WO2013106824A1 - Epherin receptor targeting agents - Google Patents

Epherin receptor targeting agents Download PDF

Info

Publication number
WO2013106824A1
WO2013106824A1 PCT/US2013/021434 US2013021434W WO2013106824A1 WO 2013106824 A1 WO2013106824 A1 WO 2013106824A1 US 2013021434 W US2013021434 W US 2013021434W WO 2013106824 A1 WO2013106824 A1 WO 2013106824A1
Authority
WO
WIPO (PCT)
Prior art keywords
imaging
agent
peptide
ephb4
hauns
Prior art date
Application number
PCT/US2013/021434
Other languages
French (fr)
Inventor
Chiyi Xiong
Qizhen Cao
Anil Sood
Chun Li
Original Assignee
Board Of Regents, The University Of Texas System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Board Of Regents, The University Of Texas System filed Critical Board Of Regents, The University Of Texas System
Publication of WO2013106824A1 publication Critical patent/WO2013106824A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
    • A61K51/1244—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles
    • A61K51/1251—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles micro- or nanospheres, micro- or nanobeads, micro- or nanocapsules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • 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
    • G01R33/56—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution
    • 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

Definitions

  • the present invention relates generally to the field of molecular imaging and nanomedicine. More particularly, it concerns cancer targeting agents and methods for targeted imaging and therapy of cancer cells.
  • radiolabeled peptides have been developed as nuclear imaging agents for tumor detection and noninvasive assessment of receptor expression in solid tumors.
  • small radiolabeled somatostatin peptidyl analogs with a molecular weight of -1.5 kDa have been successfully utilized in the clinic for localizing neuroendocrine tumors expressing somatostatin receptors (Bakker et ah, 1991 ; Hammond et ah, 1993).
  • Cyclic Arg-Gly-Asp (RGD) peptide that strongly binds to integrin ⁇ 3 receptors is currently under clinical investigation (Haubner et ah, 2009).
  • peptide-based imaging agents including melanocyte-stimulating hormone (MSH) analog (Chen et ah, 2000), substance P (Hargreaves, 2002), calcitonin (Blower et ah, 1998), atrial natriuretic peptide (ANP) (Liu et ah, 2010), bombesin/gastrin-releasing peptide (GRP), cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), and neuropeptide-Y (NPY), have also been identified and characterized for tumor receptor imaging (Schottelius and Wester, 2009).
  • MSH melanocyte-stimulating hormone
  • peptides that can be used for the noninvasive detection of prostate, ovarian, melanoma, and colon cancer remain elusive (Schottelius and Wester, 2009).
  • Many sold tumors, including prostate, ovarian, melanoma, and colon cancer overexpress EphB4 receptor, a member of the Ephrin receptor tyrosine kinase family.
  • Noninvasive imaging of EphB4 using peptide-based imaging agents could potentially increase early detection rates, monitor response to therapy directed again EphB4, and improve patient outcomes.
  • specific targeting of therapeutic agents to EphB4 expressing cancers could greatly improve the efficacy and specificity of cancer therapy.
  • an Ephrin receptor targeting agent comprising at least a first peptide comprising the amino acid sequence TNYLFSPNGPIA (SEQ ID NO: 2) conjugated to a therapeutic agent or an imaging agent.
  • the first peptide is defined as a peptide that binds specifically or preferentially to Eph receptor EphB4.
  • the peptide can comprise a sequence TNYLFSPNGPIARAW (SEQ ID NO: 1) or a sequence identical to SEQ ID NO: l, but comprising 1 or 2 amino acid substitutions, insertions or deletions.
  • the first peptide is a cyclic peptide, such as a peptide comprising a lactam bridge (e.g., a cyclic peptide comprising the structure shown in FIG. 25).
  • a targeting agent of the embodiments further comprises a second peptide targeting agent.
  • the second peptide can bind to the same target as the first peptide or to a second target.
  • the second peptide binds to an EphA2 receptor.
  • a targeting agent is provided that binds to EphA2 and EphB4 receptors.
  • the second peptide comprises an amino acid sequence of SEQ ID NO: 4.
  • a first and second peptide of the embodiments are comprised in a fusion protein.
  • the first and second peptides are connected by a linker. Examples of linkers include, without limitation, linker peptides, carbohydrate polymers and fatty acids. In some cases, the linker is a polyethylene glycol (PEG) moiety.
  • the first peptide of a targeting agent is conjugated to a radioisotope, a nanoparticle, a toxin, a chemotherapeutic agent, a fluorescent dye or a combination thereof.
  • the conjugate may be through covalent linkage or a non-covalent association, such as via chelator moiety (e.g., 1,4,7, 10-tetraazadodecane-N, N',N",N"'-tetraacetic acid (DOTA)).
  • the first peptide is conjugated to an imaging agent, such as a SPECT imaging agent, PET imaging agent an MRI contrast agent, or a fluorescent dye.
  • the first peptide is conjugated to a radioisotope, such as a gamma emitter, a positron emitter or a beta-emitter.
  • a radioisotope such as a gamma emitter, a positron emitter or a beta-emitter.
  • the radioisotope is conjugated to the first peptide through a chelating moiety (e.g., DOTA).
  • the radioisotope is conjugated to the first peptide as part of a molecule that comprises the radioisotope.
  • radioisotopes for use in accordance with the invention include, without limitation, astatine-21 1, chromium-51, cobalt-57, cobalt-58, copper-60, copper-61, copper-62, copper-64, copper-66, copper-67, Eu-152, gallium-67, gallium-68, indium-I l l, iron-59, lutetium-177m, rhenium-186, rhenium-188, selenium-75, strontium-89, technicium- 99m, thorium-227, and/or yttrium-90.
  • a first peptide of the embodiments is conjugated to a therapeutic agent, such as chemotherapeutic agent, a radioisotope, or a therapeutic nanoparticle.
  • a therapeutic agent such as chemotherapeutic agent, a radioisotope, or a therapeutic nanoparticle.
  • the chemotherapeutic agent is an anthracycline antibiotic, such as doxorubicin (Dox).
  • therapeutic nanoparticles include, without limitation, nanoparticles that may be used to apply a photothermal ablation therapy, such as a hollow gold nanosphere (HAuNS).
  • a nanoparticle of the embodiments is further loaded with or conjugated to a chemotherapeutic agent, such as Dox.
  • a method of imaging a subject comprising: (a) administering an effective amount of a targeting agent of the embodiments to the subject (e.g., a EphB4 and/or EphA2 targeting agent); and (b) imaging the subject to detect the presence of the targeting agent.
  • a targeting agent of the embodiments e.g., a EphB4 and/or EphA2 targeting agent
  • imaging the subject can comprise positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), or photoacoustic imaging.
  • SPECT single photon emission computed tomography
  • MRI magnetic resonance imaging
  • a method of treating a subject comprising administering an effective amount of a targeting agent of the embodiments to the subject.
  • a method of the embodiment can further comprise administering one or more secondary therapy to the patient.
  • the secondary therapy can be a surgical therapy, chemotherapy, radiation therapy or hormonal therapy.
  • the secondary therapy can facilitate the activity the targeting agent.
  • a targeting agent that comprises a nanoparticle such as a HAuNS
  • a photothermal ablation therapy can be applied to heat the tissues surrounding the targeting agent.
  • a nanoparticle can be loaded with a chemotherapy agent (e.g., Dox) such that heating of the nanoparticle activates local release of the chemotherapy agent.
  • a chemotherapy agent e.g., Dox
  • a method for dual imaging and therapy of a subject comprising administering an effective amount of a targeting agent of the embodiments to a subject (i.e., wherein the targeting agent comprises a therapeutic and imaging agent); and imaging the subject.
  • a subject refers to an animal, such a human, canine or feline subject.
  • the subject has or is suspected of a cancer, such as an EphB4- or EphA2-overexpressing cancer.
  • an imaging probe for noninvasive imaging of EphB4 receptors comprising a heterodimer peptide, wherein said peptide has motifs which target both EphA2 and EphB4.
  • the heterodimer bispecific polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 or 2 and 4 (e.g., the amino acid sequence of SEQ ID NO: 1 and 4).
  • the heterodimer, bispecific polypeptide comprises an EphA2-targeting motif and an EphB4- targeting motif separated by a linker, such as PEG linker.
  • the heterodimer polypeptide comprises from amino- to carboxy-terminus the amino acid sequence of SEQ ID NO: 1 ; a PEG linker; and the amino acid sequence of SEQ ID NO: 4.
  • a method of monitoring a cancer patient comprising the steps of (i) injecting the patient with a heterodimer peptide of the embodiments; (ii) imaging the patient using a PET/CT scan imaging device wherein the device produces a PET image; (iii) acquiring the PET image of a tumor in the patient; (iv) determining tumor-to-muscle ratio in the patient; and (v) administering a cancer treatment to the patient based on the tumor-to-muscle ratio.
  • a method is provided of identifying EphB4 receptor expression in a subject in need thereof comprising administering to the subject a therapeutic amount of the heterodimer peptide of the embodiments, and determining uptake or binding of the peptide in said subject.
  • embodiments of the present invention contemplate the use of targeting nanoparticles in combination with physiological and physical approaches, such as tumor priming, vascular disruption, degradation of the extracellular matrix, and vessel normalization.
  • a method of reducing renal uptake of a radiolabeled agent in a subject comprising administering the radio-labeled agent in conjunction with metformin.
  • the metformin can be administered before, after or essentially simultaneously with the radio-labeled agent.
  • the radio-labeled agent is a radio-labeled therapeutic or imaging agent (e.g., an Eph Receptor targeting agent of the embodiments).
  • a pharmaceutical composition comprising a radio-labeled therapeutic or imaging agent and metformin formulated together in a pharmaceutically acceptable carrier.
  • Such a composition may be used for instance to reduce renal uptake of a radio-labeled agent in subject (e.g., a subject being treated or imaged with the agent).
  • FIG. 1 Structure of 64 Cu-DOTA-TNYL-RAW and nat Cu-DOTA -TNYL-RAW peptides.
  • FIG. 2 SPR sensorgrams of nat Cu-DOTA-TNYL-RAW and a scrambled peptide on sensor chips coated with EphB4.
  • the peptides were injected as ten two-fold concentration series from 1.6 nM to 800 nM and were analyzed in duplicate binding cycles. Data sets (shown in black) are overlaid with curves fit to a 1 : 1 mass transfer interaction model (gray lines).
  • the vertical axes in response units represent binding of each peptide to immobilized EphB4.
  • FIG. 3 Uptake of ⁇ Cu-DOTA -TNYL-RAW in EphB4-expressing PC-3M and CT26 cells and EphB4-negative A549 cells. The cell-to-medium uptake ratio is expressed as [cprn ⁇ g protein in pellet]/[cpm ⁇ g medium]. ⁇ Cu-DOTA-TNYL-RAW exhibited increased uptake over time in PC-3M and CT26 cells. This uptake was blocked by the parent TNYL-RAW peptide.
  • FIG. 3A CT26 cells.
  • FIG. 3B PC-3M cells.
  • FIG. 3C A549 cells.
  • FIG. 5 Representative ⁇ /CT images showing blocking of ⁇ Cu-DOTA- TNYL-RAW uptake in CT26 tumors at 4 h and in PC-3M tumors at 24 h after radiotracer injection.
  • 64 Cu-DOTA-TNYL-RAW was co-injected with cold TNYL-RAW (50 ⁇ g/mouse).
  • FIG. 7A The reaction scheme for the synthesis of nat Cu/ 64 Cu-DOTA- TNYL-RAW.
  • FIG. 8 SPR sensorgrams of TNYL-RAW and DOTA-TNYL-RAW peptides on sensor chips coated with EphB4.
  • the peptides were injected as ten two-fold concentration series from 1.6 nM to 800 nM and were analyzed in duplicate binding cycles. Data sets (shown in black) are overlaid with curves fit to a 1 : 1 mass transfer interaction model (gray lines).
  • the vertical axes in response units represent binding of each peptide to immobilized EphB4.
  • FIG. 9 Reaction scheme for the synthesis of SH-PEG-c(TNYL-RAW) and its conjugation to HAuNS.
  • FIG. 10 Cellular uptake of T-DOX@HAuNS.
  • FIG. 10A Representative photomicrographs of Hey cells after incubation with T-DOX@HAuNS for 2 h. The scattering signal from the HAuNS was visualized using a dark-field condenser, and fluorescence was from DOX. Cell nuclei were counterstained with DAPI. Bar, 20 ⁇ .
  • FIG. 10B Quantitative cellular uptake of nanoparticles in Hey and A549 cells after 3 h incubation with m In-labeled T-DOX@HAuNS and T-DOX@HAuNS plus free c(TNYL-RAW) (blocking). **P ⁇ 0.005;*P ⁇ 0.05.
  • FIG. 11 T-DOX@HAuNS pharmacokinetics, biodistribution, and tumor uptake.
  • FIG. 1 IB Biodistribution of U 1 ln-labeled T-DOX@HAuNS, T-DOX@HAuNS with blocking, and DOX@HAuNS in nude mice at 24 h after injection.
  • FIG. 11C Biodistribution of U 1 ln-labeled T-DOX@HAuNS, T-DOX@HAuNS with blocking, and DOX@HAuNS in nude mice at 24 h after injection.
  • FIG. 11C Biodistribution of U 1 ln-labeled T-DOX@HAuNS, T-DOX
  • FIG. 12 NIR-induced temperature change in tumors injected with HAuNS.
  • FIG. 13 Antitumor activity of various treatments against Hey tumors.
  • FIG. 13B Average tumor weights (left panel) and photographs (right panel) of tumors from different treatment groups. Tumors were removed on day 22 for all groups except the saline-plus-laser group, in which tumors were removed on day 9.
  • FIG. 13C Representative photomicrographs of hematoxylin and eosin-stained slides from scar tissue from a mouse treated with T-DOX@HAuNS-plus-laser or tumors from mice treated with saline or DOX@HAuNS-plus-laser on day 22 after treatment.
  • FIG. 14 Physical properties of HAuNS.
  • FIG. 14A Absorption spectrum of HAuNS.
  • FIG. 14B TEM image of HAuNS.
  • FIG. 14C High-resolution TEM of a single HAuNS.
  • FIG. 15A SPR sensorgrams of c(TNYL-RAW) peptide on sensor chips coated with EphB4. Peptides were injected as ten two-fold concentration series from 1.6 nM to 800 nM and were analyzed in duplicate binding cycles. Data sets (shown in black) are overlaid with curves fit to a 1 : 1 mass transfer interaction model (gray lines). The vertical axes in response units represent binding of each peptide to immobilized EphB4.
  • FIG. 15B Comparison of peptide stability in mouse plasma. 64 Cu- DOTA-TNYL-RAW or 64 Cu-DOTA-c(TNYL-RAW) was incubated in mouse plasma at 37°C.
  • a 100 ⁇ , of plasma was removed from the incubation solution at 0, 2, 4, 8, 12, and 24 h time points and subjected to solid phase extraction on a CI 8 cartridge SPE column (Waters, Milford, MA). Then 20 ⁇ of the extract was analyzed by reversed phase-high-performance liquid chromatography (RP-HPLC) on an Agilent 1100 system (C-18, Vydac, 4.6 x 250 mm, 10 ⁇ ) equipped with a radiodetector. The system was eluted with a linear gradient of 10%- 90% acetonitrile in a 0.1% aqueous trifluoroacetic acid solution over 35 min at a flow rate of 1.0 mL/min.
  • RP-HPLC reversed phase-high-performance liquid chromatography
  • FIG. 16 Western blot analysis of EphB4 expression in MDA-MB-231, A549, A2780, and Hey cell lines.
  • FIG. 17 Cell viability as a function of equivalent DOX concentration.
  • A549 (FIG. 17A) and A2780 (FIG. 17B) cells were treated with T-DOX@HAuNS, DOX@HAuNS, or free DOX. The viability of cells was determined using MTT assay. Circles are T-DOX@HAuNS; Squares are DOX@HAuNA; Triangles are Free DOX.
  • FIG. 18 Percentage of body weight change after various treatments. All data are presented as mean ⁇ standard deviation. Diamonds are Saline + Laser; Squares are HAuNS + Laser; Triangles are DOX@HAuNS + Laser; X is T-DOX@HAuNS + Laser.
  • FIG. 19A Structure of heterodimer peptides with motifs targeting both EphA2 and EphB4 receptors.
  • FIG. 19B Characterization of heterodimer peptides by ESI-MS.
  • FIG. 19C Characterization of heterodimer peptides by HPLC.
  • FIG. 20 Surface competition assays.
  • FIG. 20A Surface competition assays were performed with YSA-TNYL-RAW dimer and ephrinB2 with the surface coated with EphB4 using an increasing concentration of heterodimer and a constant concentration of ephrinB2, the natural ligand of EphB4.
  • FIG. 20B Surface competition assays were also performed with YSA-TNYL-RAW dimer and EphA2 with the surface coated with ephrinAl, the natural ligand of EphA2, using an increasing concentration of heterodimer and a constant concentration of EphA2. The highest concentration corresponds to the bottom line on the sensorgrams while the lowest corresponds to the top line.
  • FIG. 21 Cell binding and inhibition in ovarian cancer Hey cells.
  • FIG. 21 A Cell binding of 64CU-DOTA-YSA-TNYL-RAW dimer over time.
  • FIG. 2 IB Cell binding at a single time point with various blocking agents. The bars, from left to right, are as follows: No blocking, Block w/ Dimer, Block w/ TNYL-RAW, Block w/ YSA.
  • FIG. 22 Biodistribution of 64 Cu-labeled YSA-TNYL-RAW bispecific heterodimer targeting both EphA2 and EphB4 receptors in ovarian cancer xenografts.
  • FIG. 23 Structure of the dual-labeled EphB4-binding peptide DOTA-TNYL-
  • FIG. 24 Immunohistochemistry of tumor sections confirming the binding of ⁇ Cu-DOTA-TNYL-CyS.S to tumor cells and tumor microvessels.
  • U251 and U87 tumors were stained and imaged for CD 31 and EphB4.
  • FIG. 25 Structure of EphB4-targeting cyclic peptide c(TNYL-RAW).
  • FIG. 26 Surface plasmon resonance curves for binding of c(TNYL-RAW) to
  • FIG. 27 Stability of peptides in mouse serum as assayed by LC-MS. Cyclic peptide c(TNYL-RAW) was more stable in mouse serum than its corresponding linear peptide. Top line is c(TNYL-RAW); bottom line is TNYL-RAW.
  • FIG. 28 FIG. 28A. ⁇ /CT images of nude mice bearing subcutaneous human melanoma A375SM xenografts using 7.4 MBq (200 ⁇ ) of 68 Ga-NOTA-c(TNYL- RAW) peptide. Reduced retention in the liver and the spleen, as well as minimal retention in the lungs and GI tract, were shown by ⁇ /CT imaging.
  • FIG. 28B Uptake of 68 Ga-NOTA- c(TNYL-RAW) in different organs.
  • NOTA radiometal chelator l,4,7-tetraazacyclododecane-N,N',N"-tetraacetic acid
  • Radionuclide- and fluorescent dye-labeled peptides and a method of using the peptides in imaging EphB4 receptors and Ephrin family of receptors with the radionuclide and/or fluorescent dye labeled peptides are likewise provided.
  • the disclosed peptides can be potentially used for early detection of cancer and for monitoring response to treatment directed at EphB4 receptors.
  • the imaging properties in disease models include, but are not limited to, dosimetry and toxicity.
  • EphB4 receptor a member of the Ephrin receptor tyrosine kinase family.
  • Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed again EphB4, and improve patient outcomes.
  • a series of peptide-based imaging agents are disclosed with high receptor binding affinity for nuclear imaging of EphB4 receptors.
  • the EphB4-binding peptide TNYLF SPNGPIARA W (TNYL-RAW) was conjugated with 1,4,7, 10-tetraazadodecane-N, N',N",N"'-tetraacetic acid (DOTA).
  • DOTA-TNYL-RAW was labeled with ⁇ Cu with high labeling efficiency.
  • 64 Cu-DOTA-TNYL-RAW displayed high binding affinity to EphB4 (Kd- 2 nM), was selectively taken up by CT26 and PC-3M cells, but not by A549 cells.
  • Binding of FITC-TNYL-RAW and ⁇ Cu-DOTA-TNYL-RAW to CT26 and PC-3M cells could be blocked by an excess amount of TNYL-RAW.
  • 64 Cu-DOTA-TNYL- RAW showed significantly higher uptake in PC-3M tumors than in A549 tumor, with percentages of injected dose per gram of tumor (%ID/g) values of 0.84 ⁇ 0.09 and 0.44 ⁇ 0.09 at 24 hr after radiotracer injection, respectively ⁇ /CT imaging clearly revealed deposition of ⁇ Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors.
  • EphA2 and EphB4 receptors are over-expressed in a variety of solid tumors including cancers in the ovarian, breast, colorectal, brain, and prostate. These receptors are also expressed in angiogenic blood vessels. Therefore, members of the Ephrin receptor family are attractive targets for cancer imaging and therapy (Pasquale, Nat Rev. Cancer, 10: 165, 2010).
  • imaging probes that simultaneously bind to both EphA2 and EphB4 can potentially increase early detection rates and be used to monitor response to therapy directed against EphA2 and EphB4.
  • a dual labeling approach is provided by introducing both a radionucle and a near-infrared dye to EphB4-targeting peptide that allow dual modal imaging of the receptors.
  • imaging probes can provide increased information content and are useful for both diagnostic imaging and guiding surgery intraoperatively.
  • PEG polyethylene glycol
  • YSA-TNYL was then labeled with the positron emitter ⁇ Cu through 1,4,7, 10-tetraazacyclododecane-N, N', N", N"'-tetraacetic acid (DOTA) chelator.
  • DOTA 10-tetraazacyclododecane-N, N', N", N"'-tetraacetic acid
  • SPR Surface Plasmon Resonance
  • YSA-TNYL-DOTA peptide had comparable binding affinity to EphB4 compared with TNYL peptide and higher binding affinity to EphA2 than YSA peptide.
  • YSA-TNYL-DOTA- 64 Cu dimer showed significantly higher tumor uptake value compared with monomeric 64 Cu-DOTA- TNYL and monomeric 64 Cu-DOTA-YSA monomer analogs at all time points examined.
  • the tumor uptake of YSA-TNYL-DOTA- 64 Cu could be partially blocked with an excess amount of cold TNYL, YSA, or mixture of TNYLRAW and YSA peptides.
  • the heterodimer YSA-TNYL- DOTA- Cu Compared with 64 Cu- DOTATNYL and 64 Cu-DOTA-YSA monomeric tracers, the heterodimer YSA-TNYL- DOTA- Cu also showed improved pharmacokinetics, resulting in a significantly higher target-to-background ratio.
  • this class of radiotracers directed at both EphA2 and EphB4 should be useful imaging probes for early tumor detection and noninvasive characterization of Ephrin receptors.
  • 64 Cu-DOTA and Cy5.5 dye were introduced to TNYL peptide to synthesize dual-tracer imaging probe. In vitro, dual labeled TNYL displayed significantly higher binding to U251 glioma cells over-expressing EphB4 than to U87 cells that express low level of EphB4.
  • this invention discloses new classes of peptide imaging agents suitable for noninvasive detection of tumor cells overexpressing EphB4 receptors (and in some cases EphA2 receptors).
  • the disclosed peptides can be potentially used for early cancer detection and monitoring of treatment response.
  • the concept can be potentially used for guiding cancer surgery under fluorescent imaging.
  • the same concept can be applied to the detection of other receptors in the Ephrin receptor family.
  • this invention discloses a novel 64 Cu-labeled peptide with high receptor binding affinity (i.e., low nanomolar 3 ⁇ 4 values) for PET imaging of EphB4 receptors.
  • the expression of EphB4 receptors can be noninvasively interrogated by ⁇ /CT using the disclosed peptide, 64 Cu-DOTA-TNYL-RAW.
  • Photothermal ablation is an emerging technique that uses near-infrared laser light-generated heat to destroy tumor cells.
  • complete tumor eradication by PTA therapy alone is difficult because heterogeneous heat distribution can lead to sub-lethal thermal dose in some areas of the tumor.
  • Successful PTA therapy requires selective delivery of photothermal conducting nanoparticles to mediate effective PTA of tumor cells, and the ability to combine PTA with other therapy modalities.
  • Multifunctional doxorubicin (DOX)- loaded hollow gold nanospheres were synthesized that target EphB4, a member of the Eph family of receptor tyrosine kinases overexpressed on the cell membrane of multiple tumors and angiogenic blood vessels.
  • T represents a cyclic peptide targeting EphB4 plus laser regressed completely with only residual scar tissue by 22 days following injection, and none of the treatment groups experienced a loss in body weight.
  • Concerted chemo-photothermal therapy with a single nanodevice capable of mediating simultaneous PTA and local drug release may have promise as a new anticancer therapy.
  • this invention discloses an optimized cyclic peptide with high stability and low background suitable for noninvasive detection of tumor cells overexpressing EphB4 receptors.
  • the disclosed peptides can be potentially used for early cancer detection and monitoring of treatment response.
  • the same peptide can also be used for targeted delivery nanoparticles and therapeutic agents.
  • the cyclic peptide can be labeled with any positron emitter or gamma emitter for PET and SPECT imaging, respectively.
  • the peptide can also be labeled with a fluorescent dye to guide surgery.
  • this invention discloses a method of nuclear imaging for reducing renal uptake of radiotracers.
  • the method can be potentially used to reduce renal toxicity of radiolabeled compounds used in radionuclide therapy and imaging.
  • Eph receptors are the largest family of receptor tyrosine kinases
  • Eph receptor tyrosine kinases and their ligands regulate a wide range of cell contact-dependent signaling that can effect cell proliferation, migration, morphology, adhesion, and invasion (Pitulescu et al. Genes & Dev., 24:2480-2492, 2010). This can occur through Eph signaling which alters the actin cytoskeleton organization and integrins and intercellular adhesion molecules processes. Eph-ephrin interactions are important for many biological roles including axon growth and maturation, cell positioning in the gastoinestinal tract, blood vessel morphogenesis and angiogenic sprouting, insulin secretion, bone remodeling, and immune function.
