells to deplete the library of background phage, and a mixture of BTIC isolates (BTIC 12, 25, 42, 50) from different patients for the selection.
  • A. Peptides from Phage Biopanned Against BTIC Isolates Reveal Heterogeneity within Cultured Neurospheres.
  • The most abundant phage that was isolated was called 7A (25/50) and encoded the 12-mer sequence N-PSPHRQRQHILR-C (SEQ ID NO: 11). In addition, 5 other independent phage (10C, E10, 3F, C1 and 3B) were isolated more than once and comprised 50% (25/50) of the subclones screened, shown in Table 3.
  • To confirm the preferential binding of 7A to the BTICs rather than the cells used for subtraction, neurospheres and U87MG cells were stained in suspension with biotin-labeled synthetic peptide. Two of the BTIC lines showed a high percentage of positive cells within the neurosphere, while normal stem cells (not tumorigenic and not used in the depletion or selection) and U87MG cells were predominantly negative, as expected (FIG. 5B). Examination of the interaction of all selected peptides across a panel of BTIC lines underscored the heterogeneity within each cell population, and established a platform for correlating the ability to bind particular peptide sequences with either cell behavior or patient outcome. FIG. 8 shows binding specificities of BTIC-specific peptide hits on a representative selection of BTICs.
  • TABLE 3
    BTIC-binding peptides
    7A PSPHRQRQHILR (SEQ ID NO: 11) 25/50
    10C QTIRIIIRRSRT (SEQ ID NO: 12)  6/50
    E10 SLHMRHKRKPRR (SEQ ID NO: 13)  4/50
    3F SSRSMQRTLIIS (SEQ ID NO: 14)  2/50
    C1 IRSIRMRRILIL (SEQ ID NO: 15)  2/50
    3B KTSMRPLILIHI (SEQ ID NO: 16)  2/50
  • As seen in FIG. 8, E10 binds to both U87MG and BTIC's, and has further been found to bind to both U87R (HIGC) and U87T (main tumor) cells. Thus, E10 can serve as a general marker for all three cell phenotypes in a GBM tumor.
    • IV. Homing Properties of the U87R and BTIC Binding Peptides
  • The ability of the selected phage or their corresponding peptides was examined for their ability to ‘home’ to their respective U87R or BTIC glioma cell targets in vivo, according to their ability to detect glioma cells in an orthotopic xenograft model. In the case of H10, SCID mice carrying U87R or U87T xenografts were injected with the test phage for ten minutes, which was subsequently perfused with PBS to remove any unbound particles. Serial sections of the brains were immunohistochemically stained for M13 phage, demonstrating that H10 homed specifically to the U87R, but not the U87T tumors (FIG. 6A).
  • To determine if 7A would also demonstrate binding specificity to a subset of cells within neurospheres in vivo, SCID mice carrying BTIC xenografts were injected with 7A phage, and the brains cryosectioned for anti-phage immunohistochemistry. As seen with ex vivo staining of neurospheres, only a subset of cells within the main tumor masses of BTIC 12 and 25 showed positive staining (FIG. 6B). Additionally, no staining was detected in the surrounding tissue as well as in xenografts made of non-target cells, specifically the U251N human glioblastoma line. Perfusion of a BTIC 25 xenograft with a control phage (Al, randomly selected subclone) showed minimal staining, again confirming the selectivity and binding specificity of the 7A displayed peptide.
  • The studies reported above indicate that 7A phage/peptide recognizes only a subset of cells within an individual BTIC line (FIG. 5, FIG. 6B). It was therefore of interest to determine whether there were unique properties of these cells of which 7A binding may be an indicator. The BTIC 25 cells were subcloned by limiting dilution and screened for peptide binding. The population of cells enriched for 7A binding was distinctly different from the non-binding counterparts, forming neurospheres in culture as opposed to adherent colonies (FIG. 7). Additionally, the 7A binding subpopulation did not proliferate well in orthotopic xenografts (3 months growth time), and could only be detected in small numbers within the subventricular zone of the brain, a region in the brain known to host a neural stem cell niche. The 7A non-binding subpopulation were very tumorigenic and formed very large tumor masses with limited invasion into surrounding tissue. Therefore, the 7A peptide distinguishes a population of cells associated with neurosphere formation, low proliferative activity in vivo, and a preference to locate within the subventricular zone. The fact that both populations were derived from a BTIC culture indicates that 7A is able to track the progression of cells within this reservoir from hidden precursor to recurred tumor.
    • V. Peptide Composition and Peptide Conjugates
  • In one aspect, the invention includes a peptide composition for targeting either (i) a highly invasive glioma cells (HIGC) subtype of human GBM cells, e.g., the U87R subtype of U87GM cells, or (ii) a brain tumor initiating cell (BTIC) subtype of human GBM cells, as characterized above. The peptide in the composition is 12-20 amino acid residues in length and contains one of the sequences identified as SEQ ID NOS-1-17, or a sequence that is at least 90% homologous with the given sequence. That is, the peptide has the same sequence as one of SEQ ID NOS: 1-17, or a sequence that differs from the given sequence by at most one amino acid residue. The peptide may contain only the given sequence of amino acids, or may contain additional N- and/or C-terminal residues up to a total of 20 residues. Thus, for example, the peptide corresponding to SEQ ID NO: 7, TNSIWYTAPYMF (H10), may have this exact sequence, the same sequence but with a single amino acid substitution, addition or deletion at any of the 12 residue positions, or a peptide having a total of up to eight additional residues at one or both of the N- or C-terminals. For example, SEQ ID NO:17 (KKGTNSIWYTAPYMF) contains the 12 residues of SEQ ID NO: 7 (H10), plus three N-terminal residues (KKG) which make the H10 peptide substantially more water soluble by virtue of the two additional lysine residues.
