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WO2007083175A1 - Molécules antivirales bifonctionnelles, procédés pour les produire et procédés pour traiter un cancer induit par virus au moyen de celles-ci - Google Patents

Molécules antivirales bifonctionnelles, procédés pour les produire et procédés pour traiter un cancer induit par virus au moyen de celles-ci Download PDF

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WO2007083175A1
WO2007083175A1 PCT/IB2006/001431 IB2006001431W WO2007083175A1 WO 2007083175 A1 WO2007083175 A1 WO 2007083175A1 IB 2006001431 W IB2006001431 W IB 2006001431W WO 2007083175 A1 WO2007083175 A1 WO 2007083175A1
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seq
polypeptide
channel
antibody
cell
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Inventor
Xiao-Qing Qiu
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West China Hospital of Sichuan University
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West China Hospital of Sichuan University
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Priority to PCT/IB2006/001431 priority Critical patent/WO2007083175A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • C07K16/088—Varicella-zoster virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • HIV human immunodeficiency virus
  • herpes simplex virus hepatitis B and C virus
  • Epstein-Barr virus exhibit similar capabilities. Accordingly, a need exists for compounds and methods of treating viral infections and drug resistant virus.
  • cytotoxic agents would have specificity for cancer and tumor cells while not affecting normal cells.
  • the instant invention is based on the discovery that a bifunctional molecule comprising a targeting agent and a channel-forming moiety can selectively kill cells, e.g., cancer cells.
  • the invention provides a novel class of preferred compounds that kill cells.
  • the instant invention provides a molecule comprising a targeting agent attached to a channel-forming moiety.
  • the molecule is a polypeptide.
  • the channel-forming moiety is a channel-forming peptide, a channel-forming domain, or fragment thereof.
  • the channel-forming peptide can be ⁇ -hemolysin, delta toxin, diphtheria toxin, anthrax toxin, and El family colicin.
  • the colicin is El, Ia, Ib, A, K or N.
  • the colicin is colicin Ia.
  • the channel-forming fragment is a channel-forming colicin comprising amino acid residues from about 1 to about amino acid 626 of colicin Ia (SEQ ID
  • the channel-forming domain comprises amino acid residues 451-626 (SEQ ID NO:3) having the nucleic acid sequence of SEQ ID NO:4.
  • the targeting agent is selected from the group consisting of a ligand, an antibody, an antibody fragment, a reconstituted antibody mimetic, and a phage segment.
  • the targeting agent is C-terminal to the channel-forming moiety.
  • the targeting agent is N-terminal to the channel-forming moiety.
  • the targeting agent is an antibody or fragment thereof, or a reconstituted antibody mimetic.
  • the antibody, a fragment thereof, or a reconstituted antibody mimetic is a specific for a polypeptide expressed by a cell, e.g., a cancer cell.
  • the antibody is an engineered antibody variant or a reconstituted antibody mimetic.
  • the invention provides a polypeptide comprising an antibody and a channel-forming colicin, or a channel-forming fragment thereof, of colicin.
  • the colicin comprises residues 1-626 of colicin Ia.
  • the channel-forming domain comprises residues 451-626 of colicin Ia.
  • the invention provides a polypeptide comprising the Epstein-Barr virus gp350/220 envelope glycoprotein engineered antibody variant, or a reconstituted antibody mimetic, and a channel-forming domain of colicin.
  • the channel-forming domain of colicin comprises residues from about residue 1 to about residue
  • the channel-forming domain comprises amino acid residues 451-626.
  • polypeptides of the invention may have one or more non-natural amino acid residues, e.g., amino acid analogs, or mimetics.
  • the non-natural amino acid residues are D-isomers of natural amino acid residues.
  • the invention provides a nucleic acid molecule that encodes a polypeptides of the invention.
  • the invention provides a vector, e.g., an expression vector, comprising a nucleic acid molecule that encodes a polypeptide of the invention.
  • the invention provides a host cell comprising a vector of the invention.
  • the host cell is a bacterial cell, e.g., E. coli, or a yeast or mammalian host cell.
  • the invention further provides methods of producing the polypeptide of the invention.
  • the methods comprise the steps of culturing the host cells of the invention such that the polypeptides are produced.
  • the methods of the invention may involve purifying said polypeptide.
  • the invention provides a method of producing a molecule of the invention wherein the targeting agent and the channel-forming moiety are produced separately and covalently linked after production.
  • the channel-forming moiety and/or the targeting agent are produced recombinantly.
  • the invention provides methods of treating a subject having cancer, comprising administering to the subject an effective amount of a polypeptide comprising an targeting agent and a channel-forming moiety thereby treating the subject.
  • the targeting agent is selected from the group consisting of an antibody, a fragment thereof, an engineering antibody variant, and a reconstituted antibody mimetic.