  • Eph receptors are divided into an EphA and an EphB class that bind to glycosylphosphatidylinositol-linked ephrin-A ligands and the transmembrane ephrin-B ligands, respectively.
  • EphB4 receptors play important roles in a variety of biological processes, including cell aggregation and migration, neural development, embryogenesis and angiogenesis, and vascular development (Dodelet and Pasquale, 2000; Noren et al, 2004; Erber et al, 2006; Wang et al, 1998).
  • EphB4 selectively binds to its endogenous ligand, ephrin-B2, to promote cell signaling required for cancer progression and angiogenesis and has been shown to be profoundly upregulated in numerous cancer types, such as prostate, colon, lung, gastric, bladder, ovarian, and breast cancers (Xia et al, 2006; Davalos et al, 2006; Xia et al, 2005; Takai et al, 2001; Kumar et al, 2009; Stephenson et al, 2001 ; Kumar et al, 2006).
  • Overexpression of EphB4 in cancer cells is associated with tumorigenesis and angiogenesis by stimulating reverse signaling through ephrin-B2.
  • EphB4 forward signaling has been shown to inhibit cellular proliferation (Noren et al, 2004).
  • Eph receptors are typically divided into a globular ligand-binding domain, a cysteine-rich region, and two fibronectin type III repeats in the extracellular region, a short transmembrane region with several conserved tyrosine residues and the tyrosine kinase domain, a sterile a motif (SAM) protein-protein interaction domain, and a C-terminal PDZ- binding motif in the intracellular region. Eph receptors are divided into two classes, EphA and EphB.
  • Eph ligands which are also cell-surface associated proteins, divided into two classes, GPI-anchored ephrin-A and transmembrane ephrin-B.
  • Ephrin-B molecules contain a cytoplasmic domain with several highly conserved tyrosine phosphorylation sites and a C-terminal PDZ motif.
  • EphA receptors bind to ephrin- A
  • EphB receptors bind to ephrin-B, but cross-signaling can occur.
  • Eph and ephrins are capable of bi-directional signaling through trans interactions, though interactions in cis (i.e., between molecules expressed in the same cell) appear to inhibit receptor activation.
  • a targeting peptide of the embodiments is conjugated to a radioisotope.
  • the targeting agent comprises a chelating moiety and a radionuclide chelate.
  • the peptide can be chelated to a radionuclide, such as a technetium ion, a copper ion, an indium ion, a thallium ion, a gallium ion, an arsenic ion, a rhenium ion, a holmium ion, a yttrium ion, a samarium ion, a selenium ion, a strontium ion, a gadolinium ion, a bismuth ion, an iron ion, a manganese ion, a lutetium ion, a cobalt ion, a platinum ion
  • radionuclides examples include, but are not limited to, 99m Tc, 188 Re, 186 Re, 153 Sm, 166 Ho, 90 Y, 89 Sr, 67 Ga, 68 Ga, m In, 183 Gd, 59 Fe, 225 Ac, 212 Bi, 211 At, 45 Ti, 177 Lu, 60 Cu, 61 Cu, 67 Cu, and 64 Cu.
  • Chelating moieties for use according to the invention include, but are not limited to, a acyclic polyamioncarboxylate, a diposphine, a Schiff base, a bis(thiosemicarbazone), a cyclic polyamine, a cyclic polyaminocarboxylate, a cross-bridged cyclic polyamine, a cross-bridged cyclicpolyamioncarboxylate, a l,3,5-cis,cis- triamioncyclohexane derivative, a sarcophagine, or a sepulchrate.
  • the chelating moiety can be l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), 1,4,7,10- tetraazacyclododecane-l,4,7, 10-tetraacetic acid (DOTA), diethylenetriaminetetraacetic acid (DTTA), Diethylenetriaminopentaacetic acid (DTP A), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,8,1 1-tetraazacyclotetradecane- 1,4,8,1 1 -tetraacetic acid (TETA), 1,4,8,1 l-tetraazacyclotetradecane-l,8-diacetic acid (TE2A) Mercaptoacetyltriglycine (MAG 3 ) or 4,5-bis(2-mercaptoacetamido)pentanoic acid, metformin, or phenformine.
  • Chemotherapeutic agents
  • a targeting peptide of the embodiments is conjugated to or associated with a chemotherapeutic agent.
  • chemotherapeutic agents of use as conjugates (or for loading in peptide-conjugated nanoparticles) include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin;
  • nanoparticle refers to any particles having dimensions in the 1-1,000 nm range.
  • nanoparticles have dimensions in the 2-200 nm range, preferably in the 5-150 nm range, and even more preferably in the 10- 100 nm range.
  • the nanoparticles may be conjugated to a targeting peptide of the embodiments.
  • Example nanoparticles include for example polymeric micelles, polyethylene glycol, carbon nanotubes, gold nanostructures, such as gold nanoshells, gold nanorods, gold nanocages, and hollow gold nanospheres, as detailed herein, and copper sulfide nanoparticles (Haram et al, 1996; Xu et al, 2009; Huang et al, 2010).
  • CuS NPs have been synthesized and demonstrated for photothermal destruction of tumor cells in vitro using a NIR laser beam centered at 808 nm.
  • Targeting agents of the embodiments may be used in an imaging or detection method for diagnosis or localization of tumor or angiogenic tissues. Any optical or nuclear imaging method may be contemplated, such as PET, SPECT, CT, or photoacoustic tomography.
  • the integrated radioactive isotope in the nanoparticle may exert a radiotherapeutic effect on the tissue incorporating such nanoparticle.
  • a photothermal ablation therapy may be administered to the tissue having the targeting agents to enhance the therapeutic effect.
  • Targeting agents may be used in PET.
  • Positron emission tomography PET is a powerful and widely used diagnostic tool that has the advantages of high sensitivity (down to the picomolar level) and ability to provide quantitative imaging analyses of in vivo abnormalities (Scheinin et al, 1999; Eckelman, 2003; Welch et al, 2009).
  • PET may be used in certain aspects to trace nanoparticles in vivo.
  • Certain targeting agents may also be used in SPET.
  • Single photon emission computed tomography SPECT, or less commonly, SPET
  • SPET is a nuclear medicine tomographic imaging technique using gamma rays. It is very similar to conventional nuclear medicine planar imaging using a gamma camera. However, it is able to provide true 3D information. This information is typically presented as cross-sectional slices through the patient, but can be freely reformatted or manipulated as required.
  • the SPET basic technique requires injection of a gamma-emitting radioisotope called radionuclide) into the bloodstream of the patient.
  • a radioisotope is conjugated to a targeting agent, which allow it to be concentrated in ways of medical interest for disease detection.
  • a targeting agent comprising a marker radioisotope, which is of interest for its radioactive properties, has been attached to a targeting ligand, which is of interest for its chemical binding properties to certain types of tissues.
  • This marriage allows the combination of ligand and radioisotope (the radiopharmaceutical) to be carried and bound to a place of interest in the body, which then (due to the gamma-emission of the isotope) allows the ligand concentration to be seen by a gamma-camera.
  • Targeting agents may also be used in MRI. Magnetic resonance imaging
  • MRI Magnetic resonance Imaging
  • contrast materials such as intravenous DOTA-Gd contrast are used. This is useful to highlight structures such as blood vessels that otherwise would be difficult to delineate from their surroundings. Using contrast material can also help to obtain functional information about tissues.
  • Certain targeting agents may also be used in photoacoustic tomography.
  • Photoacoustic tomography or photoacoustic computed tomography (PACT) is a materials analysis technique based on the reconstruction of an internal photoacoustic source distribution from measurements acquired by scanning ultrasound detectors over a surface that encloses the source under study.
  • the PA source is produced inside the object by the thermal expansion that results from a small temperature rise, which is caused by the absorption of externally applied radiation of pulsed electromagnetic (EM) waves.
  • EM pulsed electromagnetic
  • This technique has great potential for applications in the biomedical field because of the advantages of ultrasonic resolution in combination with EM absorption contrast.
  • PAT is also called optoacoustic tomography (OAT).
  • each temporal PA signal measured at various detection positions, provides one-dimensional radial information about the PA source relative to the detector position; 2D surface scans offer other 2D lateral information about the PA source. Combining the temporal and spatial measurements affords sufficient information for a complete reconstruction of a 3D PA source. Because the PA signal received by each ultrasound detector is the integral of the ultrasound waves over the sensing aperture of the detector, the reconstruction algorithms depend on the detector apertures as well as the scanning geometries. Small-aperture detectors are often used to approximate point detectors, which receive PA signals originating from spherical shells, centered at each point detector, with radii determined by the acoustic times of flight.
  • Targeting agents may also be used in photothermal ablation therapy.
  • Photothermal ablation (PTA) therapy has gained increasing attention in recent years as a minimally invasive alternative to conventional approaches to cancer treatment such as surgery and chemotherapy (Amin et ah, 1993; Nolsoe et ah, 1993; Fiedler et ah, 2001 ; Vogeland Venugopalan, 2003).
  • NPs with unique optical properties primarily gold nanostructures, such as gold nanoshells (Hirsch et ah, 2003; Loo e ah, 2005), gold nanorods (Dickerson et ah, 2008; Park et ah, 2010), gold nanocages (Chen et ah, 2007; Au et ah, 2008), and hollow gold nanospheres (Lu et ah, 2010; Melancon et al obsession 2008; (Lu et ah, 2009), but also carbon nanotubes (Chakravarty et ah, 2008 Burke et ah, 2009)— have been investigated as photothermal coupling agents to enhance the efficacy of PTA therapy.
  • gold nanostructures such as gold nanoshells (Hirsch et ah, 2003; Loo e ah, 2005), gold nanorods (Dickerson et ah, 2008; Park et ah, 2010), gold nanocages
  • plasmonic nanomaterials exhibit strong absorption in the near-infrared (MR) region (wavelength 700-1 100 nm) and offer an opportunity to convert optical energy to thermal energy, enabling deposition of otherwise benign optical energy into tumors for thermal ablation of tumor cells.
  • MR near-infrared
  • EphB4 receptor a member of the Ephrin receptor tyrosine kinase family.
  • Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed again EphB4, and improve patient outcomes.
  • Disclosed herein is a series of peptide-based imaging agents with high receptor binding affinity for nuclear imaging of EphB4 receptors.
  • the EphB4-binding peptide TN YLF SPNGPIARA W TNYL-RAW; SEQ ID NO: 1 was conjugated with l,4,7, 10-tetraazadodecane-N,N',N",N" '-tetraacetic acid (DOTA).
  • DOTA-TNYL-RAW was labeled with 64 Cu with high labeling efficiency.
  • EphB4 receptors can be noninvasively interrogated by ⁇ /CT using 64 Cu-DOTA -TNYL-RAW.
  • Pasquale and colleagues identified, using phage display technology, several 12-mer peptides that selectively bind to individual Eph receptors. Tyr- Asn-Tyr-Leu-Phe-Ser-Pro-Asn-Gly-Pro-Ile-Ala (TNYLFSPNGPIA; SEQ ID NO: 2), an EphB4 binding peptide from the initial screening, was further modified to include a RAW moiety at the carboxyl terminus.
  • TNYLFSPNGPIARAW Tyr-Asn-Tyr-Leu-Phe-Ser-Pro-Asn- Gly-Pro-Ile-Ala-Arg-Ala-Trp
  • TNYL-RAW can be used as a receptor ligand for the noninvasive imaging of EphB4.
  • the present disclosure shows that 64 Cu-labeled TNYL-RAW is a promising radiotracer for PET imaging of EphB4 receptor expression in both human prostate and colon cancer xenograft models.
  • l,4,7,10-Tetraazadodecane-N,N',N",N"'-tetraacetic acid was obtained from Macrocyclics (Dallas, TX).
  • the BIACore sensor chip CM5 amine coupling kit, HBSEP running buffer (0.01 M HEPES [4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid], pH 7.4, 0.15 M NaCl, 3 mM ethylenediaminetetraacetic acid [EDTA], and 0.005% [v/v] surfactant P20 solution), and regeneration buffer were purchased from BIACORE, Inc. (Piscataway, NJ).
  • 4',6-Diamidino-2-phenylindole (DAPI) was obtained from Sigma-Aldrich (St. Louis, MO).
  • the 64 Cu-labeled peptide was further purified, if necessary, by reverse-phase high- performance liquid chromatography (RP-HPLC) on an Agilent 1 100 system (C-18, Vydac, 4.6 x 250 mm, 10 ⁇ ) eluted with a linear gradient of 10%-90% acetonitrile in a 0.1% aqueous TFA solution over 35 min at a flow rate of 1.0 mL/min.
  • the solvent was then removed, reconstituted in saline, and passed through a 0.22- ⁇ filter for use in the animal experiments.
  • Natural copper chloride (“ ⁇ CuC ⁇ ) was used to synthesize nat Cu-DOTA-TNYL- RAW under identical conditions, and its identity was confirmed by high-resolution electrospray ionization mass spectrometry (HRMS-ESI). 64 Cu-DOTA-TNYL-RAW was co- injected with nat Cu-DOTA-TNYL-RAW into the above-mentioned HPLC system equipped with both ultraviolet and radiodetectors to confirm its identity.
  • HRMS-ESI high-resolution electrospray ionization mass spectrometry
  • the stock solution (100 ⁇ g/mL) of EphB4/Fc in phosphate-buffered saline (PBS) was diluted to 25, 12.5, and 6.25 ⁇ g/mL with 10 mM sodium acetate buffer at pH 4.5 and immobilized to a CM5 sensor chip using the amine coupling reaction following manufacturer-provided procedures (BIACORE). Briefly, the surfaces of the chips in flow cells (FC)-l, -2, -3, and -4 were activated by exposing them to a mixture of 200 mM N-ethyl-N'-dimethylaminopropyl carbodiimide and 50 mM N-hydroxysuccinimide for 7 min.
  • FC flow cells
  • FC-1 was used as a reference surface and was directly deactivated by injecting 1 M ethanolamine at pH 8.5 for 7 min.
  • the other three flow cells were injected with 25 ⁇ g/mL, 12.5 ⁇ g/mL, and 6.25 ⁇ g/mL EphB4/Fc, respectively, followed by injection of 1 M ethanolamine to block the remaining activated ester groups on the surface.
  • the chip was allowed to stabilize for at least 2 h in HBSEP running buffer before injecting test analytes.
  • SPR Surface plasmon resonance
  • the CT26 murine colon cancer cell line and A549 human lung adenocarcinoma epithelial cell line were purchased from the American Type Culture Collection (Manassas, VA).
  • the PC-3M human prostate cancer cell line was obtained from Professor Dominic Fan (MD Anderson Cancer Center, Houston, TX).
  • PC-3M, CT26, or A549 cells were seeded (1 x 10 5 /well) in Lab-Tek II chambered slides (Nalge Nunc International, Naperville, IL) supplemented with RPMI-1640 medium plus 10% FBS one day before the experiment.
  • the cells were incubated with 100 ⁇ ⁇ of phenol-free RPMI-1640 culture medium containing 10 ⁇ of FITC-TNYL-RAW or scrambled FITC-scTNYL-RAW (AGPFNTYLRTNAWSP; SEQ ID NO: 3) for 20 min at room temperature.
  • 10 ⁇ of FITC-TNYL-RAW and 1 mM of TNYL-RAW were added to the cells.
  • cells were incubated with 10 of PE-conjugated rat anti-human anti-EphB4 monoclonal antibody (R&D Systems, Minneapolis, MN) in 100 ⁇ ⁇ of phenol red-free RPMI-1640 medium.
  • the cells were washed and fixed with 4% paraformaldehyde for 15 min at room temperature.
  • the cell nuclei were counterstained with DAPI.
  • the slides were mounted and visualized under a Zeiss Axiovert Z.1 fluorescent microscope (Zeiss, Jena, Germany).
  • the tubes were briefly vortexed and 100 ⁇ ⁇ of the cell suspension was transferred into a microcentrifuge tube containing 500 ⁇ ⁇ of a 75:25 mixture of silicon oil (density 1.05; Sigma-Aldrich) and mineral oil (density 0.872; Acros, Geel, Belgium). The mixture was centrifuged at 14,000 rpm for 5 min. After the tubes were frozen in liquid nitrogen, the bottom tips containing the cell pellet were cut off. The cell pellets and the supernatants were counted with Packard Cobra Quantum ⁇ -counter (GMI, Ramsey, Minnesota). The protein content in a 100- ⁇ , cell suspension was quantified using the Bio-Rad protein assay kit according to the manufacturer's protocol. The radioactivity in the cell pellets and media was counted, and the data were expressed as activity ratios of the cell pellet to the medium ([cpm ⁇ ig protein in pellet]/[cpm ⁇ g medium]). The experiments were performed in pentaplicate.
  • the spatial resolution of the PET system is approximately 1.4 mm.
  • Tumor-bearing mice were anesthetized with isoflurane (2% in oxygen) and placed in a prone position.
  • the CT imaging parameters were as follows: X-ray voltage, 80 kVp; anode current, 500 mA; exposure time of each of the 360 rotational steps, 300-350 ms. Images were acquired at 1, 4, and 24 h after intravenous administration of 64 Cu-DOTA-TNYL-RAW. Images were reconstructed using the two-dimensional ordered subsets expectation maximization algorithm. PET and CT image fusion and image analysis were performed using Inveon Research Workplace (Siemens Preclinical Solutions, Knoxville, TN).
  • VOI 3- dimensional region of interest
  • a circular VOI was drawn on the muscle of the legs.
  • VOI was also drawn on a standard (radiotracer solution containing 1% of the injection dose) placed along with the animals.
  • the mean activities within the VOI of the tumor and muscle were calculated in IRW workstation (Siemens).
  • the organs of interest were excised and weighed and their radioactivity counted using an automatic gamma counter. (GMI, Ramsey, Minnesota). The stomach and intestines were not emptied prior to radioactivity measurements. The percentage of injected dose per gram of tissue (%ID/g) was calculated by dividing the %ID/organ by the weight of the organ. Values were expressed as mean ⁇ standard deviation (SD).
  • DOTA-TNYL-RAW is shown in FIG. 1.
  • the reaction scheme for the synthesis of nat Cu/ 64 Cu- DOTA-TNYL-RAW is shown in FIG. 7A.
  • 64 Cu-DOTA-TNYL-RAW used in the in vitro and in vivo experiments was typically 7.4-14.8 MBq/nmol (0.2-0.4 Ci/ ⁇ ) at the end of synthesis.
  • 64 Cu-DOTA-TNYL-RAW was stable in DMEM culture medium containing 10% FBS for up to 24 h at 37°C. Approximately 30% of the ⁇ Cu-DOTA-TNYL- RAW was degraded after 2 h of incubation in mouse serum at 37°C.
  • TNYL-RAW peptides bound to EphB4 receptors with nM affinity.
  • FIG. 2 shows representative sensorgrams obtained from SPR analyses of nat Cu-DOTA- TNYL-RAW and a scrambled peptide, with fitting curves obtained using a global 1 : 1 mass transfer model (gray lines).
  • SPR sensorgrams of TNYL-RAW and DOTA-TNYL-RAW peptides are presented in FIG. 8.
  • the corresponding binding kinetics and affinity data are summarized in Table 1.
  • TNYL had an equilibrium dissociation constant (KD) of 3.06 nM. Conjugation of DOTA to the N-terminus of the peptide increased the KD value to 23.3 nM.
  • KD equilibrium dissociation constant
  • EphB4 in vitro Two EphB4-positive cell lines (PC-3M and CT26) and one EphB4-negative cell line (A549) were used for an in vitro binding study. Immunohistostaining with PE- conjugated rat anti-human EphB4 monoclonal antibody confirmed the expression of EphB4 on the surface of PC-3M, CT26, and A549 cells.
  • PC-3M, CT26, and A549 cells were treated with FITC-TNYL-RAW (10 ⁇ ) or a scrambled peptide FITC-sc-TNYL-RAW (10 ⁇ ) for 20 min at room temperature.
  • FITC-TNYL- RAW 10 ⁇ was co-incubated with TNYL-RAW (1 mM). The cell nuclei were counterstained with DAPI. Stained cells were imaged by fluorescence microscopy. PC-3M and CT26 cells, but not A549 cells, were readily stained with FITC-TNYL-RAW. A FITC- labeled scrambled TNYL-RAW peptide (FITC-sc-TNYL-RAW) did not show detectable binding to PC-3M and CT26 cells. The binding of FITC-TNYL-RAW to PC-3M and CT26 cells was efficiently blocked by an excess amount of unlabeled TNYL-RAW peptide.
  • 64 Cu-DOTA-TNYL-RAW peptide selectively binds to EphB4-positive ceUs.
  • 64 Cu-DOTA-TNYL-RAW had increased uptake with time in EphB4-positive PC-3M (FIG. 3B) and CT26 (FIG. 3 A) cell lines, but not in EphB4-negative A549 cells (FIG. 3C).
  • Co-incubation with cold TNYL-RAW peptide completely abolished the binding of 64 Cu- DOTA-TNYL-RAW to PC-3M at all time points tested (FIG. 3B) and reduced its binding to CT26 cells by ⁇ 10-fold (FIG. 3A).
  • uPET/CT imaging FIG.
  • the uptake values of ⁇ Cu-DOTA-TNYL- RAW in PC-3M tumors were 1.4, 3.2, and 3.6 %ID/g at 1, 4, and 24 h postinjection, respectively.
  • the accumulation of 64 Cu-DOTA-TNYL-RAW in A549 tumor was low at all time points examined, reaching a level of 1.7, 1.5, and 1.2 %ID/g at 1, 4, and 24 h after radiotracer administration. These values were only slightly higher than those recorded for muscle tissue in the same animals.
  • Ex vivo immunohistochemical staining confirmed the expression of EphB4 throughout the CT26 and PC-3M tumors, whereas A549 tumors did not express EphB4 receptors.
  • FIG. 5 compares ⁇ images obtained in the presence and absence of a large excess of cold TNYL-RAW at 4 h after radiotracer injection in a CT26 tumor-bearing mouse and at 24 h after radiotracer injection in a PC-3M tumor-bearing mouse.
  • CT26 and PC-3M tumor models the co-administration of cold TNYL-RAW caused a 77% and 81% reduction in 64 Cu-DOTA-TNYL-RAW accumulation in tumors, respectively.
  • the tumor-to-muscle ratio was reduced 56.7% in CT26 tumors at 4 h postinjection and 47.6% in PC-3M tumors at 24 h postinjection when ⁇ Cu-DOTA-TNYL-RAW was co-injected with cold TNYL-RAW peptide (FIG. 6B).
  • the results of tissue sampling study corroborated with the pattern of 64 Cu-DOTA -TNYL-RAW biodistribution determined by non-invasive in vivo PET/CT imaging.
  • TNYL-RAW peptide had a dissociation constant (K D ) of 3.09 nM, which is comparable to that reported in the literature (KD 1 ⁇ 2 nM) (Koolpe et al, 2005). TNYL-RAW also had a slow dissociation rate (-1.3 x 10 "3 [s-1]), which is a better indicator than simple binding affinity for in vivo molecular imaging applications (Berezov et al, 2001). Previous studies showed that the N-terminal residues of the peptide could be modified without affecting the stability of the binding complex with EphB4 (Chrencik et al, 2006).
  • the level of EphB4 receptor expression should be the predominant factor influencing the magnitude of Cu-DOTA-TNYL-RAW accumulation and retention in tumor tissue.
  • the difference in the degradation of 64 Cu-DOTA-TNYL-RAW peptide-based radiotracer in different tumor microenvironments may also contribute to the difference in tumor retention of the radiotracer.
  • the stability of radioactive compounds is critical because the molecular integrity of the radiopharmaceutical must be maintained for an adequate time in the blood circulation during biodistribution and imaging studies.
  • ⁇ Cu-DOTA-TNYL-RAW was stable in DMEM with 10% FBS for at least 24 h and was stable in mouse serum for up to 2 h, after which slow degradation was observed. Nevertheless, these ⁇ /CT studies revealed that 64 Cu-DOTA-TNYL-RAW possess sufficient in vivo stability for tumor imaging.
  • the 64 Cu-DOTA-TNYL-RAW biodistribution data obtained by both non-invasive PET/CT imaging and by tissue sampling indicate that the liver and the kidney were the major organs for physiological uptake and clearance of this radiotracer.
  • hepatobiliary clearance is a major route for hydrophobic peptides (Rusckowski et ah, 2001).
  • TNYL-RAW peptide contains several hydrophobic amino acids (Asn, He, Leu, Phe, Pro, Ala, Trp) that could contribute to the high liver uptake of 64 Cu-DOTA-TNYL-RAW.
  • Photothermal ablation (PTA) therapy is a recently developed technique that uses near-infrared (MR) laser light-generated heat to destroy tumor cells.
  • MR near-infrared
  • PTA has gained popularity recently because a specific amount of photo-energy is delivered directly into the tumor mass without causing systemic effects, thus promising minimally invasive intervention as an alternative to surgery (Bardhan et al, 2011 ; Melancon et al, 2009).
  • PTA therapy alone is unlikely to kill all tumor cells because the heat distribution is non-uniform, especially in areas peripheral to large blood vessels where heat can be rapidly dissipated by circulating blood.
  • light-absorbing photothermal conducting nanoparticles are introduced.
  • MR laser-modulated photothermal effects can not only enable PTA of tumor cells but also trigger release of anticancer agents. Such a multimodal approach, which permits simultaneous PTA therapy and chemotherapy, should provide an opportunity for complete eradication of tumor cells.
  • HAuNS Hollow gold nanospheres
  • DOX doxorubicin
  • DOX@HAuNS DOX-loaded HAuNS
  • EphB4 is a particularly promising target for tumor-specific delivery of DOX@HAuNS.
  • DOX@HAuNS were selectively targeted to EphB4-positive tumors, and concerted chemo-photothermal therapy mediated by EphB4-targeting DOX@HAuNS induced remarkable antitumor efficacy with reduced systemic toxicity. These results support the concept of integrating multiple functions into a single nanodevice to mediate simultaneous PTA and local drug release.
  • Methoxy-polyethylene glycol (PEG)-SH (molecular weight, 5,000) and NH 2 -PEG-COOH (molecular weight, 5,000) were purchased from Nektar (San Francisco, CA).
