WO2004096254A2 - Compositions et procedes pour le traitement des tumeurs - Google Patents

Compositions et procedes pour le traitement des tumeurs Download PDF

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Publication number
WO2004096254A2
WO2004096254A2 PCT/IB2004/001954 IB2004001954W WO2004096254A2 WO 2004096254 A2 WO2004096254 A2 WO 2004096254A2 IB 2004001954 W IB2004001954 W IB 2004001954W WO 2004096254 A2 WO2004096254 A2 WO 2004096254A2
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tumor
composition
human
transferrin
egf
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WO2004096254A3 (fr
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Jasbir Sandhu
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XPRESSION ANTIBODY THERAPEUTICS Inc
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XPRESSION ANTIBODY THERAPEUTICS Inc
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Priority claimed from US10/429,662 external-priority patent/US20040220085A1/en
Priority claimed from US10/429,654 external-priority patent/US20040220390A1/en
Priority claimed from US10/429,659 external-priority patent/US20040219103A1/en
Priority claimed from US10/429,515 external-priority patent/US20040219099A1/en
Priority claimed from US10/429,497 external-priority patent/US20040219098A1/en
Priority claimed from US10/429,482 external-priority patent/US20040219097A1/en
Priority claimed from US10/429,653 external-priority patent/US20040219102A1/en
Priority claimed from US10/429,598 external-priority patent/US20040219100A1/en
Priority claimed from US10/429,655 external-priority patent/US20040220121A1/en
Application filed by XPRESSION ANTIBODY THERAPEUTICS Inc filed Critical XPRESSION ANTIBODY THERAPEUTICS Inc
Publication of WO2004096254A2 publication Critical patent/WO2004096254A2/fr
Anticipated expiration legal-status Critical
Publication of WO2004096254A3 publication Critical patent/WO2004096254A3/fr
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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
    • A61K47/66—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 the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/665—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 the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • solid tumors such as carcinomas
  • Surgical intervention may remove the primary tumor.
  • smaller groups of tumor cells may have already migrated to distant sites in the body where they can reestablish tumor masses.
  • surgical intervention and radiation may initially control localized disease, systemic therapy becomes necessary to alleviate metastatic disease.
  • Another problem associated with the use of anti-tumor agents to inhibit growth of tumors is low selectivity of the anti-tumor agents.
  • Another side effect associated with cancer therapies is the toxic effect of the anti-tumor agent on the normal host tissues that are the most rapidly dividing, such as the bone marrow, gut mucosa and cells of the lymphoid system.
  • Anti-tumor agents also exert a variety of other adverse effects, including neurotoxicity; negative effects on sexuality and gonadal function; and cardiac, pulmonary, pancreatic and hepatic toxicities; vascular and hypersensititivy reactions, and dermatological reactions.
  • Hematologic toxicity is the most dangerous form of side effect for many of the anti-tumor agents used in clinical practice. The most common hematologic toxicity is neutropenia, with an attendant high risk of infection. Life-threatening thrombocytopenia and bleeding may also occur. Cancer therapy may also induce qualitative defects in the function of both polymorphonuclear leukocytes and platelets.
  • Neurotoxicity can result from cancer treatment, such as, arachnoiditis; myelopathy or encephalomyelopathy; chronic encephalopathies and the somnolence syndrome; acute encephalopathies; peripheral neuropathies; and acute cerebellar syndromes or ataxia.
  • compositions and methods that are suitable for selectively delivering anti-tumor agents to tumor tissues are desirable.
  • Compositions and methods for identifying tumor tissues are also desirable.
  • the instant invention provides a composition that targets or selectively delivers anti-tumor agents to both tumor cells and the endothelial cells of the tumor vasculature.
  • the present composition comprises transferrin, the anti-tumor agent, and at least one other ligand that selectively binds to tumor cells, or to the endothelial cells of the tumor vasculature.
  • the composition comprises an anti-tumor agent and a ligand that binds to and is internalized by tumor cells, each of which is operatively linked to transferrin.
  • the composition comprises an anti- tumor agent and a ligand that binds to and is internalized by the endothelial cells of the tumor vasculature, each of which is operatively linked to transferrin.
  • the composition comprises an anti-tumor agent, a ligand that binds to and is internalized by tumor cells, and a ligand that binds to and is internalized by the endothelial cells of the tumor vasculature, each of which is operatively linked to transferrin.
  • the composition comprises two conjugates: one of which comprises transferrin operatively linked to an antitumor agent and a ligand that binds to tumor cells, the other of which comprises transferrin operatively linked to an anti-tumor agent and a ligand that binds to endothelial cells of the tumor vasculature.
  • the anti-tumor agent is a radionuclide.
  • the composition comprises at least two anti-tumor agents, one of which is a radionuclide.
  • the ligand that binds to and is internalized by tumor cells is epidermal growth factor (EGF), and the ligand that binds to and is internalized by endothelial cells of the tumor vasculature is vascular endothelial growth factor (VEGF).
  • EGF epidermal growth factor
  • VEGF vascular endothelial growth factor
  • the multi-targeting ability of the instant compositions allows for increased sensitivity for imaging and increased efficacy for the reduction of tumor burden when compared with compositions available in the prior art.
  • the present invention also provides a method of inhibiting growth of tumor cells in a mammalian subject, particularly a human subject. The method comprises administering a composition of the present invention to the subject.
  • the ligands are polypeptides that are native to humans, thereby reducing the immunogenicity of the present compositions.
  • Particularly good examples of such polypeptides include human EGF and human VEGF.
  • the present invention also provides a method of visualizing tumor tissue in a subject.
  • the method comprises administering a composition that comprises radiolabled transferrin operatively linked to a ligand that binds to tumor cells, or a ligand that binds to the endothelial cells of the tumor vasculature, or, preferably a ligand that binds to tumor cells and a ligand that binds to endothelial cells of the tumor vasculature, detecting the radiolabel, and visualizing the tumor tissue.
  • the present invention also relates to pharmaceutical compositions comprising a composition of the present invention and a pharmacologically effective amount of a carrier.
  • FIGURE 1 shows a diagrammatic presentation of three embodiments of the present composition.
  • a raiolabled transferrin molecule is operatively linked to epidermal growth factor (EGF); in another embodiment a radiolabeled transferrin molecule is operatively linked to vascular endothelial growth factor (VEGF); in another embodiment a radiolabeled transferrin molecule is operatively linked to EGF and VEGF.