  • the reconstituted antibody mimetic or variants was a complementarity determining regions pair (VHCDRI and V L CDR3) selected from the antibody fragment against EBV gp350/220 envelope glycoprotein.
  • the channel-forming moiety is selected from the group consisting of ⁇ -hemolysin, delta toxin, diphtheria toxin, anthrax toxin, and El family colicin, or a fragment thereof.
  • the colicin, or the fragment of colicin is selected from the group consisting of El , Ia, Ib, A, K or N.
  • the colicin is colicin Ia.
  • the fragment of colicin Ia comprises amino acid residues 1-626 or 451-626.
  • the invention provides a method of treating a subject having a viral associated cancer comprising administering to the subject an effective amount of a polypeptide comprising an targeting agent and a channel-forming moiety thereby treating said subject.
  • Figure 1 schematically depicts the structure of a recombinant plasmid that contains a complementarity determining region pair as a targeting domain which is selected from a HB-168 monoclonal antibody fragment against Epstein-Barr virus gp350/220 envelope antigen and linked to a VHCDRl /VHCDR2 linker of that fragment
  • Figure 2 schematically depicts the structure of a recombinant plasmid that contains a targeting domain derived from bacteriophage Ml 3.
  • Figure 3A-B depict images of EBV-infected Burkitt's malignant lymphoma cells after 72 hours of treatment with Ph-CNCV.
  • Figure 3 A is a CCD image of cells treated with stock solution of pheromonicin as a control.
  • Figure 3B is a CCD image of cells treated with 50 ⁇ g/ml of Ph-CNCV Cells are stained with 50 nM acridinorange/600nM propidium iodide. Cellular swelling, mitochondria degeneration and cell rupture were detected along with the Ph-CNCV.
  • Figure 4A-B depict images of human EBV-infected Burkitt's malignant lymphoma cells after 72 hours of treatment with Ph-EBV.
  • Figure 4A is a CCD image of cells treated with stock solution of pheromonicin as- a control.
  • Figure 4B is a CCD image of cells treated with 50 ⁇ g/ml of Ph-EBV. Cells are stained with 50 nM acridinorange/600nM propidium iodide. Cellular swelling, mitochondria degeneration and cell rupture were detected along with the Ph-EBV.
  • Figure 5A-B depict images of human EBV-uninfected Burkitt's malignant lymphoma cells after 72 hours of treatment with Ph-EBV.
  • Figure 5 A is a CCD image of cells treated with stock solution of pheromonicin as a control.
  • Figure 5B is a CCD image of cells treated with 50 ⁇ g/ml of Ph-EBV. Cells are stained with 50 nM acridinorange/600nM propidium iodide. Ph-EBV has no effects on EBV-uninfected lymphoma cells.
  • Figure 6A-B depict images of human EBV-infected AIDS related body cavity based lymphoma cells after 72 hours of treatment with Ph-EBV.
  • Figure 6A is a CCD image of cells treated with stock solution of pheromonicin as a control.
  • Figure 6B is a CCD image of cells treated with 50 ⁇ g/ml of Ph-EBV. Cells are stained with 50 nM acridinorange/600nM propidium iodide. Cellular swelling, mitochondria degeneration and cell rupture were detected along with the Ph-EBV.
  • Figure 7A-B depict images of SMMC-7721 human hepatocellular cancer cells after 72 hours of treatment with Ph-EBV.
  • Figure 7A is a CCD image of cells treated with stock solution of pheromonicin as a control.
  • Figure 7B is a CCD image of cells treated with 50 ⁇ g/ml of Ph-EBV. Cells are stained with 50 nM acridinorange/600nM propidium iodide. Ph-EBV has no effects on hepatoma cells.
  • Figure 8A-B depict images of ATCC 3T3 mouse fibroblast cells after 72 hours of treatment with Ph-EBV.
  • Figure 8 A is a CCD image of cells treated with stock solution of pheromonicin as a control.
  • Figure 8B is a CCD image of cells treated with 50 ⁇ g/ml of Ph-EBV. Cells are stained with 50 nM acridinorange/600nM propidium iodide. Ph-EBV has no effects on fibroblast cells.
  • Figure 9A-B depict images of ECV-304 human umbilical cord vein endothelium cells after 72 hours of treatment with Ph-EBV.
  • Figure 1 IA is a CCD imag ⁇ of cells treated with stock solution of pheromonicin as a control.
  • Figure 9B is a CCD image of cells treated with 50 ⁇ g/ml of Ph-EBV. Cells are stained with 50 nM acridinorange/600nM propidium iodide. Ph-EBV has no effects on endothelium cells.