    • A. Peptides
  • The peptide of the invention is formed by conventional solid-phase or recombinant DNA synthetic methods, using amino acids having the natural L-isomer form, the D-amino acid form, or a mixture of the two. Peptides having an all D-form or partial D-form composition are expected to be more resistant to proteolytic breakdown under biological conditions, e.g., when administered to a patient. Solid-phase peptide synthesis methods for preparing peptides composed of all L-amino acids, all D-amino acids or a mixture of D- and L-amino acids utilizing activated D- or L-form amino acid reagents are described, for example, in Guichard, G., et al., Proc. Nat. Acad. Sci. USA Vol. 91, pp. 9765-9769, October 1994). Alternatively, the peptides may be composed of D-amino acids synthesized in a reverse-sequence direction, that is, in a carboxy to amine end direction, to produce a so-called retro-inverso (RI) pilin peptide. Methods for synthesizing RI-form peptides are detailed, for example, in Fletcher, M. D. and Campbell, M. M., Partially Modified Retro-Inverso Peptides: Development, Synthesis, and Conformational Behavior, Chem Rev, 1998, 98:763-795, which is incorporated herein by reference.
  • For use in targeting HIGCs, the peptide contains a sequence selected from the group consisting of SEQ ID NOS: 1-10, 13, and 17, including SEQ ID NOS: 2 (M32), 7 (H10), and 9 (M24), of which SEQ ID NO: 7 (H10) is exemplary, 13 binds to both BTIC's, HIGC's and a population of differentiated GMB tumor cells, and 17 is modified for enhanced solubility. For use in targeting BTICs, the peptide contains a sequence selected from the group consisting of SEQ ID NOS: 11-16, preferably SEQ ID NOS: 11(7A), 14 (3F) and 16 (3B), or which SEQ ID NO: 11 is exemplary.
    • B. Peptide Conjugates and Composition
  • For use as a diagnostic reagent, for detecting HIGC or BTIC subtypes in a patient with a human GBM tumor, the peptide may be coupled to a contrast agent, such as fluorescent moiety, for use in imaging GBM cell types in vitro, or a contrast agent, for use in imaging GBM cell types in vivo. Major imaging technologies that presently utilize contrast agents for in vivo imaging include x-ray/Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Positron Emission
  • Tomography (PET), Single Photon Emission Computed Tomography (SPECT), and ultrasound technologies. Among widely used contrast agents are (i) gadolinium-based contrast agents, iron oxide contrast agents, and manganese contrast agents used in MRI; 64Cu diacetyl-bis(N4-methylthiosemicarbazone), 18F-fluorodeoxyglucose, 18F-fluoride, 3′-deoxy-3′-[18F]fluorothymidine (FLT), 18F-fluoromisonidazole, gallium, Technetium-99m, and thallium, used in PET or scintigraphy; and barium, gastrografin, and iodine contrast agents used in x-Ray imaging. The contrast agent or coordination complex carrying the agent, e.g., a radio-isotopic metal, may be covalently attached to the peptide according to well known methods, e.g., through a covalently attached ligand coordination complex.
  • For use as a therapeutic agent, to inhibit or destroy HIGC or BTIC cells specifically, the peptide may be coupled to one or a variety of anti-tumor agents, including, for example, alkylating agents, anti-metabolites, plant alkaloids and terpenoids, e.g. taxol, topoisomerases, proeosome inhibitors, and monoclonal antibodies. The anti-tumor agents may be covalently attached to the peptide according to well-known methods. In another embodiment, the peptides are attached to the surfaces of particles, e.g., liposomes, that carry the anti-tumor agent in encapsulated form.
  • In still another embodiment, the peptide of the invention is formed as a fusion peptide with a peptide carrier, such as AngioPep-2 or AngioPep-7 (SEQ ID NOS: 18 and 19, respectively) that is capable of facilitating passage of the peptide across the blood brain barrier (BBB) (62).
  • The composition of the invention may include (i) the peptide alone, (ii) the peptide in a suitable carrier, e.g., sterile physiological saline, in liquid or dried form, (iii) the peptide in coupled form as described above, or (iv) the peptide formulated in a suitable peptide-delivery nanoparticle, such as encapsulated within nanoparticles of poly(lactide-co-glycolide) copolymer, cyclodextrin nanoparticles, or cetyl alcohol/polysorbate. Nanoparticles useful for delivering drugs across the BBB barrier are well known in the art (e.g., 68-71). The nanoparticles are preferably in the 30-100 nm size range and may be coated with polyethyleneglycol for enhanced circulation time in the bloodstream. The particles may be suspended in an injectable medium, e.g., sterile physiological saline, or supplied in dehydrated form.
  • The peptide composition may contain multiple GBM binding peptides, and/or peptide conjugates, for example, at least one peptide or peptide conjugate that binds preferentially to BTIC's and at least one peptide or peptide conjugate that binds preferentially to HIGC's.
    • C. Carrier Peptide
  • Immunohistochemical staining of mouse brains carrying U87R tumors that had been perfused with H10 phage showed positive signal that was either localized within the tumor mass or peritumoral, demonstrating that H10 has the ability to cross the blood brain barrier (BBB). This feature is exploited, according to another aspect of the invention, in a novel composition comprising a conjugate of the H10 peptide, as a carrier peptide, and a neuropharmaceutical or anti-cancer agent that is to be delivered to the brain. The agent may include any pharmaceutical agent used, or potentially useful, in treating a neurological condition, such as depression, anxiety, schizophrenia, hyperactivity, Alzheimer's disease, or Parkinson's disease, or any anti-tumor compound, such as from the classes named above, that would be effective against cancers of the brain or spine if the blood brain barrier could be breached. The agent is coupled to the peptide according to known methods, such those involving direct attachment of the compound to an activated hydroxyl, sulfide, amine or carboxyl groups on the peptide or attachment of the peptide to a corresponding activated group on the compound, or by the use of a bifunctional coupling reagent.