  • EGF epidermal growth factor
  • VEGF vascular endothelial growth factor
  • FIGURE 2 shows a diagrammatic presentation of two other embodiments of the present composition.
  • a transferrin molecule is operatively linked to
  • a transferrin molecule is operatively linked to VEGF and a drug.
  • FIGURE 3 shows a graphical presentation of Breast Cancer Bone Metastatsis
  • FIGURES 4A-4B show immunohistochemistry of BCBM specific for EGFR
  • Figure 4A shows a histologic section stained with antibody (TS40) specific for the human cell surface EGFR.
  • Figure 4B is a micrograph showing an isolated EGFR + breast cancer cell in the bone marrow.
  • FIGURE 5 is a micrograph showing blood vessels of human origin in the
  • FIGURE 6 shows a graphical presentation of the inhibition of breast cancer growth achieved by treatment with a composition comprising radiolabled transferrin operatively linked to VEGF and EGF.
  • FIGURE 7 shows imaging test results in mice.
  • FIGURE 8 shows a graphical presentation comparing inhibition of breast cancer growth achieved by treatment with various compositions including a composition comprising radiolabeled transferrin operatively linked to EGF, a composition comprising radiolabeled transferrin operatively linked to VEGF, and a composition comprising radiolabled transferrin operatively linked to EGF and VEGF.
  • FIGURE 9 shows a graphical presentation comparing inhibition of breast cancer growth achieved by treatment with EGF- 11 'in-labeled transferrin and by treatment with 1 "in-labeled EGF.
  • FIGURE 10 shows a graphical presentation of the inhibition of breast cancer growth achieved by treatment with a composition containing VEGF 1 "in-labeled transferrin and EGF 1 "in-labeled transferrin, by treatment with VEGF 1 "in-labeled transferrin alone and by treatment with EGF 1 "in-labeled transferrin alone.
  • FIGURE 11 shows a graphical presentation of the inhibition of breast cancer growth achieved by treatment with a composition comprising doxorubicin and VEGF operatively linked to transferrin and a composition comprising doxorubicin and EGF operatively linked to transferrin.
  • an "anti-tumor agent” refers to any substance that is capable of inhibiting the proliferation of or killing tumor cells.
  • anti-tumor agents include alkylating agents, anti-tumor antibiotics, plant alkaloids, anti- metabolites and hormonal agonists and antagonists (see US 6,495,553, issued to Shepard, for a background discussion of anti-tumor agents).
  • anti-tumor agent also encompasses radionuclides.
  • radionuclides can be used both to inhibit the proliferation or kill cells of tumor tissues and/or to enhance imaging and diagnosis of tumor tissues.
  • Illustrative, albeit non-limiting examples of radionuclides known and commonly used in the art for radioactive labeling are 123 I, 125 I, I,
  • Alkylating agents are very reactive compounds which have the ability to either substitute alkyl groups for hydrogen atoms or to cause methylation and chloroethylation of DNA and proteins. Alkylation of nucleic acids is a critical cytotoxic action as it interferes with DNA replication and RNA transcription.
  • alkylating agents are mechlorethamine, chloroambucil, melphalan, cyclophosphamide, ifosfamide, thiotepa, busulfan, dacarbazine, carnustine, lomustine, cisplatin, carboplatin, procarbazine and altretamine.
  • Anti-tumor antibiotics are natural products of the soil fungus, Streptomyces.
  • antibiotics are capable of binding DNA, usually through intercalation, to result in the unwinding of the DNA helix. The unwinding impairs the ability of DNA to function as a template for nucleic acid synthesis. These antibiotics are also capable of forming damaging free radicals and chelating metal ions. Additionally, anti-tumor antibiotics may inhibit topoisomerase II, an enzyme important for cell division.
  • anti-tumor antibiotics are doxorubicin, daunorubicin, idarubicin, mitoxantrone, bleomycin, dactinomycin, carminomycin, detorubicin, epirubicin, esorubicin, adriamycin, mitomycin C, plicamycin and streptozocin.
  • Vinca alkaloids such as vincristine and vinblastine
  • Vinca alkaloids are capable of binding microtubular proteins of dividing cells. This binding alters the structure of tubulin addition and loss at the ends of mitotic spindles, ultimately resulting in mitotic arrest and cell death. Similar microtubular proteins are found in nervous tissue, thus vinca alkaloids are also neurotoxic.
  • Paclitaxel taxol
  • Epipodophyllotoxins such as etoposide and teniposide, are capable of inhibiting topoisomerase II, an enzyme important for cell division.
  • Anti-metabolites are structural analogs of normal metabolites that are required for cell function and replication. Anti-metabolites function by interacting with cellular enzymes. Illustrative, albeit non-limiting examples of anti-metabolites are methotrexate, 5- fluorouracil (5-FU), floxuridine (FUDR), cytarabine, 6-mercaptopurine (6-MP), 6- thioguanine, deoxycoformycin, fludarabine, 2-chlorodeoxyadenosine, and hydroxyurea. [0033] Many types of cancer are affected by hormonal changes, thus, endocrine manipulation is an effective therapy for several forms of neoplastic disease. A wide variety of hormones and hormone antagonists have been developed for potential use in cancer treatment.
  • hormonal agents are diethylstilbestrol, tumoxifen, megestrol acetate, dexamethasone, prednisone, aminoglutethimide, leuprolide, goserelin, flutamide, and octreotide acetate.
  • angiogenesis refers to the process by which tissues become vascularized. Angiogenesis involves the proteolytic degradation of the basement membrane on which the endothelial cells reside followed by the chemotactic migration and mitosis of the endothelial cells to support a new capillary shoot.
  • bioactivity refers to the ability of a ligand to bind to its complementary receptor thus enabling internalization of the ligand into the cellular interior.
  • biologically active portion refers to the portion of a ligand that has the ability to bind to its complementary receptor thus enabling internalization of the ligand into the cellular interior.
  • host suspected of having a tumor refers to a host wherein the presence of a tumor is not confirmed.
  • the phrase "host having a tumor” refers to a host wherein the presence of a tumor has been diagnosed or confirmed.
  • a composition of the instant invention is administered to a host having a tumor for therapeutic purposes.