  • Sodium citrate (>99%), cobalt chloride hexahydrate (99.99%), sodium borohydride (99%), and chloroauric acid trihydrate (American Chemical Society reagent grade) were purchased from Thermo Fisher Scientific (Waltham, MA) and were used as received. All amino acid derivatives and coupling reagents were purchased from EMD Chemicals (Philadelphia, PA), Bachem Americas (Torrance, CA), and Chem-Impex International (Wood Dale, IL).
  • PL-DMA resin was obtained from EMD Chemicals. DOX, (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4',6-diamidino-2- phenylindole (DAPI), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Reagent-grade solvents were used without further purification unless otherwise specified.
  • the BIACore sensor chip CM5 For the surface plasmon resonance (SPR) assay, the BIACore sensor chip CM5, amine-coupling kit, HBSEP running buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.005% [v/v] surfactant P20 solution), and regeneration buffer were purchased from GE Healthcare (Waukesha, WI). 3 H-labeled DOX was purchased from Moravek Biochemicals Inc. (Brea, CA).
  • HAuNS were synthesized according to a previously reported method (You et ah, 2010). Briefly, cobalt nanoparticles were first synthesized by deoxygenating deionized water containing 4.5 mL of 1 M sodium borohydride, 2.8 mL of 0.1 M sodium citrate, and 1.0 mL of 0.4 M cobalt chloride. After chloroauric acid was added into the solution containing cobalt nanoparticles, the cobalt immediately reduced the gold ions onto the surface of the nanoparticles and was simultaneously oxidized to cobalt oxide. Any remaining cobalt was further oxidized by air, resulting in the final product, HAuNS.
  • the size of the HAuNS was determined using dynamic light scattering on a Brookhaven 90Plus particle size analyzer (Holtsville, NY). The UV-visible spectra were recorded on a Beckman Coulter DU-800 UV- visible spectrometer (Brea, CA). The morphology of the HAuNS was examined using a JEM 1010 transmission electron microscope (JEOL USA, Peabody, MA).
  • DOX loading For DOX loading, free DOX (5 mg) in water (5 mL) was added to c(TNYL-RAW)-HAuNS (40 OD) in 5 mL of 2.8 mM citrate solution, and the mixture was stirred at room temperature for 24 h. The resulting DOX-loaded c(TNYL-RAW)-HAuNS (T- DOX@HAuNS) were purified by 3 repeated centrifugation and washing steps.
  • Fluorenylmethyloxycarbonyl chloride (Fmoc)-Asp-Wang resin)OAll was formed by esterification with DIPCDI (3 equiv.) and 4-dimethylaminopyridine (DMAP) (0.5 equiv.).
  • Fmoc-Lys(Boc)-Thr(tBu)-Asn(Trt)-Tyr(tBu)-Leu-Phe-Ser(tBu)-Pro-Asn(Trt)-Gly- Pro-ILe-Ala-Arg(Pbf)-Ala-Trp(Boc)-Asp(resin)OAll was synthesized using an Fmoc solid- phase strategy.
  • EphB4/Fc extracellular domain of human EphB4 fused to the carboxy -terminal Fc region of human IgGl was immobilized to CM5 sensor chips using the amine coupling reaction according to the manufacturer's instructions (GE Healthcare). Briefly, the surfaces of the chips in flow cells 1, 2, 3, and 4 were activated by exposing the chips to a mixture of 200 mM N-ethyl-N'- dimethylaminopropyl carbodiimide and 50 mM N-hydroxysuccinimide (NHS) for 7 min. Flow cell 1 was used as a reference surface and was directly deactivated by injecting 1 M ethanolamine at pH 8.5 for 7 min.
  • EphB4/Fc solutions of different concentrations were obtained by diluting a stock solution of EphB4/Fc (100 ⁇ g/mL) in phosphate-buffered saline (PBS) with 10 mM sodium acetate buffer (pH 4.5).
  • PBS phosphate-buffered saline
  • pH 4.5 10 mM sodium acetate buffer
  • Binding assays were performed at 25°C in HBSEP running buffer.
  • c(TNYL-RAW) was diluted in HBSEP buffer, filtered, degassed, and injected at concentrations between 1.6 nM and 800 nM at a flow rate of 30 ⁇ / ⁇ .
  • Peptides were injected into the HBSEP buffer over 4 min, and injection was followed by a 4-min dissociation period.
  • the chips were regenerated using a 1-min pulse of 10 mM glycine (pH 2.2) after each binding circle. Each cycle consisted of a 1-min waiting period to allow monitoring of the baseline binding stability. For subtraction of bulk effects caused by changes in the buffer composition or nonspecific binding, a double-referencing technique was employed.
  • Radiolabeling of HAuNS with m In To conjugate a radiometal chelator to c(TNYL-RAW)-HAuNS, 4-aminobenzyl-diethylenetriaminepentaacetic acid thioctamide (DTPA-TA, 10 mg/mL; 5.0 ⁇ ) (Lu et al, 2007) was mixed with 1.0 mL of aqueous solution of HAuNS (200 OD/mL) for 6 h at room temperature. SH-PEG-c(TNYL- RAW) was then added to the DTPA-TA-conjugated HAuNS as described in the previous section.
  • DTPA-TA 4-aminobenzyl-diethylenetriaminepentaacetic acid thioctamide
  • m In-labeled c(TNYL- RAW)-HAuNS were loaded with DOX to create 1 "in-labeled T-DOX@HAuNS.
  • the radiolabeling efficiency and the stability of m In-labeled T-DOX@HAuNS were analyzed using instant thin-layer chromatography.
  • the labeling efficiency was >95%.
  • MDA-MB-231 human breast carcinoma
  • Hey human ovarian carcinoma
  • A549 human lung adenocarcinoma
  • the cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Life Technologies, Inc., Carlsbad, CA) at 37°C in a humidified atmosphere containing 5% CO 2 .
  • A2780 human ovarian carcinoma cells were kindly provided by Dr. Stephen J. Williams (Fox Chase Cancer Center, Philadelphia, PA). These cells were maintained at 37°C in RPMI-1640 medium containing 10% fetal bovine serum and insulin (0.25 units/mL).
  • the cells were scraped off the dish, suspended in PBS, and the radioactivity of the cell suspension was then measured with a gamma counter. Protein concentration in cell suspension was quantified using the Bio-Rad protein assay kit (Richmond, CA). The data are expressed as radioactivity (dpm per ⁇ g protein).
  • EphB4 expression was probed with mouse anti-EphB4 antibody and Alexa Fluor 680-conjugated goat anti-mouse IgG (Invitrogen).
  • ⁇ - Actin was used as a control to indicate the loading and transfer efficiency. Protein bands were visualized with a LI-COR Odyssey system (Lincoln, NE).
  • a separate experiment was carried out to optically observe the binding between the antibody against EphB4 and the studied cell lines. Cells were cultured on 20-mm glass cover slips and incubated with primary antibody (mouse anti-EphB4 antibody) for 1 h at 4 °C.
  • the cells were then washed with PBS three times and incubated with secondary antibody (Alexa Fluor 680-conjugated goat anti-mouse IgG).
  • secondary antibody Alexa Fluor 680-conjugated goat anti-mouse IgG.
  • the cell-coated cover slip was then repeatedly rinsed with PBS and mounted for microscopic examination using an Axio Observer Zl fluorescence microscope (Carl Zeiss Microimaging GmbH, Germany).
  • Cytotoxicity was measured using an MTT assay according to the manufacturer's suggested procedures. EphB4-positive A2780 cells and EphB4-negative A549 cells were exposed to free DOX, DOX@HAuNS, or T-DOX@- HAuNS for 72 h. The data are expressed as the percentage of surviving cells and are reported as the mean values of three measurements.
  • the percentage of the injected dose per gram of blood was calculated.
  • the blood pharmacokinetic parameters for the radiotracer were analyzed using a noncompartmental model with WinNonlin 5.0.1 software (Pharsight, Sunnyvale, CA).
  • mice Female nude mice (Harlan, Indianapolis, IN) bearing 6-8 mm subcutaneous A2780, MDA-MB-231, or Hey tumors were intravenously injected with U 1 ln-labeled T-DOX@-HAuNS or m In- labeled DOX@HAuNS (20 ⁇ / ⁇ in 0.2 mL).
  • the mice were intravenously injected with a mixture of lu In-labeled T-DOX@-HAuNS (20 ⁇ / ⁇ in 0.2 mL) and an excess of free c(TNYL-RAW) (0.3 ⁇ ). Mice were killed at 24 h after injection.
  • SPECT Single photon emission computed tomography
  • mice bearing subcutaneous Hey tumors were intravenously injected with m In-labeled T- DOX@HAuNS or DOX@HAuNS (8.0 mCi/kg, 0.525 mg/mL [25 OD]).
  • mice were injected intravenously with a mixture of m In-labeled DOX@c(TNYL- RAW)-HAuNS and free c(TNYL-RAW) (0.3 ⁇ ).
  • the mice were placed in a prone position and anesthetized with 0.5%-2.0% isoflurane gas (Iso-Thesia, Rockville, NY) in oxygen.
  • SPECT images were generated at 24 h after injection. After imaging, the mice were killed, and their tumors were removed. The tumors were snap-frozen and cut into 5- ⁇ slices that were then used for autoradiography analysis on a Fujifilm FLA-5100 imaging system (Stamford, CT).
  • mice bearing Hey tumors were injected intravenously with saline (5.0 mL/kg) or T-DOX@-HAuNS (5.0 mL/kg of 50 OD HAuNS).
  • the tumor was irradiated with an NIR laser (3 W/cm 2 ) for 5 min; Diomed 15 Plus, UK) through the skin surface.
  • Temperature was measured with two thermocouples inserted into the tumor. Care was taken to ensure the thermocouple was not directly exposed to the laser beam.
  • the DOX release mediated by the photothermal effect in the Hey tumors was studied using m In- and 3 H-labeled T-DOX@-HAuNS, in which the HAuNS were labeled with U 1 ln and the DOX with 3 H.
  • Tumors were irradiated by NIR laser light (3 W/cm 2 for 5 min) at 1 h after intratumoral injection of the dual-labeled nanoparticles ( 3 H: 10 ⁇ ; m In: 20 ⁇ ) into the center of the tumor.
  • the mice were killed 5 min after laser irradiation, and the tumors were removed, snap-frozen, and sliced into 10- ⁇ sections. Injected tumors that did not receive NIR laser treatment were similarly prepared and used as controls.
  • Fujifilm FLA-5100 imaging system Briefly, the sections of the tumors were exposed to phosphorous screen film (an SR imaging plate) for 15 min at -10°C, and the m In autoradiograph was obtained by scanning the film. After the m In was completely decayed (stored at -80°C for 5 weeks), the same sections were exposed to phosphorous screen film (a TR imaging plate) for 3 days at -10°C, and the 3 H autoradiograph was obtained by scanning the film. Concurrently, control specimens from tumors that were not laser-treated were subjected to the same procedures. The autoradiographic distribution of m In-HAuNS and 3 H- DOX was compared by overlaying the two autoradiograms.
  • Hey tumors were generated by subcutaneous injection of Hey cells (5.0 x 10 6 cells/mouse). When the mean tumor volume reached -200 mm 3 , mice were divided into four groups consisting of 6-8 mice each.
  • mice were killed by CO 2 asphyxiation, and the tumors were collected and weighed. Parts of the tumors were fixed in formalin and cut into 5- ⁇ slices for hematoxylin and eosin staining. Body weight was measured weekly to assess systemic toxicity.
  • the targeting ligand cyclic peptide c(TNYL-RAW) is a second- generation EphB4-binding antagonist.
  • the peptide had an equilibrium dissociation constant (Ka) of 4.4 nM as determined by surface plasmon resonance sensorgram (FIG. 15 A).
  • Ka equilibrium dissociation constant
  • No degradation of ⁇ Cu-labeled c(TNYL-RAW) was observed by high-performance liquid chromatography after incubation of the peptide in mouse plasma over a period of 24 h, whereas ⁇ Cu-labeled linear TNYL-RAW was degraded as soon as 2 h after incubation (FIG. 15B).
  • c(TNYL-RAW) was linked to SATA-PEG-NHS through an activated ester.
  • SH-PEG-c(TNYL-RAW) was conjugated to HAuNS in an aqueous solution via S-Au bonding (FIG. 9).
  • the amount of c(TNYL-RAW) conjugated to the HAuNS was determined by quantitative amino acid analysis after complete dissolution of c(TNYL-RAW)-conjugated HAuNS.
  • the conjugation efficiency was 13.7% and there were about 880 molecules of c(TNYL-RAW) on each HAuNS nanoparticle.
  • DOX was readily loaded into c(TNYL-RAW)-conjugated HAuNS using a previously reported method to give T-DOX@HAuNS (You et al, 2010). DOX loading efficiency was over 90%, and DOX content was 30% (w/w).
  • FIG. 10A shows representative photomicrographs of fluorescence and dark-field images of Hey cells incubated with T-DOX@HAuNS.
  • the nanoparticles were readily taken up by the tumor cells.
  • the fluorescence signal from the DOX was colocalized with the signal from the HAuNS, indicating that the DOX remained associated with the HAuNS after T-DOX@HAuNS were internalized.
  • significantly more T-DOX@HAuNS was internalized in the cells with high EphB4 receptor expression (Hey) than in the cells with low EphB4 receptor expression (A549) (P ⁇ 0.05, FIG. 10B).
  • FIG. 1 1A shows the mean blood activity time profile of U 1 ln-labeled T-DOX@HAuNS and DOX@HAuNS.
  • the pharmacokinetic parameters are summarized in Table 2.
  • FIG. 1 IB shows the biodistribution of T-DOX@HAuNS, T-DOX@HAuNS with blocking by free c(TNYL-RAW), and DOX@HAuNS in nude mice at 24 h after injection. Most nanoparticles were taken up by the liver, spleen, and kidney.
  • C maximum blood concentration
  • AUCo- ⁇ area under the blood drug concentration-time curve extrapolated to infinite time
  • CL systemic clearance
  • Vd volume of distribution
  • Vss volume of distribution at steady-state
  • MRT mean residence time.
  • microSPECT/CT images showed significant blood activity in the liver and spleen for both U 1 ln-labeled T-DOX@HAuNS and DOX@HAuNS in nude mice bearing Hey tumors after intravenous injection of m In-labeled T-DOX@HAuNS, m In-labeled T- DOX@HAuNS plus free c(TNYL-RAW), and m In-labeled DOX@HAuNS.
  • TNYL-RAW free c
  • thermocouple In vivo antitumor activity.
  • the temperature measured by the thermocouple within the tumor reached ⁇ 53°C after 5 min of MR laser exposure on the tumor surface at an output power of 3 W/cm 2 in mice injected with T-DOX@HAuNS (FIG. 12). No change in temperature was noted under the same conditions in the tumors of control mice.
  • Dual-tracer autoradiography showed that immediately after NIR laser irradiation, 3 H- DOX was released and dispersed into the area surrounding the site where T-DOX@HAuNS was introduced. Conversely, 3 H-DOX was mostly colocalized with u l In-HAuNS in mice that did not undergo NIR laser treatment.
  • FIG. 13A shows the Hey tumor growth curves after intravenous injections of saline, HAuNS (5.0 mL/kg of 1.25 mg HAuNS/mL saline [50 OD], no DOX, no targeting), DOX@HAuNS (10 mg equivalent DOX/kg, 5.0 mL/kg of 1.25 mg HAuNS/mL), and T-DOX@HAuNS (10 mg equivalent DOX/kg, 5.0 mL/kg of 1.25 mg HAuNS/mL).
  • Mice in each group received NIR laser treatment (2.0 W/cm 2 for 3 min) 24 h after injection.
  • mice in the saline-plus-laser group were killed on day 9 after injection because most of the tumors in this group were -1500 mm 3 at that time. Mice in the other three groups were killed on day 22.
  • Treatment with T-DOX@HAuNS-plus-laser showed significantly enhanced antitumor activity compared with saline-plus-laser, HAuNS-plus-laser, and DOX@HAuNS- plus-laser.
  • the tumors in this group of mice became whitish immediately after treatment, suggesting disruption of blood perfusion.
  • the tumors in six of the eight mice treated with T-DOX@HAuNS-plus-laser regressed completely and became scar tissue by 22 days after the injection. Histological analysis showed the presence of scar tissue and a lack of residual tumor cells in mice treated with T-DOX@HAuNS-plus-laser (FIG. 13C). Tumors in the nontargeted DOX@HAuNS-plus-laser group became loose and discrete, suggesting damage to tumor cells. However, tumors could not be completely eradicated without targeting ligand. Tumors in the saline-plus-laser group appeared to be intact.
  • T-DOX@HAuNS had significantly greater cytotoxic effects in tumor cells with high EphB4 receptor expression than DOX@HAuNS and free DOX did in vitro. Moreover, T-DOX@HAuNS demonstrated enhanced antitumor activity when combined with NIR laser irradiation than DOX@HAuNS plus laser treatment in vivo. These findings indicate that c(TNYL-RAW) peptide is highly effective for selective delivery of T-DOX@HAuNS to tumors with high expression of EphB4.
  • T-DOX@HAuNS The significantly enhanced antitumor activity of T-DOX@HAuNS could be attributed to 1) increased accumulation of the nanoparticles in tumors, 2) controlled release of DOX mediated by NIR laser irradiation, and 3) synergistic interaction between chemotherapy and PTA therapy, both of which were activated concurrently by NIR laser.
  • c(TNYL-RAW) peptide with high EphB4 binding affinity and high plasma stability was conjugated to HAuNS through a PEG linker, which ensured availability of the peptide to the target receptor (FIG. 9). Additional monofunctional SH-PEG chains were introduced together with SH-PEG-c(TNYL-RAW) to ensure that all available gold surface was covered by PEG. This process was used to create DOX@HAuNS and ligand conjugated T-DOX@HAuNS, both of which had high colloidal stability.
  • DOX@HAuNS could be partially blocked by free c(TNYL-RAW) peptide, confirming that cell uptake of T-DOX@HAuNS was mediated by EphB4 (FIGS. 10 and 1 1).
  • T- DOX@HAuNS displayed significantly higher accumulation than nontargeted DOX@HAuNS in all three tumor models evaluated.
  • DOX@HAuNS and T-DOX@HAuNS exhibited similar pharmacokinetic behaviors (FIG. 1 1A, Table 2), the difference in tumor uptake between targeted and nontargeted HAuNS is unlikely a result of enhanced permeability and retention effect.
  • these data support successful EphB4 receptor- mediated targeted delivery of T-DOX@HAuNS after intravenous injection.
  • T-DOX@HAuNS The temperature in tumors of mice that received an intravenous injection of T-DOX@HAuNS reached ⁇ 53°C after 5 min of continuous -wave NIR laser exposure at 3 W/cm 2 (FIG. 12). This temperature is sufficient for causing irreversible damage to cancer cells (Melancon et ah, 201 1). As expected, there was no temperature change in the tumors of mice that did not receive the nanoparticle injection followed by NIR irradiation. Therefore, T-DOX@HAuNS medicated efficient photothermal effect. In aqueous solution, the release of DOX from DOX@HAuNS could be activated by NIR laser irradiation (You et ah, 2010).
  • DOX@HAuNS after a single dose at 60 mg equivalent DOX/kg had no cardiotoxicity compared to liposomal DOX (two doses at a total dose of 30 mg DOX/kg) and free DOX (single dose of 15 mg/kg).
  • 100% of both liposomal DOX- and free DOX -treated mice had a vacuolar cardiomyopathy.
  • the histopathologic features in the heart were similar to those observed in the saline-treated control mice and no abnormal features were observed.
  • EphA2 and EphB4 receptors are over-expressed in a variety of solid tumors, including ovarian, breast, colorectal, brain, and prostate. These receptors are also expressed in angiogenic blood vessels. Therefore, members of the Ephrin receptor family are attractive targets for cancer imaging and therapy (Pasquale, 2010).
  • EphA2 and EphB4 are attractive targets for cancer imaging and therapy (Pasquale, 2010).
  • peptidyl heterodimers with high receptor binding affinity to EphA2 and EphB4 suitable for molecular imaging of Ephrin receptors.
  • the heterodimers exemplified by YSA- TNYL-DOTA - 64 Cu had significantly higher target-to-background ratio than its corresponding monomeric imaging probes 64 Cu-DOTA-TNYL-RAW and 64 Cu-DOTA-YSA targeting EphB4 and EphA2, respectively.
  • imaging probes that simultaneously bind to both EphA2 and EphB4 can potentially increase early detection rates and be used to monitor response to therapy directed against EphA2 and EphB4.
  • a peptidyl heterodimer, YSAYPDSVPMMS(SEQ ID NO: 4)-PEG- TNYLFSPNGPIARAW(SEQ ID NO: 1) was synthesized by linking the two peptides YSAYPDSVPMMS (YSA; SEQ ID NO: 4) targeting EphA2 and TNYLF SPNGPIARA W (TNYL; SEQ ID NO: 1) targeting EphB4 together with a polyethylene glycol (PEG) linker.
  • YSA-TNYL was then labeled with the positron emitter ⁇ Cu through 1,4,7, 10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) chelator (FIG. 19).
  • the receptor-binding characteristics and tumor-targeting efficacy of heterodimer YSA-TNYL were evaluated in vitro with Surface Plasmon Resonance (SPR) sensor chip technology (Tables 3-5) and in vivo using ⁇ imaging.
  • SPR Surface Plasmon Resonance
  • the KD decreased from about 10 ⁇ 9 to about 10 "11 when both EphB4 and EphA2 receptors were coated on the sensor chip, indicating that the binding affinity of the heterodimer increased when both EphB4 and EphA2 receptors were coated on the sensor chip.
  • Binding of the YSA-TNYL heterodimer to EphB4 and EphA2 was blocked by their natural ligands.
  • Surface competition assays were performed with YSA- TNYL-RAW dimer and ephrinB2 with the surface coated with EphB4 using an increasing concentration of heterodimer and a constant concentration of ephrinB2, the natural ligand of EphB4 (FIG. 20A, highest concentration is the bottom line on the sensorgram; lowest is the top).
  • YSA-TNYL-RAW dimer and EphA2 were also performed with YSA-TNYL-RAW dimer and EphA2 with the surface coated with ephrinAl, the natural ligand of EphA2, using an increasing concentration of heterodimer and a constant concentration of EphA2 (FIG. 20B, highest concentration is the bottom line on the sensorgram; lowest is the top).
  • YSA-TNYL-DOTA- 64 Cu dimer showed high uptake in tumor cells over a period of 2-hr incubation (FIG. 21). The tumor uptake of YSA-TNYL-DOTA- 64 Cu could be partially blocked with an excess amount of cold TNYL, YSA, or mixture of TNYL-RAW and YSA peptides.
  • the inventors also performed ⁇ imaging of EphA2 and EphB4 receptors in ovarian cancer xenografts using 64 Cu-labeled bispecific YSA-TNYL-RAW heterodimer.
  • the dimeric radiotracer exhibited higher tumor-to-target ratio (FIG. 22) and improved imaging properties as compared to its corresponding monomeric radiotracers.
  • KD 2 which represents binding between the bispecific peptide with EphB4
  • EphA2 was two orders of magnitude lower when both EphB4 and EphA2 were coated onto the sensor chip than when only EphB4 was coated onto the sensor chip, suggesting that binding of EphA2 enhanced the binding of the specific heterodimer to EphB4.
  • Example 4 Dual Labeled Peptides for Targeting of EphB4 Receptors
  • a dual labeling approach by introducing both a radionuclide and a near-infrared dye to an EphB4-targeting peptide that allows for dual modal imaging of the receptors.
  • imaging probes can provide increased information content and are useful for both diagnostic imaging and guiding surgery intraoperatively.
  • TNYL peptide 64 Cu-DOTA and Cy5.5 dye were introduced to TNYL peptide to synthesize a dual-tracer imaging probe.
  • dual labeled TNYL displayed significantly higher binding to U251 glioma cells over-expressing EphB4 (FIG. 23) than to U87 cells that express low levels of EphB4.
  • the binding of dual labeled TNYL-RAW to U251 cells could be blocked by a large excess of unlabeled peptide.
  • a cyclic peptide with high receptor binding affinity for EphB4 with high in vivo stability suitable for molecular imaging of Epherin receptors and for targeted drug delivery was disclosed.
  • the purpose of this study was to design, synthesis, and evaluate peptidomimetics with enhanced in vivo stability and low background through conformation constrain.
  • the lactam formation of the side chain to side chain peptide cyclization (FIG. 25) was utilized to improve stability of peptides in the presence of peptidases and proteinases.
  • SPR surface plasma resonance
  • the lead cyclic peptide was conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) and labeled with 68 Ga.
  • NOTA 1,4,7-triazacyclononanetriacetic acid
  • Biodistribution, and small-animal PET/CT studies were performed in nude mice bearing EphB4-positive human melanoma.
  • the inventors identified candidate peptides with binding affinity ( 3 ⁇ 4) in the lower nanomolar range (4.4 nM; FIG. 26) with significantly improved stability in mouse plasma (FIG. 27) and reduced retention in the liver and the spleen (FIG. 28B).
  • ⁇ /CT studies demonstrated clear visualization of EphB4 expressing tumors (FIGS. 28A).
  • 68 Ga labeled cyclic(TNYL-RAW) peptide can be used as a PET/CT tracer to image tumor expression of EphB4, with a high tumor-to-background ratio.
  • DOX@HAuNS doxorubicin-loaded hollow gold nanospheres
  • the inventors demonstrated targeted delivery of nanoparticles conjugated to the cyclic(TNYL-RAW) speptide that targets EphB4, T- DOX@HAuNS. Increased uptake of targeted nanoparticles T-DOX@HAuNS was observed in three EphB4-positive tumors both in vitro and in vivo.
  • Treatment with T- DOX@HAuNS followed by near-infrared laser irradiation resulted in significantly decreased tumor growth when compared to treatments with non-targeted DOX@HAuNS plus laser or HAuNS plus laser.
  • the tumors in six of the eight mice treated with T-DOX@HAuNS plus laser regressed completely with only residual scar tissue by 22 days following injection, and none of the treatment groups experienced a loss in body weight.
  • the inventors have found a more than 3 -fold reduction in renal uptake of 68 Ga-labeled cyclic(TNYL-RAW) 68 Ga-NOTA-c(TNYL-RAW) when the radiotracer was co- injected with metformin (FIG. 29). These data indicate that metformin can be potentially used with radiolabeled compounds in radiotherapy and imaging to reduce rental toxicity.
  • EphB4 controls blood vascular morphogenesis during postnatal angiogenesis.
  • EphB receptor-binding peptides identified by phage display enable design of an antagonist with ephrin-like affinity. J. Biol. Chem., 280: 17301-1731 1, 2005.
  • EphB4 receptor suppresses breast cancer cell tumorigenicity through an
  • Receptor protein tyrosine kinase EphB4 is up-regulated in colon cancer.
  • EphB4 receptor tyrosine kinase is expressed in bladder cancer and provides signals for cell survival. Oncogene, 25:769-780, 2006.