    • VI. Diagnostic and Therapeutic Methods
  • Research into GBM has expanded from the biology of developed tumors into the subsets of cells currently believed to be the initiators and disease reservoirs for these tumors. The current view is that any given brain tumor consists of a mixture of cells with varying stages of “sternness” or differentiation, with each subpopulation retaining inherent potentials to contribute to relapse [1, 54]. New microarray data have begun to better delineate profiles for predicting patient outcome, and the nature of recurrent disease [1]. It is becoming evident that not all of these subpopulations are treatable by current therapeutics, ultimately resulting in relapsed disease. Further complicating the scenario, these cell populations may also be stimulated by chemo- and radiotherapy to evolve into more aggressive phenotypes [55, 56]. One goal of the present invention is to contribute to the practical solution of eliminating the identified subtypes of GBM and associated disease reservoirs.
  • To that end, the invention provides methods for diagnosis and/or treating patients with human GBM tumors, and in particular, provides an improvement in the treatment of GBM tumors in human patients, by characterizing and/or treating subpopulations of tumor cells that are likely causes of tumor recurrence. The patient may have been previously treated with one or more known treatment modalities, such as chemotherapy, x-radiation therapy and/or surgical resection. The initial treatment may cause a significant reduction in tumor size or growth, but the tumor may likely recur due to the presence of a HIGC subpopulation of cells that may reside outside the treatment region, or through a BTIC subpopulation of cells capable of reseeding the tumor with actively dividing GBM cells
    • A. Method of Characterizing a GBM Tumor
  • In one aspect, the invention provides a method of characterizing a glioblastoma multiforme (GBM) tumor in a patient according to the density and distribution of different cell phenotypes within the tumor, including the HIGC and BTIC phenotypes, associated with tumor reservoir that may escape current treatment methods.
  • The method involves generating one or more images of the patient's brain tumor that indicates the density and distribution of both HIGC and BTIC subtypes within the tumor region, and optionally, the main differentiated tumor cells making up the majority of the tumor. Where a single image is formed, the patient is administered a cocktail (multiple-peptide composition) of two or more single-peptide compositions, one containing a peptide that preferentially targets HIGC cells and is coupled to a first contrast agent, and the second containing a peptide that targets BTIC cells and is coupled to a second contrast agent, where the two contrast agents allow visualization of binding to each peptide to its target cell type. Where two or more separate images are formed, for visualizing the distribution of the two or more cell types separately, an individual image is formed after administration of each peptide composition. In this embodiment, the same contrast agent may be used for each image. The separate images can be integrated into a single image by known image processing techniques.
  • The techniques that may be used include magnetic resonance imaging (MRI), positron emission tomography (PET), scintigraphy, computed tomography (CT), and x-ray. For each technique, a variety of suitable contrast agents are available, including (i) gadolinium-based contrast agents, iron oxide contrast agents, and manganese contrast agents used in MRI; 64Cu diacetyl-bis(N4-methylthiosemicarbazone), 18F-fluorodeoxyglucose, 18F-fluoride, 3′-deoxy-3′-[18F]fluorothymidine (FLT), 18F-fluoromisonidazole, gallium, Technetium-99m, and thallium, used in PET or scintigraphy; and barium, gastrografin, and iodine contrast agents used in x-ray imaging. The contrast agents may be coupled to the associated peptide by standard methods, such as direct covalent coupling, or for radio-imaging metals, covalent coupling of a metal chelate to the peptide.
  • With the one or more images thus generated, the physician can readily assess the density and distribution, e.g., localization of the first and second peptides and their associated contrast agent within the tumor and at the margins of the tumor.
  • FIGS. 13-19 demonstrate in vivo targeting to BTIC- and HIGC-specific peptides to specific cells of a human GBM tumor implanted in a mouse brain. In the studies shown in FIGS. 13A and 13B, brain tumors were implanted in mice with a patient-derived BTIC cell line BT025 identified as above by its binding to the 7A peptide. After tumor growth from the implanted cells, the tumors were removed, sectioned and stained for microscopic visualization. In the studies shown in FIGS. 14-19, a fluorescent peptide was administered intravenously, either by intracorotid injection or via tail vein into mice having an implanted GBM tumor from the BT025 cell line (FIGS. 14-17) or the U87MG cell line (FIGS. 18 and 19), and after localization of the peptide, the animal were sacrificed and their brain tumors removed for sectioning and visualization by fluorescence microscopy. The results of these studies demonstrate that, for each peptide tested, a fluorescent conjugate of the peptide, when administered intravenously, was able to target specific brain tumor cells.
  • FIG. 13A shows high magnification images of thin sections from five mouse brains that were previously injected with the BT025 cell line, and stained with antibodies specific to an antigen present only in human nuclei, and a dark brown detection reagent. The figure confirms the diffusion of 7A binding cells through the brain tissue.
  • FIG. 13B shows thin sections from five separate mouse brains injected with the BT025 subclones. The toluidine blue staining clearly shows massive tumor growth and disruption of normal tissue architecture. Thus, 7A binding in vitro prior to injection of the cells into mouse brains can predict the in vivo behaviour of the different BT025 cell subpopulations.
