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WO2020014539A1 - Methods and compositions for targeting cancer cells for treatment - Google Patents

Methods and compositions for targeting cancer cells for treatment Download PDF

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Publication number
WO2020014539A1
WO2020014539A1 PCT/US2019/041484 US2019041484W WO2020014539A1 WO 2020014539 A1 WO2020014539 A1 WO 2020014539A1 US 2019041484 W US2019041484 W US 2019041484W WO 2020014539 A1 WO2020014539 A1 WO 2020014539A1
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WIPO (PCT)
Prior art keywords
seq
deletion
site
pharmaceutical composition
cancer
Prior art date
Application number
PCT/US2019/041484
Other languages
French (fr)
Inventor
Michael J. Abrams
Christopher Larson
Tony R. REID
Bryan T. Oronsky
Original Assignee
Epicentrx, Inc.
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Publication date
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Publication of WO2020014539A1 publication Critical patent/WO2020014539A1/en

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    • 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
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • 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
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
    • A61K39/001195—Prostate specific membrane antigen [PSMA]
    • 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
    • A61K47/66—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • 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
    • A61K51/0489—Phosphates or phosphonates, e.g. bone-seeking phosphonates
    • 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
    • A61K51/0495—Pretargeting
    • 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
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10332—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10371—Demonstrated in vivo effect
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the field of the invention is methods and compositions for treating cancer.
  • viruses have shown promise as oncolytic agents that not only directly destroy malignant cells via an infection-to-reproduction-to-lysis chain reaction but also indirectly induce anti-tumor immunity. These immune stimulatory properties have been augmented with the insertion of therapeutic transgenes that are copied and expressed each time the virus replicates.
  • Previously developed oncolytic viruses include the oncolytic serotype 5 adenovirus (Ad5) referred to as TAV-255 that is transcriptionally attenuated in normal cells but transcriptionally active in cancer cells (see, PCT Publication No. W02010/101921). It is believed that the mechanism by which the TAV-255 vector achieves such tumor selectivity is through targeted deletion of three transcriptional factor (TF) binding sites for the transcription factors Pea3 and E2F, proteins that regulate adenovirus expression of Ela, the earliest gene to be transcribed after virus entry into the host cell, through binding to specific DNA sequences.
  • TF transcriptional factor
  • the invention is based, in part, upon the discovery that a delivery vehicle, e.g., a recombinant oncolytic virus, can be used to selectively express a protein defining a binding site or a protein that mediates the production of a biomolecule defining a binding site in a cancer cell, and thereby target the cancer cell for a treatment by a therapeutic agent that binds to the binding site.
  • a delivery vehicle e.g., a recombinant oncolytic virus described herein, can be used to selectively express a bone morphogenetic protein (BMP) in a cancer cell and mediate the production of bone tissue in a tumor.
  • BMP bone morphogenetic protein
  • a BMP in a cancer cell allows for treatment of a cancer by targeting a therapeutic agent that binds to bone tissue, e.g., a bone targeting radionuclide, to a tumor.
  • a therapeutic agent that binds to bone tissue
  • the delivery vehicle e.g., the recombinant oncolytic virus
  • the invention provides a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject: (a) an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and (b) an effective amount of a therapeutic agent that binds to the binding site.
  • the biomolecule defining a binding site is bone tissue, and/or the protein that mediates the production of the biomolecule defining a binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR), osteocalcin, osteopontin, bone sialoprotein, osteonectin, osteoprotegerin (OPG) and alkaline phosphatase.
  • the protein defining the binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR) and prostate specific membrane antigen (PSMA).
  • the therapeutic agent comprises a radionuclide or an antibody.
  • the radionuclide is a bone targeting radionuclide, e.g., 89 Sr (e.g., 89 Sr chloride), 223 Ra (e.g, 223 Ra dichloride), 153 Sm (e.g, 153 Sm-EDTMP), 177 Lu, 90 Y, 186 Re (e.g., 186 Re HEDP), 117m Sn (e.g, 117m Sn DTP A), and 32 P.
  • the antibody is an antibody drug conjugate or an antibody radionuclide conjugate.
  • the invention provides a method of treating bone loss and/or hypercalcemia in a subject in need thereof, the method comprising administering to the subject an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding a protein that mediates the production of bone tissue.
  • the subject has cancer.
  • the invention provides a method of generating bone tissue in a subject, the method comprising administering to the subject an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding a protein that mediates the production of bone tissue.
  • the protein that mediates the production of bone is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR), osteocalcin, osteopontin, bone sialoprotein, osteonectin, osteoprotegerin (OPG) and alkaline phosphatase.
  • BMP bone morphogenetic protein
  • BMPR bone morphogenetic protein receptor
  • osteocalcin osteopontin
  • bone sialoprotein osteonectin
  • osteoprotegerin OPG
  • alkaline phosphatase alkaline phosphatase
  • the BMP is selected from BMP -2, BMP-4, BMP-6, and BMP-7.
  • the BMP comprises SEQ ID NO: 1 or a functional fragment thereof, SEQ ID NO: 2 or a functional fragment thereof, SEQ ID NO: 3 or a functional fragment thereof, or SEQ ID NO: 4 or a functional fragment thereof, or an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
  • the delivery vehicle is a recombinant oncolytic virus.
  • the recombinant oncolytic virus is selected from an adenovirus, fowlpox virus, lentivirus, herpes virus, vesicular stomatitis virus (VSV), maraba virus, polio virus, and alpha virus.
  • the recombinant virus is a recombinant oncolytic adenovirus, e.g., a type 5 adenovirus (Ad5).
  • the exogenous nucleotide sequence is inserted into an Elb-l9K insertion site located between the start site of Elb-l9K and the start site of Elb-55K.
  • the Elb-l9K insertion site is located between the start site of Elb-l9K and the stop site of Elb-l9K.
  • the Elb-l9K insertion site comprises a deletion of from about 100 to about 305, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 305, about 150 to about 300, about 150 to about 250, or about 150 to about 200 nucleotides adjacent the start site of Elb-l9K.
  • the Elb-l9K insertion site comprises a deletion of about 200 nucleotides, e.g., 203 nucleotides adjacent the start site of Elb-l9K.
  • the Elb-l9K insertion site comprises a deletion corresponding to nucleotides 1714-1916 of the Ad5 genome (SEQ ID NO: 5), or, the exogenous nucleotide sequence is inserted between nucleotides corresponding to 1714 and 1916 of the Ad5 genome (SEQ ID NO: 5).
  • the exogenous nucleotide sequence is inserted between CTGACCTC (SEQ ID NO: 6) and TCACCAGG (SEQ ID NO: 7), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CTGACCTC (SEQ ID NO: 6), the exogenous nucleotide sequence, and
  • the exogenous nucleotide sequence is inserted into an E3 insertion site located between the stop site of pVIII and the start site of Fiber.
  • the E3 insertion site comprises a deletion of from about 500 to about 3185, from about 500 to about 3000, from about 500 to about 2500, from about 500 to about 2000, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 3185, from about 1000 to about 3000, from about 1000 to about 2500, from about 1000 to about 2000, from about 1000 to about 1500, from about 1500 to about 3185, from about 1500 to about 3000, from about 1500 to about 2000, from about 2000 to about 3185, from about 2000 to about 3000, from about 2000 to about 2500, from about 2500 to about 3185, from about 2500 to about 3000, or from about 3000 to about 3185 nucleotides.
  • the E3 insertion site is located between the stop site of E3-10.5K and the stop site of E3-14.7K. In certain embodiments, the E3 insertion site comprises a deletion of from about 500 to about 1551, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 1551, from about 1000 to about 1500, or from about 1500 to about 1551 nucleotides adjacent the stop site of E3-10.5K. In certain embodiments, the E3 insertion site comprises a deletion of about 1050 nucleotides adjacent the stop site of E3-10.5K, e.g., the E3 insertion site comprises a deletion of 1063 or 1064 nucleotides adjacent the stop site of E3-10.5K.
  • the E3 insertion site comprises a deletion corresponding to the Ad5 dl309 E3 deletion. In certain embodiments, the E3 insertion site comprises a deletion corresponding to nucleotides 29773-30836 of the Ad5 genome (SEQ ID NO: 5), or, the exogenous nucleotide sequence is inserted between nucleotides corresponding to 29773 and 30836 of the Ad5 genome (SEQ ID NO: 5).