  • Figure 10A-F shows the killing effects of Ph-EBV against xenograft inoculated with tumor cells in immunodeficiency mice as solid tumor models.
  • Figure 1OA depicts exposed xenografts after 16-day intraperitoneal Ph-EBV stock solution treatment (0.5 ml/mouse/day).
  • Figure 1OB depicts exposed xenografts after 16-day intraperitoneal Ph-EB V treatment ((35O ⁇ g/mouse/day)).
  • Figure 1OC depicts a 400X microscope image of EBV-uninfected xenograft in stock solution-treated mouse.
  • Figure 1OD depicts a 400X microscope image of EBV-infected xenograft in stock solution-treated mouse.
  • Figure 1OE depicts a 400X microscope image of EBV-uninfected xenograft in Ph-EB V-treated mouse.
  • Figure 1OF depicts a 400X microscope image of EBV-infected xenograft in Ph-EB V-treated mouse.
  • Figure 1 IA-K shows the killing effects of Ph-EBV against solid tumors.
  • Figure 1 IA depicts an exposed Burkitt Lymphoma xenograft after 20-day intraperitoneal Ph-EBV stock solution treatment (0.5 ml/mouse/day).
  • Figure HB depicts a IOOX microscope image of the xenograft.
  • Figure 11C depicts exposed Burkitt Lymphoma xenograft after 20-day intraperitoneal Ph-EBV treatment (350 ⁇ g/mouse/day).
  • Figure HD depicts a IOOX microscope image of the xenograft.
  • Figure HE depicts exposed AIDS related body cavity based lymphoma xenograft after 20-day intraperitoneal Ph-EBV stock solution treatment (0.5 ml/mouse/day).
  • Figure HF depicts a IOOX microscope image of the xenograft.
  • Figure HG depicts an exposed AIDS related body cavity based lymphoma xenograft after 20-day intraperitoneal Ph-EBV treatment (350 ⁇ g/mouse/day).
  • Figure 1 IH depicts a IOOX microscope image of the xenograft.
  • Figure 111 depicts exposed nasopharyngeal cancer xenograft after 20-day intraperitoneal Ph-EBV stock solution treatment (0.5 ml/mouse/day).
  • Figure HJ depicts a IOOX microscope image of the xenograft.
  • Figure 11K depicts exposed nasopharyngeal cancer after 20-day intraperitoneal Ph-EBV treatment (350 ⁇ g/mouse/day).
  • Figure HL depicts IOOX microscope image of the xenograft. Inset: enlarged view of rectangle area where nasopharyngeal cells were undergoing coagulative necrosis (arrows).
  • Figures 12A-G show the targeting distribution of circulating FITC-labeled Ph-EBV, Ph-SA or HB-168 IgG molecules in immunodeficiency mice with EBV-infected and EBV-uninfected Burkitt Lymphoma xenografts.
  • Xenografts of a BALB/C nude mouse were inoculated either with EBV-infected (right side) or with EBV-uninfected (left side) Burkitt Lymphoma cells at respective forelimb armpits of the same mouse.
  • Figure 12A depicts the FITC-labeled Ph-EBV in vivo image at 1 hour after intraperitoneal Ph-EBV given.
  • Figure 12B depicts the image at 2 hours after intraperitoneal Ph-EBV given.
  • Figure 12C depicts the image at 3 hours after intraperitoneal Ph-EBV given.
  • Figure 12D depicts the image at 6 hours after intraperitoneal Ph-EBV given.
  • Figure 12E depicts the image at 3 hours after intraperitoneal HB-168 given.
  • Figure 12F depicts the image at 6 hours after intraperitoneal HB-168 given.
  • Figure 12G depicts the image at 6 hours after intraperitoneal Ph-SA given.
  • Figure 13A-D shows the presence or absence damage in visceral organs of regular mouse after 30-day intraperitoneal Ph-EBV treatment (700 ⁇ g/mouse/day).
  • Figure 13 A depicts a IOOX microscope image of liver.
  • Figure 13B depicts a IOOX microscope image of intestine.
  • Figure 13C depicts a IOOX microscope image of kidney.
  • Figure 13D depicts IOOX microscope image of spleen.
  • targeting agent is intended to include molecules, e.g., small molecules, peptides, and polypeptides, that specifically recognize given types of cells. These agents can bind to a polypeptide or carbohydrate expressed by a cell, e.g., on the surface of a cancer cell.
  • Nonlimiting examples of the targeting agents of the invention include antibodies, engineered antibody variants, reconstituted antibody mimetics, fragments of antibodies, single domain antibodies, phage segments or small molecules. These targeting agents can recognize a polypeptide expressed by a cancerous cell or a viral peptide expressed by a cancerous cell.