Abstract

Peptide targeting agents are provided that may be used as imaging and/or therapeutic agents for tumor cells overexpressing Ephrin receptors. In particular, the agents can be used for targeting cells expressing EphB4 and/or EphA2 receptors. Methods for non-invasive imaging and anticancer therapy using the targeting agents are likewise provided.

Description

DESCRIPTION
EPHERIN RECEPTOR TARGETING AGENTS
BACKGROUND OF THE INVENTION
[0001] This application claims the benefit of United States Provisional Patent Application Nos. 61/586,282, filed January 13, 2012, and 61/666,474, filed June 29, 2012, the entirety of which are incorporated herein by reference.
[0002] The invention was made in part with government support under Grant No. RC2 GM092599 awarded by the National Institutes of Health. The government has certain rights in the invention. 1. Field of the Invention
[0003] The present invention relates generally to the field of molecular imaging and nanomedicine. More particularly, it concerns cancer targeting agents and methods for targeted imaging and therapy of cancer cells.
2. Description of Related Art
[0004] Over the past two decades, various radiolabeled peptides have been developed as nuclear imaging agents for tumor detection and noninvasive assessment of receptor expression in solid tumors. For example, small radiolabeled somatostatin peptidyl analogs with a molecular weight of -1.5 kDa have been successfully utilized in the clinic for localizing neuroendocrine tumors expressing somatostatin receptors (Bakker et ah, 1991 ; Hammond et ah, 1993). Cyclic Arg-Gly-Asp (RGD) peptide that strongly binds to integrin ανβ3 receptors is currently under clinical investigation (Haubner et ah, 2009). Several other peptide-based imaging agents, including melanocyte-stimulating hormone (MSH) analog (Chen et ah, 2000), substance P (Hargreaves, 2002), calcitonin (Blower et ah, 1998), atrial natriuretic peptide (ANP) (Liu et ah, 2010), bombesin/gastrin-releasing peptide (GRP), cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), and neuropeptide-Y (NPY), have also been identified and characterized for tumor receptor imaging (Schottelius and Wester, 2009). In spite of these efforts, peptides that can be used for the noninvasive detection of prostate, ovarian, melanoma, and colon cancer remain elusive (Schottelius and Wester, 2009). [0005] Many sold tumors, including prostate, ovarian, melanoma, and colon cancer, overexpress EphB4 receptor, a member of the Ephrin receptor tyrosine kinase family. Noninvasive imaging of EphB4 using peptide-based imaging agents could potentially increase early detection rates, monitor response to therapy directed again EphB4, and improve patient outcomes. Likewise, specific targeting of therapeutic agents to EphB4 expressing cancers could greatly improve the efficacy and specificity of cancer therapy.
SUMMARY OF THE INVENTION
[0006] In a first embodiment there is provided an Ephrin receptor targeting agent comprising at least a first peptide comprising the amino acid sequence TNYLFSPNGPIA (SEQ ID NO: 2) conjugated to a therapeutic agent or an imaging agent. In some aspects, the first peptide is defined as a peptide that binds specifically or preferentially to Eph receptor EphB4. For example, the peptide can comprise a sequence TNYLFSPNGPIARAW (SEQ ID NO: 1) or a sequence identical to SEQ ID NO: l, but comprising 1 or 2 amino acid substitutions, insertions or deletions. In still a further aspect, the first peptide is a cyclic peptide, such as a peptide comprising a lactam bridge (e.g., a cyclic peptide comprising the structure shown in FIG. 25).
[0007] In a further embodiment, a targeting agent of the embodiments further comprises a second peptide targeting agent. For example, the second peptide can bind to the same target as the first peptide or to a second target. In some aspects, the second peptide binds to an EphA2 receptor. Accordingly, in certain aspects, a targeting agent is provided that binds to EphA2 and EphB4 receptors. For instance, in some cases, the second peptide comprises an amino acid sequence of SEQ ID NO: 4. In certain aspects, a first and second peptide of the embodiments are comprised in a fusion protein. In still further aspects, the first and second peptides are connected by a linker. Examples of linkers include, without limitation, linker peptides, carbohydrate polymers and fatty acids. In some cases, the linker is a polyethylene glycol (PEG) moiety.
[0008] In further aspects of the embodiments, the first peptide of a targeting agent is conjugated to a radioisotope, a nanoparticle, a toxin, a chemotherapeutic agent, a fluorescent dye or a combination thereof. For example, the conjugate may be through covalent linkage or a non-covalent association, such as via chelator moiety (e.g., 1,4,7, 10-tetraazadodecane-N, N',N",N"'-tetraacetic acid (DOTA)). In certain aspects, the first peptide is conjugated to an imaging agent, such as a SPECT imaging agent, PET imaging agent an MRI contrast agent, or a fluorescent dye.
[0009] In still further aspects, the first peptide is conjugated to a radioisotope, such as a gamma emitter, a positron emitter or a beta-emitter. In certain aspects, the radioisotope is conjugated to the first peptide through a chelating moiety (e.g., DOTA). In other aspects, the radioisotope is conjugated to the first peptide as part of a molecule that comprises the radioisotope. Example, radioisotopes for use in accordance with the invention include, without limitation, astatine-21 1, chromium-51, cobalt-57, cobalt-58, copper-60, copper-61, copper-62, copper-64, copper-66, copper-67, Eu-152, gallium-67, gallium-68, indium-I l l, iron-59, lutetium-177m, rhenium-186, rhenium-188, selenium-75, strontium-89, technicium- 99m, thorium-227, and/or yttrium-90.
[0010] In still further aspects, a first peptide of the embodiments is conjugated to a therapeutic agent, such as chemotherapeutic agent, a radioisotope, or a therapeutic nanoparticle. For example, in some aspects, the chemotherapeutic agent is an anthracycline antibiotic, such as doxorubicin (Dox). Examples of therapeutic nanoparticles include, without limitation, nanoparticles that may be used to apply a photothermal ablation therapy, such as a hollow gold nanosphere (HAuNS). In some aspects, a nanoparticle of the embodiments is further loaded with or conjugated to a chemotherapeutic agent, such as Dox.
[0011] In a further embodiment a method of imaging a subject is provided comprising: (a) administering an effective amount of a targeting agent of the embodiments to the subject (e.g., a EphB4 and/or EphA2 targeting agent); and (b) imaging the subject to detect the presence of the targeting agent. For example, imaging the subject can comprise positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), or photoacoustic imaging. [0012] In still a further embodiment a method of treating a subject is provided comprising administering an effective amount of a targeting agent of the embodiments to the subject. In certain aspects, a method of the embodiment can further comprise administering one or more secondary therapy to the patient. For example, the secondary therapy can be a surgical therapy, chemotherapy, radiation therapy or hormonal therapy. In some aspects, the secondary therapy can facilitate the activity the targeting agent. For example, in the cases, a targeting agent that comprises a nanoparticle, such as a HAuNS, and a photothermal ablation therapy can be applied to heat the tissues surrounding the targeting agent. Likewise, in some cases, a nanoparticle can be loaded with a chemotherapy agent (e.g., Dox) such that heating of the nanoparticle activates local release of the chemotherapy agent.
[0013] In still a further embodiment a method for dual imaging and therapy of a subject is provided comprising administering an effective amount of a targeting agent of the embodiments to a subject (i.e., wherein the targeting agent comprises a therapeutic and imaging agent); and imaging the subject.
[0014] As used here a subject refers to an animal, such a human, canine or feline subject. In some aspects, the subject has or is suspected of a cancer, such as an EphB4- or EphA2-overexpressing cancer.
[0015] Thus, in some embodiments, there is provided an imaging probe for noninvasive imaging of EphB4 receptors comprising a heterodimer peptide, wherein said peptide has motifs which target both EphA2 and EphB4. For example, the heterodimer bispecific polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 or 2 and 4 (e.g., the amino acid sequence of SEQ ID NO: 1 and 4). Thus, in some aspects, the heterodimer, bispecific polypeptide comprises an EphA2-targeting motif and an EphB4- targeting motif separated by a linker, such as PEG linker. In some very specific aspects, the heterodimer polypeptide comprises from amino- to carboxy-terminus the amino acid sequence of SEQ ID NO: 1 ; a PEG linker; and the amino acid sequence of SEQ ID NO: 4. [0016] In a further embodiment a method of monitoring a cancer patient is provided comprising the steps of (i) injecting the patient with a heterodimer peptide of the embodiments; (ii) imaging the patient using a PET/CT scan imaging device wherein the device produces a PET image; (iii) acquiring the PET image of a tumor in the patient; (iv) determining tumor-to-muscle ratio in the patient; and (v) administering a cancer treatment to the patient based on the tumor-to-muscle ratio.
[0017] In still a further embodiment, a method is provided of identifying EphB4 receptor expression in a subject in need thereof comprising administering to the subject a therapeutic amount of the heterodimer peptide of the embodiments, and determining uptake or binding of the peptide in said subject. [0018] In another aspect, embodiments of the present invention contemplate the use of targeting nanoparticles in combination with physiological and physical approaches, such as tumor priming, vascular disruption, degradation of the extracellular matrix, and vessel normalization. [0019] In a still a further embodiment a method of reducing renal uptake of a radiolabeled agent in a subject is provided comprising administering the radio-labeled agent in conjunction with metformin. For example, the metformin can be administered before, after or essentially simultaneously with the radio-labeled agent. In some aspects, the radio-labeled agent is a radio-labeled therapeutic or imaging agent (e.g., an Eph Receptor targeting agent of the embodiments). Thus, in some aspects, there is provided a pharmaceutical composition comprising a radio-labeled therapeutic or imaging agent and metformin formulated together in a pharmaceutically acceptable carrier. Such a composition may be used for instance to reduce renal uptake of a radio-labeled agent in subject (e.g., a subject being treated or imaged with the agent). [0020] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.
[0021] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.
[0022] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. [0023] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0025] FIG. 1. Structure of 64Cu-DOTA-TNYL-RAW and natCu-DOTA -TNYL-RAW peptides.
[0026] FIG. 2. SPR sensorgrams of natCu-DOTA-TNYL-RAW and a scrambled peptide on sensor chips coated with EphB4. The peptides were injected as ten two-fold concentration series from 1.6 nM to 800 nM and were analyzed in duplicate binding cycles. Data sets (shown in black) are overlaid with curves fit to a 1 : 1 mass transfer interaction model (gray lines). The vertical axes in response units represent binding of each peptide to immobilized EphB4.
[0027] FIG. 3. Uptake of ^Cu-DOTA -TNYL-RAW in EphB4-expressing PC-3M and CT26 cells and EphB4-negative A549 cells. The cell-to-medium uptake ratio is expressed as [cprn^g protein in pellet]/[cpm^g medium]. ^Cu-DOTA-TNYL-RAW exhibited increased uptake over time in PC-3M and CT26 cells. This uptake was blocked by the parent TNYL-RAW peptide. FIG. 3A: CT26 cells. FIG. 3B: PC-3M cells. FIG. 3C: A549 cells. [0028] FIG. 4. Representative μΡΕΤ/CT images of mice bearing CT26, PC-3M, and
A549 tumors after intravenous administration of 64Cu-DOTA-TNYL-RAW (n = 4).
[0029] FIG. 5. Representative μΡΕΤ/CT images showing blocking of ^Cu-DOTA- TNYL-RAW uptake in CT26 tumors at 4 h and in PC-3M tumors at 24 h after radiotracer injection. For the blocking experiment, 64Cu-DOTA-TNYL-RAW was co-injected with cold TNYL-RAW (50 μg/mouse).
[0030] FIG. 6. FIG. 6A. Biodistribution of 64Cu-DOTA-TNYL-RAW in mice bearing PC-3M and A549 tumors at 24 h after injection. Data are presented as percent of injected dose per gram of tissue (%ID/g) and are expressed as mean ± standard deviation (n = 4). FIG. 6B. Tumor-to-muscle-uptake ratio. In the blocking group, mice were injected with 64Cu- DOTA-TNYL-RAW (200 μϋϊ) and cold TNYL-RAW peptide (20 μ^ηιοη8ε). Data are presented as mean ± standard deviation (n = 6). Data were obtained at 4 h after injection in mice with CT26 tumors and at 24 h after injection in mice with PC-3M tumors.
[0031] FIG. 7. FIG. 7A. The reaction scheme for the synthesis of natCu/64Cu-DOTA- TNYL-RAW. FIG. 7B. Mass spectrometric confirmation of the formation of the natCu(II)- labeled complex natCu-DOTA-TNYL-RAW. The m/z for [M+Cu]2+ = 1076.9957 (C96H143Cu 26027, calculated [M+Cu]2+: 1076.9917). FIG. 7C. Under the same HPLC conditions, natCu-DOTA-TNYL-RAW (UV detector) and 64Cu-DOTA-TNYL-RAW (radiodetector) had almost identical retention times (12.5 min and 12.6 min, respectively). [0032] FIG. 8. SPR sensorgrams of TNYL-RAW and DOTA-TNYL-RAW peptides on sensor chips coated with EphB4. The peptides were injected as ten two-fold concentration series from 1.6 nM to 800 nM and were analyzed in duplicate binding cycles. Data sets (shown in black) are overlaid with curves fit to a 1 : 1 mass transfer interaction model (gray lines). The vertical axes in response units represent binding of each peptide to immobilized EphB4.
[0033] FIG. 9. Reaction scheme for the synthesis of SH-PEG-c(TNYL-RAW) and its conjugation to HAuNS.
[0034] FIG. 10. Cellular uptake of T-DOX@HAuNS. FIG. 10A. Representative photomicrographs of Hey cells after incubation with T-DOX@HAuNS for 2 h. The scattering signal from the HAuNS was visualized using a dark-field condenser, and fluorescence was from DOX. Cell nuclei were counterstained with DAPI. Bar, 20 μιη. FIG. 10B. Quantitative cellular uptake of nanoparticles in Hey and A549 cells after 3 h incubation with mIn-labeled T-DOX@HAuNS and T-DOX@HAuNS plus free c(TNYL-RAW) (blocking). **P < 0.005;*P < 0.05. [0035] FIG. 11. T-DOX@HAuNS pharmacokinetics, biodistribution, and tumor uptake. FIG. 11 A. Activity-time profiles of mIn-labeled T-DOX@HAuNS and DOX@HAuNS in Swiss mice. The data are expressed as percentage of the injected dose per gram of blood (%ID/g) and are presented as mean ± standard deviation (n = 8). FIG. 1 IB. Biodistribution of U 1ln-labeled T-DOX@HAuNS, T-DOX@HAuNS with blocking, and DOX@HAuNS in nude mice at 24 h after injection. FIG. 11C. Comparison of nanoparticle uptake in Hey, A2780, and MDA-MB-231 tumors at 24 h postinjection. The data are expressed as percentage of the injected dose per gram of tissue (%ID/g) and are presented as mean ± standard deviation (n = 6). **P < 0.005; *P < 0.05.
[0036] FIG. 12. NIR-induced temperature change in tumors injected with HAuNS.
[0037] FIG. 13. Antitumor activity of various treatments against Hey tumors. FIG. 13 A. Tumor growth curves for mice treated with saline plus laser (n = 6), HAuNS plus laser (n = 6), DOX@HAuNS plus laser (n = 7), and T-DOX@HAuNS plus laser (n = 8). R laser irradiation (2.0 W/cm2 for 3 min) was commenced at 24 h after injection. FIG. 13B. Average tumor weights (left panel) and photographs (right panel) of tumors from different treatment groups. Tumors were removed on day 22 for all groups except the saline-plus-laser group, in which tumors were removed on day 9. Six tumors in the T-DOX@HAuNS group regressed completely by day 22 after treatment, and only the scar tissues at the site of tumor inoculation were removed and photographed (circle, right panel). FIG. 13C. Representative photomicrographs of hematoxylin and eosin-stained slides from scar tissue from a mouse treated with T-DOX@HAuNS-plus-laser or tumors from mice treated with saline or DOX@HAuNS-plus-laser on day 22 after treatment.
[0038] FIG. 14. Physical properties of HAuNS. FIG. 14A. Absorption spectrum of HAuNS. FIG. 14B. TEM image of HAuNS. FIG. 14C. High-resolution TEM of a single HAuNS.
[0039] FIG. 15. FIG. 15A. SPR sensorgrams of c(TNYL-RAW) peptide on sensor chips coated with EphB4. Peptides were injected as ten two-fold concentration series from 1.6 nM to 800 nM and were analyzed in duplicate binding cycles. Data sets (shown in black) are overlaid with curves fit to a 1 : 1 mass transfer interaction model (gray lines). The vertical axes in response units represent binding of each peptide to immobilized EphB4. c(TNYL- RAW) peptide bound to EphB4 with an association rate (kon) of 1.09 x 106 [M^S 1], a dissociation rate (k0fj) of 4.82 x 10"3 [s 1], and an equilibrium dissociation constant (¾ = kofjlkon) of 4.4 10"9 [M]. FIG. 15B. Comparison of peptide stability in mouse plasma. 64Cu- DOTA-TNYL-RAW or 64Cu-DOTA-c(TNYL-RAW) was incubated in mouse plasma at 37°C. A 100 μΐ, of plasma was removed from the incubation solution at 0, 2, 4, 8, 12, and 24 h time points and subjected to solid phase extraction on a CI 8 cartridge SPE column (Waters, Milford, MA). Then 20 μϊ^ of the extract was analyzed by reversed phase-high-performance liquid chromatography (RP-HPLC) on an Agilent 1100 system (C-18, Vydac, 4.6 x 250 mm, 10 μηι) equipped with a radiodetector. The system was eluted with a linear gradient of 10%- 90% acetonitrile in a 0.1% aqueous trifluoroacetic acid solution over 35 min at a flow rate of 1.0 mL/min.
[0040] FIG. 16. Western blot analysis of EphB4 expression in MDA-MB-231, A549, A2780, and Hey cell lines.
[0041] FIG. 17. Cell viability as a function of equivalent DOX concentration. A549 (FIG. 17A) and A2780 (FIG. 17B) cells were treated with T-DOX@HAuNS, DOX@HAuNS, or free DOX. The viability of cells was determined using MTT assay. Circles are T-DOX@HAuNS; Squares are DOX@HAuNA; Triangles are Free DOX. [0042] FIG. 18. Percentage of body weight change after various treatments. All data are presented as mean ± standard deviation. Diamonds are Saline + Laser; Squares are HAuNS + Laser; Triangles are DOX@HAuNS + Laser; X is T-DOX@HAuNS + Laser.
[0043] FIG. 19. FIG. 19A. Structure of heterodimer peptides with motifs targeting both EphA2 and EphB4 receptors. FIG. 19B. Characterization of heterodimer peptides by ESI-MS. FIG. 19C. Characterization of heterodimer peptides by HPLC.
[0044] FIG. 20. Surface competition assays. FIG. 20A. Surface competition assays were performed with YSA-TNYL-RAW dimer and ephrinB2 with the surface coated with EphB4 using an increasing concentration of heterodimer and a constant concentration of ephrinB2, the natural ligand of EphB4. FIG. 20B. Surface competition assays were also performed with YSA-TNYL-RAW dimer and EphA2 with the surface coated with ephrinAl, the natural ligand of EphA2, using an increasing concentration of heterodimer and a constant concentration of EphA2. The highest concentration corresponds to the bottom line on the sensorgrams while the lowest corresponds to the top line.
[0045] FIG. 21. Cell binding and inhibition in ovarian cancer Hey cells. FIG. 21 A. Cell binding of 64CU-DOTA-YSA-TNYL-RAW dimer over time. FIG. 2 IB. Cell binding at a single time point with various blocking agents. The bars, from left to right, are as follows: No blocking, Block w/ Dimer, Block w/ TNYL-RAW, Block w/ YSA.
[0046] FIG. 22. Biodistribution of 64Cu-labeled YSA-TNYL-RAW bispecific heterodimer targeting both EphA2 and EphB4 receptors in ovarian cancer xenografts. [0047] FIG. 23. Structure of the dual-labeled EphB4-binding peptide DOTA-TNYL-
Cy5.5.
[0048] FIG. 24. Immunohistochemistry of tumor sections confirming the binding of ^Cu-DOTA-TNYL-CyS.S to tumor cells and tumor microvessels. U251 and U87 tumors were stained and imaged for CD 31 and EphB4.
[0049] FIG. 25. Structure of EphB4-targeting cyclic peptide c(TNYL-RAW).
[0050] FIG. 26. Surface plasmon resonance curves for binding of c(TNYL-RAW) to
EphB4.
[0051] FIG. 27. Stability of peptides in mouse serum as assayed by LC-MS. Cyclic peptide c(TNYL-RAW) was more stable in mouse serum than its corresponding linear peptide. Top line is c(TNYL-RAW); bottom line is TNYL-RAW.
[0052] FIG. 28. FIG. 28A. μΡΕΤ/CT images of nude mice bearing subcutaneous human melanoma A375SM xenografts using 7.4 MBq (200 μϋϊ) of 68Ga-NOTA-c(TNYL- RAW) peptide. Reduced retention in the liver and the spleen, as well as minimal retention in the lungs and GI tract, were shown by μΡΕΤ/CT imaging. FIG. 28B. Uptake of 68Ga-NOTA- c(TNYL-RAW) in different organs. The peptide was labeled with 68Ga through radiometal chelator l,4,7-tetraazacyclododecane-N,N',N"-tetraacetic acid (NOTA). Data presented were obtained at 4 h postinjection by quantifying radioactivity in the region-of- interest from μΡΕΤ/CT images. Ml = mouse #1 ; M2 = mouse #2. [0053] FIG. 29. Biodistribution of 68Ga-NOTA-c(TNYL-RAW) injected with and without co-injection with metformin. The data presented were obtained from μΡΕΤ/CT imaged of melanoma A375SM xenografts. Images were acquired 1 h after intravenous injection of 68Ga-NOTA-c(TNYL-RAW) together with metformin at a dose of 40 mg metformin/kg. Metformin is a clinically used drug. Co-injection with metformin reduced kidney uptake while in the mean time increased tumor uptake of the radiotracer. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. The Present Invention
[0054] Provided herein are a new class of peptides for imaging cells and patients expressing EphB4 receptors. Radionuclide- and fluorescent dye-labeled peptides and a method of using the peptides in imaging EphB4 receptors and Ephrin family of receptors with the radionuclide and/or fluorescent dye labeled peptides are likewise provided. The disclosed peptides can be potentially used for early detection of cancer and for monitoring response to treatment directed at EphB4 receptors. The imaging properties in disease models include, but are not limited to, dosimetry and toxicity.
[0055] Many solid tumors overexpress EphB4 receptor, a member of the Ephrin receptor tyrosine kinase family. Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed again EphB4, and improve patient outcomes. A series of peptide-based imaging agents are disclosed with high receptor binding affinity for nuclear imaging of EphB4 receptors.
[0056] In the first generation imaging probes, the EphB4-binding peptide TNYLF SPNGPIARA W (TNYL-RAW) was conjugated with 1,4,7, 10-tetraazadodecane-N, N',N",N"'-tetraacetic acid (DOTA). DOTA-TNYL-RAW was labeled with ^Cu with high labeling efficiency. In vitro, 64Cu-DOTA-TNYL-RAW displayed high binding affinity to EphB4 (Kd- 2 nM), was selectively taken up by CT26 and PC-3M cells, but not by A549 cells. Binding of FITC-TNYL-RAW and ^Cu-DOTA-TNYL-RAW to CT26 and PC-3M cells could be blocked by an excess amount of TNYL-RAW. In vivo, 64Cu-DOTA-TNYL- RAW showed significantly higher uptake in PC-3M tumors than in A549 tumor, with percentages of injected dose per gram of tumor (%ID/g) values of 0.84±0.09 and 0.44±0.09 at 24 hr after radiotracer injection, respectively μΡΕΤ/CT imaging clearly revealed deposition of ^Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors. Furthermore, uptake of 64Cu-DOTA-TNYL-RAW in both CT26 and PC-3M tumors could be blocked by cold TNYL-RAW. Thus, it was demonstrated that the expression of EphB4 receptors can be noninvasively interrogated by μΡΕΤ/CT using 64Cu-DOTA-TNYLRAW.
[0057] In the second generation imaging probes, the stability and the pharmacokinetics of the 1st generation peptides were further improved, leading to imaging probes with improved imaging properties [0058] Both EphA2 and EphB4 receptors are over-expressed in a variety of solid tumors including cancers in the ovarian, breast, colorectal, brain, and prostate. These receptors are also expressed in angiogenic blood vessels. Therefore, members of the Ephrin receptor family are attractive targets for cancer imaging and therapy (Pasquale, Nat Rev. Cancer, 10: 165, 2010). A series of peptidyl heterodimers as disclosed here with high receptor binding affinity to EphA2 and EphB4 suitable for molecular imaging of Ephrin receptors. It is demonstrated that the heterodimers exemplified by YSA-TNYL-DOTA-64Cu had significantly higher target-tobackground ratio than its corresponding monomeric imaging probes 64Cu-DOTA-TNYL-RAW and 64Cu-DOTAYSA targeting EphB4 and EphA2, respectively. Thus, imaging probes that simultaneously bind to both EphA2 and EphB4 can potentially increase early detection rates and be used to monitor response to therapy directed against EphA2 and EphB4. Moreover, a dual labeling approach is provided by introducing both a radionucle and a near-infrared dye to EphB4-targeting peptide that allow dual modal imaging of the receptors. Such imaging probes can provide increased information content and are useful for both diagnostic imaging and guiding surgery intraoperatively.
[0059] A peptidyl heterodimer, YSAYPDSVPMMS(SEQ ID NO: 4)-PEG- TNYLF SPNGPIARA W (SEQ ID NO: 1) (YSA-TNYL), was synthesized by linking the two peptides YSAYPDSVPMMS (YSA; SEQ ID N: 4) targeting EphA2 and TNYLF SPNGPIARA W (TNYL; SEQ ID NO: 1) targeting EphB4 together with a polyethylene glycol (PEG) linker. YSA-TNYL was then labeled with the positron emitter ^Cu through 1,4,7, 10-tetraazacyclododecane-N, N', N", N"'-tetraacetic acid (DOTA) chelator. The receptor-binding characteristics and tumor-targeting efficacy of heterodimer YSA-TNYL was evaluated in vitro with Surface Plasmon Resonance (SPR) sensor chip technology and in vivo using μΡΕΤ imaging. YSA-TNYL-DOTA peptide had comparable binding affinity to EphB4 compared with TNYL peptide and higher binding affinity to EphA2 than YSA peptide.
[0060] In human ovarian tumor HEY xenografts, YSA-TNYL-DOTA-64Cu dimer showed significantly higher tumor uptake value compared with monomeric 64Cu-DOTA- TNYL and monomeric 64Cu-DOTA-YSA monomer analogs at all time points examined. The tumor uptake of YSA-TNYL-DOTA-64Cu could be partially blocked with an excess amount of cold TNYL, YSA, or mixture of TNYLRAW and YSA peptides. Compared with 64Cu- DOTATNYL and 64Cu-DOTA-YSA monomeric tracers, the heterodimer YSA-TNYL- DOTA- Cu also showed improved pharmacokinetics, resulting in a significantly higher target-to-background ratio. Thus, this class of radiotracers directed at both EphA2 and EphB4 should be useful imaging probes for early tumor detection and noninvasive characterization of Ephrin receptors. [0061] 64Cu-DOTA and Cy5.5 dye were introduced to TNYL peptide to synthesize dual-tracer imaging probe. In vitro, dual labeled TNYL displayed significantly higher binding to U251 glioma cells over-expressing EphB4 than to U87 cells that express low level of EphB4. The binding of dual labeled TNYL-RAW to U251 cells could be blocked by a large excess of unlabeled peptide. In vivo, μΡΕΤ/CT and near-infrared fluorescence optical imaging clearly showed the uptake of the dual labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of nude mice after intravenous injection. The specific uptake of dual labeled peptide in both tumors was confirmed by blocking experiments. In U87 tumors, Cy5.5-labeled peptide was found co-localized only with CD31- and EphB4-expressing tumor blood vessels. In U251 tumors, 64Cu-DOTA-TNYLCy5.5 was found to bind to both brain tumor cells and angiogenic blood vessels expressing EphB4 receptors.
[0062] Thus, in certain embodiments, this invention discloses new classes of peptide imaging agents suitable for noninvasive detection of tumor cells overexpressing EphB4 receptors (and in some cases EphA2 receptors). The disclosed peptides can be potentially used for early cancer detection and monitoring of treatment response. The concept can be potentially used for guiding cancer surgery under fluorescent imaging. The same concept can be applied to the detection of other receptors in the Ephrin receptor family.
[0063] In some aspects, this invention discloses a novel 64Cu-labeled peptide with high receptor binding affinity (i.e., low nanomolar ¾ values) for PET imaging of EphB4 receptors. The expression of EphB4 receptors can be noninvasively interrogated by μΡΕΤ/CT using the disclosed peptide, 64Cu-DOTA-TNYL-RAW.
[0064] Photothermal ablation (PTA) is an emerging technique that uses near-infrared laser light-generated heat to destroy tumor cells. However, complete tumor eradication by PTA therapy alone is difficult because heterogeneous heat distribution can lead to sub-lethal thermal dose in some areas of the tumor. Successful PTA therapy requires selective delivery of photothermal conducting nanoparticles to mediate effective PTA of tumor cells, and the ability to combine PTA with other therapy modalities. Multifunctional doxorubicin (DOX)- loaded hollow gold nanospheres (DOX@HAuNS) were synthesized that target EphB4, a member of the Eph family of receptor tyrosine kinases overexpressed on the cell membrane of multiple tumors and angiogenic blood vessels. The tumors in six of eight mice treated with T-DOX@HAuNS where T represents a cyclic peptide targeting EphB4 plus laser regressed completely with only residual scar tissue by 22 days following injection, and none of the treatment groups experienced a loss in body weight. Concerted chemo-photothermal therapy with a single nanodevice capable of mediating simultaneous PTA and local drug release may have promise as a new anticancer therapy.
[0065] In some other aspects, this invention discloses an optimized cyclic peptide with high stability and low background suitable for noninvasive detection of tumor cells overexpressing EphB4 receptors. The disclosed peptides can be potentially used for early cancer detection and monitoring of treatment response. The same peptide can also be used for targeted delivery nanoparticles and therapeutic agents. The cyclic peptide can be labeled with any positron emitter or gamma emitter for PET and SPECT imaging, respectively. The peptide can also be labeled with a fluorescent dye to guide surgery.
[0066] In some aspects, this invention discloses a method of nuclear imaging for reducing renal uptake of radiotracers. The method can be potentially used to reduce renal toxicity of radiolabeled compounds used in radionuclide therapy and imaging.