  • selective delivery is defined as delivery which is targeted to a specific cell type for the purpose of avoiding uniform or even delivery to all cell types.
  • ligand refers to a molecule that exhibits specific binding of high affinity for another molecule and upon binding with that molecule is internalized into the cellular interior.
  • An illustrative, albeit non-limiting example of how the term "ligand” is used in the context of the instant specification is a protein ligand binding to a cell surface receptor, such as EGF binding to the EGFR.
  • receptor refers to a molecule that exhibits specific binding of high affinity for its complementary ligand.
  • An illustrative, albeit non-limiting example of how the term “receptor” is used in the context of the instant specification is a cell surface receptor binding to a ligand, such as the EGFR binding to the EGF.
  • complementary receptor refers to the receptor a ligand specifically binds with high affinity, for example, the EGFR is the complementary receptor for EGF.
  • target refers to a specific molecule expressed on the cellular surface such as a receptor to which a specific moiety can be directed, for example the
  • EGFR is a target for EGF.
  • targeting agent refers to a specific molecule that binds to a complementary molecule expressed on the cellular surface such as a ligand, for example EGF is a targeting agent for the EGFR.
  • VEGF refers to a glycosylated polypeptide that serves as a mitogen to stimulate vascular development. VEGF imparts activity by binding to vascular endothelial cell plasma membrane-spanning tyrosine kinase receptors (VEGFR's) which then activates signal transduction.
  • VAGFR's vascular endothelial cell plasma membrane-spanning tyrosine kinase receptors
  • VEGFR refers to a vascular endothelial cell plasma membrane-spanning tyrosine kinase receptor which binds VEGF thus exerting a mitogenic signal to stimulate vascularization of tissues.
  • vascular endothelial growth factor encompasses VEGF and isolated peptide fragments or biologically active portions thereof, analogues of VEGF and any biologically active portion thereof and any molecules and portions of molecules having the biological activity of VEGF.
  • EGF refers to a mitogenic polypeptide that exhibits growth stimulatory effects for epidermal and epithelial cells.
  • EGF imparts activity by binding to epidermal and/or epithelial cell plasma membrane-spanning tyrosine kinase receptors (EGFR's) which then activates signal transduction.
  • EGFR epidermal and/or epithelial cell plasma membrane-spanning tyrosine kinase receptors
  • EGFR refers to a epidermal and/or epithelial cell plasma membrane-spanning tyrosine kinase receptor which binds EGF thus exerting a mitogenic signal.
  • epidermal growth factor encompasses EGF and isolated peptide fragments or biologically active portions thereof, analogues of EGF and any biologically active portion thereof and any molecules and portions of molecules having the biological activity of EGF.
  • the term "transferrin” refers to a vertebrate glycoprotein that functions to bind and transport iron. Since it functions as a transporter of iron, the transferrin molecule has iron-binding sites. Radioactive ions can be bound in the iron-binding sites of the transferrin ligand of the compositions of the present invention where they can serve as an imaging agent or a cytotoxic agent.
  • transferrin receptor refers to a receptor expressed on the surface of cells functioning to capture and bind iron saturated transferrin. Expression of the transferrin receptor is increased in cells which are actively proliferating.
  • transferrin encompasses transferrin and isolated peptide fragments or biologically active portions thereof, analogues of transferrin and any biologically active portion thereof and any molecules and portions of molecules having the biological activity of transferrin.
  • vascular endothelial growth factor acts as a vector for delivery of anti-tumor agents to the endothelial cells of the tumor vasculature and epidermal growth factor (EGF) acts as a vector for delivery of anti-tumor agents to the tumor cells.
  • EGF epidermal growth factor
  • Transferrin acts as a dual- functioning vector for delivery of anti-tumor agents to both the tumor cells and the endothelial cells of the tumor vasculature.
  • the term "host” refers to any animal having a tumor, including a human patient.
  • tumor tissue refers to all of the cellular types which contribute to formation of a tumor mass, including tumor cells and endothelial cells, for example, the tumor tissue includes tumor cells and blood vessels.
  • tumor mass refers to a foci of tumor tissue.
  • tumor vessels all refer to the circulatory vessels which supply the tumor tissue with blood.
  • linker refers to the molecules which join the ligands of the composition of the instant invention together to form a single compound; for example, EGF-PEG attached to biotin links streptavidin attached to transferrin.
  • the phrase "operatively linked” means that the linkage does not destroy the functions of each of the separate elements of the composition of the instant invention, for example, when linked together by a linker to form the single composition of the instant invention the ligands retain the ability to bind their complementary receptors.
  • carrier refers to a pharmaceutically inert substance that facilitates delivery of an active agent to a host, for example, as is shown in the experiments described herein, saline functions as a carrier for delivery of the composition of the instant invention to the mouse host.
  • the phrase "pharmacologically effective amount of a carrier” refers to an amount of a carrier that is sufficient to effectively deliver an active agent to a host.
  • composition refers to the compositions of the instant invention combined with a pharmacologically effective amount of a carrier.
  • tumor cell neoplastic cell
  • cancer cell neoplastic cell
  • selective delivery is defined as delivery which is targeted to a specific cell type for the purpose of avoiding uniform or even delivery to all cell types.
  • the term “selective concentration” is defined as concentrating a substance, such as an anti-tumor agent, to a specific area for the purpose of avoiding uniform or even concentration of a substance in all areas.
  • dose is defined as the amount of a substance administered at one time. A dose should be administered in mg per kg of body weight of the host to be which it is to be administered.
  • the term "therapeutic index" is defined with regard to dose and indicates safety of a substance.
  • a dose is administered in an amount having a specified effect on a stated fraction of experimental animals tested.
  • the therapeutic index is defined by the fraction LD 50 /ED 5 o wherein LD 50 represents the dose causing death in 50% of experimental animals and ED 50 represents the dose at which 50% of the experimental animals show an effect.
  • the methods of the instant invention concentrate anti-tumor agents to the tumor tissue and simultaneously reduce concentration of anti-tumor agents in non-diseased tissues, resulting in increased death of tumor tissue and decreased death of non-diseased tissues.
  • epidermal growth factor refers to epidermal growth factor
  • EGFR epidermal growth factor receptor
  • VEGF vascular endothelial growth factor
  • VEGFR vascular endothelial growth factor receptor
  • BCBM breast cancer bone metastatsis
  • PEG polyethylene glygol
  • TF transferrin
  • SA streptavidin
  • TF/SA refers to a compound comprising transferrin linked to streptavidin.