  • FIG. 14A shows the ability of fluorescently labeled 7A peptide (SEQ ID NO: 11, preferential for BTIC binding) to home in a live mouse to a large implanted tumor grown from BT025 cells. The peptide was administered to the mouse via intracarotid injection and allowed to circulate for approximately half an hour to allow clearance of background signal before the mouse was sacrificed and the brain removed for imaging using an Olympus OV100 whole mouse imaging system. By non-magnified inspection, the large outgrowth of the tumor is obvious. (FIG. 14A) The fluorescent signal appears strongly in part of the tumor, but clearly does not detect all tumor cells present.
  • FIGS. 14B and 14C are lower and higher magnification photomicrographs of a thin section taken from the brain in FIG. 14A after it was bisected close to the tumor implantation site and stained for human nuclear antigen (green) and total DNA (blue) to outline the positioning of the cells. The red peptide signal is visible on cells immediately adjacent to the main tumor mass as well as spread diffusely in the surrounding tissue. FIG. 14C in particular, shows that the 7A peptide binds primarily to cells that are diffusely spread outside the main tumor mass.
  • FIGS. 15A and 15B show similar sections views, where the animal was injected with a fluorescently labeled 3B peptide (SEQ ID NO: 16, also preferential for BTIC cell binding). FIG. 15B shows that 3B binds to cells that are both within and outside the main tumor mass. This indicates that 3B can detect both proliferative and diffusely migratory tumor cell populations.
  • FIGS. 16A and 16B show similar views, where the animal was injected with a fluorescently labeled 3F peptide (SEQ ID NO: 14, also preferential for BTIC cell binding). FIG. 16B shows that when thin sectioned, most of the red signal appears outside of the main tumor mass, but close to the green signal for human nuclear antigen on cells that have diffusely migrated in the surrounding tissue. FIGS. 17A and 17B shows that similar results can be obtained when the peptide is administered by tail vein injection rather than intracarotid.
  • FIGS. 18A and 18B-18D show similar views, where the animal was injected with a fluorescently labeled modified H10 peptide (SEQ ID NO: 17, preferential for HIGC binding), in animals carrying a tumor initiated with the U87MG human glioblastoma cell line. For these studies, the tumors were not grown as large, and a far-red fluorescent signal is not obvious from inspection without sectioning (FIG. 18A). FIG. 18B-18C (different magnifications of the same section) show that the modified H10 peptide binds primarily to cells that are diffusely spread outside the main tumor mass, as would be expected of HIGC's. A similar result was seen when the peptide was injected via a tail vein (FIG. 18D).
  • FIGS. 19A and 19B and 19D show similar views, where the animal was injected with a fluorescently labeled modified E10 peptide (SEQ ID NO: 13, which binds to both BTIC's and HIGC's), in animals carrying a tumor initiated with the U87MG human glioblastoma cell line. For these studies, the tumors were not grown as large, and a far-red fluorescent signal is not obvious from inspection without sectioning (FIG. 19A). FIG. 19B shows that E10 binds to a portion of cells within the main tumor mass, and FIG. 19C shows that E10 also binds to cells outside of the main tumor mass. Similar results were obtained when the peptide was administered via the tail vein.
  • The studies reported above show that peptide conjugates specific for BTICs (7A, 3B, and 3F), for HIGC's (modified H10), and for both cell types (E10), can localize in human GBM tissue in vivo to provide information on the density and localization of the various cell types in vivo, and that targeting can be carried out by intravenous administration of the peptide conjugate.
  • It can be appreciated from the images obtained how the method can be used to determine the dimensions and coordinates of the tumor, both the main tumor and the tumor margin that may contain reservoir cells, particularly HIGC's but also BTIC's. This information, in turn, will allow the surgeon to better define the margins of the tumor that need to be removed in order to reduce the risk of relapse, and will allow the radiation oncologist to more accurately define the brain volume and coordinates that need to be included in the radiation treatment.
  • Since the progress of a treatment regimen will need to take into account the presence and distribution of reservoirs tumor cells, the method can also be used to monitor the effectiveness of treatment in eliminating or reducing reservoir cells. In this embodiment, the brain may be imaged in accordance with the method at an initial time zero, then reimaged at one or more times over the course of the treatment, to track the effect of the tumor on reservoir cells.
  • Finally, the treatment method may be used to tailoring a chemotherapy for GBM tumor therapy, using the different expression profiles of different tumor-cell phenotypes within the tumor. FIG. 20 show gene clustering of microarray data generated from mRNA isolated from three independently isolated 7A binding cell subclones and three independently isolated 7A non-binding subclones. The gene expression profiles of all the 7A binding subclones clustered together (demonstrating similar gene expression), as did the 7A non-binding subclones. This strengthens the notion that 7A distinguishes between BTIC subtypes that are transcriptionally unique, and whose behavior in vivo can be predicted. The gene expression data may allow the oncologist to examine the different gene expression profiles of cells in identifying chemotherapeutic agents that are most effective against particular gene expression profiles.
    • B. Gene Expression Profiling
  • In accordance with another aspect of the invention, the discovery of peptides that bind preferentially to particular peptides to GBM cell subtypes that in turn have different gene expression profiles provides a novel method for characterizing a tumor in terms of its component cells. According to this method, the oncologist would correlate the identified cell type with the known gene expression pattern of that cell type, then determine the presence and/or distribution and/or number of cells of each particular type with the peptides of the invention, as a basis for tailoring the therapy to a known identified gene expression profile.
    • C. Therapy
  • In still another aspect, the peptide composition of the invention are useful for targeting therapeutic agents to particular tumor cell types for tumor treatment. As one general strategy, the oncologist might adopt one known therapy, e.g., surgery, radiation, or chemotherapy or a combination of these, for treating the tumor, and secondarily use the peptide composition of the invention to target therapeutic agents to the reservoir cells of the tumor, to minimize the reservoir effect in relapse after the initial treatment.