II. Ephrin receptors
[0067] The Eph receptors are the largest family of receptor tyrosine kinases
(Pasquale, 2005). Eph receptor tyrosine kinases and their ligands (ephrin) regulate a wide range of cell contact-dependent signaling that can effect cell proliferation, migration, morphology, adhesion, and invasion (Pitulescu et al. Genes & Dev., 24:2480-2492, 2010). This can occur through Eph signaling which alters the actin cytoskeleton organization and integrins and intercellular adhesion molecules processes. Eph-ephrin interactions are important for many biological roles including axon growth and maturation, cell positioning in the gastoinestinal tract, blood vessel morphogenesis and angiogenic sprouting, insulin secretion, bone remodeling, and immune function.
[0068] Eph receptors are divided into an EphA and an EphB class that bind to glycosylphosphatidylinositol-linked ephrin-A ligands and the transmembrane ephrin-B ligands, respectively. EphB4 receptors play important roles in a variety of biological processes, including cell aggregation and migration, neural development, embryogenesis and angiogenesis, and vascular development (Dodelet and Pasquale, 2000; Noren et al, 2004; Erber et al, 2006; Wang et al, 1998). EphB4 selectively binds to its endogenous ligand, ephrin-B2, to promote cell signaling required for cancer progression and angiogenesis and has been shown to be profoundly upregulated in numerous cancer types, such as prostate, colon, lung, gastric, bladder, ovarian, and breast cancers (Xia et al, 2006; Davalos et al, 2006; Xia et al, 2005; Takai et al, 2001; Kumar et al, 2009; Stephenson et al, 2001 ; Kumar et al, 2006). Overexpression of EphB4 in cancer cells is associated with tumorigenesis and angiogenesis by stimulating reverse signaling through ephrin-B2. EphB4 forward signaling, on the other hand, has been shown to inhibit cellular proliferation (Noren et al, 2004).
[0069] Eph receptors are typically divided into a globular ligand-binding domain, a cysteine-rich region, and two fibronectin type III repeats in the extracellular region, a short transmembrane region with several conserved tyrosine residues and the tyrosine kinase domain, a sterile a motif (SAM) protein-protein interaction domain, and a C-terminal PDZ- binding motif in the intracellular region. Eph receptors are divided into two classes, EphA and EphB. Likewise, Eph ligands (ephrins), which are also cell-surface associated proteins, divided into two classes, GPI-anchored ephrin-A and transmembrane ephrin-B. Ephrin-B molecules contain a cytoplasmic domain with several highly conserved tyrosine phosphorylation sites and a C-terminal PDZ motif. Generally, EphA receptors bind to ephrin- A, and EphB receptors bind to ephrin-B, but cross-signaling can occur. Eph and ephrins are capable of bi-directional signaling through trans interactions, though interactions in cis (i.e., between molecules expressed in the same cell) appear to inhibit receptor activation.
III. Targeting agents and Conjugates
Radioisotopes [0070] In certain aspects, a targeting peptide of the embodiments is conjugated to a radioisotope. In some aspects, the targeting agent comprises a chelating moiety and a radionuclide chelate. For example, the peptide can be chelated to a radionuclide, such as a technetium ion, a copper ion, an indium ion, a thallium ion, a gallium ion, an arsenic ion, a rhenium ion, a holmium ion, a yttrium ion, a samarium ion, a selenium ion, a strontium ion, a gadolinium ion, a bismuth ion, an iron ion, a manganese ion, a lutetium ion, a cobalt ion, a platinum ion, a calcium ion, and/or a rhodium ion. Examples of radionuclides include, but are not limited to, 99mTc, 188Re, 186Re, 153Sm, 166Ho, 90Y, 89Sr, 67Ga, 68Ga, mIn, 183Gd, 59Fe, 225 Ac, 212Bi, 211At, 45Ti, 177Lu, 60Cu, 61Cu, 67Cu, and 64Cu.
[0071] Chelating moieties for use according to the invention include, but are not limited to, a acyclic polyamioncarboxylate, a diposphine, a Schiff base, a bis(thiosemicarbazone), a cyclic polyamine, a cyclic polyaminocarboxylate, a cross-bridged cyclic polyamine, a cross-bridged cyclicpolyamioncarboxylate, a l,3,5-cis,cis- triamioncyclohexane derivative, a sarcophagine, or a sepulchrate. For example, the chelating moiety can be l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), 1,4,7,10- tetraazacyclododecane-l,4,7, 10-tetraacetic acid (DOTA), diethylenetriaminetetraacetic acid (DTTA), Diethylenetriaminopentaacetic acid (DTP A), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,8,1 1-tetraazacyclotetradecane- 1,4,8,1 1 -tetraacetic acid (TETA), 1,4,8,1 l-tetraazacyclotetradecane-l,8-diacetic acid (TE2A) Mercaptoacetyltriglycine (MAG3) or 4,5-bis(2-mercaptoacetamido)pentanoic acid, metformin, or phenformine. Chemotherapeutic agents
[0072] In some aspects, a targeting peptide of the embodiments is conjugated to or associated with a chemotherapeutic agent. Examples of chemotherapeutic agents of use as conjugates (or for loading in peptide-conjugated nanoparticles) include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, paclitaxel, docetaxel, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate; toll-like receptor agonists such as CpG; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
Nanoparticles
[0073] As used herein, the term "nanoparticle" (NP) refers to any particles having dimensions in the 1-1,000 nm range. In some embodiments, nanoparticles have dimensions in the 2-200 nm range, preferably in the 5-150 nm range, and even more preferably in the 10- 100 nm range. In certain aspects, the nanoparticles may be conjugated to a targeting peptide of the embodiments.
[0074] Example nanoparticles include for example polymeric micelles, polyethylene glycol, carbon nanotubes, gold nanostructures, such as gold nanoshells, gold nanorods, gold nanocages, and hollow gold nanospheres, as detailed herein, and copper sulfide nanoparticles (Haram et al, 1996; Xu et al, 2009; Huang et al, 2010). For example, CuS NPs have been synthesized and demonstrated for photothermal destruction of tumor cells in vitro using a NIR laser beam centered at 808 nm.
IV. Methods of Using Targeting Agents
[0075] Targeting agents of the embodiments may be used in an imaging or detection method for diagnosis or localization of tumor or angiogenic tissues. Any optical or nuclear imaging method may be contemplated, such as PET, SPECT, CT, or photoacoustic tomography. The integrated radioactive isotope in the nanoparticle may exert a radiotherapeutic effect on the tissue incorporating such nanoparticle. In addition, a photothermal ablation therapy may be administered to the tissue having the targeting agents to enhance the therapeutic effect.
[0076] Targeting agents may be used in PET. Positron emission tomography (PET) is a powerful and widely used diagnostic tool that has the advantages of high sensitivity (down to the picomolar level) and ability to provide quantitative imaging analyses of in vivo abnormalities (Scheinin et al, 1999; Eckelman, 2003; Welch et al, 2009). ^Cu (T1/2 = 12.7 h; β+, 0.653 MeV [17.8%]; β", 0.579 MeV [38.4%]) has decay characteristics that allow for both PET imaging and targeted radiotherapy for cancer (Shokeen and Anderson, 2009). It has been investigated as a promising radiotracer for real-time PET monitoring of regional drug concentration, pharmacokinetics, and dosimetry during radiotherapy (Shokeen and Anderson, 2009; Lu et ah, 2010). PET may be used in certain aspects to trace nanoparticles in vivo.
[0077] Certain targeting agents may also be used in SPET. Single photon emission computed tomography (SPECT, or less commonly, SPET) is a nuclear medicine tomographic imaging technique using gamma rays. It is very similar to conventional nuclear medicine planar imaging using a gamma camera. However, it is able to provide true 3D information. This information is typically presented as cross-sectional slices through the patient, but can be freely reformatted or manipulated as required. [0078] The SPET basic technique requires injection of a gamma-emitting radioisotope called radionuclide) into the bloodstream of the patient. In certain aspects, a radioisotope is conjugated to a targeting agent, which allow it to be concentrated in ways of medical interest for disease detection. In other aspects, a targeting agent comprising a marker radioisotope, which is of interest for its radioactive properties, has been attached to a targeting ligand, which is of interest for its chemical binding properties to certain types of tissues. This marriage allows the combination of ligand and radioisotope (the radiopharmaceutical) to be carried and bound to a place of interest in the body, which then (due to the gamma-emission of the isotope) allows the ligand concentration to be seen by a gamma-camera. [0079] Targeting agents may also be used in MRI. Magnetic resonance imaging
(MRI) is a medical imaging method routinely used in the clinic. Sometimes contrast materials such as intravenous DOTA-Gd contrast are used. This is useful to highlight structures such as blood vessels that otherwise would be difficult to delineate from their surroundings. Using contrast material can also help to obtain functional information about tissues. [0080] Certain targeting agents may also be used in photoacoustic tomography.
Photoacoustic tomography (PAT), or photoacoustic computed tomography (PACT), is a materials analysis technique based on the reconstruction of an internal photoacoustic source distribution from measurements acquired by scanning ultrasound detectors over a surface that encloses the source under study. The PA source is produced inside the object by the thermal expansion that results from a small temperature rise, which is caused by the absorption of externally applied radiation of pulsed electromagnetic (EM) waves. This technique has great potential for applications in the biomedical field because of the advantages of ultrasonic resolution in combination with EM absorption contrast. PAT is also called optoacoustic tomography (OAT).
[0081] In photoacoustic tomography (PAT), each temporal PA signal, measured at various detection positions, provides one-dimensional radial information about the PA source relative to the detector position; 2D surface scans offer other 2D lateral information about the PA source. Combining the temporal and spatial measurements affords sufficient information for a complete reconstruction of a 3D PA source. Because the PA signal received by each ultrasound detector is the integral of the ultrasound waves over the sensing aperture of the detector, the reconstruction algorithms depend on the detector apertures as well as the scanning geometries. Small-aperture detectors are often used to approximate point detectors, which receive PA signals originating from spherical shells, centered at each point detector, with radii determined by the acoustic times of flight. The three geometries commonly used are planar, cylindrical, and spherical surfaces. Both Fourier- and time-domain reconstruction formulas with point-detector measurements for these geometries have been well established. Besides, algorithms based on other detection methods, such as large-aperture (plane), line, or circle detectors have also been derived.
[0082] Targeting agents may also be used in photothermal ablation therapy. Photothermal ablation (PTA) therapy has gained increasing attention in recent years as a minimally invasive alternative to conventional approaches to cancer treatment such as surgery and chemotherapy (Amin et ah, 1993; Nolsoe et ah, 1993; Fiedler et ah, 2001 ; Vogeland Venugopalan, 2003). NPs with unique optical properties— primarily gold nanostructures, such as gold nanoshells (Hirsch et ah, 2003; Loo e ah, 2005), gold nanorods (Dickerson et ah, 2008; Park et ah, 2010), gold nanocages (Chen et ah, 2007; Au et ah, 2008), and hollow gold nanospheres (Lu et ah, 2010; Melancon et al„ 2008; (Lu et ah, 2009), but also carbon nanotubes (Chakravarty et ah, 2008 Burke et ah, 2009)— have been investigated as photothermal coupling agents to enhance the efficacy of PTA therapy. These plasmonic nanomaterials exhibit strong absorption in the near-infrared (MR) region (wavelength 700-1 100 nm) and offer an opportunity to convert optical energy to thermal energy, enabling deposition of otherwise benign optical energy into tumors for thermal ablation of tumor cells. V. Examples
[0083] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 - In vivo μΡΕΤ/CT Imaging of EphB4 Receptors using 64Cu-Labeled
Peptides
[0084] Many solid tumors overexpress EphB4 receptor, a member of the Ephrin receptor tyrosine kinase family. Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed again EphB4, and improve patient outcomes. Disclosed herein is a series of peptide-based imaging agents with high receptor binding affinity for nuclear imaging of EphB4 receptors. In the first generation imaging probes, the EphB4-binding peptide TN YLF SPNGPIARA W (TNYL-RAW; SEQ ID NO: 1) was conjugated with l,4,7, 10-tetraazadodecane-N,N',N",N" '-tetraacetic acid (DOTA). DOTA-TNYL-RAW was labeled with 64Cu with high labeling efficiency. In vitro, 64Cu- DOTA-TNYL-RAW displayed high binding affinity to EphB4 (Kd = 2 nM) and was selectively taken up by CT26 and PC-3M cells, but not by A549 cells. Binding of FITC- TNYL-RAW and 64Cu-DOTA-TNYL-RAW to CT26 and PC-3M cells was blocked by an excess amount of TNYL-RAW. In vivo, 64Cu-DOTA-TNYL-RAW showed significantly higher uptake in PC-3M tumors than in A549 tumors, with percentages of injected dose per gram of tumor (%ID/g) values of 0 84 ± 0.09 and 0.44 ± 0.09 at 24 hr after radiotracer injection, respectively. μΡΕΤ/CT imaging clearly revealed deposition of ^Cu-DOTA-TNYL- RAW in CT26 and PC-3M tumors but not in A549 tumors. Furthermore, uptake of 64Cu- DOTA-TNYL-RAW in both CT26 and PC-3M tumors could be blocked by cold TNYL- RAW. Thus, the expression of EphB4 receptors can be noninvasively interrogated by μΡΕΤ/CT using 64Cu-DOTA -TNYL-RAW. [0085] Pasquale and colleagues (Koolpe et ah, 2005) identified, using phage display technology, several 12-mer peptides that selectively bind to individual Eph receptors. Tyr- Asn-Tyr-Leu-Phe-Ser-Pro-Asn-Gly-Pro-Ile-Ala (TNYLFSPNGPIA; SEQ ID NO: 2), an EphB4 binding peptide from the initial screening, was further modified to include a RAW moiety at the carboxyl terminus. The resulting peptide, Tyr-Asn-Tyr-Leu-Phe-Ser-Pro-Asn- Gly-Pro-Ile-Ala-Arg-Ala-Trp (TNYLFSPNGPIARAW, designated as TNYL-RAW; SEQ ID NO: 1), was shown to be a potent antagonist of EphB4, with a fifty percent inhibition concentration (IC50) of ~15 nM for the binding of ephrin-B2 to murine EphB4 receptors.
[0086] The inventors investigated whether TNYL-RAW can be used as a receptor ligand for the noninvasive imaging of EphB4. The present disclosure shows that 64Cu-labeled TNYL-RAW is a promising radiotracer for PET imaging of EphB4 receptor expression in both human prostate and colon cancer xenograft models.
[0087] Materials and Methods
[0088] Materials. 64Cu was produced on a CS-15 biomedical cyclotron at Washington University School of Medicine (St. Louis, MO). Recombinant EphB4/Fc chimera, phycoerythrin (PE)-conjugated rat anti-human EphB4 monoclonal antibody, and rabbit anti-EphB4 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit antibody conjugated with near-infrared dye was purchased from Li- COR (Lincoln, NE). The protein assay kit was obtained from Bio-Rad (Hercules, CA). l,4,7,10-Tetraazadodecane-N,N',N",N"'-tetraacetic acid (DOTA) was obtained from Macrocyclics (Dallas, TX). The BIACore sensor chip CM5, amine coupling kit, HBSEP running buffer (0.01 M HEPES [4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid], pH 7.4, 0.15 M NaCl, 3 mM ethylenediaminetetraacetic acid [EDTA], and 0.005% [v/v] surfactant P20 solution), and regeneration buffer were purchased from BIACORE, Inc. (Piscataway, NJ). 4',6-Diamidino-2-phenylindole (DAPI) was obtained from Sigma-Aldrich (St. Louis, MO).
[0089] Radiolabeling of DOTA-TNYL-RAW. 64CuCl2 (74-148 MBq mCi]) in 0.1 M sodium acetate (pH 5.2) was added to 10 μg of DOTA-TNYL-RAW in water. The reaction mixture was incubated at 70°C for 1 h. The progress of the reaction was monitored by RP-HPLC with a radiodetector. The reaction was terminated with the addition of EDTA. The 64Cu-labeled peptide was further purified, if necessary, by reverse-phase high- performance liquid chromatography (RP-HPLC) on an Agilent 1 100 system (C-18, Vydac, 4.6 x 250 mm, 10 μιη) eluted with a linear gradient of 10%-90% acetonitrile in a 0.1% aqueous TFA solution over 35 min at a flow rate of 1.0 mL/min. 64Cu-DOTA-TNYL-RAW (retention time, tR = 12.5 min) was collected in 1- to 2-mL fractions. The solvent was then removed, reconstituted in saline, and passed through a 0.22-μιη filter for use in the animal experiments. Natural copper chloride ("^CuC^) was used to synthesize natCu-DOTA-TNYL- RAW under identical conditions, and its identity was confirmed by high-resolution electrospray ionization mass spectrometry (HRMS-ESI). 64Cu-DOTA-TNYL-RAW was co- injected with natCu-DOTA-TNYL-RAW into the above-mentioned HPLC system equipped with both ultraviolet and radiodetectors to confirm its identity.
[0090] Stability of radiolabeled ^Cu-D OT A-TNYL-RA W. 64Cu-DOTA-TNYL- RAW was incubated in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) or mouse serum at 37°C. Aliquots were removed at 1, 2, 4, 6, 12, and 24 h and analyzed by RP-HPLC with a radiodetector to assess their stability. [0091] Immobilization of EphB4 receptor to sensor chip. The stock solution (100 μg/mL) of EphB4/Fc in phosphate-buffered saline (PBS) was diluted to 25, 12.5, and 6.25 μg/mL with 10 mM sodium acetate buffer at pH 4.5 and immobilized to a CM5 sensor chip using the amine coupling reaction following manufacturer-provided procedures (BIACORE). Briefly, the surfaces of the chips in flow cells (FC)-l, -2, -3, and -4 were activated by exposing them to a mixture of 200 mM N-ethyl-N'-dimethylaminopropyl carbodiimide and 50 mM N-hydroxysuccinimide for 7 min. FC-1 was used as a reference surface and was directly deactivated by injecting 1 M ethanolamine at pH 8.5 for 7 min. The other three flow cells were injected with 25 μg/mL, 12.5 μg/mL, and 6.25 μg/mL EphB4/Fc, respectively, followed by injection of 1 M ethanolamine to block the remaining activated ester groups on the surface. The chip was allowed to stabilize for at least 2 h in HBSEP running buffer before injecting test analytes.
[0092] Surface plasmon resonance (SPR) assay of receptor binding affinity. Binding assays were performed at 25°C in HBSEP running buffer. The peptides were diluted in HBSEP buffer, filtered, degassed, and injected at concentrations between 1.6 nM and 800 nM at a flow rate of 30 μΕ/ηήη. The injection time of peptides into the HBSEP buffer was 4 min, followed by a 4-min dissociation period. The chips were regenerated using a 1-min pulse of 10 mM glycine (pH 2.2) after each binding circle. Each cycle consisted of a 1-min waiting period to allow monitoring of the baseline binding stability. For subtraction of bulk effects, caused by changes in the buffer composition or nonspecific binding, double-referencing was performed. Therefore, all analyzed samples were additionally injected onto an uncoated reference surface, including a sample of the running buffer, which was also tested on the EphB4/Fc coated flow cell. Data were evaluated with BIAevaluation software (version 3.0, BIACORE), applying a simple 1 : 1 binding mass transfer model. The obtained sensorgrams were fitted globally over the whole range of injected concentrations for both the association and dissociation phases. Equilibrium dissociation constants were then calculated from the rate constants (KD = koff/kon) (Cooper, 2002). [0093] Fluorescence microscopy. The CT26 murine colon cancer cell line and A549 human lung adenocarcinoma epithelial cell line were purchased from the American Type Culture Collection (Manassas, VA). The PC-3M human prostate cancer cell line was obtained from Professor Dominic Fan (MD Anderson Cancer Center, Houston, TX). PC-3M, CT26, or A549 cells were seeded (1 x 105/well) in Lab-Tek II chambered slides (Nalge Nunc International, Naperville, IL) supplemented with RPMI-1640 medium plus 10% FBS one day before the experiment. The cells were incubated with 100 μϊ^ of phenol-free RPMI-1640 culture medium containing 10 μΜ of FITC-TNYL-RAW or scrambled FITC-scTNYL-RAW (AGPFNTYLRTNAWSP; SEQ ID NO: 3) for 20 min at room temperature. For the blocking experiment, 10 μΜ of FITC-TNYL-RAW and 1 mM of TNYL-RAW were added to the cells. For detection of EphB4 expression, cells were incubated with 10 of PE-conjugated rat anti-human anti-EphB4 monoclonal antibody (R&D Systems, Minneapolis, MN) in 100 μϊ^ of phenol red-free RPMI-1640 medium. The cells were washed and fixed with 4% paraformaldehyde for 15 min at room temperature. The cell nuclei were counterstained with DAPI. After washing with PBS, the slides were mounted and visualized under a Zeiss Axiovert Z.1 fluorescent microscope (Zeiss, Jena, Germany).
[0094] Cell binding. For the cell binding study, CT26, PC-3M, and A549 cells were grown in 6-cm Petri dishes as described before (Bauer et al, 2005). The culture medium was replaced with 2 mL of RPMI 1640 without FBS containing ^Cu-DOTA-TNYL-RAW (-40 μΟί/ιηΕ, 100 nM), and cells were incubated at room temperature for 30, 60, or 120 min. For the blocking experiment, ^Cu-DOTA-TNYL-RAW peptide (-40 μΟ/mL, 100 nM) was co- incubated with TNYL-RAW (10 μΜ) under the same conditions. Thereafter, the monolayers were scraped and transferred into 5-mL tubes. The tubes were briefly vortexed and 100 μΐ^ of the cell suspension was transferred into a microcentrifuge tube containing 500 μΐ^ of a 75:25 mixture of silicon oil (density 1.05; Sigma-Aldrich) and mineral oil (density 0.872; Acros, Geel, Belgium). The mixture was centrifuged at 14,000 rpm for 5 min. After the tubes were frozen in liquid nitrogen, the bottom tips containing the cell pellet were cut off. The cell pellets and the supernatants were counted with Packard Cobra Quantum γ-counter (GMI, Ramsey, Minnesota). The protein content in a 100-μΙ, cell suspension was quantified using the Bio-Rad protein assay kit according to the manufacturer's protocol. The radioactivity in the cell pellets and media was counted, and the data were expressed as activity ratios of the cell pellet to the medium ([cpm^ig protein in pellet]/[cpm^g medium]). The experiments were performed in pentaplicate.
[0095] Animal models. All animal studies were performed under the guidelines and approval of the Institutional Animal Care and Use Committee. Athymic nude mice (4-6 weeks old, both sexes) were obtained from Harlan Laboratories (Charles River, Wilmington, Massachusetts). CT26, PC-3M, and A549 cells were grown in DMEM/F 12 supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere with 5% CO2. The cells were harvested by trypsinization. After centrifugation of cell suspension at 5000 rpm for 5 min, the culture medium was aspirated and the cell pellet was re-suspended in culture medium for subcutaneous injection in the right front leg of each mouse. When tumors were about 1 cm in diameter, micro-PET/CT (μΡΕΤ/CT) imaging and biodistribution analyses were performed.
[0096] uPET/CT imaging. Mice bearing PC-3M tumors (n = 4), CT26 tumors (n = 4; EphB4 positive), or A549 tumors (n = 4; EphB4 negative) were imaged with μΡΕΤ/CT at various times after intravenous injection of 200 μθϊ 64Cu-DOTA-TNYL-RAW (0.5-1 μg). For the blocking experiment, the tumor-bearing mice (n = 2) were co-injected with 200 μ& ^Cu-DOTA-TNYL-RAW and cold TNYL-RAW peptide at a dose of 50 μg/mouse. Images were acquired using an Inveon PET/CT system (Siemens, Knoxville, TN). The spatial resolution of the PET system is approximately 1.4 mm. Tumor-bearing mice were anesthetized with isoflurane (2% in oxygen) and placed in a prone position. The CT imaging parameters were as follows: X-ray voltage, 80 kVp; anode current, 500 mA; exposure time of each of the 360 rotational steps, 300-350 ms. Images were acquired at 1, 4, and 24 h after intravenous administration of 64Cu-DOTA-TNYL-RAW. Images were reconstructed using the two-dimensional ordered subsets expectation maximization algorithm. PET and CT image fusion and image analysis were performed using Inveon Research Workplace (Siemens Preclinical Solutions, Knoxville, TN). For tumor voxel intensity calculation, an irregular 3- dimensional region of interest (VOI) was manually drawn covering the whole tumor on CT and then copied to the corresponding PET images. A circular VOI (approximately 20 mm3) was drawn on the muscle of the legs. VOI was also drawn on a standard (radiotracer solution containing 1% of the injection dose) placed along with the animals. The mean activities within the VOI of the tumor and muscle were calculated in IRW workstation (Siemens).
[0097] Assessment of biodistribution by tissue sampling. In a separate experiment, athymic nude mice bearing PC-3M, CT26, and A549 xenografts (n = 4/group) were injected with 0.74 MBq (20 μθί, 50 ng) of 64Cu-DOTA-TNYL-RAW peptide to evaluate the biodistribution of the radiotracer. For the blocking experiment, the tumor-bearing mice (n = 2) were co-injected with 20 μθί 64Cu-DOTA-TNYL-RAW and 200-fold cold TNYL-RAW peptide. The animals were sacrificed at 4 and 24 h after injection. The organs of interest were excised and weighed and their radioactivity counted using an automatic gamma counter. (GMI, Ramsey, Minnesota). The stomach and intestines were not emptied prior to radioactivity measurements. The percentage of injected dose per gram of tissue (%ID/g) was calculated by dividing the %ID/organ by the weight of the organ. Values were expressed as mean ± standard deviation (SD).
[0098] Immunohistochemical analysis. After the μΡΕΤ/CT studies were completed, the mice were sacrificed and their tumors were excised, snap-frozen, and cut into 4-μιη sections. For immunohistochemical analysis, frozen tumor sections were fixed in 4% paraformaldehyde solution for 20 min at room temperature and washed with PBS three times. Tumor sections were blocked with 10% goat serum for 30 min at 37°C, then the slides were then incubated with rabbit anti-EphB4 antibody (U-200, 1 : 100 dilution) in PBS at 37°C overnight. After incubation with primary antibody, slides were washed with PBS three times and incubated with secondary goat anti-rabbit antibody conjugated with Alexa Flour 488 (1 :500 dilution; Invitrogen). Slides were washed again in PBS and counterstained with DAPI. Microphotographs were taken under a Zeiss Axiovert Z. l fluorescence microscope with the same conditions and displayed at the same scale to make sure that the relative brightness observed in the images reflected the difference in EphB4 expression level. [0099] Results
[00100] Chemistry, radiochemistry, and stability. The structure of natCu/64Cu-
DOTA-TNYL-RAW is shown in FIG. 1. The reaction scheme for the synthesis of natCu/64Cu- DOTA-TNYL-RAW is shown in FIG. 7A. RAW was eluted as a single peak with a retention time of 13.8 min, m/z = 1705.7764 for [M+H]+ (C80H117 22O20, calculated [M+H]+: 1705.8814). DOTA-TNYL-RAW was eluted as a single peak with a retention time of 12.6 min, m/z = 2092.0554 for [M+H]+ (C96H143N26O27, calculated [M+H]+: 2092.0616). FITC- TNYL-RAW was eluted as a single peak with a retention time of 15.6 min, m/z = 2094.9003 for [M+H]+ (CioiHm zsOzsS, calculated [M+H]+: 2094.9172). [00101] The formation of the natCu(II)-labeled complex natCu-D OTA-TNYL-
RAW was confirmed by mass spectrometry (FIG. 7B), with m/z = 1076.9957 for [M+Cu]2+ (C96H143CU 26O27, calculated [M+Cu]2+: 1076.9917). Under the same HPLC conditions, natCu-DOTA-TNYL-RAW (UV detector) and 64Cu-DOTA-TNYL-RAW (radiodetector) had almost identical retention times (12.5 min and 12.6 min, respectively) (FIG. 7C). The radiochemical purity, defined as the ratio of the main product peak to all peaks, was determined by HPLC to be >95%. The specific activity of 64Cu-DOTA-TNYL-RAW used in the in vitro and in vivo experiments was typically 7.4-14.8 MBq/nmol (0.2-0.4 Ci/μιηοι) at the end of synthesis. 64Cu-DOTA-TNYL-RAW was stable in DMEM culture medium containing 10% FBS for up to 24 h at 37°C. Approximately 30% of the ^Cu-DOTA-TNYL- RAW was degraded after 2 h of incubation in mouse serum at 37°C.
[00102] TNYL-RAW peptides bound to EphB4 receptors with nM affinity.
FIG. 2 shows representative sensorgrams obtained from SPR analyses of natCu-DOTA- TNYL-RAW and a scrambled peptide, with fitting curves obtained using a global 1 : 1 mass transfer model (gray lines). SPR sensorgrams of TNYL-RAW and DOTA-TNYL-RAW peptides are presented in FIG. 8. The corresponding binding kinetics and affinity data are summarized in Table 1. TNYL had an equilibrium dissociation constant (KD) of 3.06 nM. Conjugation of DOTA to the N-terminus of the peptide increased the KD value to 23.3 nM. Chelation of Cu2+ to DOTA-TNYL-RAW restored the binding affinity of the resulting metal complex, with a KD value of 1.98 nM. No binding to EphB4 was detected with a scrambled peptide (AGPFNTYLRTNAWSP; SEQ ID NO: 3). [00103] Table 1. Association (kon) and dissociation (k0ff) rate and equilibrium dissociation (¾) constants of peptides interacting with immobilized EphB4 receptors obtained from SPR analysis.
Figure imgf000029_0001
[00104] FITC-TNYL-RAW selectively bound to tumor cells overexpressing
EphB4 in vitro. Two EphB4-positive cell lines (PC-3M and CT26) and one EphB4-negative cell line (A549) were used for an in vitro binding study. Immunohistostaining with PE- conjugated rat anti-human EphB4 monoclonal antibody confirmed the expression of EphB4 on the surface of PC-3M, CT26, and A549 cells. PC-3M, CT26, and A549 cells were treated with FITC-TNYL-RAW (10 μΜ) or a scrambled peptide FITC-sc-TNYL-RAW (10 μΜ) for 20 min at room temperature. Cells were also stained with PE-conjugated anti-EphB4 antibody for the expression of EphB4 receptors. For the blocking experiment, FITC-TNYL- RAW (10 μΜ) was co-incubated with TNYL-RAW (1 mM). The cell nuclei were counterstained with DAPI. Stained cells were imaged by fluorescence microscopy. PC-3M and CT26 cells, but not A549 cells, were readily stained with FITC-TNYL-RAW. A FITC- labeled scrambled TNYL-RAW peptide (FITC-sc-TNYL-RAW) did not show detectable binding to PC-3M and CT26 cells. The binding of FITC-TNYL-RAW to PC-3M and CT26 cells was efficiently blocked by an excess amount of unlabeled TNYL-RAW peptide.