  • MMS m-maleimidobenzoyl N- hydroxysuccinimide ester
  • HPLC high performance liquid chromatography
  • RP-HPLC reverse phase high performance liquid chromatography
  • PBS phosphate buffered saline
  • TFA trifluoroacetic acid
  • PBS phosphate buffered saline
  • SCID refers to a type of transgenic mouse that is sever combined immuno-deficient.
  • 5-FU refers to the anti-tumor agent 5- fluorouracil.
  • FUDR refers to the anti-tumor agent floxuridine.
  • 6-MP refers to the anti-tumor agent 6- mercaptopurine.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a carrier and a biologically effective amount of a delivery composition comprising one or more anti-tumor agents and a ligand that binds to tumor cells, or a ligand that binds to endothelial cells of the tumor vasculature, or both a ligand that binds to tumor cells and a ligand that binds to endothelial cells of the tumor vasculature, each of which is operatively linked to transferrin.
  • the acceptable carrier is a physiologically acceptable diluent or adjuvant.
  • physiologically acceptable means a non-toxic material that does not interfere with the effectiveness of the delivery composition or the anti-tumor agent.
  • the characteristics of the carrier will depend on the route of administration and particular compound or combination of compounds in the composition. Illustrative, albeit non-limiting examples of carriers known in the art and suitable for use with the instant invention are water, saline solutions and dextrose solutions. A particularly preferred carrier is saline, the use of which is illustrated in the examples herein. [0092] Preparation of such formulations is within the level of skill in the art.
  • the composition may further contain other agents which either enhance the activity of the anti- tumor agent or complement its activity.
  • the composition may further comprise fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
  • the present invention provides methods of delivering an anti-tumor agent to tumor cells and tumor tissues in a subject, particularly a human subject and methods of enhancing the therapeutic index of the anti-tumor agent.
  • the methods comprise administering a composition of the present invention to a subject having a tumor or suspected of having a tumor.
  • compositions may, for example, be administered orally, intra-vascularly, intraperitoneally, intranasal, intrabronchial, subcutaneously, intramuscularly or topically (including aerosol). If the tumor cells are localized, local administration may be used.
  • delivery via local injection contemplates the use of a syringe, catheter or similar device, which delivers the pharmaceutical composition to the target site, i.e., to an area exhibiting cellular proliferative disease.
  • Delivery may be direct, i.e., intratumoral, or nearly direct, i.e., intralesional, that is, to an area that is sufficiently close to a tumor so that the active agent exhibits the desired pharmacological activity with respect to the tumor itself.
  • the pharmaceutical composition is preferably delivered intralesionally or intratumorally.
  • the pharmaceutical composition is administered once or repeatedly in a therapeutically effective amount.
  • therapeutically effective amount means the total amount of the anti-tumor agent of the pharmaceutical formulation or method that is sufficient to show a meaningful subject or patient benefit, i.e., a reduction in tumor size, arrest, or inhibition of tumor growth and/or motility or metastasis, and/or an increase in apoptosis, and/or a reduction the symptoms related to the presence of the tumor.
  • the attending physician will administer low doses of the composition and observe the patient's response. Larger doses of composition may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It may.
  • a therapeutically effective amount of one or more of the therapeutic compositions of the invention to one individual as a single treatment episode.
  • the attending physician will decide the amount of therapeutic composition with which to treat each individual patient.
  • Illustrative, albeit non-limiting examples of periods of time allowed between doses are hours, days and weeks. A particularly preferred period of time between doses is one week, the use of which is illustrated in the examples herein.
  • a therapeutic dose is administered each selected period of time until a statistically significant inhibition of tumor growth is achieved. The amount of inhibition is determined by comparison of tumor growth in treated animals with tumor growth in control animals which have not received treatments.
  • Multiple doses of the present pharmaceutical composition may be administered over a period of time for the purpose of treatment and extend the periods of treatment normally seen with anti-tumor agents since the methods of the instant invention reduce side effects.
  • the period of time between doses is selected based upon the needs of the host receiving treatment.
  • the present invention also provides methods of visualizing tumor tissues.
  • the methods comprise administering a targeting composition comprising transferrin operatively linked to a radionuclide and a ligand that selectively binds to tumor cells (e.g. EGF), or a ligand that selectively binds to endothelial cells of the tumor vasculature (e.g. VEGF), or a ligand that selectively binds to tumor cells and a ligand that binds to endothelial cells of the tumor vasculature.
  • the radionuclides are bound in the iron-binding sites of the transferrin molecule. It is anticipated that one dose of the composition will be administered for the purpose of imaging.
  • radionuclides known and commonly used in the art for radioactive labeling are 123 I, 125 I, 130 I, 131 I, 133 I, 135 I, 47 Sc, 72 As, 72 Se, 90 Y, 88 Y, 97 Ru, 100 Pd, 101m Rh, " 9 Sb, 128 BA, 197 Hg, 211 At, 212 Bi, 153 Sm, 169 Eu, 212 Pb, 109 Pd, " 'in, 67 Ga, 68 Ga, 67 Cu, 75 Br, 76 Br, 99m Tc, "C, 13 N, 15 O and 18 F.
  • a particularly preferred radiolabel is ' ' 'in, the use of which is illustrated in the examples herein.
  • Homo sapiens (human) VEGF 165 (vasculature endothelial growth factor isoform 165) nucleic acid sequence is disclosed as SEQ ID NO:l and translates into VEGF165 protein disclosed as amino acid sequence SEQ ID NO:2.
  • Homo sapiens (human) transferrin nucleic acid sequence is disclosed as SEQ ID NO: 3 and translates into transferrin protein disclosed as amino acid sequence SEQ ID NO:4.
  • Homo sapiens (human) EGF (epidermal growth factor) nucleic acid sequence is disclosed as SEQ ID NO: 5 and translates into EGF protein disclosed as amino acid sequence SEQ ID NO:6.
  • elements are either linked directly through chemical conjugation (for example through reaction with an amine or sulfhydryl group) or are linked indirectly through molecules termed linkers.
  • linkers When selecting a linker it is important to choose the appropriate length and flexibility of a linker in order to reduce steric hindrance between the elements of the composition. For example, if an element of a composition is brought into close physical proximity of another element by linkage, the function of either or both elements can be affected.