  • Exemplary, non-limiting, cell produced polypeptides include Her2, estrogen receptor, progesterone receptor, STEAP, carcinoembryonic antigen, prostate carcinoma tumor antigen and T-antigen.
  • the targeting agent may be a ligand that binds to a receptor expressed by a cancer cell, e.g., estrogen receptor.
  • the term "channel-forming moiety" is intended to include transmembrane polypeptides that are capable of inserting into a lipid bilayer thereby creating a gating passageway, from inside of the cell compartment to outside of the cell compartment.
  • the gating passageway is not specific in term of what is permitted to pass through the channel.
  • channel-forming moieties are polypeptides that naturally form channels in lipid bilayers, i.e., ⁇ -hemolysin, delta toxin, diphtheria toxin, anthrax toxin, and El family colicin
  • Channel-forming moieties can also be fragments of naturally occurring polypeptides that retain the ability to insert into a lipid bilayer and form a channel.
  • One of skill in the art would be able to isolate many fragments of naturally occurring polypeptides that have the ability to form a channel. Those channel-forming fragments are intended to be used in the methods and compositions of the instant invention.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen binding sites which specifically bind (immunoreacts with) an antigen.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme, such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies.
  • the term "reconstituted antibody mimetic” refers to a targeting agent comprising one or more antibody complementarity determining regions (CDRs) covalently linked to, for example, a polypeptide linker.
  • V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2, or VLCDR3 regions from light or heavy chains of an antibody fragment may be covalently attached to confer binding affinity, i.e., sufficient binding ability to allow the molecules of the invention to target a cell and allow for insertion of the channel-forming domain into the lipid bilayer.
  • the CDRs may be linked together directly, by using random peptides, or by using me natural linking peptides present in antibody heavy or light chains.
  • the CDR regions are generally from an antibody that specifically recognizes an epitope presented by a target cell.
  • the reconstituted antibody mimetic or variants are a complementarity determining region pair (VHCDRI and VLCDR3) selected from the antibody fragment linked by natural VHCDR1/VHCDR2 linker.
  • VHCDRI and VLCDR3 complementarity determining region pair
  • the terms "protein” and “polypeptide” are used interchangeably herein.
  • the term "peptide” is used herein to refer to a chain of two or more amino acids or amino acid analogs (including non-naturally occurring amino acids and amino acid analogues), with adjacent amino acids joined by peptide (-NHCO-) bonds.
  • peptides in accordance with the invention include oligopeptides, polypeptides, proteins, and peptidomimetics.
  • fragment refers to any portion of a natural, recombinant or synthetic polypeptide. A fragment can be made synthetically, enzymatically, or recombinantly.
  • pharmaceutical composition includes preparations suitable for administration to mammals, e.g., humans. When the compounds of the present invention are administered as pharmaceuticals to mammals, e.g., humans, they can be given as is or as a pharmaceutical composition containing, for example, 0.1 to 99.5% of active ingredient in combination with a pharmaceutical acceptable carrier. Pharmaceutical compositions of the current invention may further contain, for example a biocide, antimicrobial, or antibiotic.
  • the term "cell proliferative disorder” is intended to include diseases or disorders characterized by abnormal cell growth, e.g., cancer.
  • abnormal cell growth is intended to include cell growth which is undesirable or inappropriate.
  • Abnormal cell growth also includes proliferation which is undesirable or inappropriate (e.g., unregulated cell proliferation or undesirably rapid cell proliferation).
  • Abnormal cell growth can be benign and result in benign masses of tissues or cells, or benign tumors.
  • Abnormal cell growth can also be malignant and result in malignancies, malignant masses of tissues or cells, or malignant tumors. Many art-recognized conditions and disorders are associated with malignancies, malignant masses, and malignant tumors including cancer and carcinoma.
  • tumor is intended to encompass both in vitro and in vivo tumors that form in the body. Tumors may be associated with benign abnormal cell growth (e.g., benign tumors) or malignant cell growth (e.g., malignant tumors).
  • benign abnormal cell growth e.g., benign tumors
  • malignant cell growth e.g., malignant tumors.
  • Cancer includes a malignant neoplasm characterized by deregulated or uncontrolled cell growth.
  • the term “cancer” includes primary malignant tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor) and secondary malignant tumors (e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site pf the original tumor).
  • Cancer may develop in, for example, the following tissues: larynx, prostate, stomach, skin, oral cavity, pharynx, esophagus, liver, lung, head, neck, bronchus, pancreas, small intestine, colon, rectum, breast, bladder, uterus, brain, lymph system, blood, ovaries, kidneys, or soft tissue.
  • Anaplasia Malignant neoplasms often contain numerous mitotic cells. These cells are typically abnormal. Such mitotic aberrations account for some of the karyotypic abnormalities found in most cancers. Bizarre multinucleated cells are also seen in some cancers, especially those which are highly anaplastic. "Dysplasia” refers to a pre-malignant state in which a tissue demonstrates histologic and cytologic features intermediate between normal and anaplastic. Dysplasia is often reversible.