  • the exogenous nucleotide sequence is inserted between CAGTATGA (SEQ ID NO: 8) and TAATAAAAAA (SEQ ID NO: 9), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CAGTATGA (SEQ ID NO: 8), the exogenous nucleotide sequence, and TAATAAAAAA (SEQ ID NO: 9).
  • the recombinant oncolytic adenovirus may comprise a deletion of at least one Pea3 binding site, or a functional portion thereof, e.g., the virus may comprise a deletion of nucleotides
  • the recombinant adenovirus may comprise a deletion of nucleotides corresponding to 195-244 of the Ad5 genome (SEQ ID NO: 5), and/or the recombinant adenovirus may comprise the sequence GGTGTTTTGG (SEQ ID NO: 10).
  • the recombinant oncolytic adenovirus may comprise a deletion of at least one Pea3 binding site, or a functional portion thereof, and not comprise a deletion of an E2F binding site.
  • the recombinant oncolytic adenovirus may comprise a deletion of at least one E2F binding site, or a functional portion thereof. In certain embodiments, the recombinant oncolytic adenovirus may comprise a deletion of at least one E2F binding site, or a functional portion thereof, and not comprise a deletion of a Pea3 binding site.
  • the recombinant oncolytic adenovirus may comprise an Ela promoter having a deletion of a functional TATA box, e.g., the deletion of an entire TATA box.
  • the virus may comprise a deletion of nucleotides corresponding to -29 to -26, -33 to -26, -44 to +52, or -148 to +52 of the Ela promoter.
  • the deletion comprises a deletion of nucleotides corresponding to 353-552 of the Ad5 genome (SEQ ID NO: 5), and/or the Ela promoter comprises the sequence CTAGGACTG (SEQ ID NO: 11).
  • the exogenous nucleotide sequence is not operably linked to an exogenous promoter sequence.
  • the recombinant oncolytic adenovirus may selectively replicate in a hyperproliferative cell.
  • the recombinant oncolytic adenovirus may selectively express the protein defining a binding site or the protein that mediates the production of a biomolecule defining a binding site in a hyperproliferative cell.
  • the hyperproliferative cell may be a cancer cell, e.g., a lung cancer cell, a colon cancer cell, and a pancreatic cancer cell.
  • the recombinant oncolytic adenovirus comprises a nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the recombinant oncolytic virus further comprises an exogenous nucleotide sequence encoding a
  • costimulatory molecule an antiangiogenic and/or antivascular molecule, an anti-fibrotic molecule, an anti-inflammatory or pro-inflammatory molecule, a monoclonal antibody or fragment thereof, a pro-apoptotic molecule, an enzyme, an immunomodulatory molecule, a cytokine, or a cytokine inhibitor (e.g., a cytokine trap).
  • the delivery vehicle e.g. , a recombinant oncolytic virus
  • the delivery vehicle e.g., a recombinant oncolytic virus
  • the therapeutic agent are administered in combination with one or more therapies selected from surgery, radiation, chemotherapy, immunotherapy, hormone therapy, and virotherapy.
  • the cancer is selected from melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, head and neck cancer, breast cancer, anal cancer, cervical cancer, non-small cell lung cancer, mesothelioma, small cell lung cancer, renal cell carcinoma, prostate cancer, gastroesophageal cancer, colorectal cancer, testicular cancer, bladder cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, brain and central nervous system cancer, thyroid cancer, parathyroid cancer (e.g., parathyroid carcinoma), endometrial cancer, neuroendocrine cancer, lymphoma (e.g., Hodgkin and non- Hodgkin), leukemia, merkel cell carcinoma, gastrointestinal stromal tumors, multiple myeloma, uterine cancer, a sarcoma, kidney cancer, ocular cancer, pancreatic cancer, and a germ cell cancer (e.g., ovarian germ cell cancer).
  • melanoma
  • the delivery vehicle and/or the therapeutic agent are administered in combination with a bisphosphonate, hyaluronic acid, vitamin D, calcium, testosterone, estrogen, progesterone, a selective estrogen modulator (SERM), calcitonin, a RANK or RANKL inhibitor (e.g., denosumab), pulsed ultrasound (e.g., low intensity pulsed ultrasound (LIPU)), electrical stimulation,
  • a bisphosphonate, hyaluronic acid, vitamin D, calcium, testosterone, estrogen, progesterone a selective estrogen modulator (SERM), calcitonin, a RANK or RANKL inhibitor (e.g., denosumab), pulsed ultrasound (e.g., low intensity pulsed ultrasound (LIPU)), electrical stimulation
  • SERM selective estrogen modulator
  • ESWT extracorporeal shockwave therapy
  • PEMF pulsed electromagnetic field
  • the effective amount of the recombinant oncolytic virus is 10 2 -10 15 plaque forming units (pfiis).
  • the subject can, e.g., be a human, e.g., a pediatric human, or an animal.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (a) an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and (b) optionally, an effective amount of a therapeutic agent that binds to the binding site.
  • the biomolecule defining a binding site is bone tissue, and/or the protein that mediates the production of the biomolecule defining a binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR), osteocalcin, osteopontin, bone sialoprotein, osteonectin, osteoprotegerin (OPG) and alkaline phosphatase.
  • BMP bone morphogenetic protein
  • BMPR bone morphogenetic protein receptor
  • osteocalcin osteopontin
  • bone sialoprotein osteonectin
  • osteoprotegerin OPG
  • alkaline phosphatase alkaline phosphatase
  • the protein defining the binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR) and prostate specific membrane antigen (PSMA).
  • BMP bone morphogenetic protein
  • BMPR bone morphogenetic protein receptor
  • PSMA prostate specific membrane antigen
  • the BMP comprises SEQ ID NO: 1 or a functional fragment thereof, SEQ ID NO: 2 or a functional fragment thereof, SEQ ID NO: 3 or a functional fragment thereof, or SEQ ID NO: 4 or a functional fragment thereof, or an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
  • the therapeutic agent comprises a radionuclide or an antibody.
  • the radionuclide is a bone targeting radionuclide, e.g., 89 Sr (e.g., 89 Sr chloride), 223 Ra (e.g., 223 Ra dichloride), 153 Sm (e.g, 153 Sm-EDTMP), 177 Lu, 90 Y, 186 Re (e.g., 186 Re HEDP), 117m Sn (e.g, 117m Sn DTP A), and 32 P.
  • the antibody is an antibody drug conjugate or an antibody radionuclide conjugate.
  • the delivery vehicle is a recombinant oncolytic virus.
  • the recombinant oncolytic virus is selected from an adenovirus, fowlpox virus, lentivirus, herpes virus, vesicular stomatitis virus (VSV), maraba virus, polio virus, and alpha virus.
  • the recombinant virus is a recombinant oncolytic adenovirus, e.g., a type 5 adenovirus (Ad5).
  • the exogenous nucleotide sequence is inserted into an Elb-l9K insertion site located between the start site of Elb-l9K and the start site of Elb-55K.
  • the Elb-l9K insertion site is located between the start site of Elb-l9K and the stop site of Elb-l9K.
  • the Elb-l9K insertion site comprises a deletion of from about 100 to about 305, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 305, about 150 to about 300, about 150 to about 250, or about 150 to about 200 nucleotides adjacent the start site of Elb-l9K.
  • the Elb-l9K insertion site comprises a deletion of about 200 nucleotides, e.g., 203 nucleotides adjacent the start site of Elb-l9K.
  • the Elb-l9K insertion site comprises a deletion corresponding to nucleotides 1714-1916 of the Ad5 genome (SEQ ID NO: 5), or, the exogenous nucleotide sequence is inserted between nucleotides corresponding to 1714 and 1916 of the Ad5 genome (SEQ ID NO: 5).
  • the exogenous nucleotide sequence is inserted between CTGACCTC (SEQ ID NO: 6) and TCACCAGG (SEQ ID NO: 7), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CTGACCTC (SEQ ID NO: 6), the exogenous nucleotide sequence, and
  • the exogenous nucleotide sequence is inserted into an E3 insertion site located between the stop site of pVIII and the start site of Fiber.