  • Each element of the composition must retain its bioactivity, for example in the instant invention, each ligand must retain its ability to bind to its complementary receptor after linkage with the other two ligands of the composition.
  • linkers are glycols, alcohols and peptides. Particularly prefe ⁇ ed linkers are PEG (polyethylene glycol) and the peptide linker shown as SEQ ID NO: 8 (use of each of these linkers is illustrated in the exampled described herein).
  • EGF and VEGF are crosslinked to a biotinylated polylinker by carrying out the following protocol.
  • the polylinker used consists of 15 amino acid residues shown as SEQ ID NO:8.
  • the cDNA sequence encoding this polylinker is shown as SEQ ID NO:7.
  • the first glycine residue at the N-terminal was biotinylated.
  • EDG l-Ethyl-3-(3- Dimethylaminopropyl) carbodiimide Hydrochloride
  • NHS N-Hydroxysuccinimide
  • EDC and 0.6 mg of NHS were added to 1 mg/ml of the polylinker peptide solution (in activation buffer: 0.1 M MES (2-[N-morpholino] ethane sulfonic acid), 0.5 M ⁇ aCl, pH 6.0) to a final concentration of EDC and ⁇ HS of 2 mM and 5 mM respectively.
  • activation buffer 0.1 M MES (2-[N-morpholino] ethane sulfonic acid), 0.5 M ⁇ aCl, pH 6.0
  • the reaction mixture was then held for 15 minutes at room temperature.
  • 2-mercaptoethanol was then added (to a final concentration of 20 mM).
  • the reaction mixture was then run through P2 gel filtration mini-column and eluted by the activation buffer. Fractions containing the protein were then pooled together.
  • 2-Iminothiolane hydrochloride (Traut's reagent) in pH 8.0, 0.16 M borate. Following 90 minutes at room temperature, the thiolated TF was desalted and concentrated by Centricon microconcentrators. Ellman's reagent (Pierce) was then used to demonstrate that a single thiol group was inserted on the surface of TF. 7 mg of streptavidin (SA) (in PBS) was actived by adding to a 20:1 molar ratio of m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS) (stock at lmg/ml in dimethylformamide).
  • SA streptavidin
  • MBS m-maleimidobenzoyl N-hydroxysuccinimide ester
  • the activated SA was desalted on a microconcentrator and immediately, the activated SA was added to a 10 molar excess of thiolated TF. They were mixed and then incubated at room temperature for 3 hours. Purification of the TF/SA compound was done by HPLC using TSK-G3000 column. The number of biotin binding sites for TF/SA compound was determined with 3 H-biotin biding assay.
  • VEGF-Linker-Biotin and EGF-Linker-Biotin are each added to TF/SA by carrying out the following protocol.
  • the compound of VEGF-Linker-biotin and EGF-Linker- biotin and TF/SA was prepared by mixing 5 nmol of VEGF-Linker-biotin (or 5 nmol of EGF - Linker-biotin) with 8 nmol of TF/SA (1 :1.6 molar ratio). HPLC was then used to purify the VEGF-Linker-biotin-TF-SA-EGF-Linker-biotin compound.
  • reaction mixture was then applied to a TSK-gel G-3000 SW L HPLC gel filtration column, followed by elution in a 0.01 M Na 2 HPO 4 /0.15 M NaCl/pH 7.4/0.05% Tween-20 at a flow rate fo 0.5 mL/min for 40 minutes, and 0.5 mL fractions were collected.
  • 2 mCi '"in acetate was mixed with the compound in 10 mM HEPES, 15 mM NaHCO3 pH 7.4 buffer for 1 hour at room temperature.
  • VEGF and EGF can also be linked to transferrin using PEG by ca ⁇ ying out the following protocol.
  • NHS-PEG3400-biotin (20 nmol in 310 ⁇ l of 0.05 M NaHCO3) was added in a 1 :1 molar ratio to either VEGF or EGF (16 nmol is 250 ⁇ l of 0.05 M NaHCO3) followed by incubation at room temperature for 60 minutes.
  • EGF and/or VEGF were purified by HPLC.
  • the EGF (and/or VEGF) -NHS-PEG3400-biotin and TF/SA compounds were then mixed (1 :1.6 molar ratio).
  • the compounds EGF (and/or VEGF) -NHS-PEG3400-biotin-TF-SA was purified by HPLC and labeled with " 'in by mixing with '"in acetate and purified on a P-2 size-exclusion mini- column.
  • the specific activity of ' "in-EGF (and/or VEGF) -PEG3400-biotin-TF-SA compounds were about 100-400 mCi/mg.
  • mice Severe combined immuno-deficient C.B.-17 scid/scid (SCLD) mice were bred and maintained according to the protocol of Sandu et al. (Critical Reviews in Biotechnology 16(1):95-118 1996). Mice were used when 6-8 weeks old and were pre-treated with a dose of
  • composition of the instant invention is effective when used to target either an EGFR + tumor or an EGFR ' tumor since the transferrin moiety targets those tumor cells that are EGFR ' .
  • a bone metastatic focus of a primary EGFR + breast tumor was used in the experimental examples herein described.
  • the composition of the instant invention can be administered to a host having any tumor comprising cells which are positive for the expression of at least one of the cell surface receptors described herein (the transferrin receptor, the EGFR and the VEGFR).
  • BCBM Breast cancer bone metastasis
  • EGFR expression in breast cancer bone metastasis biopsies ranged from 1-1300 fmol/mg membrane protein (approximately 400-1,000,000 receptors/cell) and was associated with high relapse rate and poor long term survival.
  • Normal epithelial cells express ⁇ 10 4 receptors/cells.
  • HBL-100 For the normal breast cell line HBL-100, 8000 EGFR/cell has been reported.
  • EGFR in breast cancer cell lines has a reported range of 800 EGFR cell for MCF-7 cells to 10 6 EGFR cell for MDA-MB-468 cells.
  • the liver is the only normal tissue exhibiting moderate levels of EGFR (8X10 4 to 3x5 5 receptors/cell) likely reflecting is role in the elimination of EGF from the blood. Utilizing the Auger electron emitter '"in was used in the initial experiments to illustrate the utility of the invention using EGF- 1 "in-labeled transferrin compounds.