  • Anaplasia refers to the histological features of cancer. These features include derangement of the normal tissue architecture, the crowding of cells, lack of cellular orientation termed dyspolarity, cellular heterogeneity in size and shape termed “pleomorphism.”
  • the cytologic features of anaplasia include an increased nuclear-cytoplasmic ratio (the nuclear-cytoplasmic ratio can be over 50% for malignant cells), nuclear pleomorphism, clumping of the nuclear chromatin along the nuclear membrane, increased staining of the nuclear chromatin, simplified endoplasmic reticulum, increased free ribosomes, pleomorphism of mitochondria, decrease in size and number of organelles, enlarged and increased numbers of nucleoli, and sometimes the presence of intermediate filaments.
  • Neoplasmsis or "neoplastic transformation” is the pathologic process that results in the formation and growth of a neoplasm, tissue mass, or tumor. Such process includes uncontrolled cell growth, including either benign or malignant tumors. Neoplasms include abnormal masses of tissue, the growth of which exceeds and is uncoordinated with that of the normal tissues and persists in the same excessive manner after cessation of the stimuli which evoked the change. Neoplasms may show a partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue. [0058] Neoplasms tend to morphologically and functionally resemble the tissue from which they originated.
  • neoplasms arising within the islet tissue of the pancreas resemble the islet tissue, contain secretory granules, and secrete insulin.
  • Clinical features of a neoplasm may result from the function of the tissue from which it originated.
  • benign neoplasms may attain enormous size, they remain discrete and distinct from the adjacent non-neoplastic tissue.
  • Benign tumors are generally well circumscribed and round, have a capsule, and have a grey or white color, and a uniform texture.
  • malignant tumor generally have fingerlike projections, irregular margins, are not circumscribed, and have a variable color and texture. Benign tumors grow by pushing on adjacent tissue as they grow. As the benign tumor enlarges it compresses adjacent tissue, sometimes causing atrophy. The junction between a benign tumor and surrounding tissue may be converted to a fibrous connective tissue capsule allowing for easy surgical remove of benign tumors.
  • malignant tumors are locally invasive and grow into the adjacent tissues usually giving rise to irregular margins that are not encapsulated making it necessary to remove a wide margin of normal tissue for the surgical removal of malignant tumors.
  • Benign neoplasms tends to grow more slowly than malignant tumors. Benign neoplasms also tend to be less autonomous than malignant tumors. Benign neoplasms tend to closely histologically resemble the tissue from which they originated. More highly differentiated cancers, cancers that resemble the tissue from which they originated, tend to have a better prognosis than poorly differentiated cancers. Malignant tumors are more likely than benign tumors to have an aberrant function (i.e. the secretion of abnormal or excessive quantities of hormones).
  • subject includes organisms which can suffer from cancer. The term subject includes mammals, e.g., horses, monkeys, bears, dogs, cats, mice, rabbits, cattle, squirrels. Rats, and, preferably, humans.
  • the present invention provides molecules, e.g., fusion molecules, comprising a targeting agent and a channel-forming moiety.
  • the targeting agent can be a small molecule, peptide, e.g., an antibody, or peptidomimetic.
  • the polypeptides of the invention are threefold: a polypeptides comprising a targeting agent, a polypeptide comprising a channel-forming moiety, and a polypeptide comprising a targeting agent and a channel-forming moiety.
  • the channel-forming moiety is a channel-forming peptide, a channel-forming domain, or fragment thereof.
  • the channel-forming peptide can be ⁇ -hemolysin, delta toxin, diphtheria toxin, anthrax toxin, and El family colicin.
  • the colicin is El , Ia, Ib, A, K or N.
  • the colicin is colicin Ia.
  • the channel-forming fragment is a channel-forming colicin comprising amino acid residues from about 1 to about amino acid 626 of colicin Ia (SEQ ID NO: 1)
  • the channel-forming domain comprises amino acid residues 451-626 (SEQ ID NO:3) having the nucleic acid sequence of SEQ ID NO:4.
  • Targeting agents of the invention serve to bring a fusion molecule in close proximity to the surface of a cell, e.g., a cancer cell.
  • One class of targeting agents of the invention are small molecules that bind to receptors expressed on the surface of an cell.
  • a second class of targeting agents are peptides, or polypeptides, that specifically bind to proteins on a cell surface, e.g., an antibody.
  • the peptides are molecules that bind to a cell surface receptor.
  • the peptides are molecules that bind to a cell surface antigen, e.g., antibodies, reconstituted antibodies, ScFvs, or fragments thereof.