  • the E3 insertion site comprises a deletion of from about 500 to about 3185, from about 500 to about 3000, from about 500 to about 2500, from about 500 to about 2000, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 3185, from about 1000 to about 3000, from about 1000 to about 2500, from about 1000 to about 2000, from about 1000 to about 1500, from about 1500 to about 3185, from about 1500 to about 3000, from about 1500 to about 2000, from about 2000 to about 3185, from about 2000 to about 3000, from about 2000 to about 2500, from about 2500 to about 3185, from about 2500 to about 3000, or from about 3000 to about 3185 nucleotides.
  • the E3 insertion site is located between the stop site of E3-10.5K and the stop site of E3-14.7K. In certain embodiments, the E3 insertion site comprises a deletion of from about 500 to about 1551, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 1551, from about 1000 to about 1500, or from about 1500 to about 1551 nucleotides adjacent the stop site of E3-10.5K. In certain embodiments, the E3 insertion site comprises a deletion of about 1050 nucleotides adjacent the stop site of E3-10.5K, e.g., the E3 insertion site comprises a deletion of 1063 or 1064 nucleotides adjacent the stop site of E3-10.5K.
  • the E3 insertion site comprises a deletion corresponding to the Ad5 dl309 E3 deletion. In certain embodiments, the E3 insertion site comprises a deletion corresponding to nucleotides 29773-30836 of the Ad5 genome (SEQ ID NO: 5), or, the exogenous nucleotide sequence is inserted between nucleotides corresponding to 29773 and 30836 of the Ad5 genome (SEQ ID NO: 5).
  • the exogenous nucleotide sequence is inserted between CAGTATGA (SEQ ID NO: 8) and TAATAAAAAA (SEQ ID NO: 9), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CAGTATGA (SEQ ID NO: 8), the exogenous nucleotide sequence, and TAATAAAAAA (SEQ ID NO: 9).
  • the recombinant oncolytic adenovirus may comprise a deletion of at least one Pea3 binding site, or a functional portion thereof, e.g., the virus may comprise a deletion of nucleotides corresponding to about -300 to about -250 upstream of the initiation site of E la or a deletion of nucleotides corresponding to -305 or -304 to -255 upstream of the initiation site of Ela.
  • the recombinant adenovirus may comprise a deletion of nucleotides corresponding to 195-244 of the Ad5 genome (SEQ ID NO: 5), and/or the recombinant adenovirus may comprise the sequence GGTGTTTTGG (SEQ ID NO: 10).
  • the recombinant oncolytic adenovirus may comprise a deletion of at least one Pea3 binding site, or a functional portion thereof, and not comprise a deletion of an E2F binding site.
  • the recombinant oncolytic adenovirus may comprise a deletion of at least one E2F binding site, or a functional portion thereof.
  • the recombinant oncolytic adenovirus may comprise a deletion of at least one E2F binding site, or a functional portion thereof, and not comprise a deletion of a Pea3 binding site.
  • the recombinant oncolytic adenovirus may comprise an Ela promoter having a deletion of a functional TATA box, e.g., the deletion of an entire TATA box.
  • the virus may comprise a deletion of nucleotides corresponding to -29 to -26, -33 to -26, -44 to +52, or -148 to +52 of the Ela promoter.
  • the deletion comprises a deletion of nucleotides corresponding to 353-552 of the Ad5 genome (SEQ ID NO: 5), and/or the Ela promoter comprises the sequence CTAGGACTG (SEQ ID NO: 11).
  • the exogenous nucleotide sequence is not operably linked to an exogenous promoter sequence.
  • the recombinant oncolytic adenovirus may selectively replicate in a hyperproliferative cell.
  • the recombinant oncolytic adenovirus may selectively express the protein defining a binding site or the protein that mediates the production of a biomolecule defining a binding site in a hyperproliferative cell.
  • the hyperproliferative cell may be a cancer cell, e.g., a lung cancer cell, a colon cancer cell, and a pancreatic cancer cell.
  • the recombinant oncolytic adenovirus comprises a nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the recombinant oncolytic virus further comprises an exogenous nucleotide sequence encoding a
  • the composition further comprises a bisphosphonate, hyaluronic acid, vitamin D, calcium, testosterone, estrogen, progesterone, a selective estrogen modulator (SERM), calcitonin, or a RANK or RANKL inhibitor (e.g., denosumab).
  • SERM selective estrogen modulator
  • FIGURE 1 is a bar graph showing the expression of BMP-2 by the TAV-BMP-2 virus in A549 cells as determined by ELISA.
  • FIGURE 2 is a bar graph showing the expression of BMP-7 by the TAV-BMP-7 virus in A549 cells as determined by ELISA.
  • FIGURE 3 depicts crystal violet staining of A549 cells at the indicated time points following infection with the TAV- A 19k, TAV-BMP-2, and TAV-BMP-7 viruses.
  • FIGURE 4 depicts tumor volumes of mice carrying subcutaneous ADS-12 tumors treated with vehicle, TAVA19k. or TAV-BMP-7 viruses. Lines show the mean tumor volume of 10 mice in each treatment group and error bars show SEM.
  • the invention is based, in part, upon the discovery that a delivery vehicle, e.g., a recombinant oncolytic virus, can be used to selectively express a protein defining a binding site or a protein that mediates the production of a biomolecule defining a binding site in a cancer cell, and thereby target the cancer cell for a treatment by a therapeutic agent that binds to the binding site.
  • a delivery vehicle e.g., a recombinant oncolytic virus
  • a delivery vehicle e.g., a recombinant oncolytic virus described herein
  • a delivery vehicle can be used to selectively express a bone morphogenetic protein (BMP) in a cancer cell, and cause a cancer to acquire a bone cell phenotype, e.g., an osteoblast-like cell phenotype, and/or mediate the production of bone tissue in a tumor.
  • BMP bone morphogenetic protein
  • Certain cancers and metastases are associated with increased levels of BMP expression, and cancer cells that acquire a bone cell phenotype have a greater likelihood of surviving and proliferating in bone.
  • the invention provides a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject: (a) an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and (b) an effective amount of a therapeutic agent that binds to the binding site.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (a) an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and (b) optionally, an effective amount of a therapeutic agent that binds to the binding site.
  • the invention provides methods for treating bone loss and/or hypercalcemia or promoting the growth of bone in a subject in need thereof using a delivery vehicle (e.g., a recombinant oncolytic virus) described herein.
  • a delivery vehicle e.g., a recombinant oncolytic virus
  • Delivery vehicles suitable for use in the invention include, e.g., viruses, bacteria (e.g., Listeria monocytogenes, Salmonella enterica or Serovar typhi murium), nanoparticles, liposomes, exosomes, or microemulsions.
  • viruses e.g., viruses, bacteria (e.g., Listeria monocytogenes, Salmonella enterica or Serovar typhi murium), nanoparticles, liposomes, exosomes, or microemulsions.
  • viruses are used herein to refer any of the obligate intracellular parasites having no protein-synthesizing or energy-generating mechanism.
  • the viral genome may be RNA or DNA.
  • the viruses useful in the practice of the present invention include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picomoviridiae, herpesviridiae, poxyiridae, or adenoviridiae.
  • a recombinantly modified virus is referred to herein as a“recombinant virus.”
  • a recombinant virus may, e.g., be modified by recombinant DNA techniques to be replication deficient, conditionally replicating, or replication competent, and/or be modified by recombinant DNA techniques to include expression of exogenous transgenes.
  • Chimeric viral vectors which exploit advantageous elements of each of the parent vector properties (See, e.g., Feng et al. (1997) NATURE BIOTECHNOLOGY 15:866-870) may also be useful in the practice of the present invention. Although it is generally favored to employ a virus from the species to be treated, in some instances it may be advantageous to use vectors derived from different species that possess favorable pathogenic features.
  • equine herpes virus vectors for human gene therapy are described in PCT Publication No. WO 98/27216.
  • the vectors are described as useful for the treatment of humans as the equine virus is not pathogenic to humans.
  • ovine adenoviral vectors may be used in human gene therapy as they are claimed to avoid the antibodies against the human adenoviral vectors.
  • Such vectors are described in PCT Publication No. WO 97/06826.
  • viruses useful in the practice of the invention include adenovirus, fowlpox virus, lentivirus, herpes virus, vesicular stomatitis virus (VSV), maraba virus, polio virus, and alpha virus.
  • the recombinant virus is an adenovirus.
  • Adenoviruses are medium-sized (90-100 nm), non-enveloped (naked), icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. Adenoviruses replicate in the nucleus of mammalian cells using the host's replication machinery.