  • the EGF- 1 "in-labeled transferrin (0.25-80 ng) was incubated with 1.5 x 10 6 cells/dish JJ5 Breast Cancer (prepared from BCBM JJ5) cells in 1 ml of 0.1% human serum albumin in 35 mm multiwell culture dishes at 37°C for 30 minutes. The cells were transfe ⁇ ed to a centrifuge tube and centrifuged. The cell pellet was separated from the supernatant and counted in a g-scintillation counter to determine bound (B) and free (F) radioactivity. Non-specific binding was determined by conducting the assay in 100 nM hEGF.
  • the kinetics of binding was determined by incubating 1 ng of EGF- '"in-labeled transferrin compound with 3 x 10 6 JJ5 Breast Cancer cells at 37°C and determining the proportion of radioactivity bound to the cells at various times up to 24 hours. Internalized fraction was measured by determining the proportion of radioactivity which could not be displaced from the cell surface by 100 nM hEGF. Cell-associated binding (surface-binding and intracellular accumulation) was expressed as a percentage of medium radioactivity bound per mg of cell study protein.
  • JJ5 cells was 8 x 10 8 L/mol and the number of binding sites was 2.7 x 10 6 .
  • EFG-" 1 In-labeled transfe ⁇ in compound was rapidly bound by the breast cancer cells and retained for at least 24 hours. Over a 24 hour period at 37°C, ⁇ 8% was lost from the cells in vitro.
  • JJ5 breast cancer cells prepared from BCBM JJ5 expressing approximately
  • EGF- 1 in-labeled transferrin compound (3.4 pCi/cell) achieved a 83% growth inhibition of the JJ5 cells compared to the medium control, whereas "'in oxine (3.5 pCi/cell) which enters all the cells resulted in 91% growth inhibition.
  • CYTOTOXICITY ASSAY OF EGF- 1 IN-LABELED TRANSFERRIN COMPOUND AGAINST JJ5 BREAST CANCER CELLS
  • JJ5 breast cancer cells were incubated with increasing amounts of EFF- "in- labeled transferrin compound or '"in-oxine, centrifuged to remove free ligand, assayed and then seeded into 50 mm culture dishes. The number of cells seeded was varied from 3xl0 4 to 3xl0 6 cells to obtain approximately 400 viable colonies/dish taking into account the plating efficiency and the expected level of cytotoxicity. Control dishes contained JJ5 breast cancer cells which were incubated with normal saline. Growth medium was added and the cells were cultured at 37°C/5% CO 2 for 14 days.
  • the growth medium was removed and the colonies were stained with methyl ene blue (1% in a 1 :1 mixture of ethanol and water) then washed twice.
  • the number of colonies per dish was counted using a manual colony counter (Manostat Corp).
  • the plating efficiency was calculated by dividing the number of colonies observed by the number of cells seeded in each dish.
  • the surviving fraction at increasing amounts of EGF- 1 "in-labeled transferrin compound or '"in-oxine was calculated by dividing the plating efficiency for dishes containing treated cells with that observed for control dishes with normal saline.
  • EGF- 1 "in-labeled transferrin compounds (8 pCi/cell) resulted in a 99% reduction in cell survival for JJ5 cells
  • '"in-oxine was also radiotoxic with greater han 99% cell killing at ⁇ 6 pCi/cell.
  • EGF- 1 "in-labeled transfe ⁇ in compositions are rapidly internalized by cancer cells.
  • the internalization process for EGF- " 'in-labeled transferrin compositions involves an active transport mechanism utilizing the EGFR binding domain of the compound, rather than simple diffusion across the cell membrane.
  • This active transport mechanism for the composition probably also includes nuclear translocation, as for the case of EGF, which allows for a maximal radiation dose of Auger electrons to be delivered to the cell's DNA.
  • the compositions of the instant invention employ human polypeptides and are not immunogenic in humans.
  • EFG-" 'in-labeled transferrin compositions have been shown to retain '"in over a 24 hour period at 37°C, with ⁇ 8% of '"in radioactivity was lost from cells in vitro. These characteristics are important for cell killing.
  • the arrow in Figure 4B points out an isolated EGFR + cell in the bone ma ⁇ ow.
  • Mean ( ⁇ SDEV) expression levels of EGF receptor was measured on breast cancer cells from tumor JJ5 by radioligand binding assay 24 and were in the range of 2.7 ( ⁇ 0.8) xlO 6 receptors/cell.
  • mice were treated intraperitoneally with EGF- "'in-labeled transferrin (200 uCi) once a week for 5 weeks.
  • Experimental group 5 BCBM-SCID mice were treated intraperitoneally with ' "in-labeled VEGF (200 uCi) once a week for 5 weeks.
  • Experimental group 6 BCBM-SCID mice were treated intraperitoneally with '"in-labled EGF (200 uCi) once a week for 5 weeks.
  • the BCBM were resected from control and experimental mice and tumor weight and volume determined. Maximum inhibition of tumor growth is obtained by targeting the tumor cells and the tumor blood vessels with a single compound.
  • bar #1 represents the tumor volume seen in control mice
  • bar #2 represents the tumor volume seen in mice administered VEGF- 1 " In-labeled transfe ⁇ in compounds
  • bar #3 represents the tumor volume seen in mice administered VEGF- 1 " In-labeled transferrin-EGF compounds (the composition of the instant invention)
  • bar #4 represents the tumor volume seen in mice administered EGF- '"in-labeled transfe ⁇ in
  • bar #5 represents the tumor volume seen in mice administered '"in-labeled VEGF
  • bar #6 represents the tumor volume seen in mice administered '"in-labeled EGF.
  • the P values representing the statistical significance of inhibition of tumor growth as compared with the tumor growth of the control are as follows: bar #3 0.0054; bar #2 0.0202; bar #4 0.0129; bar #5 0.3610 and bar #6 0.1328.
  • a maximum, statistically significant amount of inhibition of tumor growth (BCBM) was seen in mice treated with the composition of the instant invention (VEGF- 1 "in-labeled transferrin-EGF comounds; shown by bar #3 in Figure 8).
  • Figure 6 shows a bar graph similar to the bar graph of Figure 8 comparing the inhibition of breast cancer growth achieved by treatment with the composition of the instant invention (VEGF- "'in-labeled transferrin-EGF compounds).
  • bar #1 represents the tumor volume seen in control mice and bar #2 represents the tumor volume seen in mice administered the composition of the instant invention (VEGF- 1 ' ' In-labeled transferrin-EGF).