  • a preferred polypeptide that is capable of acting as a targeting agent is an antibody or a reconstituted antibody mimetic.
  • Antibody fragments e.g., Fab fragments or reconstituted antibody mimetics, preferably retain the ability to specifically recognize a target cell.
  • VHCDRI and V L CDR3 selected from the antibody fragment linked by natural
  • VHCDRI /VBCDKZ linker is capable of acting as such a targeting agent.
  • Reconstituted antibody mimetic molecules that specifically recognize an antigen on the cell surface of a cell can be used as the targeting agent.
  • reconstituted antibody mimetic molecules that recognize viral peptides e.g., herpes virus particles can be used as a targeting moiety of the invention.
  • Exemplary, non-limiting mimetic molecules that are contemplated for use in the methods and compositions of the invention are the variants of complementarity determining regions pairs selected from the antibody fragment against Epstein-Barr virus gp350/220 envelope glycoprotein.
  • the reconstituted antibody mimetic comprises a VHCDR1/V L CDR3 pair of ATCC HB-168 monoclonal IgG fragment linked to the HB-168
  • VHCDRI /V H CDR2 linker to produce V H CDR1- V H CDR1/CDR2 linker- V L CDR3 having the amino acid sequence of SEQ ID NO:5 (SFGMHWVRQAPEKGLEWVAGQGY
  • SYPYT and the nucleic acid sequence of SEQ ID NO: 6. See Example 1 below.
  • the reconstituted antibody mimetic comprises V H CDRI- VHCDR1/CDR2 linker- VHCDR3 having the amino acid sequence of
  • SEQ ID NO: 13 and a nucleic acid sequence of SEQ ID NO: 14.
  • the reconstituted antibody mimetic comprises V H CDR1- V H CDR2-V L CDR2 having the amino acid sequence of SEQ ID NO: 15 and a nucleic acid sequence of SEQ ID NO: 16.
  • the reconstituted antibody mimetic comprises V L CDRI- V H CDRI /CDR2 linker- VHCDRI having the amino acid sequence of
  • SEQ ID NO: 17 and a nucleic acid sequence of SEQ ID NO: 18.
  • the reconstituted antibody mimetic comprises V L CDR1- V L CDR1/CDR2 linker-V H CDR3 having the amino acid sequence of
  • SEQ ID NO: 19 and a nucleic acid sequence of SEQ ID NO:20.
  • the reconstituted antibody mimetic comprises V H CDRI having the amino acid sequence of SEQ ID NO:21 and a nucleic acid sequence of SEQ ID NO:22.
  • Another preferred class of targeting agents are antibodies against target cell surface polypeptides that are differentially expressed in cancer cells.
  • a number of polypeptides are known that are expressed at higher levels in cancer cells than in normal non-cancerous cells.
  • Exemplary polypeptides that are differentially expressed in one or more cancerous tissues are Her2, estrogen receptor, progesterone receptor, STEAP, carcinoembryonic antigen, prostate carcinoma tumor antigen and T-antigen.
  • the molecules comprising a targeting agent and a channelrforming moiety are polypeptides having the amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO: 11, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35 and SEQ ID NO:37.
  • polypeptides are encoded by nucleic acid sequences selected from the group consisting of SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36 and SEQ ID NO:38, respectively.
  • the polypeptides of the invention comprising a targeting agent and a channel forming moiety have a molecular weight from about 25,000 daltons to about 32,000 daltons.
  • vectors preferably expression vectors, containing a nucleic acid molecule encoding a polypeptide of the invention (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector is another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors are often in the form of plasmids. In the present specification, "plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g
  • the recombinant expression vectors of the invention comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid molecule in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cell to be used for expression, which are operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Acedemic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., polypeptides comprising a targeting domain and a channel-forming polypeptide).
  • the recombinant expression vectors of the invention can be designed for expression of the polypeptides of the invention in prokaryotic or eukaryotic cells.
  • the polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vector) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Acedemic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Such enzymes, and their cognate recognition sequences including Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • GST glutathione S-transferase
  • Suitable inducible non-fusion E.coli expression vectors include pTrc(Amann et al., (1988) Gene 69:301-315) and pET 1 Id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Acedemic Press, San Diego, CA (1990) 69-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion pomoter.
  • Target gene expression from the pET Hd vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral IUSTA polymerase (T7gnl). This viral polymerase is supplied by host strains BL21(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E.coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Acedemic Press, San Diego, CA (1990) 119-128).
  • Another strategy is to alter the nucleic acid sequences of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E.coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSec 1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJGRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Co ⁇ . San Diego, CA), and picZ (Invitrogen Corp. San Diego, CA).