  • adenovirus refers to any virus in the genus Adenoviridiae including, but not limited to, human, bovine, ovine, equine, canine, porcine, murine, and simian adenovirus subgenera.
  • human adenoviruses includes the A-F subgenera as well as the individual serotypes thereof, the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Adl la and Adl lp), 12, 13, 14, 15, 16, 17, 18, 19, l9a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91.
  • Preferred are recombinant viruses derived from human adenovirus types 2 and 5.
  • all adenovirus type 5 nucleotide numbers are relative to the NCBI reference sequence AC_000008.1, which is depicted herein in SEQ ID NO: 5.
  • the adenovirus replication cycle has two phases: an early phase, during which 4 transcription units (El, E2, E3, and E4) are expressed, and a late phase which occurs after the onset of viral DNA synthesis, and during which late transcripts are expressed primarily from the major late promoter (MLP).
  • MLP major late promoter
  • the late messages encode most of the virus's structural proteins.
  • the gene products of El, E2 and E4 are responsible for transcriptional activation, cell transformation, viral DNA replication, as well as other viral functions, and are necessary tor viral growth.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid sequence is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a gene if it affects the transcription of the gene.
  • Operably linked nucleotide sequences are typically contiguous. However, as enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome.
  • the virus has one or more modifications to a regulatory sequence or promoter.
  • a modification to a regulatory sequence or promoter comprises a deletion, substitution, or addition of one or more nucleotides compared to the wild-type sequence of the regulatory sequence or promoter.
  • the modification of a regulatory sequence or promoter comprises a modification of sequence of a transcription factor binding site to reduce affinity for the transcription factor, for example, by deleting a portion thereof, or by inserting a single point mutation into the binding site.
  • the additional modified regulatory sequence enhances expression in neoplastic cells, but attenuates expression in normal cells.
  • the modified regulatory sequence is operably linked to a sequence encoding a protein.
  • at least one of the adenoviral Ela and Elb genes (coding regions) is operably linked to a modified regulatory sequence.
  • the Ela gene is operably linked to the modified regulatory sequence.
  • the Ela regulatory sequence contains five binding sites for the transcription factor Pea3, designated Pea3 I, Pea3 II, Pea3 III, Pea3 IV, and Pea3 V, where Pea3 I is the Pea3 binding site most proximal to the Ela start site, and Pea3 V is most distal.
  • the Ela regulatory sequence also contains binding sites for the transcription factor E2F, hereby designated E2F I and E2F II, where E2F I is the E2F binding site most proximal to the Ela start site, and E2F II is more distal. From the Ela start site, the binding sites are arranged: Pea3 I, E2F I, Pea3 II, E2F II, Pea3 III, Pea3 IV, and Pea3 V.
  • At least one of these seven binding sites, or a functional portion thereof, is deleted.
  • a "functional portion” is a portion of the binding site that, when deleted, decreases or even eliminates the functionality, e.g. binding affinity, of the binding site to its respective transcription factor (Pea3 or E2F) by, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to the complete sequence.
  • one or more entire binding sites are deleted.
  • a functional portion of one or more binding sites is deleted.
  • a "deleted binding site” encompasses both the deletion of an entire binding site and the deletion of a functional portion thereof. When two or more binding sites are deleted, any combination of entire binding site deletion and functional portion deletion may be used.
  • At least one Pea3 binding site, or a functional portion thereof, is deleted.
  • the deleted Pea3 binding site can be Pea3 I, Pea3 II, Pea3 III, Pea3 IV, and/or Pea3 V.
  • the deleted Pea3 binding site is Pea3 II, Pea3 III, Pea3 IV, and/or Pea3 V.
  • the deleted Pea3 binding site is Pea3 IV and/or Pea3 V.
  • the deleted Pea3 binding site is Pea3 II and/or Pea3
  • the deleted Pea3 binding site is both Pea3 II and Pea3 III. In certain embodiments, the Pea3 I binding site, or a functional portion thereof, is retained.
  • At least one E2F binding site, or a functional portion thereof is deleted. In certain embodiments, at least one E2F binding site, or a functional portion thereof, is retained. In certain embodiments, the retained E2F binding site is E2F I and/or E2F II. In certain embodiments, the retained E2F binding site is E2F II. In certain embodiments, the total deletion consists essentially of one or more of Pea3 II, Pea3 III, Pea3
  • the virus has a deletion of a 50 base pair region located from -304 to -255 upstream of the Ela initiation site, e.g., corresponding to 195-244 of the Ad5 genome (SEQ ID NO: 5), hereafter referred to as the TAV-255 deletion.
  • the TAV-255 deletion results in an Ela promoter that comprises the sequence GGTGTTTTGG (SEQ ID NO: 10).
  • the virus comprises an Ela promoter having a deletion of a functional TATA box, e.g., the deletion of an entire TATA box.
  • a “functional TATA box” refers to a TATA box that is capable of binding to a TATA box binding protein (TBP), e.g., a TATA box that has at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or at least 40%, of the TBP binding activity of a corresponding wild-type TATA box sequence.
  • TBP TATA box binding protein
  • a“non-functional TATA box” refers to a TATA box that, e.g., has less than 30%, less than 20%, less than 10%, or 0% of the TBP binding activity of a corresponding wild-type TATA box sequence.
  • Assays for determining whether a TBP binds to a TATA box are known in the art. Exemplary binding assays include electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and DNAse footprinting assays.
  • the virus comprises a deletion of nucleotides corresponding to -27 to -24, -31 to -24, -44 to +54, or -146 to +54 of the adenovirus type 5 Ela promoter, which correspond, respectively, to nucleotides 472 to 475, 468 to 475, 455 to 552, and 353 to 552 of the Ad5 genome (SEQ ID NO: 5).
  • the virus comprises a deletion of nucleotides corresponding to -29 to -26, -33 to -26, -44 to +52, or -148 to +52 of the adenovirus type 5 Ela promoter.
  • the virus comprises a deletion of nucleotides corresponding to 353 to 552 of the Ad5 genome (SEQ ID NO: 5).
  • the virus comprises a polynucleotide deletion that results in an adenovirus comprising the sequence CTAGGACTG (SEQ ID NO: 11), AGTGCCCG (SEQ ID NO: 19), or TATTCCCG (SEQ ID NO: 20), which result from joining the two polynucleotide sequences that would otherwise flank the deleted polynucleotide sequence.
  • the virus comprises a polynucleotide deletion that results in an adenovirus comprising the sequence CTAGGACTG (SEQ ID NO: 11) .
  • the virus comprises an Ela promoter having a deletion of a functional CAAT box, e.g., the deletion of an entire CAAT box.
  • a “functional CAAT box” refers to a CAAT box that is capable of binding to a C/EBP or NF-Y protein, e.g., a CAAT box that has at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or at least 40%, of the a C/EBP or NF-Y binding activity of a corresponding wild-type CAAT box sequence.
  • a“non-functional CAAT box” refers to a CAAT box that, e.g., has less than 30%, less than 20%, less than 10%, or 0% of the a C/EBP or NF-Y binding activity of a corresponding wild-type CAAT box sequence.
  • Assays for determining whether a C/EBP or NF-Y protein binds to a CAAT box are known in the art. Exemplary binding assays include electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and DNAse footprinting assays.
  • the virus comprises a deletion of nucleotides corresponding to -76 to -68 of the adenovirus type 5 Ela promoter, which corresponds to nucleotides 423 to 431 of the Ad5 genome (SEQ ID NO: 5).
  • the virus comprises a polynucleotide deletion that results in an adenovirus comprising the sequence TTCCGTGGCG (SEQ ID NO: 21), which results from joining the two polynucleotide sequences that would otherwise flank the deleted polynucleotide sequence.
  • the adenoviral Elb-l9k gene functions primarily as an anti-apoptotic gene and is a homolog of the cellular anti-apoptotic gene, BCL-2. Since host cell death prior to maturation of the progeny viral particles would restrict viral replication, Elb-l9k is expressed as part of the El cassette to prevent premature cell death thereby allowing the infection to proceed and yield mature virions. Accordingly, in certain embodiments, a recombinant virus is provided that includes an Elb-l9K insertion site, e.g., the adenovirus has an exogenous nucleotide sequence inserted into an Elb-l9K insertion site.