  • Example 2 ANIMAL IMAGING
  • Mice bearing subcutaneous xenografts were injected intravenously in the tail vein with VEGF-" 'in labeled transferrin-EGF compounds (200 ⁇ Ci).
  • Tissue samples were weighed and counted along with a standard of the injectate in gamma counter (Packard Auto Gamma 5650, Packard Instruments, Downer's Grove, IL) using a window of 150 ⁇ 270 keV for '"in.
  • the tumor and normal tissue uptake of the compounds was expressed as percent injected dose per gram of tissue (%H>/g) and as tumor: normal tissue ratios.
  • BCBM JJ5 was used in the imaging studies since it was shown to produce the highest tumor volumes in SCID mice (see Figure 3).
  • BCBM JJ5 has tumor cells that express high levels of EGF receptor and the blood vessels in the tumor are of human origin and thus express human VEGF receptors.
  • VEGF-'"ln-labeled transfe ⁇ in-EGF (the composition of the instant invention) was used to image JJ5 tumors.
  • This radioactive compound targets EGF receptors on the tumor cells, VEGF receptors on the tumor blood vessels and transferrin receptors on both the tumor cells and tumor blood vessels.
  • This multi-targeting approach provided images of the tumor. When constructs targeting either the EGF receptor or the VEGF receptor were used alone, no imaging of the JJ5 tumors was observed.
  • Figure 7 shows imaging test results obtained in mice.
  • the image at the top left represents radioactive distribution in a mouse at 0 hours after injection
  • the image at the top right represents radioactive distribution in a mouse at 1 hour after injection
  • the image at the bottom left represents radioactive distribution in a mouse at 24 hours after injection
  • the image at the bottom right represents radioactive distribution localized in the tumor only 48 hours after injection.
  • the radiolabeled compound collected in several areas of the body but mostly in tumor site as this site is visualized as "hotspots". This radiolabeled compound does not have any toxicity in the mice and the tumor imaging results show that the radiopharmaceutical has great potential for in vivo localization and biological characterization of cancers.
  • the BCBM were resected from control and experimental mice and tumor weight and volume determined.
  • bar #1 represents the tumor volume seen in control mice
  • bar #2 represents the tumor volume seen in mice administered EGF- 1 "in-labeled tranferrin compositions
  • bar #3 represents the tumor volume seen in mice administered '"in-labeled EGF.
  • the P values representing the statistical significance of inhibition of tumor growth as compared with the tumor growth of the control are as follows: bar #2 0.0129 and bar #3 0.1328.
  • Example 4 EFFECT OF COMPOSITION ON BCBM GROWTH
  • bar #1 represents the tumor volume seen in mice administered VEGF- l u In-labeled transferrin alone
  • bar #2 represents the tumor volume seen in mice administed EGF- 1 "in-labeled transferrin alone
  • bar #3 represents the tumor volume seen in mice administered a composition containing VEGF- 1 "in labeled transferrin and EGF ' "in labeled transferrin
  • bar #4 represents the tumor volume seen in control mice.
  • the P values representing the statistical significance of inhibition of tumor growth as compared with the tumor growth of the control are as follows: bar #1 0.0202; bar #2 0.0129 and bar #3 0.006.
  • compositions of the instant invention are able to act as both a multi-targeting visualizing agent and a multi-targeting therapeutic agent.
  • the instant invention provides a composition comprising a single non-immunogenic compound that can be used to target both the tumor cells and the endothelial cells of the tumor vasculature.
  • the instant invention provides a novel multi-targeted approach to cancer diagnostics, imaging and therapy.
  • Example 5 CELL CULTURE STUDIES USING COMPOSITION CONTAINING DOXORUBICIN
  • JJ5 breast cancer cells expressing approximately 10 6 epidermal growth factor receptors/cell were incubated with EGF-NHS-PED3400-Biotin-TF-SA-Doxorubicin [EGF- Transferrin-Doxorubicin], or EGF-NHS-PED3400-Biotin-TF-SA or Doxorubicin centrifuged to remove test material, and then seeded (10 6 cells/dish) into 35 mm culture dishes. Growth medium was added and the cells were cultured at 37°C/5% CO2 for 4 days, the cells were then recovered by trypsinization and counted in a hemocytometer.
  • Control dishes contained cells cultured in growth medium containing EGF-NHS-PEG3400-Biotin-TF-SA. Cytotoxicity Assay of JJ5 breast cancer cells expressing approximately 10 6 epidermal growth factor receptors/cell were incubated with increasing amounts of EGF-NHS-PEG3400-Biotin- TF-SA-Doxorubicin or Doxorubicin, centrifuged to remove free test material, assayed and then seeded into 50 mm culture dishes. The number of cells seeded was varied from 3 x 10 4 to 3 x 10 6 cells to obtain approximately 350 viable colonies/dish taking into account the plating efficiency and the expected level of cytotoxicity. Control dishes contained JJ5 breast cancer cells which were incubated with normal saline.
  • the plating efficiency was calculated by dividing the number of colonies observed by the number of cells seeded in each dish. The surviving fraction at increasing amounts of EGF-NHS-PEG3400-Biotin-TF-SA-Doxorubicin or Doxorubicin was calculated by dividing the plating efficiency for dishes containing treated cells with that observed for control dishes with normal saline.
  • EGF-NHS-PEG3400- Biotin-TF-SA-Doxorubicin achieved 86% growth inhibition of the JJ5 cells compared to the medium control whereas Doxorubicin which enters all the cells resulted in 95% growth inhibition.
  • SCID mice were treated intraperitoneally with PBS solution once a week for 5 weeks.
  • Experimental group 2 BDBM-SCED mice were treated intraperitoneally with free doxorubicin (lOOug) once a week for 5 weeks.
  • Experimental group 3 BCBM-SCID mice were treated intraperitoneally with VEGF-transferrin-doxorubicin (lOOug) once a week for 5 weeks.
  • Experimental group 4 BCBM-SCID mice were treated intraperitoneally with EGF- transferrin-doxorubicin (lOOug) once a week for 5 weeks.
  • Experimental group 5 BCBM- SCID mice were treated intraperitoneally with VEGF-transferrin (lOOug) once a week for 5 weeks.