[00105] 64Cu-DOTA-TNYL-RAW peptide selectively binds to EphB4-positive ceUs. 64Cu-DOTA-TNYL-RAW had increased uptake with time in EphB4-positive PC-3M (FIG. 3B) and CT26 (FIG. 3 A) cell lines, but not in EphB4-negative A549 cells (FIG. 3C). Co-incubation with cold TNYL-RAW peptide completely abolished the binding of 64Cu- DOTA-TNYL-RAW to PC-3M at all time points tested (FIG. 3B) and reduced its binding to CT26 cells by ~10-fold (FIG. 3A). [00106] uPET/CT imaging. FIG. 4 shows μΡΕΤ/CT imaging of both coronal and transverse slices that contain tumor. Tumors were clearly visualized at 1 h and 4 h (CT26) and at 4 h and 24 h (PC-3M) after the 64Cu-DOTA-TNYL-RAW injection. In contrast, A549 tumors were barely discernible after radiotracer injection. The uptake of 64Cu-DOTA-TNYL-RAW in CT26 tumors was rapid, reaching 1.3 and 2.6 %ID/g at 1 h and 4 h postinjection, respectively. By 24 h after injection, the level of tumor radioactivity had declined to the body background level (FIG. 4). The uptake values of ^Cu-DOTA-TNYL- RAW in PC-3M tumors were 1.4, 3.2, and 3.6 %ID/g at 1, 4, and 24 h postinjection, respectively. The accumulation of 64Cu-DOTA-TNYL-RAW in A549 tumor was low at all time points examined, reaching a level of 1.7, 1.5, and 1.2 %ID/g at 1, 4, and 24 h after radiotracer administration. These values were only slightly higher than those recorded for muscle tissue in the same animals. Ex vivo immunohistochemical staining confirmed the expression of EphB4 throughout the CT26 and PC-3M tumors, whereas A549 tumors did not express EphB4 receptors.
[00107] FIG. 5 compares μΡΕΤ images obtained in the presence and absence of a large excess of cold TNYL-RAW at 4 h after radiotracer injection in a CT26 tumor-bearing mouse and at 24 h after radiotracer injection in a PC-3M tumor-bearing mouse. In CT26 and PC-3M tumor models, the co-administration of cold TNYL-RAW caused a 77% and 81% reduction in 64Cu-DOTA-TNYL-RAW accumulation in tumors, respectively.
[00108] Biodistribution of 64Cu-DOTA-TNYL-RAW assessed by tissue sampling. Biodistribution of 64Cu-DOTA-TNYL-RAW at 24 h after radiotracer injection in nude mice bearing PC-3M prostate and A549 lung cancer xenografts is summarized in FIG. 6A. There was significantly higher uptake of 64Cu-DOTA-TNYL-RAW in PC-3M than in A549 tumors (0.84 %ID/g vs. 0.44 %ID/g, SD = 0.09, 0.09 respectively, p = 0.000015). The liver, spleen, and kidney were the major organs with highest radiotracer retention (FIG. 6A). The tumor-to-muscle ratio was reduced 56.7% in CT26 tumors at 4 h postinjection and 47.6% in PC-3M tumors at 24 h postinjection when ^Cu-DOTA-TNYL-RAW was co-injected with cold TNYL-RAW peptide (FIG. 6B). The results of tissue sampling study corroborated with the pattern of 64Cu-DOTA -TNYL-RAW biodistribution determined by non-invasive in vivo PET/CT imaging.
[00109] Discussion
[00110] The ability to identify patients with significant receptor EphB4 receptor expression in tumors and/or to monitor changes in EphB4 expression during treatment using noninvasive imaging techniques would markedly enhance the selection and evaluation of patients treated with anticancer drugs directed at EphB4. With this aim, the inventors developed 64Cu-DOTA-TNYL-RAW, a novel PET radioligand for EphB4 receptor imaging, based on an EphB4 angonist peptide with binding to EphB4 receptors in the low nM range (Koolpe et al, 2005). [00111] SPR analysis showed that TNYL-RAW peptide had a dissociation constant (KD) of 3.09 nM, which is comparable to that reported in the literature (KD 1~2 nM) (Koolpe et al, 2005). TNYL-RAW also had a slow dissociation rate (-1.3 x 10"3 [s-1]), which is a better indicator than simple binding affinity for in vivo molecular imaging applications (Berezov et al, 2001). Previous studies showed that the N-terminal residues of the peptide could be modified without affecting the stability of the binding complex with EphB4 (Chrencik et al, 2006). However, when the radiometal chelator DOTA was introduced at the N-terminus of TNYL-RAW, an 8-fold reduction in KD was observed. Introduction of Cu2+ to DOTA-TNYL-RAW restored the binding affinity of TNYL-RAW (Table 1). Therefore, subtle structural change at the N-terminus of the peptide can still affect the binding of the peptide to EphB4 receptors. Because no interaction between a scrambled peptide and EphB4 was observed, the binding of TNYL-RAW to EphB4 is sequence-specific.
[00112] Most colon cancer and prostate cancer cell lines overexpress EphB4 receptors (Xia et al, 2005; Liu et al, 2002; Huang et al, 2007). Both the CT26 colon cancer and PC-3M prostate cancer cell lines used in these studies were positive for EphB4 expression. Taken together, the results of our binding and uptake studies (FIG. 3) indicate that ^Cu-DOTA-TNYL-RAW exhibits specific, high-affinity binding to EphB4 on the surface of cancer cells, with low nonspecific interaction. This makes 64Cu-DOTA-TNYL- RAW a suitable probe for noninvasive imaging of EPhB4 receptors.
[00113] In vivo μΡΕΤ/CT imaging studies revealed that ^Cu-DOTA-TNYL- RAW has very favorable tumor-homing characteristics in CT26 and PC-3M tumor xenografts in mice. In contrast, the radiotracer showed only minimal uptake in A549 tumors at all time points (FIG. 4). Ex vivo immunohistostaining of tumor xenografts confirmed that both CT26 and PC-3M tumors expressed high levels of EphB4, while A549 tumors had negligible EphB4 levels. In CT26 tumors, 64Cu-DOTA-TNYL-RAW was washed out of the tumor after 4 h, but it was significantly retained by PC-3M tumors for up to 24 h (FIG. 4). Given the fact that the peptide-EphB4 complex has a relatively slow off-rate (Kcff = 1.31 x 10"2 [S 1]), the level of EphB4 receptor expression should be the predominant factor influencing the magnitude of Cu-DOTA-TNYL-RAW accumulation and retention in tumor tissue. However, the difference in the degradation of 64Cu-DOTA-TNYL-RAW peptide-based radiotracer in different tumor microenvironments may also contribute to the difference in tumor retention of the radiotracer. [00114] The stability of radioactive compounds is critical because the molecular integrity of the radiopharmaceutical must be maintained for an adequate time in the blood circulation during biodistribution and imaging studies. ^Cu-DOTA-TNYL-RAW was stable in DMEM with 10% FBS for at least 24 h and was stable in mouse serum for up to 2 h, after which slow degradation was observed. Nevertheless, these μΡΕΤ/CT studies revealed that 64Cu-DOTA-TNYL-RAW possess sufficient in vivo stability for tumor imaging.
[00115] To establish the specificity of tumor uptake of 64Cu-DOTA-TNYL-
RAW in vivo, blocking studies were performed by co-injection of the radiotracer with a large excess of non-radioactive TNYL-RAW peptide. PET images revealed significant reductions in tumor uptake of ^Cu-DOTA-TNYL-RAW in both CT26 and PC-3M tumors in the presence of cold TNYL-RAW (FIG. 5). Quantitative analysis of radiotracer concentration showed that tumor-to-muscle ratios were reduced 56.7% in CT26 tumors at 4 h postinjection and 47.6% in PC-3M tumors at 24 h postinjection when 64Cu-DOTA-TNYL-RAW was co- injected with cold TNYL-RAW peptide (FIG. 6B). These results, together with the findings that EphB4-negative A549 tumors were hardly discernible in PET images acquired with ^Cu-DOTA-TNYL-RAW (FIG. 4) and that there was significantly higher uptake of 64Cu- DOTA-TNYL-RAW in EphB4-positive PC-3M tumors than in EphB4-negative A549 tumors (FIG. 6A), support the notion that the uptake of ^Cu-DOTA-TNYL-RAW in colon and prostate cancer xenografts in mice was specific and mediated by EphB4 receptors.
[00116] The 64Cu-DOTA-TNYL-RAW biodistribution data obtained by both non-invasive PET/CT imaging and by tissue sampling indicate that the liver and the kidney were the major organs for physiological uptake and clearance of this radiotracer. In general, hepatobiliary clearance is a major route for hydrophobic peptides (Rusckowski et ah, 2001). TNYL-RAW peptide contains several hydrophobic amino acids (Asn, He, Leu, Phe, Pro, Ala, Trp) that could contribute to the high liver uptake of 64Cu-DOTA-TNYL-RAW. The high level of accumulation of the radiotracer in the kidneys may be attributed to lysosomal degradation of the peptide within renal cells (Tsai et ah, 2001). Example 2 - Effective Photothermal Chemotherapy Using Doxorubicin-Loaded Gold Nanospheres That Target EphB4 Receptors in Tumors
[00117] Photothermal ablation (PTA) therapy is a recently developed technique that uses near-infrared (MR) laser light-generated heat to destroy tumor cells. PTA has gained popularity recently because a specific amount of photo-energy is delivered directly into the tumor mass without causing systemic effects, thus promising minimally invasive intervention as an alternative to surgery (Bardhan et al, 2011 ; Melancon et al, 2009). However, PTA therapy alone is unlikely to kill all tumor cells because the heat distribution is non-uniform, especially in areas peripheral to large blood vessels where heat can be rapidly dissipated by circulating blood. To improve the efficacy and tumor selectivity of laser- induced PTA, light-absorbing photothermal conducting nanoparticles are introduced. In principle, MR laser-modulated photothermal effects can not only enable PTA of tumor cells but also trigger release of anticancer agents. Such a multimodal approach, which permits simultaneous PTA therapy and chemotherapy, should provide an opportunity for complete eradication of tumor cells.
[00118] Hollow gold nanospheres (HAuNS) are a novel gold nanostructure ideally suited for PTA because of their unique combination of small size (30-50 nm), biocompatibility, and strong and tunable absorption in the entire NIR region (Melancon et al , 2008; Lu et al, 2009). Owing to their hollow interior, exceptionally high doxorubicin (DOX) payload to HAuNS (up to 60% by weight, 4-fold of the payload achievable with solid gold nanoparticles of the similar size) could be achieved with HAuNS (You et al, 2010). To maximize the therapeutic efficacy of DOX-loaded HAuNS (DOX@HAuNS), it is essential that DOX@HAuNS is selectively delivered to the tumor. EphB4 is a particularly promising target for tumor-specific delivery of DOX@HAuNS. [00119] Using c(TNYL-RAW) as a homing ligand, DOX@HAuNS were selectively targeted to EphB4-positive tumors, and concerted chemo-photothermal therapy mediated by EphB4-targeting DOX@HAuNS induced remarkable antitumor efficacy with reduced systemic toxicity. These results support the concept of integrating multiple functions into a single nanodevice to mediate simultaneous PTA and local drug release. [00120] Materials and Methods
[00121] Reagents. Indium-I l l chloride (1πΙη(¾, Iso-Tex Diagnostics
(Friendswood, TX). Methoxy-polyethylene glycol (PEG)-SH (molecular weight, 5,000) and NH2-PEG-COOH (molecular weight, 5,000) were purchased from Nektar (San Francisco, CA). Sodium citrate (>99%), cobalt chloride hexahydrate (99.99%), sodium borohydride (99%), and chloroauric acid trihydrate (American Chemical Society reagent grade) were purchased from Thermo Fisher Scientific (Waltham, MA) and were used as received. All amino acid derivatives and coupling reagents were purchased from EMD Chemicals (Philadelphia, PA), Bachem Americas (Torrance, CA), and Chem-Impex International (Wood Dale, IL). PL-DMA resin was obtained from EMD Chemicals. DOX, (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4',6-diamidino-2- phenylindole (DAPI), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Reagent-grade solvents were used without further purification unless otherwise specified. For the surface plasmon resonance (SPR) assay, the BIACore sensor chip CM5, amine-coupling kit, HBSEP running buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.005% [v/v] surfactant P20 solution), and regeneration buffer were purchased from GE Healthcare (Waukesha, WI). 3H-labeled DOX was purchased from Moravek Biochemicals Inc. (Brea, CA).
[00122] Synthesis of SH-PEG-c(TNYL-RAW). The heterofunctional PEG linker SATA-PEG-CONHS containing a protected sulfohydryl group (S-acetylthioacetate, SAT A) and NHS activated ester on each end of the PEG chain was first synthesized. Briefly, NH2-PEG-COOH (0.5 g, 0.25 mmol) and 1.2 equivalent N-succinimidyl-S-acetylthioacetate (SATA) were dissolved in 2 mL anhydrous DCM, and then 3 equivalents of dry DIPEA was added dropwise. The reaction solution was stirred at room temperature overnight. After all organic solvent was removed under a vacuum, the crude product was purified by gel filtration with a PD-10 column. The collected aqueous solution was lyophilized to yield SATA-PEG- COOH, which was further activated with l-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and NHS to make SATA-PEG-CONHS. c(TNYL-RAW) was synthesized on solid support. The peptide was conjugated to SATA-PEG5000-CONHS in a 1 : 1 molar ratio in DCM to obtain SATA-PEG-c(TNYL-RAW). The product was purified by PD-10 column and then treated with 0.5 M NH2OH to obtain SH-PEG-c(TNYL-RAW) before conjugation with HAuNS. [00123] Synthesis of DOX-loaded. c(TNYL-RAW)-coniugated HAuNS.
HAuNS were synthesized according to a previously reported method (You et ah, 2010). Briefly, cobalt nanoparticles were first synthesized by deoxygenating deionized water containing 4.5 mL of 1 M sodium borohydride, 2.8 mL of 0.1 M sodium citrate, and 1.0 mL of 0.4 M cobalt chloride. After chloroauric acid was added into the solution containing cobalt nanoparticles, the cobalt immediately reduced the gold ions onto the surface of the nanoparticles and was simultaneously oxidized to cobalt oxide. Any remaining cobalt was further oxidized by air, resulting in the final product, HAuNS. The size of the HAuNS was determined using dynamic light scattering on a Brookhaven 90Plus particle size analyzer (Holtsville, NY). The UV-visible spectra were recorded on a Beckman Coulter DU-800 UV- visible spectrometer (Brea, CA). The morphology of the HAuNS was examined using a JEM 1010 transmission electron microscope (JEOL USA, Peabody, MA).
[00124] To obtain c(TNYL-RAW)-conjugated HAuNS, SH-PEG-c(TNYL-
RAW) (50 nmol) was mixed with 1.0 mL HAuNS solution (200 optical density [OD], 5.0 mg/mL) and stirred overnight. Then, SH-PEG (3 μιηοΐ) was added to the solution. After another overnight stirring, c(TNYL-RAW)-conjugated HAuNS was purified on a PD-10 column.
[00125] For DOX loading, free DOX (5 mg) in water (5 mL) was added to c(TNYL-RAW)-HAuNS (40 OD) in 5 mL of 2.8 mM citrate solution, and the mixture was stirred at room temperature for 24 h. The resulting DOX-loaded c(TNYL-RAW)-HAuNS (T- DOX@HAuNS) were purified by 3 repeated centrifugation and washing steps.
[00126] Synthesis of cyclic peptide c(TNYL-RAW) (Lys-Thr-Asn-Tyr-Leu-
Phe-Ser-Pro-Asn-Gly-Pro-ILe-Ala-Arg-Ala-Trp-Asp). PL-DMA resin (1 g) was treated with ethylenediamine (30 mL) overnight and washed with dimethylformamide. 4-(4- Hydoxymethyl-3-methoxyphenoxy) acetic acid linker (3 equivalents) was attached to resin in the presence of diisopropylcarbodiimide (DIPCDI) (3 equiv.) and hydroxybenzotriazole (HOBt) (3 equiv.). Fluorenylmethyloxycarbonyl chloride (Fmoc)-Asp-Wang resin)OAll was formed by esterification with DIPCDI (3 equiv.) and 4-dimethylaminopyridine (DMAP) (0.5 equiv.). Fmoc-Lys(Boc)-Thr(tBu)-Asn(Trt)-Tyr(tBu)-Leu-Phe-Ser(tBu)-Pro-Asn(Trt)-Gly- Pro-ILe-Ala-Arg(Pbf)-Ala-Trp(Boc)-Asp(resin)OAll was synthesized using an Fmoc solid- phase strategy. Amino acids (3 equiv.) were coupled stepwise on the solid support in the presence of DIPCDI (3 equiv.) and HOBt (3 equiv.) coupling reagents. After removal of the allyl-protecting groups using Pdu[P(C6H5)3]4 (3 equiv.) in DCM/NMP/AcOH (dichloromethane/N-methylpyrrolidone/acetic acid) (37/1/2, v/v/v) and Fmoc -protecting groups using 20% piperidine in dimethylformamide (DMF), head-and-tail cyclization on the solid support was carried out in DMF using benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate (Py-BOP) (3 equiv.), HOBt (3 equiv.), and Ν,Ν-diisopropylethylamine (DIPEA) (6 equiv.) as coupling agents. Deprotection and cleavage of the conjugates from the solid support were carried out simultaneously by treatment with trifluoroacetic acid/water/triethylsilane (TFA/H20/TES, 95/1/4, v/v/v) to yield c(TNYL-RAW). The product was purified by high-performance liquid chromatography and validated by electrospray ionization mass spectrometry (m/z = 1931.0085 for [M+H]+[C9oHi32 25023, calculated [M+H]+ = 1930.9928]).
[00127] SPR assay of receptor binding affinity. EphB4/Fc (extracellular domain of human EphB4 fused to the carboxy -terminal Fc region of human IgGl was immobilized to CM5 sensor chips using the amine coupling reaction according to the manufacturer's instructions (GE Healthcare). Briefly, the surfaces of the chips in flow cells 1, 2, 3, and 4 were activated by exposing the chips to a mixture of 200 mM N-ethyl-N'- dimethylaminopropyl carbodiimide and 50 mM N-hydroxysuccinimide (NHS) for 7 min. Flow cell 1 was used as a reference surface and was directly deactivated by injecting 1 M ethanolamine at pH 8.5 for 7 min. The other three flow cells were injected with 25 μg/mL, 12.5 μg/mL, and 6.25 μg/mL EphB4/Fc per flow cell, respectively, followed by injection of 1 M ethanolamine to block the remaining activated ester groups on the surface. EphB4/Fc solutions of different concentrations (25, 12.5, and 6.25 μg/mL) were obtained by diluting a stock solution of EphB4/Fc (100 μg/mL) in phosphate-buffered saline (PBS) with 10 mM sodium acetate buffer (pH 4.5). The chip was allowed to stabilize for at least 2 h in HBSEP running buffer before injecting the test analytes.
[00128] Binding assays were performed at 25°C in HBSEP running buffer. c(TNYL-RAW) was diluted in HBSEP buffer, filtered, degassed, and injected at concentrations between 1.6 nM and 800 nM at a flow rate of 30 μΕ/ηιίη. Peptides were injected into the HBSEP buffer over 4 min, and injection was followed by a 4-min dissociation period. The chips were regenerated using a 1-min pulse of 10 mM glycine (pH 2.2) after each binding circle. Each cycle consisted of a 1-min waiting period to allow monitoring of the baseline binding stability. For subtraction of bulk effects caused by changes in the buffer composition or nonspecific binding, a double-referencing technique was employed. In this technique, all analyzed samples, including a sample of the running buffer, were additionally injected onto a reference cell capped with ethanolamine but without the EphB4 coating. Data were evaluated with BIAevaluation software (version 3.0, GE Healthcare), by applying a simple 1 : 1 binding mass transfer model. The resultant sensorgrams were fitted globally over the whole range of injected concentrations for both the association and dissociation phases. Equilibrium dissociation constants were then calculated from the experimental rate constants (¾ = k0ff/kon).
[00129] Radiolabeling of HAuNS with mIn. To conjugate a radiometal chelator to c(TNYL-RAW)-HAuNS, 4-aminobenzyl-diethylenetriaminepentaacetic acid thioctamide (DTPA-TA, 10 mg/mL; 5.0 μί) (Lu et al, 2007) was mixed with 1.0 mL of aqueous solution of HAuNS (200 OD/mL) for 6 h at room temperature. SH-PEG-c(TNYL- RAW) was then added to the DTPA-TA-conjugated HAuNS as described in the previous section. For radiolabeling with mIn, aliquots of DTPA-TA-conjugated c(TNYL-RAW)- HAuNS in 0.1 M sodium acetate solution (pH 5.4) were mixed with an aqueous solution of u lInCl3 (5 mCi) for 1 h. U 1ln-labeled c(TNYL-RAW)-HAuNS were purified by centrifugation at 10,000 rpm for 10 min and washed three times with PBS. The nanoparticles were then resuspended in sodium citrate solution (3.5 mM). Finally, mIn-labeled c(TNYL- RAW)-HAuNS were loaded with DOX to create 1 "in-labeled T-DOX@HAuNS. The radiolabeling efficiency and the stability of mIn-labeled T-DOX@HAuNS were analyzed using instant thin-layer chromatography. The paper strips were developed with PBS (pH 7.4) containing 4.0 mM EDTA, and the radioactivity was quantified using a Bioscan IAR-2000 thin-layer chromatography imaging scanner (Washington, DC). Free mIn3+ moved to the solvent front (Rf = 0.9-1.0), and the nanoparticles remained at the original spot (Rf = 0.0). The labeling efficiency was >95%.
[00130] Cell culture. MDA-MB-231 (human breast carcinoma), Hey (human ovarian carcinoma), and A549 (human lung adenocarcinoma) cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Life Technologies, Inc., Carlsbad, CA) at 37°C in a humidified atmosphere containing 5% CO2. A2780 human ovarian carcinoma cells were kindly provided by Dr. Stephen J. Williams (Fox Chase Cancer Center, Philadelphia, PA). These cells were maintained at 37°C in RPMI-1640 medium containing 10% fetal bovine serum and insulin (0.25 units/mL).
[00131] Cell uptake of HAuNS. Cells were transferred and cultured onto 20- mm glass cover slips in a 24-well plate and allowed to grow for 2 days. The medium was replaced with 1 mL of fresh culture medium containing T-DOX@HAuNS. After incubation for 2 h, cell nuclei were stained with DAPI. The cell monolayer on the cover slip was removed, repeatedly rinsed with PBS, and then mounted for microscopic examination. The cellular fluorescence and dark-field light scattering images were examined under a Zeiss Axio Observer Zl fluorescence microscope equipped with a dark- field condenser. [00132] Quantitative cellular uptake was determined using mIn-labeled nanoparticles. Hey and A549 cells (~1 x 106/dish) were cultured in 60 mm x 15 mm dishes. Cells were incubated with mIn-labeled T-DOX@HAuNS or mIn-labeled DOX@HAuNS (1.5 μθϊ/άϊβη) for 3 h at room temperature in culture medium lacking fetal bovine serum and phenol red. For the blocking experiments, cells were incubated with free c(TNYL-RAW) (1.0 μΜ) for 0.5 h and then with mIn-labeled T-DOX@HAuNS or mIn-labeled DOX@HAuNS under the same conditions. Cells were rinsed five times with PBS (pH 7.4). After the PBS was removed, the cells were scraped off the dish, suspended in PBS, and the radioactivity of the cell suspension was then measured with a gamma counter. Protein concentration in cell suspension was quantified using the Bio-Rad protein assay kit (Richmond, CA). The data are expressed as radioactivity (dpm per μg protein).
[00133] Analysis of EphB4 expression. For western blot analysis, cells were harvested at 70% confluence by adding CelLytic M cell lysis buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN), followed by centrifugation at 13,500 rpm. The supernatants were collected, and protein concentrations were measured with Coomassie protein assay reagent (Thermo Fisher Scientific). Equal amounts of protein from each experimental group were run on a 4-12% NuPAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (Invitrogen, Carlsbad, CA) and transferred to a nitrocellulose membrane. The EphB4 expression was probed with mouse anti-EphB4 antibody and Alexa Fluor 680-conjugated goat anti-mouse IgG (Invitrogen). β- Actin was used as a control to indicate the loading and transfer efficiency. Protein bands were visualized with a LI-COR Odyssey system (Lincoln, NE). [00134] A separate experiment was carried out to optically observe the binding between the antibody against EphB4 and the studied cell lines. Cells were cultured on 20-mm glass cover slips and incubated with primary antibody (mouse anti-EphB4 antibody) for 1 h at 4 °C. The cells were then washed with PBS three times and incubated with secondary antibody (Alexa Fluor 680-conjugated goat anti-mouse IgG). The cell-coated cover slip was then repeatedly rinsed with PBS and mounted for microscopic examination using an Axio Observer Zl fluorescence microscope (Carl Zeiss Microimaging GmbH, Germany).
[00135] Cytotoxicity. Cytotoxicity was measured using an MTT assay according to the manufacturer's suggested procedures. EphB4-positive A2780 cells and EphB4-negative A549 cells were exposed to free DOX, DOX@HAuNS, or T-DOX@- HAuNS for 72 h. The data are expressed as the percentage of surviving cells and are reported as the mean values of three measurements.
[00136] Pharmacokinetics and biodistribution. All animal studies were carried out under Institutional Animal Care and Use Committee-approved protocols. For the pharmacokinetic analysis, 8 healthy female Swiss mice (22-25 g; Charles River Laboratories, Wilmington, MA) for each group were each injected intravenously with a dose of either mIn- labeled T-DOX@-HAuNS or mIn-labeled DOX@HAuNS (both 20 μϋϊ, 1.0 mg/mL [40 OD] in 0.2 mL citrate buffered solution). At predetermined intervals up to 48 h, blood samples (10 μΚ) were taken from the tail vein, and the radioactivity of each sample was measured with a gamma counter. The percentage of the injected dose per gram of blood (%ID/g) was calculated. The blood pharmacokinetic parameters for the radiotracer were analyzed using a noncompartmental model with WinNonlin 5.0.1 software (Pharsight, Sunnyvale, CA).
[00137] To investigate the in vivo distribution of T-DOX@-HAuNS, female nude mice (Harlan, Indianapolis, IN) bearing 6-8 mm subcutaneous A2780, MDA-MB-231, or Hey tumors were intravenously injected with U 1ln-labeled T-DOX@-HAuNS or mIn- labeled DOX@HAuNS (20 μθί/πιοιιβε in 0.2 mL). For the blocking studies, the mice were intravenously injected with a mixture of luIn-labeled T-DOX@-HAuNS (20 μθϊ/πιοιιβε in 0.2 mL) and an excess of free c(TNYL-RAW) (0.3 μιηοΐ). Mice were killed at 24 h after injection. Various tissues, including tumors, were collected and weighted. The radioactivity for each sample was measured with a gamma counter. Uptake of nanoparticles in various tissues was calculated as %ID/g. [00138] Single photon emission computed tomography (SPECT) imaging.
Mice bearing subcutaneous Hey tumors were intravenously injected with mIn-labeled T- DOX@HAuNS or DOX@HAuNS (8.0 mCi/kg, 0.525 mg/mL [25 OD]). For the blocking studies, mice were injected intravenously with a mixture of mIn-labeled DOX@c(TNYL- RAW)-HAuNS and free c(TNYL-RAW) (0.3 μιηοΐ). The mice were placed in a prone position and anesthetized with 0.5%-2.0% isoflurane gas (Iso-Thesia, Rockville, NY) in oxygen. SPECT images were generated at 24 h after injection. After imaging, the mice were killed, and their tumors were removed. The tumors were snap-frozen and cut into 5-μιη slices that were then used for autoradiography analysis on a Fujifilm FLA-5100 imaging system (Stamford, CT).
[00139] NIR laser-triggered POX release in vivo. For measuring the photothermal effect of T-DOX@HAuNS, mice bearing Hey tumors were injected intravenously with saline (5.0 mL/kg) or T-DOX@-HAuNS (5.0 mL/kg of 50 OD HAuNS). At 24 h after injection, the tumor was irradiated with an NIR laser (3 W/cm2) for 5 min; Diomed 15 Plus, UK) through the skin surface. Temperature was measured with two thermocouples inserted into the tumor. Care was taken to ensure the thermocouple was not directly exposed to the laser beam.
[00140] The DOX release mediated by the photothermal effect in the Hey tumors was studied using mIn- and 3H-labeled T-DOX@-HAuNS, in which the HAuNS were labeled with U 1ln and the DOX with 3H. Tumors were irradiated by NIR laser light (3 W/cm2 for 5 min) at 1 h after intratumoral injection of the dual-labeled nanoparticles (3H: 10 μθί; mIn: 20 μθί) into the center of the tumor. The mice were killed 5 min after laser irradiation, and the tumors were removed, snap-frozen, and sliced into 10-μιη sections. Injected tumors that did not receive NIR laser treatment were similarly prepared and used as controls.
[00141] The radioactive signals from mIn and 3H were detected using a
Fujifilm FLA-5100 imaging system. Briefly, the sections of the tumors were exposed to phosphorous screen film (an SR imaging plate) for 15 min at -10°C, and the mIn autoradiograph was obtained by scanning the film. After the mIn was completely decayed (stored at -80°C for 5 weeks), the same sections were exposed to phosphorous screen film (a TR imaging plate) for 3 days at -10°C, and the 3H autoradiograph was obtained by scanning the film. Concurrently, control specimens from tumors that were not laser-treated were subjected to the same procedures. The autoradiographic distribution of mIn-HAuNS and 3H- DOX was compared by overlaying the two autoradiograms.
[00142] Antitumor activity in vivo. Hey tumors were generated by subcutaneous injection of Hey cells (5.0 x 106 cells/mouse). When the mean tumor volume reached -200 mm3, mice were divided into four groups consisting of 6-8 mice each. Mice in groups 1 through 4 were injected intravenously with saline (n = 6, 5.0 mL/kg), HAuNS (n = 6, 5.0 mL/kg of 1.25 mg HAuNS/mL solution), DOX@HAuNS (n = 7, 10 mg equivalent DOX/kg, 5.0 mL/kg of 1.25 mg HAuNS/mL solution), and T-DOX@HAuNS (n = 8, 10 mg equivalent DOX/kg, 5.0 mL/kg of 1.25 mg HAuNS/mL solution), respectively. All mice received MR laser irradiation through the skin surface at 24 h postinjection (2.0 W/cm2 for 3 min). The tumor dimensions were measured with a caliper, and the tumor volume was calculated according to the equation: volume = (tumor length) x (tumor width)2/2. At the end of the experiment (when the tumor size reached >1500 mm3 or 22 days after initial injection, whichever came first), mice were killed by CO2 asphyxiation, and the tumors were collected and weighed. Parts of the tumors were fixed in formalin and cut into 5-μιη slices for hematoxylin and eosin staining. Body weight was measured weekly to assess systemic toxicity.
[00143] Statistics. Pharmacokinetic data were analyzed using non- compartmental WinNonlin method. Statistical analysis was performed using the SYSTAT program. P values were obtained using the two-sample ?-test. For cellular uptake, biodistribution, and antitumor activity data, statistic analyses were performed by ANOVA, with P <0.05 considered to be statistically significant.
[00144] Results