  • the polypeptides can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include thepAc series (Smith et al. (1983) MoI. CellBiol 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • viral regulatory elements For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.F., and Maniatis, T, Molecular Cloning: A Laboratory Manual. 2" , ed., Cold Spring Harbor Laboratory ⁇ Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in art.
  • suitable tissue-specific promoters include the albumin promoter (live-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Lmmunol. 43:235-275), in particular promoters of T-cell receptors (Winoto and Baltimore (1989) EMBO J.
  • promoters are also encompassed, for example the murine box promoters (Kessek and Gruss (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • Another aspect of the invention pertains to host cells into which a nucleic acid molecule encoding a polypeptide of the invention is introduced within a recombinant expression vector or a nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a polypeptide of the invention can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • CHO Chinese hamster ovary cells
  • COS cells Chinese hamster ovary cells
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or eletroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. ⁇ Molecular Cloning: A Laboratory Manual. 2 nd , ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • a nucleic acid encoding the polypeptide of the invention can be introduced on a separate vector.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) the polypeptides of the invention.
  • the invention further provides methods for producing polypeptides using the host cells of the invention.
  • the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that a polypeptide of the invention is produced.
  • the method further comprises isolating the polypeptide from the medium or the host cell.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous sequences have been introduced into their genome or homologous recombinant animals in which endogenous sequences have been altered.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as rat or mouse, in which one or more of the cells of the animal includes transgene.
  • transgenic animals include non-human primates sheep, dogs, cows, goats, chickens, amphibians, and the like.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing a nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the cDNA sequence encoding a polypeptide of the invention can be introduced as a transgene into the genome of a non-human animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to a transgene to direct expression of a protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of a transgene in its genome and/or expression of mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a protein can further be bred to other transgenic animals carrying other transgenes.
  • transgenic non-humans animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage Pl .
  • Cre/loxP recombinase system of bacteriophage Pl .
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355.
  • l ⁇ a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the ere recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO97/07668 and WO97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • molecules of the invention may be made recombinantly using the nucleic acid molecules, vectors, and host cells described above.
  • the targeting moiety can be made synthetically, or isolated from a natural source and linked to the channel-forming moiety using methods and techniques well known to one of the skill in the art.
  • the peptides may be made, e.g., synthetically or recombinantly, to include one or more peptide analogs or mimetics.
  • Exemplary peptides can be synthesized to include D-isomers of the naturally occurring amino acid residues to increase the half life of the molecule when administered to a subject.
  • compositions suitable for administration typically comprise the active compound and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and anitfungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except in so far as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • PH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered salines (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredients plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch, a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch, a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as suc
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal spray or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polyacetic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ration between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5O/ED5O.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially form cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • the present invention provides therapeutic methods of treating a subject having a cell proliferative disorder.
  • the cell proliferative disorder is cancer.
  • the cell proliferative disorder is a viral associated cancer.
  • treating refers to the administration of a therapeutically effective amount of an active compound to a subject, for prophylactic and/or therapeutic purposes.
  • administration includes delivery to a subject, e.g., by any appropriate method which serves to deliver the drug to the site of the cancer.
  • Administration of the drug can be, e.g., oral, intravenous, or topical (as described in further detail below).
  • a subject having a cell proliferative disorder is treated with an effective amount of an active compound of the invention such that the cell proliferative disorder is treated.
  • the subject that is being treated has cancer.
  • composition of the invention is administered to a subject in combination with additional agents, e.g., other compositions useful for treating cell proliferative disorders.
  • composition of the invention can be administered to a subject in need of treatment in an effective amount using the pharmaceutical compositions described herein.
  • Ph-EBV pheromonicin-EBV
  • a second fusion peptide that has been denominated as pheromonicin-CNCV was created by incorporating a peptide chain of colicin Ia with a phage segment of filamentous bacteriophage M13, TLTTKLY (SEQ ID NO:9) having the nucleic acid sequence of SEQ ID NO : 10, and was introduced following the C-terminus of colicin Ia (1626) to form a 633 residue peptide (SEQ ID NO: 11) having the polynucleotide sequence of SEQ ID N0:12.
  • Ph-EBV and Ph-CNCV both demonstrated cytocidal effects on EBV-infected rumor cells (Ragi cell) in vitro ( Figure 3, 4 and 6, respectively). In contrast, neither peptide demonstrated cytocidal effects on normal mammalian cells and malignant cells with no virus gene transferred ( Figure 5, 7, 8, and 9, respectively).
  • Ph-EBV and Ph-CNCV were added to human Burkitt malignant lymphoma cell cultures at concentration of 50 ⁇ g/ml ( Figure 3B and 4B).