  • bar #1 represents the tumor volume seen in control mice treated with PBS solution
  • bar #2 represents the tumor volume seen in mice administered free doxorubicin
  • bar #3 represents the tumor volume seen in mice administered VEGF-transferrin-doxorubicin
  • bar #4 represents the tumor volume seen in mice administered EGF-transfe ⁇ in-doxorubicin
  • bar #5 represents tumor volume seen in mice administered VEGF-transferrin
  • bar #6 represents the tumor volume seen in mice administed EGF-tranferrin.
  • compositions of the instant invention enable selective delivery of anti-tumor agents to a host having a tumor.
  • the instant invention provides compositions capable of selectively concentrating anti-tumor agents to the tumor cells and associated tumor vasculature while simultaneously reducing the concentration of anti-tumor agents in non- diseased tissues.

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Abstract

L'invention porte sur des compositions permettant d'inhiber la croissance tumorale ou d'effectuer de l'imagerie tumorale chez un patient et sur des procédés utilisant ces compositions. Selon un mode de réalisation, la composition comprend de la transferrine, l'agent antitumoral, et au moins un autre ligand qui se lie sélectivement aux cellules tumorales, ou aux cellules endothéliales du système vasculaire de la tumeur. L'invention comprend également un procédé permettant d'inhiber la croissance des cellules tumorales chez un mammifère, en particulier chez un patient humain. Le procédé consiste à administrer une composition de l'invention à un sujet. L'invention comprend également un procédé de visualisation du tissu tumoral chez un patient. Le procédé consiste à administrer une composition qui comprend de la transferrine radiomarquée liée fonctionnellement à un ligand qui se lie à des cellules tumorales, ou un ligand qui se lie aux cellules endothéliales du système vasculaire de la tumeur, où, de préférence un ligand qui se lie aux cellules tumorales et un ligand qui se lie aux cellules endothéliales du système vasculaire de la tumeur, détectant le radiomarquage, et visualisant le tissu tumoral.
PCT/IB2004/001954 2003-05-02 2004-05-03 Compositions et procedes pour le traitement des tumeurs Ceased WO2004096254A2 (fr)

Applications Claiming Priority (19)

Application Number Priority Date Filing Date Title
US10/429,515 2003-05-02
US10/429,515 US20040219099A1 (en) 2003-05-02 2003-05-02 Method for the treatment of tumors
US10/429,497 US20040219098A1 (en) 2003-05-02 2003-05-02 Methods for the treatment of tumors
US10/429,659 2003-05-02
US10/429,654 2003-05-02
US10/429,482 US20040219097A1 (en) 2003-05-02 2003-05-02 Composition useful for the diagnosis, imaging and treatment of tumors
US10/429,482 2003-05-02
US10/429,662 US20040220085A1 (en) 2003-05-02 2003-05-02 Compositions for nucleic acid delivery
US10/429,655 2003-05-02
US10/429,659 US20040219103A1 (en) 2003-05-02 2003-05-02 Methods useful for the diagnosis, imaging and treatment of tumors
US10/429,662 2003-05-02
US10/429,497 2003-05-02
US10/429,653 US20040219102A1 (en) 2003-05-02 2003-05-02 Compositions for drug delivery
US10/429,655 US20040220121A1 (en) 2003-05-02 2003-05-02 Methods for drug delivery
US10/429,653 2003-05-02
US10/429, 2003-05-02
US10/429,654 US20040220390A1 (en) 2003-05-02 2003-05-02 Composition useful for the treatment of tumors
US10/429,598 US20040219100A1 (en) 2003-05-02 2003-05-02 Composition useful for the treatment of tumors
US10/429,598 2003-05-02

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WO2006090232A3 (fr) * 2005-02-22 2007-04-05 Ge Healthcare Ltd Complexes de gallium radiomarques, procedes de synthese et d'utilisation pour l'imagerie tep de l'expression du recepteur du facteur de croissance epidermique dans des tumeurs malignes
WO2012066581A1 (fr) * 2010-11-19 2012-05-24 Venus Remedies Limited Nouveaux conjugués pour administration ciblée de médicaments
EP2197909A4 (fr) * 2007-08-24 2014-12-03 Univ Minnesota Réactifs de ciblage de récepteurs
WO2016026942A1 (fr) * 2014-08-20 2016-02-25 Academia Sinica Méthodes pour améliorer la perméabilité de la barrière hémato-encéphalique et leurs utilisations

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US4522750A (en) * 1984-02-21 1985-06-11 Eli Lilly And Company Cytotoxic compositions of transferrin coupled to vinca alkaloids
AU3374795A (en) * 1994-08-29 1996-03-22 Prizm Pharmaceuticals, Inc. Conjugates of vascular endothelial growth factor with targeted agents
AU3724495A (en) * 1994-09-13 1996-03-29 Prizm Pharmaceuticals, Inc. Conjugates of heparin-binding epidermal growth factor-like growth factor with targeted agents
JP2004532245A (ja) * 2001-05-15 2004-10-21 ページ ダブル フォーク 癌を治療するための生体影響性化合物の標的送達
JP2005510483A (ja) * 2001-10-17 2005-04-21 ページ ダブル フォーク 選択された細胞を標的化してそれに作用する方法及び物質

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006090232A3 (fr) * 2005-02-22 2007-04-05 Ge Healthcare Ltd Complexes de gallium radiomarques, procedes de synthese et d'utilisation pour l'imagerie tep de l'expression du recepteur du facteur de croissance epidermique dans des tumeurs malignes
EP2197909A4 (fr) * 2007-08-24 2014-12-03 Univ Minnesota Réactifs de ciblage de récepteurs
US9155798B2 (en) 2007-08-24 2015-10-13 Regents Of The University Of Minnesota Receptor-targeting reagents containing epidermal factor receptor-binding agents and IL-13 receptor-binding agents or IL-4 receptor-binding agents
WO2012066581A1 (fr) * 2010-11-19 2012-05-24 Venus Remedies Limited Nouveaux conjugués pour administration ciblée de médicaments
WO2016026942A1 (fr) * 2014-08-20 2016-02-25 Academia Sinica Méthodes pour améliorer la perméabilité de la barrière hémato-encéphalique et leurs utilisations
TWI559928B (en) * 2014-08-20 2016-12-01 Academia Sinica Methods for enhancing permeability to blood-brain barrier and uses thereof

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