[00145] Synthesis and characterization of EphB4-targeted T-DOX(¾HAuNS. Prior to carrying out efficacy studies, the inventors performed a number of experiments to fully characterize the targeted DOX@HAuNS system for in vivo delivery. The average diameter of the HAuNS was 36.8 ± 1.6 nm, as determined by dynamic light scattering. Transmission electron microscopy (TEM) confirmed that the HAuNS consisted of a thin gold shell (~4 nm thickness) with a hollow interior. The extinction spectrum showed that the plasma resonance peak for HAuNS was -800 nm (FIG. 14). [00146] The targeting ligand cyclic peptide c(TNYL-RAW) is a second- generation EphB4-binding antagonist. The peptide had an equilibrium dissociation constant (Ka) of 4.4 nM as determined by surface plasmon resonance sensorgram (FIG. 15 A). No degradation of ^Cu-labeled c(TNYL-RAW) was observed by high-performance liquid chromatography after incubation of the peptide in mouse plasma over a period of 24 h, whereas ^Cu-labeled linear TNYL-RAW was degraded as soon as 2 h after incubation (FIG. 15B). c(TNYL-RAW) was linked to SATA-PEG-NHS through an activated ester. After deprotection of the SH group, SH-PEG-c(TNYL-RAW) was conjugated to HAuNS in an aqueous solution via S-Au bonding (FIG. 9). The amount of c(TNYL-RAW) conjugated to the HAuNS was determined by quantitative amino acid analysis after complete dissolution of c(TNYL-RAW)-conjugated HAuNS. The conjugation efficiency was 13.7% and there were about 880 molecules of c(TNYL-RAW) on each HAuNS nanoparticle. DOX was readily loaded into c(TNYL-RAW)-conjugated HAuNS using a previously reported method to give T-DOX@HAuNS (You et al, 2010). DOX loading efficiency was over 90%, and DOX content was 30% (w/w).
[00147] In vitro uptake in cancer cells. Next, the inventors evaluated the selectivity of uptake of c(TNYL-RAW)-conjugated DOX@HAuNS in EphB4-positive tumor cells. Western blotting analysis indicated high expression levels of EphB4 receptor in MBA- MD-231, A2780, and Hey cells, but only low expression level in A549 cells (FIG. 16A). Immunostaining using anti-EphB4 antibody confirmed strong EphB4 signals from A2780, MDA-MB-231, and Hey cells but weak signal from A549 cells. On the basis of these findings, Hey cells were selected for subsequent efficacy studies.
[00148] FIG. 10A shows representative photomicrographs of fluorescence and dark-field images of Hey cells incubated with T-DOX@HAuNS. The nanoparticles were readily taken up by the tumor cells. The fluorescence signal from the DOX was colocalized with the signal from the HAuNS, indicating that the DOX remained associated with the HAuNS after T-DOX@HAuNS were internalized. Under the same conditions, significantly more T-DOX@HAuNS was internalized in the cells with high EphB4 receptor expression (Hey) than in the cells with low EphB4 receptor expression (A549) (P < 0.05, FIG. 10B). The addition of free c(TNYL-RAW) peptide into the culture medium (Hey cells) induced a significant decrease in the cellular uptake of the nanoparticles (P < 0.005) but did not cause a change in uptake in the A549 cells that had low EphB4 receptor expression (FIG. 10B). These data indicate that T-DOX@HAuNS was taken up by EphB4-positive cells via receptor-mediated endocytosis.
[00149] Cytotoxicity. For cells with low EphB4 expression (A549), the difference in cytotoxicity between T-DOX@HAuNS and non-targeted DOX@HAuNS was minimal, demonstrating the importance of EphB4-mediated uptake (FIG. 17 A). Free DOX exhibited higher toxicity in A549 cells than both T-DOX@HAuNS and DOX@HAuNS. The lower cytotoxic potency of T-DOX@HAuNS and DOX@HAuNS can be attributed to relatively stable complexes formed between DOX and HAuNS and delayed DOX release inside cells. However, significantly higher toxicity was shown for T-DOX@HAuNS in A2780 cells than either free DOX or DOX@HAuNS (FIG. 17B). The higher cellular cytotoxicity with targeted nanoparticles can be attributed to the increased uptake of T- DOX@HAuNS in the target cells.
[00150] Pharmacokinetics, biodistribution, and SPECT imaging. FIG. 1 1A shows the mean blood activity time profile of U 1ln-labeled T-DOX@HAuNS and DOX@HAuNS. The pharmacokinetic parameters are summarized in Table 2. DOX@HAuNS had moderately higher area under the blood drug concentration-time curve extrapolated to infinite time (AUC0-∞, 322.3 ± 72 %ID h/mL) than T-DOX@HAuNS (242.8 ± 46 %ID h/mL, P = 0.046). However, there was no significant difference between the two HAuNS formulations in any other pharmacokinetic parameters (P > 0.05), suggesting that the conjugation of c(TNYL-RAW) to HAuNS did not significantly change the pharmacokinetic properties of the nanoparticles. Both types of nanoparticles (T-DOX@HAuNS and DOX@HAuNS) were almost completely eliminated from the blood at 48 h after injection. FIG. 1 IB shows the biodistribution of T-DOX@HAuNS, T-DOX@HAuNS with blocking by free c(TNYL-RAW), and DOX@HAuNS in nude mice at 24 h after injection. Most nanoparticles were taken up by the liver, spleen, and kidney. Interestingly, significantly less T-DOX@HAuNS than DOX@HAuNS accumulated in the liver (P = 0.0009) and the spleen (P = 0.0006). Conversely, T-DOX@HAuNS had significantly higher uptake in the kidney than DOX@HAuNS did (P = 0.004). With the exception of kidney uptake, co-injection with an excess of c(TNYL-RAW) did not affect the biodistribution pattern of T-DOX@HAuNS. c(TNYL-RAW) blocking reduced the kidney uptake of T-DOX@HAuNS from 17.2 %ID/g to 12.9 %ID/g (P = 0.02). This finding may be reflective of EphB4 expression in venous endothelium of the kidney (Ozgur et ah, 201 1). Table 2. Pharmacokinetic parameters of DOX@HAuNS and T-DOX@HAuNS after intravenous injection in female Swiss mice.
Figure imgf000044_0001
Analysis of variance showed no differences between the two HAuNS formulations. C, maximum blood concentration; AUCo-∞, area under the blood drug concentration-time curve extrapolated to infinite time; CL, systemic clearance; Vd, volume of distribution; Vss, volume of distribution at steady-state; MRT, mean residence time.
[00151] To demonstrate efficiency of tumor targeting, the inventors carried out biodistribution studies in tumor-bearing mice. T-DOX@HAuNS displayed significantly higher accumulation than nontargeted DOX@HAuNS in tumors with high EphB4 receptor expression, with 3.0-, 2.1-, and 1.6-fold mean increases in Hey (P = 0.0016), A2780 (P = 0.0005), and MDA-MB-231 tumors (P = 0.019), respectively (FIG. 1 1C). Furthermore, the addition of free c(T YL-RAW) peptide significantly reduced the uptake of T- DOX@HAuNS in these tumors, with 2.3-, 1.5-, and 2.0-fold decreases, respectively (FIG. 11C). These results indicated that T-DOX@HAuNS was actively targeted to tumors expressing EphB4.
[00152] microSPECT/CT images showed significant blood activity in the liver and spleen for both U 1ln-labeled T-DOX@HAuNS and DOX@HAuNS in nude mice bearing Hey tumors after intravenous injection of mIn-labeled T-DOX@HAuNS, mIn-labeled T- DOX@HAuNS plus free c(TNYL-RAW), and mIn-labeled DOX@HAuNS. By 24 h after injection, accumulation of T-DOX@HAuNS in the tumor was clearly visualized. The uptake of T-DOX@HAuNS in the Hey tumors was blocked by an excess of free c(TNYL-RAW). Similarly, tumor uptake of then non-targeted DOX@HAuNS was barely visible. Autoradiographs of slices sectioned from all 3 tumors (Hey, A2780, and MDA-MB-231) 24 h after injection showed stronger mIn radioactivity signals with T-DOX@HAuNS than with T- DOX@HAuNS plus free c(TNYL-RAW) or with DOX@HAuNS.
[00153] In vivo antitumor activity. The temperature measured by the thermocouple within the tumor reached ~53°C after 5 min of MR laser exposure on the tumor surface at an output power of 3 W/cm2 in mice injected with T-DOX@HAuNS (FIG. 12). No change in temperature was noted under the same conditions in the tumors of control mice. Dual-tracer autoradiography showed that immediately after NIR laser irradiation, 3H- DOX was released and dispersed into the area surrounding the site where T-DOX@HAuNS was introduced. Conversely, 3H-DOX was mostly colocalized with u lIn-HAuNS in mice that did not undergo NIR laser treatment.
[00154] FIG. 13A shows the Hey tumor growth curves after intravenous injections of saline, HAuNS (5.0 mL/kg of 1.25 mg HAuNS/mL saline [50 OD], no DOX, no targeting), DOX@HAuNS (10 mg equivalent DOX/kg, 5.0 mL/kg of 1.25 mg HAuNS/mL), and T-DOX@HAuNS (10 mg equivalent DOX/kg, 5.0 mL/kg of 1.25 mg HAuNS/mL). Mice in each group received NIR laser treatment (2.0 W/cm2 for 3 min) 24 h after injection. The mice in the saline-plus-laser group were killed on day 9 after injection because most of the tumors in this group were -1500 mm3 at that time. Mice in the other three groups were killed on day 22. Treatment with T-DOX@HAuNS-plus-laser showed significantly enhanced antitumor activity compared with saline-plus-laser, HAuNS-plus-laser, and DOX@HAuNS- plus-laser. The mean tumor weight in T-DOX@HAuNS-plus-laser group on day 22 after treatment was 0.0038 ± 0.0007 g (n = 8), which was significantly smaller than that of the saline-plus-laser (1.2 ± 0.51 g on day 9, n = 6; P < 0.0001), HAuNS-plus-laser (1.5 ± 0.34 g on day 22, n = 6; P < 0.0001), and DOX@HAuNS-plus-laser (0.36 ± 0.24 g on day 22, n = 7; P < 0.001) groups (FIG. 13B). The tumors in this group of mice became whitish immediately after treatment, suggesting disruption of blood perfusion. Remarkably, the tumors in six of the eight mice treated with T-DOX@HAuNS-plus-laser regressed completely and became scar tissue by 22 days after the injection. Histological analysis showed the presence of scar tissue and a lack of residual tumor cells in mice treated with T-DOX@HAuNS-plus-laser (FIG. 13C). Tumors in the nontargeted DOX@HAuNS-plus-laser group became loose and discrete, suggesting damage to tumor cells. However, tumors could not be completely eradicated without targeting ligand. Tumors in the saline-plus-laser group appeared to be intact. In the saline-plus-laser and DOX@HAuNS-plus-laser treatment groups, viable tumor cells were found throughout the tumor volumes (FIG. 13C). No apparent signs of toxicity were observed throughout the course of the study. The body weight of mice in all treatment groups steady increased during the study period (FIG. 18).
[00155] Discussion [00156] In this study, the inventors showed that T-DOX@HAuNS had significantly greater cytotoxic effects in tumor cells with high EphB4 receptor expression than DOX@HAuNS and free DOX did in vitro. Moreover, T-DOX@HAuNS demonstrated enhanced antitumor activity when combined with NIR laser irradiation than DOX@HAuNS plus laser treatment in vivo. These findings indicate that c(TNYL-RAW) peptide is highly effective for selective delivery of T-DOX@HAuNS to tumors with high expression of EphB4. The significantly enhanced antitumor activity of T-DOX@HAuNS could be attributed to 1) increased accumulation of the nanoparticles in tumors, 2) controlled release of DOX mediated by NIR laser irradiation, and 3) synergistic interaction between chemotherapy and PTA therapy, both of which were activated concurrently by NIR laser. [00157] In our nanoconstruct, c(TNYL-RAW) peptide with high EphB4 binding affinity and high plasma stability was conjugated to HAuNS through a PEG linker, which ensured availability of the peptide to the target receptor (FIG. 9). Additional monofunctional SH-PEG chains were introduced together with SH-PEG-c(TNYL-RAW) to ensure that all available gold surface was covered by PEG. This process was used to create DOX@HAuNS and ligand conjugated T-DOX@HAuNS, both of which had high colloidal stability.
[00158] Both in vitro and in vivo data showed that tumor uptake of T-
DOX@HAuNS could be partially blocked by free c(TNYL-RAW) peptide, confirming that cell uptake of T-DOX@HAuNS was mediated by EphB4 (FIGS. 10 and 1 1). In vivo, T- DOX@HAuNS displayed significantly higher accumulation than nontargeted DOX@HAuNS in all three tumor models evaluated. Because DOX@HAuNS and T-DOX@HAuNS exhibited similar pharmacokinetic behaviors (FIG. 1 1A, Table 2), the difference in tumor uptake between targeted and nontargeted HAuNS is unlikely a result of enhanced permeability and retention effect. Hence, these data support successful EphB4 receptor- mediated targeted delivery of T-DOX@HAuNS after intravenous injection. [00159] The temperature in tumors of mice that received an intravenous injection of T-DOX@HAuNS reached ~53°C after 5 min of continuous -wave NIR laser exposure at 3 W/cm2 (FIG. 12). This temperature is sufficient for causing irreversible damage to cancer cells (Melancon et ah, 201 1). As expected, there was no temperature change in the tumors of mice that did not receive the nanoparticle injection followed by NIR irradiation. Therefore, T-DOX@HAuNS medicated efficient photothermal effect. In aqueous solution, the release of DOX from DOX@HAuNS could be activated by NIR laser irradiation (You et ah, 2010). To demonstrate DOX could be released in vivo upon NIR irradiation, we used an autoradiographic technique with dual-radiotracer labeling. 3H undergoes beta decay with a 12.3 year half-life, releasing 18.6 keV of energy in the process. On the other hand, mIn emits gamma radiation with a half-life of 2.81 days at much higher energy (171 KeV and 245 KeV). Taking advantage of these differences in the decay properties between 3H and mIn, the inventors were able to separate signals from HAuNS and DOX in the tumors. Thus, immediately after NIR laser irradiation at 3 W/cm2, 3H-DOX was dissociated from mIn- HAuNS, whereas in the control tumor not exposed to NIR laser irradiation, signals from 3H- DOX colocalized perfectly with i nIn-HAuNS (FIG. 13B). These data confirm that free DOX was released from T-DOX@HAuNS and then diffused away from the nanoparticles when tumors were exposed to the NIR laser.
[00160] The antitumor efficacy of free DOX, DOX@HAuNS, DOX@HAuNS- plus-NIR laser was investigated against MDA-MB-231 tumors in the inventors previous work (You et ah, 2012). There was no significant difference in tumor size between groups treated with DOX (15 mg/kg, one injection) and DOX@HAuNS (15 mg equivalent DOX/kg per injection, 2 injections). However, DOX@HAuNS-plus-NIR laser (15 mg equivalent DOX/kg per injection, 2 injections) reduced tumor volume significantly more than the other 2 treatments. In these earlier studies, free DOX at a single dose of 15 mg/kg caused serious systemic toxicity and cardiotoxicity. Therefore, free DOX was not used as a control in the current study. Instead, non-targeted DOX@HAuNS was used as a control and compared the antitumor studies of T-DOX@HAuNS against ovarian Hey tumors with that of DOX@HAuNS. Combined T-DOX@HAuNS-plus-laser treatments demonstrated significantly better antitumor activity than combined HAuNS-plus-laser (PTA only) and nontargeted DOX@HAuNS-plus-laser treatments (FIG. 13). [00161] Significantly, this higher antitumor activity was achieved without causing significant systemic toxicity, as indicated by continued increase in body weight in mice in all treatment groups (FIG. 18). The inventors previously compared the toxicity of free DOX, liposomal DOX, and DOX@HAuNS (You et al, 2012). DOX@HAuNS after a single dose at 60 mg equivalent DOX/kg had no cardiotoxicity compared to liposomal DOX (two doses at a total dose of 30 mg DOX/kg) and free DOX (single dose of 15 mg/kg). In the heart, 100% of both liposomal DOX- and free DOX -treated mice had a vacuolar cardiomyopathy. However, for mice treated with DOX@HAuNS, the histopathologic features in the heart were similar to those observed in the saline-treated control mice and no abnormal features were observed.
Example 3 - Heterodimeric Peptides for Bispecific Targeting of EphB4 and EphA2 Receptors
[00162] Both EphA2 and EphB4 receptors are over-expressed in a variety of solid tumors, including ovarian, breast, colorectal, brain, and prostate. These receptors are also expressed in angiogenic blood vessels. Therefore, members of the Ephrin receptor family are attractive targets for cancer imaging and therapy (Pasquale, 2010). Herein is disclosed a series of peptidyl heterodimers with high receptor binding affinity to EphA2 and EphB4 suitable for molecular imaging of Ephrin receptors. The heterodimers exemplified by YSA- TNYL-DOTA -64Cu had significantly higher target-to-background ratio than its corresponding monomeric imaging probes 64Cu-DOTA-TNYL-RAW and 64Cu-DOTA-YSA targeting EphB4 and EphA2, respectively. Thus, imaging probes that simultaneously bind to both EphA2 and EphB4 can potentially increase early detection rates and be used to monitor response to therapy directed against EphA2 and EphB4.
[00163] A peptidyl heterodimer, YSAYPDSVPMMS(SEQ ID NO: 4)-PEG- TNYLFSPNGPIARAW(SEQ ID NO: 1) (YSA-TNYL), was synthesized by linking the two peptides YSAYPDSVPMMS (YSA; SEQ ID NO: 4) targeting EphA2 and TNYLF SPNGPIARA W (TNYL; SEQ ID NO: 1) targeting EphB4 together with a polyethylene glycol (PEG) linker. YSA-TNYL was then labeled with the positron emitter ^Cu through 1,4,7, 10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) chelator (FIG. 19). The receptor-binding characteristics and tumor-targeting efficacy of heterodimer YSA-TNYL were evaluated in vitro with Surface Plasmon Resonance (SPR) sensor chip technology (Tables 3-5) and in vivo using μΡΕΤ imaging. YSA-TNYL-DOTA peptide had comparable binding affinity to EphB4 compared with TNYL peptide and higher binding affinity to EphA2 than YSA peptide. The KD decreased from about 10~9 to about 10"11 when both EphB4 and EphA2 receptors were coated on the sensor chip, indicating that the binding affinity of the heterodimer increased when both EphB4 and EphA2 receptors were coated on the sensor chip.
[00164] Binding of the YSA-TNYL heterodimer to EphB4 and EphA2 was blocked by their natural ligands. Surface competition assays were performed with YSA- TNYL-RAW dimer and ephrinB2 with the surface coated with EphB4 using an increasing concentration of heterodimer and a constant concentration of ephrinB2, the natural ligand of EphB4 (FIG. 20A, highest concentration is the bottom line on the sensorgram; lowest is the top). Surface competition assays were also performed with YSA-TNYL-RAW dimer and EphA2 with the surface coated with ephrinAl, the natural ligand of EphA2, using an increasing concentration of heterodimer and a constant concentration of EphA2 (FIG. 20B, highest concentration is the bottom line on the sensorgram; lowest is the top). [00165] In human ovarian tumor HEY cells in vitro, YSA-TNYL-DOTA-64Cu dimer showed high uptake in tumor cells over a period of 2-hr incubation (FIG. 21). The tumor uptake of YSA-TNYL-DOTA-64Cu could be partially blocked with an excess amount of cold TNYL, YSA, or mixture of TNYL-RAW and YSA peptides.
[00166] The inventors also performed μΡΕΤ imaging of EphA2 and EphB4 receptors in ovarian cancer xenografts using 64Cu-labeled bispecific YSA-TNYL-RAW heterodimer. The dimeric radiotracer exhibited higher tumor-to-target ratio (FIG. 22) and improved imaging properties as compared to its corresponding monomeric radiotracers.
[00167] Compared with ^Cu-DOTA-TNYL and 64Cu-DOTA-YSA monomeric tracers, the heterodimer YSA-TNYL-DOTA-64Cu also showed improved pharmacokinetics, resulting in a significantly higher target-to-background ratio. Thus, this class of radiotracers directed at both EphA2 and EphB4 should be useful as imaging probes for early tumor detection, monitoring treatment response to therapies directed at Ephrin receptors, and noninvasive characterization of Ephrin receptors. Unexpectedly, KD 2, which represents binding between the bispecific peptide with EphB4, was two orders of magnitude lower when both EphB4 and EphA2 were coated onto the sensor chip than when only EphB4 was coated onto the sensor chip, suggesting that binding of EphA2 enhanced the binding of the specific heterodimer to EphB4.
Table 3. Peptide binding with EphB4 coated to the sensor chip.
Figure imgf000050_0001
Ta le 5. Heterodimer binding to coated mixture of EphB4 and EphA2 (1 : 1).
Figure imgf000050_0002
Example 4 - Dual Labeled Peptides for Targeting of EphB4 Receptors [00168] Herein is disclosed a dual labeling approach by introducing both a radionuclide and a near-infrared dye to an EphB4-targeting peptide that allows for dual modal imaging of the receptors. Such imaging probes can provide increased information content and are useful for both diagnostic imaging and guiding surgery intraoperatively.
[00169] 64Cu-DOTA and Cy5.5 dye were introduced to TNYL peptide to synthesize a dual-tracer imaging probe. In vitro, dual labeled TNYL displayed significantly higher binding to U251 glioma cells over-expressing EphB4 (FIG. 23) than to U87 cells that express low levels of EphB4. The binding of dual labeled TNYL-RAW to U251 cells could be blocked by a large excess of unlabeled peptide. In vivo, μΡΕΤ/CT and near-infrared fluorescence optical imaging clearly showed the uptake of the dual labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of nude mice 1 h and 24 h after intravenous injection. The specific uptake of dual labeled peptide in both tumors was confirmed by blocking experiments. In U87 tumors, Cy5.5-labeled peptide was found co- localized only with CD31- and EphB4-expressing tumor blood vessels (FIG. 24). In U251 tumors, 64Cu-DOTA-TNYL-Cy5 5 was found to bind to both brain tumor cells and angiogenic blood vessels expressing EphB4 receptors. Example 5 - μΡΕΤ/CT Imaging of EphB4 Receptors in Melanoma Xenografts with 68Ga Labeled Cyclic TNYL-RAW Peptides
[00170] Herein is disclosed a cyclic peptide with high receptor binding affinity for EphB4 with high in vivo stability suitable for molecular imaging of Epherin receptors and for targeted drug delivery. The purpose of this study was to design, synthesis, and evaluate peptidomimetics with enhanced in vivo stability and low background through conformation constrain. The lactam formation of the side chain to side chain peptide cyclization (FIG. 25) was utilized to improve stability of peptides in the presence of peptidases and proteinases. Surface plasma resonance (SPR) sensor chip technology was used to screen the resulting peptides with regard to their receptor binding affinity. The lead cyclic peptide was conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) and labeled with 68Ga. Biodistribution, and small-animal PET/CT studies were performed in nude mice bearing EphB4-positive human melanoma. The inventors identified candidate peptides with binding affinity ( ¾) in the lower nanomolar range (4.4 nM; FIG. 26) with significantly improved stability in mouse plasma (FIG. 27) and reduced retention in the liver and the spleen (FIG. 28B). μΡΕΤ/CT studies demonstrated clear visualization of EphB4 expressing tumors (FIGS. 28A). 68Ga labeled cyclic(TNYL-RAW) peptide can be used as a PET/CT tracer to image tumor expression of EphB4, with a high tumor-to-background ratio. The improved metabolic stability, along with favorable pharmacokinetic profile and high receptor binding affinity, suggest that these peptides are promising candidates as novel therapeutic and imaging agents targeted to EphB4 receptors.
[00171] Using doxorubicin (DOX)-loaded hollow gold nanospheres (DOX@HAuNS) as an example, the inventors demonstrated targeted delivery of nanoparticles conjugated to the cyclic(TNYL-RAW) speptide that targets EphB4, T- DOX@HAuNS. Increased uptake of targeted nanoparticles T-DOX@HAuNS was observed in three EphB4-positive tumors both in vitro and in vivo. [00172] In vivo release of DOX from DOX@HAuNS, triggered by near- infrared laser, was confirmed by dual radiotracer technique. Treatment with T- DOX@HAuNS followed by near-infrared laser irradiation resulted in significantly decreased tumor growth when compared to treatments with non-targeted DOX@HAuNS plus laser or HAuNS plus laser. The tumors in six of the eight mice treated with T-DOX@HAuNS plus laser regressed completely with only residual scar tissue by 22 days following injection, and none of the treatment groups experienced a loss in body weight.
[00173] Together, our findings demonstrate that concerted chemo-photothermal therapy with a single nanodevice capable of mediating simultaneous PTA and local drug release may have promise as a new anticancer therapy.
Example 6 - μΡΕΤ/CT Imaging of EphB4 Receptors in Melanoma Xenografts with Co-injection of 68Ga-Labeled Cyclic TNYL-RAW Peptides and Metformin
[0001] Herein is disclosed a method of imaging EphB4 receptors by co-injection of 68Ga-labeled cyclic TNYL-RAW with metformin, an anti-diabetic drug in the biguanide class. Most peptide-based radionuclide therapy and imaging agents are cleared through the kidneys and reabsorbed and partially retained in the proximal tubules, causing dose-limiting nephrotoxicity. Various attempts have been made to reduce renal toxicity of radiolabeled compounds. Competitive inhibition of proximal tubular reabsorption can cause reduction in the renal uptake of the radiolabeled compounds. A maximum reduction of the kidney radiation dose of approximately 50% has been achieved by co-infusion with basic (positively charged amino acids).
[0002] The inventors have found a more than 3 -fold reduction in renal uptake of 68Ga-labeled cyclic(TNYL-RAW) 68Ga-NOTA-c(TNYL-RAW) when the radiotracer was co- injected with metformin (FIG. 29). These data indicate that metformin can be potentially used with radiolabeled compounds in radiotherapy and imaging to reduce rental toxicity.
* * *
[0003] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
Bakker et ah, In vivo application of [1 1 lln-DTPA-D-Phel] -octreotide for detection of somatostatin receptor-positive tumors in rats. Life Set, 49: 1593-1601, 1991.
Bardhan et ah, Theranostic nanoshells: from probe design to imaging and treatment of cancer. Acc. Chem. Res., 44:936-946, 2011.
Bauer et ah, 1311-labeled peptides as caspase substrates for apoptosis imaging. J. Nucl. Med., 46: 1066-1074, 2005.
Berezov et ah, Disabling ErbB receptors with rationally designed exocyclic mimetics of antibodies: structure-function Analysis. J. Med. Chem., 44:2565-2574, 2001.
Blower et ah, Iodine- 123 salmon calcitonin, an imaging agent for calcitonin receptors: synthesis, biodistribution, metabolism and dosimetry in humans. Eur. J. Nucl. Med.,
25: 101-108, 1998.
Chen et ah, Melanoma-targeting properties of 99mtechnetium-labeled cyclic alpha - melanocyte-stimulating hormone peptide analogues. Cancer Res., 60:5649-5658,
2000.
Chrencik et ah, Structure and thermodynamic characterization of the ephB4/ephrin-B2 antagonist peptide complex reveals the determinants for receptor specificity. Structure, 14:321-330, 2006.
Cooper, Optical biosensors in drug discovery. Nature Reviews Drug Discovery, 1 :515, 2002. Davalos et al, EPHB4 and survival of colorectal cancer patients. Cancer Res., 66:8943-8948, 2006.
Dodelet and Pasquale, Eph receptors and ephrin ligands: embryogenesis to tumorigenesis.
Oncogene, 19:5614-5619, 2000.
Erber et ah, EphB4 controls blood vascular morphogenesis during postnatal angiogenesis.
EMBO J., 25:628-641, 2006.
Hammond et ah, Amino acid infusion blocks renal tubular uptake of an indium-labelled somatostatin analogue. Br. J. Cancer, 67: 1437-1439, 1993. Hargreaves, Imaging substance P receptors (NKl) in the living human brain using positron emission tomography. J. Clin. Psychiatry., 63: 18-24, 2002.
Haubner and Decristoforo, Radiolabeled RGD peptides and peptidomimetics for tumour targeting. Front. Biosci., 14:872-886, 2009.
Huang et al, EphB4 overexpression in B16 melanoma cells affects arterial-venous patterning in tumor angiogenesis. Cancer Res., 67:9800-9808, 2007.
Koolpe et al, EphB receptor-binding peptides identified by phage display enable design of an antagonist with ephrin-like affinity. J. Biol. Chem., 280: 17301-1731 1, 2005.
Kumar et al, Receptor tyrosine kinase EphB4 is a survival factor in breast cancer. Am. J.
Pathol, 169:279-293, 2006.
Kumar et al. , Preferential induction of EphB4 over EphB2 and its implication in colorectal cancer progression. Cancer Res., 69:3736-3745, 2009.
Liu et al, Coexpression of ephrin-Bs and their receptors in colon carcinoma. Cancer, 94:934-
939, 2002.
Liu et al, Molecular imaging of atherosclerotic plaque with 64Cu-labeled natriuretic peptide and PET. J. Nucl. Med., 51 :85-91, 2010.
Lu et al, Tumor priming enhances delivery and efficacy of nanomedicines. J. Pharmacol.
Exp. Ther., 322:80-88, 2007.
Lu et al. , Targeted photothermal ablation of murine melanomas with melanocyte-stimulating hormone analog-conjugated hollow gold nanospheres. Clin. Cancer Res., 15:876-886,
2009.
Melancon et al. , In vitro and in vivo targeting of hollow gold nanoshells directed at epidermal growth factor receptor for photothermal ablation therapy. Mol. Cancer Ther., 7: 1730- 1739, 2008.
Melancon et al, Gold-based magneto/optical nanostructures: Challenges for in vivo applications in cancer diagnostics and therapy. Mater. Res. Bull, 34:415-421, 2009. Melancon et al, Cancer theranostics with near- infrared light-activatable multimodal nanoparticles. Acc. Chem. Res., 44:947-956, 201 1.
Melancon et al, Theranostics with multifunctional magnetic gold nanoshells: photothermal therapy and t2* magnetic resonance imaging. Invest. Radiol, 46: 132-140, 2011.
Noren et al. , Interplay between EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth. Proc. Natl. Acad. Sci. U. S. A., 101 :5583-5588, 2004.
Noren et al, The EphB4 receptor suppresses breast cancer cell tumorigenicity through an
Abl-Crk pathway. Nature Cell Biology, 8:815-825, 2006. Noren and Pasquale, Paradoxes of the EphB4 receptor in cancer. Cancer Res., 67:3994-3997, 2007.
Ozgur et al. , Distribution of EphB4 and EphrinB2 in normal and malignant urogenital tissue.
Urol. Oncol, 29:78-84, 2011.
Pasquale, Eph receptor signalling casts a wide net on cell behaviour. Nat. Rev. Mol. Cell Biol, 6:462-475, 2005.
Pasquale, Nat. Rev. Cancer, 10: 165, 2010.
Rusckowski et al, A comparison in monkeys of 99mTc labeled to a peptide by 4 methods. J.
Nucl. Med., 42: 1870-1877, 2001.
Schottelius and Wester, Molecular imaging targeting peptide receptors. Methods, 48: 161-177, 2009.
Stephenson et al, Receptor protein tyrosine kinase EphB4 is up-regulated in colon cancer.
BMC Molecular Biology, 2 : 15 , 2001.
Takai et al, Expression of receptor tyrosine kinase EphB4 and its ligand ephrin-B2 is associated with malignant potential in endometrial cancer. Oncol. Rep., 8:567-573,
2001.
Tsai et al, Metabolism and renal clearance of mIn-labeled DOTA-conjugated antibody fragments. Bioconjugate Chem, 12:264-270, 2001.
Wang et al. , Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell, 93 :741-753, 1998.
Xia et al, EphB4 expression and biological significance in prostate cancer. Cancer Res.,
65:4623-4632, 2005.
Xia et al, EphB4 receptor tyrosine kinase is expressed in bladder cancer and provides signals for cell survival. Oncogene, 25:769-780, 2006.
You et al, Exceptionally high payload of doxorubicin in hollow gold nanospheres for near- infrared light-triggered drug release. ACS Nano, 4: 1033-1041, 2010.
You et al, Photothermal-chemotherapy with doxorubicin-loaded hollow gold nanospheres: A platform for near-infrared light-trigged drug release. J. Control Release, 158:319-328, 2012.