  • A number of delivery system are available for delivering the peptide composition to the brain (62-71), including intravenous administration. As noted above, the H10 peptide is itself able to pass the blood brain barrier, and so may be administered by any systemic route including intra-arterial, IV, IM, subQ, nasal, mucosal or through the lungs, either as a peptide alone or peptide coupled to an imaging or an anti-tumor agent. The studies conducted on mice tumor further shows that all of the peptides studied were able to target brain tissue following intravenous administration.
  • Other available strategies for CNS delivery may be broadly classified as either invasive (neurosurgical-based), such as convection-enhanced delivery (66), pharmacologic-based, or physiologic-based (63). Neurosurgical-based strategies include intraventricular drug infusion, intracerebral implants, and BBB disruption. Pharmacologic-based strategies include the use of nanoparticle carriers (described, for example, in 68-71). Physiologic-based strategies take advantage of the normal, endogenous pathways of either carrier-mediated transport of nutrients or receptor-mediated transport of peptides.
  • In the latter category, Drappatz et al. conducted a Phase I study of ANG1005 in patients with recurrent glioblastomas. ANG1005 consists of three paclitaxel molecules linked to a novel peptide, Angio-Pep-2, that allows it to be transported across the blood-brain barrier via the low-density lipoprotein receptor-related protein 1 receptor (62). Angio-pep-2 is 19-amino acid peptide (SEQ ID NO: 17) that binds to low density lipoprotein receptor-related protein (LRP) receptors at the BBB and has the potential to deliver drugs to brain by receptor-mediated transport. Here the BTIC- or HIGC-specific peptide, with or without associated radio-imaging or anti-tumor agent, is coupled to the Angio-pep peptide, and delivered by a systemic route.
  • Alternatively, after completing surgery to remove the primary tumor, the composition of the invention can be administered either directly in wafers or by installing a convection enhanced delivery pump to deliver the peptides on a routine schedule. Convection-enhanced delivery (CED) uses an infusion catheter whose tip is placed close to the target site. In this technique, a cannula is inserted directly into the area of the brain to be treated, and the therapeutic agent is delivered through the cannula via bulk flow, circumventing the BBB.
  • The peptide composition could alternatively be administered after brief disruption of the BBB using mannitol or other BBB disruption agent. This approach has recently been used to deliver Avastin to the brain tumors of patients (67).
  • Still another delivery method uses nasal transport routes via the olfactory nerve pathway (axonal transport) and the olfactory epithelial pathway.
  • As indicated above, the diagnostic method is carried out to image reservoirs of BTICs and/or HIGCs, typically after an initial treatment of the GBM tumor, e.g., by surgery, radiation therapy or chemotherapy. Depending on the resolution in visualizing targeted cells, the dose of diagnostic composition administered to the patient, or the route of administration, may be varied until a meaningful diagnostic result is obtained.
  • After identifying reservoirs of either cell type, the patient may be treated with the peptide in therapeutic form, to knock out or reduce populations of the reservoir cells. During this treatment, progress in reducing HIGC or BTIC populations can be checked periodically with the diagnostic method, and the therapeutic dose of the composition may be varied, if necessary, to increase the extent of inhibition or destruction of the targeted HIGCs or BTICs. Treatment is continued, e.g., by twice weekly or weekly administration of the composition, until a desired endpoint is observed.
  • The experimental procedures described below are exemplary, and in no way intended the scope of the invention as defined by the claims.
    • VII. Experimental Procedures:
  • All animal experiments were conducted in accordance with the approval documents provided by the University of Calgary Ethics Board and Animal Care Facility. Tumor and normal tissues including the isolation of brain tumor initiating cell from banked patients' samples were obtained from the Tumor Tissue Bank in Foothills Hospital, Calgary, Alberta.
  • A. Cell Culture:
  • The human glioma cell line U87 was obtained from the American Type Culture Collection, transfected with GFP and separated into the U87T and U87R subpopulations as previously described [6]. The human glioma cell line U251N and human fetal astrocytes were a kind gift from V. W. Yong (University of Calgary, Calgary, Alberta, Canada). All cells were maintained in complete media (Dulbecco's modified eagle's medium [DMEM] supplemented with 10% heat-inactivated fetal bovine serum [FBS], 0.1 mM nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and transfected cells with 400 μg/ml of G418 (Invitrogen) at 37° C. in a humidified 5% CO2 incubator. Cells were passaged by harvesting with trypsin (Gibco BRL) at 80%-90% confluence.
  • Brain tumour initiating cells (BTICs) were supplied by the BTIC Core Facility, maintained by Drs. Greg Cairncross and Samuel Weiss, after isolation by Dr. John Kelly [28] and maintained in NeuroCult media (Stem Cell Technologies) as neurospheres. Subcloning of the BTIC25 line into 7A positive and negative populations was done by limiting dilution.
  • B. Bacteriophage Culture:
  • The Ph.D.TM-12 Phage Display Peptide Library Kit (New England Biolabs) was used as per manufacturer's protocol. Briefly, phage libraries were amplified as follows: Overnight cultures of the bacterial host strain ER2738 were diluted 1/100 in LB broth with 20 μg/mL tetracycline and inoculated with 10 μL of phage (˜108-1010 pfu/μL), then cultured with shaking at 37° C. overnight. Bacteria were pelleted and the phage were precipitated from the supernatant by adding 1/6 volume of 20% w/v PEG-8000, 2.5M NaCl and storing at 4° C. overnight, followed by centrifugation for 15 minutes at 10000×g. The pellet contained phage and was reconstituted in PBS. Purified phage were put through a 0.22 μm filter if intended for mouse work.