  • the Elb-l9K insertion site is located between the start site of Elb-l9K (i.e., the nucleotide sequence encoding the start codon of Elb-l9k, e.g., corresponding to nucleotides 1714-1716 of SEQ ID NO: 5) and the start site of Elb-55K (i.e., the nucleotide sequence encoding the start codon of Elb-55k, e.g., corresponding to nucleotides 2019-2021 of SEQ ID NO: 5).
  • an insertion between two sites for example, an insertion between (i) a start site of a first gene (e.g., Elb-l9k) and a start site of a second gene, (e.g., Elb-55K), (ii) a start site of a first gene and a stop site of a second gene, (iii) a stop site of a first gene and start site of a second gene, or (iv) a stop site of first gene and a stop site of a second gene, is understood to mean that all or a portion of the nucleotides constituting a given start site or a stop site surrounding the insertion may be present or absent in the final virus. Similarly, an insertion between two nucleotides is understood to mean that the nucleotides surrounding the insertion may be present or absent in the final virus.
  • the Elb-l9K insertion site is located between the start site of Elb-l9K (i.e., the nucleotide sequence encoding the start codon of Elb-l9k, e.g., corresponding to nucleotides 1714-1716 of SEQ ID NO: 5) and the stop site of Elb-l9K (i.e., the nucleotide sequence encoding the stop codon of Elb-l9k, e.g., corresponding to nucleotides 2242-2244 of SEQ ID NO: 5).
  • start site of Elb-l9K i.e., the nucleotide sequence encoding the start codon of Elb-l9k, e.g., corresponding to nucleotides 1714-1716 of SEQ ID NO: 5
  • the stop site of Elb-l9K i.e., the nucleotide sequence encoding the stop codon of Elb-l9k, e.g., corresponding
  • the Elb-l9K insertion site comprises a deletion of from about 100 to about 305, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 305, about 150 to about 300, about 150 to about 250, or about 150 to about 200 nucleotides adjacent the start site of Elb-l9K.
  • the Elb-l9K insertion site comprises a deletion of about 200 nucleotides, e.g., 203 nucleotides adjacent the start site of Elb-l9K.
  • the Elb-l9K insertion site comprises a deletion corresponding to nucleotides 1714-1916 of the Ad5 genome (SEQ ID NO: 5), or the exogenous nucleotide sequence is inserted between nucleotides corresponding to 1714 and 1916 of the Ad5 genome (SEQ ID NO: 5).
  • the exogenous nucleotide sequence is inserted between CTGACCTC (SEQ ID NO: 6) and TCACCAGG (SEQ ID NO: 7), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CTGACCTC (SEQ ID NO: 6), the exogenous nucleotide sequence, and TCACCAGG (SEQ ID NO: 7).
  • CTGACCTC (SEQ ID NO: 6) and TCACCAGG (SEQ ID NO: 7) define unique boundary sequences for the Elb- 19K insertion site within the Ad5 genome (SEQ ID NO: 5).
  • a deletion adjacent to a site for example, a deletion adjacent to a start site of a gene or a deletion adjacent to a stop site of a gene, is understood to mean that the deletion may include a deletion of all, a portion, or none of the nucleotides constituting a given start site or a stop site.
  • an exogenous nucleotide sequence is inserted into an E3 insertion site located between the stop site of pVIII (i.e., the nucleotide sequence encoding the stop codon of pVIII, e.g., corresponding to nucleotides 27855-27857 of SEQ ID NO: 5) and the start site of Fiber (i.e., the nucleotide sequence encoding the start codon of Fiber, e.g., corresponding to nucleotides 31042-31044 of SEQ ID NO: 5).
  • the E3 insertion site comprises a deletion of from about 500 to about 3185, from about 500 to about 3000, from about 500 to about 2500, from about 500 to about 2000, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 3185, from about 1000 to about 3000, from about 1000 to about 2500, from about 1000 to about 2000, from about 1000 to about 1500, from about 1500 to about 3185, from about 1500 to about 3000, from about 1500 to about 2000, from about 2000 to about 3185, from about 2000 to about 3000, from about 2000 to about 2500, from about 2500 to about 3185, from about 2500 to about 3000, or from about 3000 to about 3185 nucleotides.
  • the E3 insertion site is located between the stop site of E3-10.5K (i.e., the nucleotide sequence encoding the stop codon of E3-10.5K, e.g., corresponding to nucleotides 29770-29772 of SEQ ID NO: 5) and the stop site of E3-14.7K (i.e., the nucleotide sequence encoding the stop codon of E3-14.7K, e.g., corresponding to nucleotides 30837-30839 of SEQ ID NO: 5).
  • the E3 insertion site comprises a deletion of from about 500 to about 1551, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 1551, from about 1000 to about 1500, or from about 1500 to about 1551 nucleotides adjacent the stop site of E3-10.5K.
  • the E3 insertion site comprises a deletion of about 1050 nucleotides adjacent the stop site of E3-10.5K, e.g., the E3 insertion site comprises a deletion of 1063 or 1064 nucleotides adjacent the stop site of E3-10.5K.
  • the E3 insertion site comprises a deletion corresponding to the Ad5 dl309 E3 deletion.
  • the E3 insertion site comprises a deletion corresponding to nucleotides 29773-30836 of the Ad5 genome (SEQ ID NO: 5), or the exogenous nucleotide sequence is inserted between nucleotides corresponding to 29773 and 30836 of the Ad5 genome (SEQ ID NO: 5).
  • the exogenous nucleotide sequence is inserted between CAGTATGA (SEQ ID NO: 8) and TAATAAAAAA (SEQ ID NO: 9), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CAGTATGA (SEQ ID NO: 8), the exogenous nucleotide sequence, and TAATAAAAAA (SEQ ID NO: 9).
  • CAGTATGA (SEQ ID NO: 8) and TAATAAAAAA (SEQ ID NO: 9) define unique boundary sequences for an E3 insertion site within the Ad5 genome (SEQ ID NO: 5).
  • the E3 insertion site is located between stop site of E3- gpl9K (i.e.. the nucleotide sequence encoding the stop codon of E3-gpl9K, e.g., corresponding to nucleotides 29215-29217 of SEQ ID NO: 5) and the stop site of E3-14.7K (i.e., the nucleotide sequence encoding the stop codon of E3-14.7K, e.g., corresponding to nucleotides 30837-30839 of SEQ ID NO: 5).
  • stop site of E3- gpl9K i.e.. the nucleotide sequence encoding the stop codon of E3-gpl9K, e.g., corresponding to nucleotides 29215-29217 of SEQ ID NO: 5
  • the stop site of E3-14.7K i.e., the nucleotide sequence encoding the stop codon of E3-14.7K, e.
  • the E3 insertion site comprises a deletion of from about 500 to about 1824, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 1824, from about 1000 to about 1500, or from about 1500 to about 1824 nucleotides adjacent the stop site of E3-gpl9K. In certain embodiments, the E3 insertion site comprises a deletion of about 1600 nucleotides adjacent the stop site of E3-gpl9K. e.g., the E3 insertion site comprises a deletion of 1622 nucleotides adjacent the stop site of E3-gpl9K.
  • the E3 insertion site comprises a deletion corresponding to nucleotides 29218-30839 of the Ad5 genome (SEQ ID NO: 5).
  • the exogenous nucleotide sequence is inserted between nucleotides corresponding to 29218 and 30839 of the Ad5 genome (SEQ ID NO: 5).
  • the exogenous nucleotide sequence is inserted between TGCCTTAA (SEQ ID NO: 17) and TAAAAAAAAAT (SEQ ID NO: 18), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, TGCCTTAA (SEQ ID NO: 17), the exogenous nucleotide sequence, and TAAAAAAAAAT (SEQ ID NO: 18).
  • TGCCTTAA (SEQ ID NO: 17) and TAAAAAAAAAT (SEQ ID NO: 18) define unique boundary sequences for an E3 insertion site within the Ad5 genome (SEQ ID NO: 5).
  • the vims comprises an E4 deletion.
  • the E4 deletion is located between the start site of E4-ORF6/7 (i.e., the nucleotide sequence encoding the start codon of E4-ORF6/7, e.g., corresponding to nucleotides 34075-34077 of SEQ ID NO: 5) and the right inverted terminal repeat (ITR; e.g., corresponding to nucleotides 35836-35938 of SEQ ID NO: 5).
  • the E4 deletion is located between the start site of E4-ORF6/7 and the start site of E4-ORF1 (i.e..