Claims

I. An Ephrin receptor targeting agent comprising a first peptide comprising the amino acid sequence of SEQ ID NO: 1 or a cyclic peptide comprising the sequence of SEQ ID NO: 1 wherein the first peptide is conjugated to a therapeutic agent or an imaging agent. 2. The method of claim 1, wherein the first peptide is a cyclic peptide.
3. The targeting agent of claim 1, wherein the first peptide is conjugated to a radioisotope, a nanoparticle, a toxin, a chemotherapeutic agent, a fluorescent dye or a combination thereof.
4. The targeting agent of claim 1 , wherein the first peptide is conjugated to a therapeutic agent.
5. The targeting agent of claim 1, wherein the first peptide is conjugated to an imaging agent.
6. The targeting agent of claim 5, wherein the imaging agent is a SPECT imaging agent, PET imaging agent or an MRI contrast agent. 7. The targeting agent of claim 3, wherein the radioisotope is a gamma emitter, a positron emitter or a beta-emitter.
8. The targeting agent of claim 3, wherein the radioisotope is conjugated to the first peptide through a chelating moiety.
9. The targeting agent of claim 8, wherein the chelating moiety is DOTA or NOTA. 10. The targeting agent of claim 3, wherein the radioisotope is Cu-64, Cu-67, In- 1 11, Tc- 99m, Ga-67, Ga-68, Y-90 or Lu-177.
I I. The targeting agent of claim 3, wherein the chemotherapeutic agent is doxorubicin.
12. The targeting agent of claim 3, wherein the nanoparticle is a hollow gold nanosphere (HAuNS). 13. The targeting agent of claim 12, wherein the HAuNS is a doxorubicin-loaded nanosphere.
14. The targeting agent of claim 1, comprising a second peptide targeting agent.
15. The targeting agent of claim 14, wherein the second peptide targeting agent comprises an amino acid sequence of SEQ ID NO: 4.
16. The targeting agent of claim 14, wherein the first and second peptides are comprised as a fusion protein.
17. The targeting agent of claim 14, wherein the first and second peptides are connected by a linker.
18. The targeting agent of claim 17, wherein the linker polyethylene glycol (PEG).
19. An imaging probe for noninvasive imaging of EphB4 receptors comprising a heterodimer peptide, wherein said peptide has motifs which target both EphA2 and EphB4.
20. The imaging probe of claim 19, wherein the heterodimer polypeptide comprises the amino acid sequence of SEQ ID NO: 1 ; a cyclic peptide comprising the sequence of SEQ ID NO: 1; or the sequence of SEQ ID NO: 4.
21. The imaging probe of claim 20, wherein the heterodimer polypeptide comprises the amino acid sequence of SEQ ID NO: 1 and 4.
22. The imaging probe of claim 20, wherein the heterodimer polypeptide comprises a cyclic peptide comprising the sequence of SEQ ID NO: 1 and a peptide comprising the sequence of SEQ ID NO: 4.
23. The imaging probe of claim 19, wherein the heterodimer polypeptide comprises an EphA2 -targeting motif and an EphB4-targeting motif separated by a linker.
24. The imaging probe of claim 23, wherein the linker is a PEG linker.
25. The imaging probe of claim 21, wherein the heterodimer polypeptide comprises from amino- to carboxy-terminus the amino acid sequence of SEQ ID NO: 1 ; a PEG linker; and the amino acid sequence of SEQ ID NO: 4. 26. A method of imaging a subject comprising:
(a) administering an effective amount of a targeting agent of claim 5 to the subject; and (b) imaging the subject to detect the presence of the targeting agent.
27. The method of claim 26, wherein imaging the agent comprises performing the imaging comprises positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), fluorescence imaging or photoacoustic imaging.
28. The method of claim 26, wherein the subject has a cancer.
29. The method of claim 28, wherein the cancer is an EphB4- or EphA2-overexpressing cancer.
30. The method of claim 28, wherein the targeting agent is conjugated to a therapeutic and imaging agent, further defined as a method of dual imaging and therapy.
31. A method of treating a subject comprising administering an effective amount of a targeting agent of claim 4 to the subject.
32. The method of claim 31 , wherein the subject has a cancer.
33. The method of claim 31, wherein the cancer is an EphB4- or EphA2-overexpressing cancer.
34. The method of claim 31, wherein the targeting agent is conjugated to a therapeutic and imaging agent, further defined as a method of dual imaging and therapy.
35. The method of claim 31, wherein the targeting agent comprises a hollow gold nanosphere (HAuNS) conjugate. 36. The method of claim 36, wherein the HAuNS is a DOX-loaded HAuNS.
37. The method of claim 35, wherein the method further comprises administering a photothermal ablation therapy to the subject.
38. The method of claim 38, wherein the photothermal ablation therapy is administered by application of a photothermal ablation therapy. 39. A composition for use in the treatment or imaging of a subject comprising a targeting agent in accordance with claim 1.
40. A method of monitoring a cancer patient comprising the steps of:
injecting the patient with the peptide of claim 19;
imaging the patient using a PET/CT scan imaging device wherein the device produces a PET image;
acquiring the PET image of a tumor in the patient;
determining tumor-to-muscle ratio in the patient; and
administering a cancer treatment to the patient based on the tumor-to-muscle ratio.
41. A method of identifying EphB4 receptor expression in a subject in need thereof comprising administering to the subject a therapeutic amount of the heterodimer peptide of claim 19, and determining uptake or binding of the peptide in said subject.
42. A method of reducing renal uptake of a radio-labeled agent in a subject comprising administering the radio-labeled agent to the subject in conjunction with metformin.
43. The method of claim 42, wherein the radio-labeled agent is a therapeutic or imaging agent.
44. The method of claim 42, wherein the radio-labeled agent is a radio-labeled targeting agent in accordance with claim 1.
45. The method of claim 42, wherein the radio-labeled agent is administered before or essentially simultaneously with the metformin. 46. The method of claim 42, wherein the radio-labeled agent is administered after the metformin.
47. A pharmaceutical composition comprising a radio-labeled therapeutic or imaging agent and metformin formulated together in a pharmaceutically acceptable carrier.
48. The composition of claim 47, wherein the radio-labeled therapeutic or imaging agent is a radio-labeled targeting agent in accordance with claim 1.
49. A composition for use in reducing renal uptake of a radio-labeled agent, the composition comprising a radio-labeled therapeutic or imaging agent and metformin formulated together in a pharmaceutically acceptable carrier.
PCT/US2013/021434 2012-01-13 2013-01-14 Epherin receptor targeting agents WO2013106824A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201261586282P 2012-01-13 2012-01-13
US61/586,282 2012-01-13
US201261666474P 2012-06-29 2012-06-29
US61/666,474 2012-06-29

Publications (1)

Publication Number Publication Date
WO2013106824A1 true WO2013106824A1 (en) 2013-07-18

Family

ID=48781984

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/021434 WO2013106824A1 (en) 2012-01-13 2013-01-14 Epherin receptor targeting agents

Country Status (1)

Country Link
WO (1) WO2013106824A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105713075A (en) * 2014-12-04 2016-06-29 北京睿德欧生物科技有限公司 EphB4 acceptor targeting polypeptide and applications thereof
CN105906691A (en) * 2016-07-05 2016-08-31 珠海诺贝尔国际生物医药研究院有限公司 Eph kinase polypeptide inhibitor and application thereof
US9974774B2 (en) 2013-07-26 2018-05-22 Race Oncology Ltd. Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
CN113727737A (en) * 2019-01-17 2021-11-30 卡斯西部储备大学 Tumor extracellular matrix oncoprotein specific peptide PET/SPECT probe
CN114262362A (en) * 2021-10-14 2022-04-01 齐鲁工业大学 EphA2 receptor targeting68Ga-NODAGA-cyclic polypeptide FG01, preparation method and application
US11964948B2 (en) 2022-06-07 2024-04-23 Actinium Pharmaceuticals, Inc. Bifunctional chelators and conjugates

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020111300A1 (en) * 1995-03-21 2002-08-15 Thomas M. Behr Methods for reduced renal uptake of protein conjugates
US20050147612A1 (en) * 2001-06-20 2005-07-07 Avner Yayon Antibodies that block receptor protein tyrosine kinase activation, methods of screening for and uses thereof
WO2008122993A1 (en) * 2007-04-09 2008-10-16 Panacea Biotec Limited Controlled release formulation of coated microparticles
US7582438B2 (en) * 2005-01-27 2009-09-01 Burnham Institute For Medical Research EphB receptor-binding peptides
US20110091562A1 (en) * 2008-06-16 2011-04-21 Georgia Tech Research Corporation Nanogels for cellular delivery of therapeutics

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020111300A1 (en) * 1995-03-21 2002-08-15 Thomas M. Behr Methods for reduced renal uptake of protein conjugates
US20050147612A1 (en) * 2001-06-20 2005-07-07 Avner Yayon Antibodies that block receptor protein tyrosine kinase activation, methods of screening for and uses thereof
US7582438B2 (en) * 2005-01-27 2009-09-01 Burnham Institute For Medical Research EphB receptor-binding peptides
WO2008122993A1 (en) * 2007-04-09 2008-10-16 Panacea Biotec Limited Controlled release formulation of coated microparticles
US20110091562A1 (en) * 2008-06-16 2011-04-21 Georgia Tech Research Corporation Nanogels for cellular delivery of therapeutics

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10548876B2 (en) 2013-07-26 2020-02-04 Race Oncology Ltd. Compositions to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
US9974774B2 (en) 2013-07-26 2018-05-22 Race Oncology Ltd. Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
US9993460B2 (en) 2013-07-26 2018-06-12 Race Oncology Ltd. Compositions to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
US10500192B2 (en) 2013-07-26 2019-12-10 Race Oncology Ltd. Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
US11135201B2 (en) 2013-07-26 2021-10-05 Race Oncology Ltd. Compositions to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
US11147800B2 (en) 2013-07-26 2021-10-19 Race Oncology Ltd. Combinatorial methods to improve the therapeutic benefit of bisantrene and analogs and derivatives thereof
CN105713075A (en) * 2014-12-04 2016-06-29 北京睿德欧生物科技有限公司 EphB4 acceptor targeting polypeptide and applications thereof
CN105906691A (en) * 2016-07-05 2016-08-31 珠海诺贝尔国际生物医药研究院有限公司 Eph kinase polypeptide inhibitor and application thereof
CN105906691B (en) * 2016-07-05 2019-09-13 珠海诺贝尔国际生物医药研究院有限公司 A kind of Eph kinase polypeptide inhibitor and its application
CN113727737A (en) * 2019-01-17 2021-11-30 卡斯西部储备大学 Tumor extracellular matrix oncoprotein specific peptide PET/SPECT probe
CN114262362A (en) * 2021-10-14 2022-04-01 齐鲁工业大学 EphA2 receptor targeting68Ga-NODAGA-cyclic polypeptide FG01, preparation method and application
CN114262362B (en) * 2021-10-14 2023-05-26 齐鲁工业大学 EphA2 receptor targeting 68 Ga-NODAGA-cyclic polypeptide FG01, preparation method and application
US11964948B2 (en) 2022-06-07 2024-04-23 Actinium Pharmaceuticals, Inc. Bifunctional chelators and conjugates

Similar Documents

Publication Publication Date Title
Psimadas et al. Molecular nanomedicine towards cancer: 111In-labeled nanoparticles
Muller Folate based radiopharmaceuticals for imaging and therapy of cancer and inflammation
KR100896983B1 (en) Peptide-Based Compounds
US20150258217A1 (en) Methods of Synthesizing and Using Peg-Like Fluorochromes
Pooja et al. Bombesin receptors as potential targets for anticancer drug delivery and imaging
Nanda et al. Bombesin analogues for gastrin-releasing peptide receptor imaging
WO2013106824A1 (en) Epherin receptor targeting agents
KR20020087973A (en) Peptide-Based Compounds
WO2009107139A1 (en) Rgd-(bacterio)chlorophyll conjugates for photodynamic therapy and imaging of necrotic tumors
US20210330819A1 (en) Design and development of neurokinin-1 receptor-binding agent delivery conjugates
CA3022354A1 (en) Methods and compositions for theranostic nanoparticles
Zhang et al. Targeted radiotherapy with tumor vascular homing trimeric GEBP11 peptide evaluated by multimodality imaging for gastric cancer
Gu et al. Folate-PEG-CKK 2-DTPA, A potential carrier for lymph-metastasized tumor targeting
Petrov et al. PSMA-targeted low-molecular double conjugates for diagnostics and therapy
KR20070029200A (en) Peptide-based compounds
Liu et al. Development of an albumin-based PSMA probe with prolonged half-life
US10449261B2 (en) Compositions targeting radiation-induced molecules and methods of use thereof
Yu et al. 99mTc-labeled iRGD for single-positron emission computed tomography imaging of triple-negative breast cancer
Wang et al. Recent progress in functional peptides designed for tumor-targeted imaging and therapy
CA3206250A1 (en) Psma-targeting ligands for multimodal applications
EP3911373A2 (en) Peptide pet/spect probes specific to oncoproteins in tumor extracellular matrix
R Sivashankari et al. Peptides to target tumor vasculature and lymphatics for improved anti-angiogenesis therapy
Ding et al. Development of gold nanorods conjugated with radiolabeled anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody as single-photon emission computed tomography/photoacoustic dual-imaging probes targeting HER2-positive tumors
US20240139351A1 (en) Targeting system with improved uptake
CN117677405A (en) Targeting system with higher absorptivity

Legal Events

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

Ref document number: 13736023

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13736023

Country of ref document: EP

Kind code of ref document: A1