  • Phage titers were determined by adding 10 μL of serially diluted phage and 200 μL of ER2738 to 3 mL of 7% agarose in LB warmed to 50° C., and poured over a warmed plate of LB agar with 20 μg/mL tetracycline 40 μg/mL XGal and 50 μg/mL IPTG. After overnight culture at 37° C., blue plaques were counted for calculation of pfu/μL. Plaques could be subcloned by removing an isolated plaque from the agar plate using a Pasteur pipette and extracting the phage in 100 μL of 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 6 mM MgSO4 at 4° C. overnight. Amplification cultures were inoculated with 20 μL of the extraction buffer, as described above.
  • C. Biopanning for U87R (Invasive Glioma)-Specific Peptide Sequences:
  • Biopanning was performed as described in [61]. Subtraction cells (e.g. U87T) were released from tissue culture plastic with Puck's EDTA, and after washing with PBS, 1×107 cells were resuspended in 1% BSA in PBS with 1.5×1011 pfu of Ph.D.-12 M13 Phage Library (New England Biolabs). The cells and phage were incubated in an eppendorf tube for 1 hour at room temperature with gentle shaking, before pelleting the cells and retaining the supernatant, which was transferred to a fresh tube with another aliquot of subtraction cells. The incubation and transfer of supernatant was performed three times, with the supernatant being transferred to an empty tube on the last round. Selection cells were released from tissue culture plastic with Puck's EDTA (e.g. U87R cells) and washed in PBS. 5×106 cells were resuspended in the 3× subtracted supernatant and incubated for 4 hours at 4° C. with slow shaking The cell pellet was washed 5× in cold 1% BSA/0.1% Tween 20 in PBS five times, changing the tube after each wash. Bound phage were eluted by rocking the cells gently for 10 minutes at 4° C. in 1 mL of 0.2M Glycine-HCl pH 2.2 with 1 mg/mL BSA, and the supernatant was immediately neutralized with 150 μL of 1M Tris-HCl pH 9.1. The final library was amplified as per manufacturer's protocol.
  • D. Biopanning for BTIC-Specific Peptide Sequences:
  • A BTIC-specific phage library was generated as described for the U87R library, this time using U87MG cells, U251N cells, and human normal fetal astrocytes to deplete the library of background phage, and a mixture of BTIC cell lines from different patients were used for the selection. Cells were released with EDTA if adherent, or if grown as neurospheres (e.g. BTIC cells) were separated into single cells by repeated pipetting through a small bore pipette tip. Otherwise, the subtractive and selective biopanning were performed as described for the U87R library.
  • E. Whole Cell ELISA:
  • 1×104 test cells were plated into a well of a 24-well dish, in 0.5 mL of media and allowed to equilibrate overnight under normal culture conditions, then the media was replaced with HEPES-buffered culture media. 5×109 pfu phage were added to the wells with gentle shaking for 1 hour at room temperature followed by three washes with PBS. Bound phage were detected with anti-M13-HRP antibody (GE Healthcare) and insoluble TMB substrate (Sigma).
  • F. Transwell Assays:
  • The membranes of 8 μm pore sized transwell inserts (Corning Costar) were coated with brain-like matrix (720 μL of 3 mg/mL collagen I (PureCol); 1804, of 10× DMEM (Invitrogen); 9 uL of 1 mg/mL human plasma fibronectin (Sigma); 9 uL of 1 mg/mL chondroitin sulfate proteoglycans (Chemicon); 16 μL of 0.15 mg/mL laminin (Chemicon)) on both sides, allowed to dry, then plated with 5×104 cells in 100 μL media in the upper chamber and 500 μL media in the lower chamber. Inclusion of collagen was essential to see the inhibitory effect of H10 (Fig. S4). 3×1010 pfu of test phage were added to the upper chambers containing the cells, which were incubated for 4 hours under normal culture conditions. At the end of the incubation, the media was aspirated and the cells were fixed and stained in 1% crystal violet in 95% ethanol for one minute. Transwells were rinsed in PBS, then cells on the upper membrane surfaces were removed with a cotton swab. Cells which had migrated to the undersurface of the membranes were counted by light microscopy, and the sum of cells in five microscopic fields was recorded for each membrane.
  • G. Confocal Microscopy:
  • Adherent cells: 13 mm coverslips were coated with neutralized 3 mg/mL Collagen I (PureCol), allowed to dry, then plated with test cells at 1×104/mL, 0.5 mL volume and allowed to equilibrate 20 minutes under normal culture conditions. Coverslips were fixed for 10 minutes in 3% formaldehyde in PBS, washed, then incubated in biotinylated peptide (3 mM stock) or primary antibody at a 1:100 dilution in 2% BSA/0.02% Tween 20 in PBS for 1 hour at room temperature. After washing in PBS, secondary antibody or streptavidin-Alexa 568 was applied at a dilution of 1:250 for 1 hour before coverslips were washed and mounted with DAKO mounting media with antifade.
  • Suspended neurospheres: 504, of densely suspended neurospheres were stained as described above, except in suspension in an eppendorf tube. The stained neurospheres were mounted in a drop of DAKO mounting media under a coverslip.
  • H. In Vivo Phage Homing
  • 3×104 U87T or 3×105 U87R cells were injected in a 34 volume into one brain hemisphere of a SCID mouse and allowed to grow for 4 or 6 weeks respectively. For specific molecular staining, unlabeled primary antibodies, FITC-transferrin or A1exa555-cholera toxin B subunit (Invitrogen) were diluted 1:100 in 2% BSA/0.02% Tween 20 in PBS and used in place of biotinylated peptides. When needed, species-specific fluorescently labeled secondary antibodies (Molecular Probes, Invitrogen) were diluted 1:500. Primary antibodies used included anti-p75 intracellular domain pAb (Promega), anti-p75 extracellular domain mAb clone ME20.4 (Cell Signaling, Millipore), and anti-transferrin receptor (Invitrogen).