  • the E4 deletion comprises a deletion of a nucleotide sequence between the start site of E4-ORF6/7 and the start site of E4-ORF1.
  • the E4 deletion comprises a deletion of from about 500 to about 2500, from about 500 to about 2000, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 2500, from about 1000 to about 2000, from about 1000 to about 1500, from about 1500 to about 2500, from about 1500 to about 2000, or from about 2000 to about 2500 nucleotides.
  • the E4 deletion comprises a deletion of from about 250 to about 1500, from about 250 to about 1250, from about 250 to about 1000, from about 250 to about 750, from about 250 to about 500, from 500 to about 1500, from about 500 to about 1250, from about 500 to about 1000, from about 500 to about 750, from 750 to about 1500, from about 750 to about 1250, from about 750 to about 1000, from about 1000 to about 1500, or from about 1000 to about 1250 nucleotides adjacent the start site of E4-ORF6/7.
  • the E4 deletion comprises a deletion of about 1450 nucleotides adjacent the start site of E4-ORF6/7, e.g., the E4 deletion comprises a deletion of about 1449 nucleotides adjacent the start site of E4-ORF6/7. In certain embodiments, the E4 deletion comprises a deletion corresponding to nucleotides 34078-35526 of the Ad5 genome (SEQ ID NO: 5).
  • the virus includes an IX-E2 insertion site, e.g., the virus has an exogenous nucleotide sequence inserted into an IX-E2 insertion site.
  • the IX-E2 insertion site is located between the nucleotide sequence encoding the stop codon of IX and the nucleotide sequence encoding the stop codon of IVa2.
  • the nucleotide sequence is inserted between nucleotides corresponding to 4029 and 4093 of the Ad5 genome (SEQ ID NO: 5).
  • the nucleotide sequence is inserted between nucleotides corresponding to 4029 and 4050, nucleotides corresponding to 4051 and 4070, or nucleotides corresponding to 4071 and 4093 of the Ad5 genome (SEQ ID NO: 5).
  • the IX-E2 insertion site comprises a deletion of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides.
  • the virus includes an L5-E4 insertion site, e.g., the virus has an exogenous nucleotide sequence inserted into an L5-E4 insertion site.
  • the L5-E4 insertion site is located between the nucleotide sequence encoding the stop codon of Fiber and the nucleotide sequence encoding the stop codon of E4-ORF6 or E40RF6/7.
  • the nucleotide sequence is inserted between nucleotides corresponding to 32785 to 32916 of the Ad5 genome (SEQ ID NO: 5).
  • the nucleotide sequence is inserted between nucleotides corresponding to 32785 and 32800, nucleotides corresponding to 32801 and 32820, nucleotides corresponding to 32821 and 32840, nucleotides corresponding to 32841 and 32860, nucleotides
  • the L5-E4 insertion site comprises a deletion of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 nucleotides.
  • the recombinant virus comprises a nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • Sequence identity may be determined in various ways that are within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • BLAST Basic Local Alignment Search Tool
  • analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al, ( 1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES.
  • BLOSUM62 matrix (Henikoff et al, (1992) PROC. NATL. ACAD. SCI. USA 89: 10915-10919, fully incorporated by reference).
  • a recombinant virus is an oncolytic virus, e.g., a virus that exhibits tumor-selective replication and/or viral mediated lysis.
  • a recombinant virus of the invention exhibits selective expression of a protein, e.g., a protein defining a binding site or the protein that mediates the production of a biomolecule defining a binding site therapeutic transgene in a hyperproliferative cell, e.g., a cancer cell, relative to a non-hyperprobferative cell.
  • the expression of the protein in a non- hyperproliferative cell is about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% , or about 5% of the expression of in a
  • the hyperproliferative cell may be a cancer cell, e.g., a carcinoma, sarcoma, leukemia, lymphoma, prostate cancer, lung cancer, gastrointestinal tract cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, stomach cancer, thyroid cancer, mesothelioma, liver cancer, kidney cancer, skin cancer, head and neck cancer, or brain cancer cell.
  • a cancer cell e.g., a carcinoma, sarcoma, leukemia, lymphoma, prostate cancer, lung cancer, gastrointestinal tract cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, stomach cancer, thyroid cancer, mesothelioma, liver cancer, kidney cancer, skin cancer, head and neck cancer, or brain cancer cell.
  • a disclosed virus is produced in a suitable host cell line using conventional techniques including culturing a transfected or infected host cell under suitable conditions so as to allow the production of infectious viral particles.
  • Nucleic acids encoding viral genes can be incorporated into plasmids and introduced into host cells through conventional transfection or transformation techniques.
  • Exemplary suitable host cells for production of disclosed viruses include human cell lines such as HeLa, Hela-S3, HEK293, 911, A549, HER96, or PER-C6 cells. Specific production and purification conditions will vary depending upon the virus and the production system employed.
  • adenovirus For adenovirus, the traditional method for the generation of viral particles is co-transfection followed by subsequent in vivo recombination of a shuttle plasmid (usually containing a small subset of tire adenoviral genome and optionally containing a potential transgene an expression cassette) and an adenoviral helper plasmid (containing most of the entire adenoviral genome).
  • a shuttle plasmid usually containing a small subset of tire adenoviral genome and optionally containing a potential transgene an expression cassette
  • adenoviral helper plasmid containing most of the entire adenoviral genome
  • Alternative technologies for the generation of adenovirus include utilization of the bacterial artificial chromosome (BAG) system, in vivo bacterial recombination in a recAT bacterial strain utilizing two plasmids containing complementary adenoviral sequences, and the yeast artificial chromosome (YAC) system.
  • BAG bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • infectious viral particles are recovered from the culture and optionally purified.
  • Typical purification steps may include plaque purification, centrifugation, e.g., cesium chloride gradient centrifugation, clarification, enzymatic treatment, e.g., benzonase or protease treatment, chromatographic steps, e.g., ion exchange chromatography or filtration steps.
  • the invention provides methods and compositions comprising: (a) an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and (b) an effective amount of a therapeutic agent that binds to the binding site.
  • the term“therapeutic agent that binds to a binding site” refers to any therapeutic agent (/. e. , any agent that imparts a therapeutic effect in a target cell, body fluid, tissue, organ, physiological system, or subject) that reacts or associates more frequently, more rapidly, with greater duration and/or with stronger affinity with a particular binding site (e.g., a binding site on a protein encoded by a delivery vehicle or a binding site on a biomolecule the production of which is mediated by a protein encoded by a delivery vehicle, e.g., bone tissue) than it does with an alternative binding site.
  • a therapeutic agent e. , any agent that imparts a therapeutic effect in a target cell, body fluid, tissue, organ, physiological system, or subject
  • a particular binding site e.g., a binding site on a protein encoded by a delivery vehicle or a binding site on a biomolecule the production of which is mediated by a protein encoded by a delivery vehicle, e.g.,
  • the therapeutic agent may, e.g., have affinity for the binding site stronger than 100 nM, 50 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM, as determined by surface plasmon resonance.
  • a biomolecule defining a binding site is understood to contemplate an individual biomolecule, e.g., a protein, and/or a collection of biomolecules, e.g., a tissue, e.g., bone tissue.
  • the protein that defines a binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR) and prostate specific membrane antigen (PSMA), and/or the therapeutic agent that binds to the protein is selected from an anti-BMP antibody, an anti-BMPR antibody, and an anti-PSMA antibody.
  • BMP bone morphogenetic protein
  • BMPR bone morphogenetic protein receptor
  • PSMA prostate specific membrane antigen
  • the BMP may, for example, be selected from BMP-2, BMP-4, BMP-6 and BMP- 7, and accordingly, the anti-BMP antibody may, for example, be selected from an anti-BMP - 2 antibody, an anti-BMP -4 antibody, an anti-BMP-6 antibody, and an anti-BMP-7 antibody.
  • the therapeutic agent is an antibody.
  • antibodies are multimeric proteins that contain four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains).
  • the immunoglobulin heavy and light chains are connected by an interchain disulfide bond.
  • the immunoglobulin heavy chains are connected by interchain disulfide bonds.
  • a light chain consists of one variable region (V L ) and one constant region (C L ).
  • the heavy chain consists of one variable region (V H ) and at least three constant regions (CHi, CH 2 and CH 3 ).