  • An equivalent amount of PBS stock solution pH7.4, 10 mM PBS, 0.2M NaCl
  • 50 ⁇ g/ml Ph-EBV killed about 60-70%
  • 50 ⁇ g/ml Ph-CNCV killed about 30-40% of the lymphoma cells after 72 hours of incubation.
  • Example 2 In vivo Elimination Effects against EBV-associated human tumor xenografts in immunodeficiency mice
  • solid tumor models were set up in two species of immunodeficiency mice by inoculation either EBV-infected or uninfected human tumor cells to identify whether pheromonicin inhibited the proliferation of solid tumors.
  • SCID Beige mice neoplasms were inoculated either with EBV-infected ATCC CCL-86 (left side) or with EBV-uninfected ATCC CRL- 1648 (right side) Burkitt Lymphoma cells respectively at forelimb armpits of the same mouse and grew up to 1 x 1 x 1 mm in 7 days. The 20-d treatment was started eight days after the tumor cell inoculation.
  • mice Microscopic examination demonstrated that mass proliferation existed in all CRL-1648 neoplasms of treated mice, CRL-1648 and CCL-86 neoplasms of controls (Fig. lOC-E). Whereas either coagulative necrosis spread to the entire cell population or only necrotic entities remained in the CCL-86 neoplasms of treated mice (Fig. 10F).
  • Fig. 10F In BALB/C nude mice, only EB V-infected ATCC CCL-86 inoculated at right forelimb armpit area. The 20-d intraperitoneal pheromonicin treatment started at the 8 th day of inoculation while the neoplasms grew up to 1 x 1 x 1 mm in all mice.
  • Autopsy found that either neoplasm were smaller than l x l x l mm in 7 of them or no visible neoplasm detected in the rest three (Fig.11 C).
  • Fig. HD Microscopic examination demonstrated that only necrotic entities remained in either visible or microscopic neoplasm with pheromonicin treatment (Fig. HD). Three in vivo assays were done for control and pheromonicin treatment respectively.
  • AIDS related body cavity based lymphoma ATCC CRL-2230 strain, EBV+)(AIDSL) and nasopharyngeal cancer (TNE-I strain, EBV+, Institute of Cancer Research, Hunan University of Medical Sciences, Changsha, Hunan, China)(NC) cells were inoculated into BALB/C nude mice respectively and mice were treated as to what performed in the BL experiment.
  • Fig. 11 G-H shows that pheromonicin presented identical elimination effects in the ATDSL neoplasm.
  • the NC neoplasm was not eliminated so keen as to what occurred in the BL and AIDSL cases.
  • encapsulated NC cells were undergoing coagulative necrosis in the shrunk NC neoplasm (Inset of Fig. 1 IL).
  • [0118] Taken together, there were 30 treated cases and 30 control cases in each BL, AIDSL and NC experiments. Total weight of all visible neoplasms in control or treated experiments (no visible neoplasm were counted as zero) was added up for quantitative analysis. There were statistically significance differences in total weight of neoplasms between control and treated BL neoplasm (p ⁇ 0.001), AIDSL neoplasm (p O.001) and NC neoplasm (p ⁇ 0.001).
  • Example 3 In vivo Penetration Effects into solid tumor model in immunodeficiency mice [0119] In vivo distribution of circulating pheromonicin molecules were observed with fluorescent-labeling imagine system in the animals with an inoculated xenograft. One hour after intraperitoneal injection, the FITC-labeled pheromonicin molecules started to accumulate inside the implanted solid tumor of EBV-induced cells. The accumulation lasted approximately 6 hours. At least half of the systemic distributed pheromonicin had been cleared by approximately 4 hours after injection through urination ( Figures A-D). Conversely, there was no accumulation in the implanted solid tumor of EBV-uninfected cells.

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Abstract

La présente invention concerne des molécules capables de tuer des cellules. Ces molécules comprennent un agent de ciblage et une partie de formation de canal. Les molécules peuvent être des polypeptides. Cette invention concerne également des séquences polynucléotidiques codant lesdits polypeptides. Dans un mode de réalisation préféré, la partie de formation de canal comprend une colicine et l'agent de ciblage est un anticorps. En outre, cette invention concerne des procédés de traitement qui consistent à administrer lesdites molécules.
PCT/IB2006/001431 2006-01-17 2006-01-17 Molécules antivirales bifonctionnelles, procédés pour les produire et procédés pour traiter un cancer induit par virus au moyen de celles-ci Ceased WO2007083175A1 (fr)

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WO2013177231A1 (fr) * 2012-05-21 2013-11-28 Massachusetts Institute Of Technology Translocation d'entités chimiques artificielles par un pore d'antigène protecteur contre l'anthrax
US9498538B2 (en) 2012-05-21 2016-11-22 Massachusetts Institute Of Technology Translocation of non-natural chemical entities through anthrax protective antigen pore

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