  • I. Sucrose/Iodixanol Gradients:
  • Cells were scraped into 1 mL of 0.25M sucrose, 140 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0, and dounce homogenized until cells were no longer visible by microscopy. Debris was pelleted for 5 min, 800×g and the supernatant was loaded on top of a 10 mL continuous 10-40% gradient, prepared by the dilution of OptiPrep (Sigma, 60% iodixanol) with 0.25M sucrose, 140 mM NaCl, 3 mM EDTA, 60 mM Tris-HCl, pH8.0. Gradients were spun in a swinging bucket rotor at 48000×g for 18 hrs and 0.5 mL fractions were collected. Total protein was precipitated from each fraction by the addition of 2 volumes of −20° C. 20 mM DTT, 15% TCA, storage at −20° C. overnight, followed by centrifugation at 4° C. for 20 minutes. Pellets were resuspended in 20 μL 1M Tris and 20 μL 2× Laemmli buffer. The entire volume was loaded into a single well for SDS-PAGE resolution and western transfer.
  • J. In Vivo Phage Homing:
  • 3×104 U87T cells, 3×105 U87R cells or 5×104 BTICs were injected in a 34 volume into one brain hemisphere of a SCID mouse and allowed to grow for 4, 6 or 12 weeks respectively. The mice were anaesthetized, then treated with 150 μL of 20% (w/v) mannitol for 15 minutes before injecting 5×109 pfu into the brain via the carotid. After allowing the phage to circulate for 10 minutes, unbound phage were flushed out of the circulatory system with 15 mL of PBS injected into the left ventricle after the right atrium was clipped. The brains were harvested, immediately frozen on dry ice and embedded in OCT for sectioning.
  • K. Immunohistochemistry:
  • Serial sections were fixed with cold acetone, and rehydrated through an ethanol gradient. Endogenous peroxidases in the sections were inactivated with 0.075% H2O2/methanol, and nonspecific binding was blocked with 10% normal goat serum in PBS. The sections were incubated with 1:100 diluted rabbit polyclonal anti-M13 antibody (in house), or 1:50 diluted mouse monoclonal anti-human nuclei (Chemicon) in blocking buffer. Following washing with PBS, the appropriate biotinylated secondary antibody (Vector Laboratories, http://www.vectorlabs.com) was applied. Avidin-biotin peroxidase complexes were then formed using the VECTASTAIN Elite ABC kit (Vector Laboratories) and detected by addition of SIGMAFAST DAB (3,39-diaminobenzidine tetrahydrochloride) (Sigma-Aldrich), which was converted to a brown reaction product by the peroxidase. Toluidine blue (for frozen sections) was used as a nuclear counterstain. Sections were then dehydrated in an ethanol/xylene series and mounted with Entellan (Electron Microscopy Sciences).
    • SEQUENCE LISTING
  • SVSVGMKPSPRP (SEQ ID NO: 1)
    (A2)
    GISLSSYLQSTQ (SEQ ID NO: 2)
    (M32)
    EHMALTYPFRPP (SEQ ID NO: 3)
    (C12)
    HWAPSMYDYVSW (SEQ ID NO: 4)
    (M5)
    RTVPDYTAHVRT (SEQ ID NO: 5)
    (M43)
    SGHQLLLNKMPN (SEQ ID NO: 6)
    (M19)
    TNSIWYTAPYMF (SEQ ID NO: 7)
    (H10)
    GMSLSRQMLWSL (SEQ ID NO: 8)
    (F2)
    HLFPQSNYGGHS (SEQ ID NO: 9)
    (M24)
    CIQLANPPRLXG (SEQ ID NO: 10)
    (M23)
    PSPHRQRQHILR (SEQ ID NO: 11)
    (7A)
    QTIRIIIRRSRT (SEQ ID NO: 12)
    (10C)
    SLHMRHKRKPRR (SEQ ID NO: 13)
    (E10)
    SSRSMQRTLIIS (SEQ ID NO: 14)
    (3F)
    IRSIRMRRILIL (SEQ ID NO: 15)
    (C1)
    KTSMRPLILIHI (SEQ ID NO: 16)
    (3B)
    KKGTNSIWYTAPYMF (SEQ ID NO: 17)
    (Modified H10)
    TFFYGGSRGKRNNFKTEEY (SEQ ID NO: 18)
    (AngioPep-2)
    TFFYGGSRGRRNNFRTEEY (SEQ ID NO: 19)
    AngioPep-7)

Claims (21)

1. A peptide composition for targeting one of (i) a highly invasive glioma cell (HIGC) subtype of human glioblastoma multiforme (GBM) cells characterized by their ability to migrate from one brain hemisphere into which the cells are injected into the contralateral hemisphere, and (ii) a brain tumor initiating cell (BTIC) subtype of human GBM cells characterized by their stem-cell like properties of being able to self renew, generate spheres without the addition of exogenous mitogens and growth factors, and induce tumor formation in vivo when placed in the brains of immuno-compromised mice, comprising
an isolated peptide having between 12-20 amino acids, and containing a sequence selected from the group consisting of SEQ ID NOS: 1-10, 13, and 17 for targeting HIGCs, and SEQ ID NOS: 11-16, for targeting BTICs.
2. The peptide composition of claim 1, wherein the peptide is composed of L-amino acids, D-amino acids, a mixture of L- and D-amino acids, or a retro-inverso peptide formed of D-amino acids arranged in reverse order.
3. The peptide composition of claim 1, for use in localizing of HIGCs or BTICs in a subject with a human GBM tumor, which further includes a contrast agent attached to the peptide.