  • the variable regions determine the binding specificity of the antibody. [0090]
  • Each variable region contains three hypervariable regions known as
  • CDRs complementarity determining regions flanked by four relatively conserved regions known as framework regions (FRs).
  • FRs framework regions
  • the extent of the FRs and CDRs has been defined (Rabat, E.A., et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, FIFTH EDITION, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. MOL. BIOL. 196:901-917).
  • the three CDRs referred to as CDRi, CDR 2 , and CDR3, contribute to the antibody binding specificity.
  • Naturally occurring antibodies have been used as starting material for engineered antibodies, such as chimeric antibodies and humanized antibodies.
  • antibody means an intact antibody (e.g., an intact monoclonal antibody) or antigen-binding fragment of an antibody, including an intact antibody or antigen-binding fragment that has been modified, engineered or chemically conjugated.
  • antibodies that have been modified or engineered are chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies).
  • antigen-binding fragments include Fab, Fab’, F(ab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies.
  • an antibody conjugated to a toxin, radioisotope, cytokine, or enzyme is an example of a chemically conjugated antibody.
  • the antibody the antibody is selected from an antibody drug conjugate and an antibody radionuclide conjugate.
  • an antibody may mediate an“effector functions,” e.g., a biological activity attributable to the Fc region of an antibody that varies with the antibody isotype.
  • effector functions include Clq binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell mediated cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.
  • the therapeutic agent is a cell, e.g., a T-cell.
  • the T-cell can be any T-cell, such as a cultured T-cell, e.g., a primary T-cell, or a T-cell from a cultured T-cell line, e.g., Jurkat, SupTi, etc., or a T-cell obtained from a mammal. If obtained from a mammal, the T-cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T-cells can also be enriched for or purified.
  • the T-cell is a human T-cell, which can be an autologous or heterologous cell.
  • the T-cell can be any type of T-cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T-cells, CD4+ helper T-cells, e.g., Thl and Th2 cells, CD4+ T-cells, CD8+ T-cells (e.g., cytotoxic T-cells), tumor infiltrating lymphocytes (TILs), memory T-cells (e.g., central memory T-cells and effector memory T-cells), naive T-cells, and the like.
  • the cells can include autologous cells derived from a subject to be treated, or alternatively allogenic cells derived from a donor.
  • a T-cell binds to a binding site through a T-cell receptor.
  • the T-cell receptor may be an endogenous or a recombinant T-cell receptor.
  • T-cell receptors comprise two chains referred to as the a- and b-chains, that form a pair on the surface of a T- cell to form a heterodimeric receptor.
  • the T-cell receptor is involved in recognition of MHC- restricted antigens.
  • Each of a- and b- chain comprises two regions, a constant region and a variable region.
  • Each variable region of the a- and b- chains defines three loops, referred to as complementary determining regions (CDRs) known as CDRi, CDR 2 , and CDR 3 that confer the T-cell receptor with antigen binding activity and binding specificity.
  • CDRs complementary determining regions
  • a T-cell binds to a binding site through a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • CARs refers to any artificial receptor including an antigen-specific binding moiety and one or more signaling chains derived from an immune receptor.
  • CARs comprise a single chain fragment variable (scFv) of an antibody specific for an antigen coupled via hinge and transmembrane regions to cytoplasmic domains of T-cell signaling molecules, e.g. a T-cell costimulatory domain (e.g., from CD28, CD 137, 0X40, ICOS, and CD27) in tandem with a T-cell triggering domain (e.g. from CD3z).
  • a T-cell expressing a chimeric antigen receptor is referred to as a CAR T-cell.
  • the protein that mediates the production of a biomolecule defining a binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR), osteocalcin, osteopontin, bone sialoprotein, osteonectin, osteoprotegerin (OPG) and alkaline phosphatase and/or the biomolecule defining a binding site is bone tissue, e.g., expression of a BMP, a BMPR, osteocalcin, osteopontin, bone sialoprotein, osteonectin, OPG or alkaline phosphatase by the delivery vehicle mediates the production of bone tissue.
  • BMP bone morphogenetic protein
  • BMPR bone morphogenetic protein receptor
  • OPG osteoprotegerin
  • alkaline phosphatase alkaline phosphatase
  • the biomolecule defining a binding site is bone tissue, e.g., expression of a BMP, a BMPR, osteocalcin, osteopontin, bone si
  • the BMP may, for example, be selected from BMP-2, BMP-4, BMP-6 or BMP-7.
  • expression of a BMP, a BMPR, osteocalcin, osteopontin, bone sialoprotein, osteonectin, OPG or alkaline phosphatase by the delivery vehicle in a cancer cell results in the cancer cell acquiring an osteoblast-like cell phenotype.
  • the therapeutic agent that binds to a biomolecule, e.g., bone is a radionuclide, i.e., an atom that emits radiation and, e.g., kills surrounding cancer cells.
  • Radionuclides may, e.g., be g-emitting radionuclides, b-emitting radionuclides, a- emitting radionuclides, and positron-emitting radionuclides.
  • Exemplary radionuclides include 32 P, 47 Sc, 64 Cu, 67 Cu, 89 Sr, 86 Y, 87 Y, 90 Y, 105 Rh, m Ag, m In, 117m Sn, 149 Pm, 153 Sm, 166 Ho, 177 Lu, I S6 RC. 188 Re, 211 At, 212 Bi, and 223 Ra.
  • the radionuclide is a bone targeting (i.e., a bone-homing) radionuclide.
  • a bone-targeting radionuclide is a calcium mimetic (e.g., Sr and Ra).
  • a bone homing radionuclide is conjugated to a ligand (e.g., ethylenediaminetetramethylene phosphonate (EDTMP), e.g., 153 Sm-EDTMP), which binds to calcium.
  • a ligand e.g., ethylenediaminetetramethylene phosphonate (EDTMP), e.g., 153 Sm-EDTMP
  • the radionuclide is selected from 89 Sr (e.g., 89 Sr chloride, i.e., MetastronTM),
  • 223 Ra e.g., 223 Ra dichloride, i.e., Xofigo ®
  • 153 Sm e.g., 153 Sm-EDTMP, i.e., 153 Sm
  • lexidronam i.e., Quadramet ®
  • 177 Lu 90 Y
  • 186 Re e.g, 186 Re HEDP
  • 117m Sn e.g, 117m Sn DTP A
  • 32 P 32 P.
  • a bone morphogenetic protein may, for example, be selected from BMP-2, BMP -4, BMP-6 or BMP-7.
  • An exemplary nucleotide sequence of BMP -2 cloned into an exemplary recombinant adenovirus delivery vehicle is as follows, where the flanking adenoviral sequence including restriction sites is underlined:
  • An exemplary nucleotide sequence of BMP -4 cloned into an exemplary recombinant adenovirus delivery vehicle is as follows, where the flanking adenoviral sequence including restriction sites is underlined:
  • An exemplary nucleotide sequence of BMP-6 cloned into an exemplary recombinant adenovirus delivery vehicle is as follows, where the flanking adenoviral sequence including restriction sites is underlined: ATCTGACCTCGTCGACATGCCGGGGCTGGGGCGGAGGGCGCAGTGGCTGTGCTGGTGGTGGG GGCTGCTGTGCAGCTGCTGCGGGCCCCCGCCGCTGCGGCCGCCCTTGCCCGCTGCCGCGGCC GCCGGGGGGCAGCTGCTGGGGGACGGCGGGAGCCCCGGCCGCACGGAGCAGCCGCC GCCGTCGCCGCAGTCCTCCTCGGGCTTCCTGTACCGGCGGCTCAAGACGCAGGAGAAGCGGG AGATGCAGAAGGAGATCTTGTCGGTGCTGGGGCTCCCGCACCGGCCCCGGCCCCTGCACGGC CTCCAACAGCCGCAGCCCCCGGCGCTCCGGCAGCAGGAGGAGCAGCAGCAGCAGCT GCCTCGCGGAGCT GCCTCGCGGAGCT G
  • An exemplary nucleotide sequence of BMP-7 cloned into an exemplary recombinant adenovirus delivery vehicle is as follows, where the flanking adenoviral sequence including restriction sites is underlined:
  • a BMP comprises SEQ ID NO: 1 or a functional fragment thereof, SEQ ID NO: 2 or a functional fragment thereof, SEQ ID NO: 3 or a functional fragment thereof, or SEQ ID NO: 4 or a functional fragment thereof, or an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
  • a functional fragment of a BMP refers to a fragment that exhibits at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity, e.g., BMP receptor binding activity, of a corresponding full length BMP protein.