4. The peptide composition of claim 1, for use in inhibiting or killing HIGCs or BTICs in a subject with a human GBM tumor, which further includes an anti-tumor agent attached to the peptide.
5. The peptide composition of claim 1, for use in targeting HIGCs, wherein the peptide contains a sequence selected from the group consisting of SEQ ID NOS: 7 (H10), 13 (E10) and 17 (modified H10);
6. The peptide composition of claim 1, for use in targeting HIGCs, wherein the peptide contains the sequence identified by SEQ ID NOS: 17 (modified H10);
7. The peptide of composition 1, for use in targeting BTICs, wherein the peptide contains a sequence selected from the group consisting SEQ ID NOS: 11 (7A), 13 (E10), 14 (3F) and 16 (3B).
8. The peptide composition of claim 7, wherein the peptide has the sequence identified by SEQ ID NOS: 11 (7A).
9. The peptide composition of claim 1, for use in delivery to a patient human GBM tumor across the blood-brain barrier, wherein the peptide is coupled to a carrier peptide having the sequence identified by SEQ ID NOS: 18 or 19.
10. The peptide composition of claim 1, wherein the peptide is encapsulated within a nanoparticle formed of poly(lactide-co-glycolide) copolymer, a cyclodextrin, or cetyl alcohol/polysorbate.
11. The peptide composition of claim 1 which includes at least one peptide that binds preferentially to HGIC's and at least one peptide that binds preferentially to BTIC's.
12. The use of the composition of claim 1, for detecting the presence of HIGC or BTIC subtypes of cells in a patient with GBM tumor, where the composition includes a detectable contrast agent coupled to the peptide.
13. The use of the peptide composition of claim 1, for inhibiting or killing HIGC or BTIC subtypes of cells in a patient with GBM tumor, where the composition includes an anti-tumor agent coupled to the peptide.
14. A method of characterizing a glioblastoma multiforme (GBM) tumor in a patient, comprising
(a) generating an image of the patient's brain tumor after administering to the patient a peptide composition containing (i) a first peptide having between 12-20 amino acids in length that has been selected for its preferential binding to a highly invasive glioma cell (HIGC) subtype of human GBM cells characterized by their ability to migrate from one brain hemisphere into which the cells are injected into the contralateral hemisphere, and coupled to first peptide, a first contrast agent that allows the first peptide, when bound to cells in the patient tumor region, to be imaged in vivo;
(b) generating an image of the patient's brain tumor after administering to the patient a peptide composition containing (i) second peptide having between 12-20 amino acids in length that has been selected for its preferential binding to a brain tumor initiating cell (BTIC) subtype of human GBM cells characterized by their stem-cell like properties of being able to self renew, generate spheres without the addition of exogenous mitogens and growth factors, and induce tumor formation in vivo when placed in the brains of immunocompromised mice, and coupled to the second peptide, a second contrast agent that allows the second peptide, when bound to cells in the patient tumor region, to be imagined in vivo;
(c) determining, from the distribution of the first and second peptides and their associated contrast observed in the image(s) generated in steps (a) and (b), at least one of:
(ci) the boundaries of the tumor, for purposes of surgical resection of the tumor,
(cii) the boundaries of the tumor for purposes of radiation therapy of the tumor,
(ciii) the expression profiles of different tumor-cell phenotypes within the tumor, for purposes of tailoring a chemotherapeutic regimen for treating the tumor; and
(civ) the change in the distribution of the first and second peptides and their associated contrast agents over a given time course in which steps (a) and (b) are repeated over time.
15. The method of claim 14, wherein steps (a) and (b) are carried out by one of:
(i) MRI, wherein the contrast agent is selected from the group consisting of a gadolinium-based contrast agent, an iron oxide contrast agent, and a manganese contrast agent;
(ii) positron emission tomography (PET) or scintigraphy, wherein the contrast agent is selected from the group consisting of 64Cu diacetyl-bis(N4-methylthiosemicarbazone), 18F-fluorodeoxyglucose, 18F-fluoride, 3′-deoxy-3′-[18F]fluorothymidine (FLT), 18F-fluoromisonidazole, gallium, Technetium-99m, and thallium; and
(iii) x-ray imaging, where the contrast agent is selected from the group consisting of barium, gastrografin, and iodine contrast agents.
16. The method of claim 14, wherein steps (a) and (b) are carried out together and the first and second contrast agents allow the distributions of bound first and second peptides to be independently determined.
17. The method of claim 14, wherein steps (a) and (b) are carried sequentially.
18. The method of claim 14, wherein the first peptide contains a sequence selected from the group consisting of SEQ ID NOS: 7 (H10), 13 (E10) and 17 (modified H10);
19. The method of claim 14, wherein the second peptide contains a sequence selected from the group consisting SEQ ID NOS: 11 (7A), 13 (E10), 14 (3F) and 16 (3B).
20. The method of claim 14, wherein said peptide compositions are administered intravenously.
21. A method of characterizing the gene expression profiles of cellular phenotypes in a human glioblastoma multiforme (GBM) tumor in a patient, comprising
(a) identifying cells in the tumor corresponding to one of (i) a differentiated GBM tumor cell, (ii) a highly invasive glioma cell (HIGC) subtype of human glioblastoma multiforme (GBM) cells characterized by their ability to migrate from one brain hemisphere into which the cells are injected into the contralateral hemisphere, and (iii) a brain tumor initiating cell (BTIC) subtype of human GBM cells characterized by their stem-cell like properties of being able to self renew, generate spheres without the addition of exogenous mitogens and growth factors, and induce tumor formation in vivo when placed in the brains of immuno-compromised mice, and
(b) correlating the identified cell type with the known gene expression pattern of that cell type.
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