  • a disclosed delivery vehicle may further comprise an exogenous nucleotide sequence that encodes for a therapeutic transgene.
  • a therapeutic transgene may encode a therapeutic nucleic acid, e.g., an antisense RNA or ribozyme RNA.
  • the therapeutic transgene may encode a therapeutic peptide or polypeptide, e.g., a costimulatory molecule, an antiangiogenic and/or antivascular molecule, an anti-fibrotic molecule, an anti-inflammatory or pro-inflammatory molecule, a monoclonal antibody or fragment thereof, a pro-apoptotic molecule, an enzyme, an immunomodulatory molecule, a cytokine, a cytokine inhibitor (e.g., a cytokine trap), an oncoprotein, a tumor suppressor, a lytic peptide, a vaccine antigen, and a molecule which complements genetic defects in somatic cells
  • a therapeutic peptide or polypeptide e.g., a costimulatory molecule, an antiangiogenic and/or antivascular molecule, an anti-fibrotic molecule, an anti-inflammatory or pro-inflammatory molecule, a monoclonal antibody or fragment thereof, a pro-apoptotic molecule, an enzyme, an immunomodulatory molecule
  • the therapeutic transgene encodes a therapeutic polypeptide, or a fragment thereof, selected from acetylcholine, an androgen-receptor, an anti-PD-l antibody heavy chain and/or light chain, an anti-PD-Ll antibody heavy chain and/or light chain, BORIS/CTCFL, BRAF, CD19, CD20, CD30, CD80, CD86, CD137, CD137L, CD154, CEA, DKKl/Wnt, EGFRvIII, FGF, gplOO, Her-2/neu, ICAM, IL-l, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-l 7, IL-23A/pl9, p40, IL-24, IL-27, IL-27A/p28, IL- 27B/EBI3, IL-35, interferon-gamma, KRAS, MAGE, MAGE-A3, MART1, melan-A
  • the therapeutic transgene encodes a cancer antigen derived from 9D7, androgen receptor, a BAGE family protein, b-catenin, BING-4, BRAF, BRCA1/2, a CAGE family protein, calcium-activated chloride channel 2, CD 19, CD20, CD30, CDK4, CEA, CML66, CT9, CT10, cyclin-Bl, EGFRvIII, Ep-CAM, EphA3, fibronectin, a GAGE family protein, gpl00/pmell7, Her-2/neu, HPV E6, HPV E7, Ig, immature laminin receptor, a MAGE family protein (e.g., MAGE-A3), MART- 1 /melan-A, MART2, MC1R, mesothelin, a mucin family protein (e.g., MUC-l), NY-ESO-l/LAGE-l, P.polypeptide, p53, podocalyx
  • a delivery vehicle e.g., a recombinant virus
  • a therapeutic agent preferably is combined with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the carrier(s) should be“acceptable” in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient.
  • Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
  • compositions containing recombinant viruses disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method.
  • a pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, intraocular, intranasal, transdermal, topical, transmucosal, rectal, oral, parenteral, subcutaneous, intramuscular, ophthalmic, epidural, intratracheal, sublingual, buccal, vaginal, and nasal administration.
  • An exemplary route of administration is IV infusion.
  • Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990).
  • Formulation components suitable for parenteral administration include 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 EDTA;
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.
  • compositions preferably are sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
  • an effective amount refers to the amount of an active component (e.g., the amount of a recombinant virus of the present invention) sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
  • a therapeutically effective amount of active component is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg, 1 mg/kg to 5 mg/kg, 10 mg/kg, 7.5 mg/kg, 5 mg/kg, or 2.5 mg/kg.
  • a therapeutically effective amount of a recombinant virus is in the range of 10 2 to 10 15 plaque forming units (pfiis), e.g., l0 2 to 10 10 , l0 2 to 10 5 , l0 5 to 10 15 , l0 5 to 10 10 , or l0 10 to l0 15 plaque forming units.
  • a therapeutically effective amount of a radionuclide virus is in the range of 1-500000 kBq/kg, e.g., 1-200000 kBq/kg, 1-150000 kBq/kg, 1-100000 kBq/kg, 1-50000 kBq/kg, 1-20000 kBq/kg, 1-10000 kBq/kg, 1-5000 kBq/kg, 1-2000 kBq/kg, 1-1000 kBq/kg, 1-500 kBq/kg, 1-100 kBq/kg, 1-50 kBq/kg, or 1-20 kBq/kg.
  • the amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the subject, the in vivo potency of the virus, the
  • the initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue- level.
  • the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment.
  • Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg.
  • Dosing frequency can vary, depending on factors such as route of administration, dosage amount, the half-life of the recombinant virus, and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks.
  • a preferred route of administration is parenteral, e.g., intravenous infusion.
  • the methods and compositions disclosed herein can be used to treat various medical indications.
  • the methods and compositions disclosed herein can be used to treat cancers.
  • the cancer cells are exposed to a therapeutically effective amount of a delivery vehicle (e.g., a recombinant virus) and/or a therapeutic agent so as to inhibit or reduce proliferation of the cancer cells.
  • a delivery vehicle e.g., a recombinant virus
  • the invention provides a method of treating a cancer in a subject.
  • the method comprises administering to the subject an effective amount of a delivery vehicle and/or a therapeutic agent to treat the cancer in the subject.
  • administering an effective amount of a delivery vehicle and/or a therapeutic agent to a subject reduces tumor load in that subject by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.
  • the methods and compositions disclosed herein can be used to treat bone loss and/or hypercalcemia, e.g., bone loss and/or hypercalcemia associated with a cancer.
  • the invention provides a method of bone loss and/or hypercalcemia in a subject, e.g., a subject with cancer.
  • the method comprises administering to the subject an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding a protein that mediates the production of bone tissue to treat the bone loss and/or hypercalcemia in a subject.
  • “treat”,“treating” and“treatment” mean the treatment of a disease in a subject, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state.
  • the terms“subject” and“patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.
  • Examples of cancers include solid tumors, soft tissue tumors, hematopoietic tumors and metastatic lesions.
  • hematopoietic tumors include, leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), e.g., transformed CLL, diffuse large B-cell lymphomas (DLBCL), follicular lymphoma, hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, or Richter’s Syndrome (Richter’s Transformation).
  • solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting head and neck (including pharynx), thyroid, lung (small cell or non-small cell lung carcinoma (NSCLC)), breast, lymphoid, gastrointestinal (e.g., oral, esophageal, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genitals and genitourinary tract (e.g., renal, urothelial, bladder, ovarian, uterine, cervical, endometrial, prostate, testicular), CNS (e.g., neural or glial cells, e.g., neuroblastoma or glioma), or skin (e.g, melanoma).
  • malignancies e.g., sarcomas, adenocarcinomas, and carcinomas
  • various organ systems such as those
  • the cancer is selected from melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, head and neck cancer, breast cancer, anal cancer, cervical cancer, non-small cell lung cancer, mesothelioma, small cell lung cancer, renal cell carcinoma, prostate cancer, gastroesophageal cancer, colorectal cancer, testicular cancer, bladder cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, brain and central nervous system cancer, thyroid cancer, parathyroid cancer (e.g, parathyroid carcinoma), endometrial cancer, neuroendocrine cancer, lymphoma (e.g., Hodgkin and non- Hodgkin), leukemia, merkel cell carcinoma, gastrointestinal stromal tumors, multiple myeloma, uterine cancer, a sarcoma, kidney cancer, ocular cancer, pancreatic cancer, and a germ cell cancer (e.g., ovarian germ cell cancer).
  • melanoma s
  • the invention also provides a method of generating bone tissue in a subject in need thereof.
  • the method comprises administering to the subject an effective amount of a delivery vehicle described herein to generate bone tissue in the subject.
  • the generation of bone tissue may, e.g., treat a non-union or fracture in the subject, or be used for a spinal fusion or dental implant in the subject.
  • a delivery vehicle e.g., a recombinant virus
  • a therapeutic agent are administered to the subject in combination with one or more therapies, e.g., surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or virotherapy.
  • a delivery vehicle e.g., a recombinant virus
  • a therapeutic agent are administered to the subject in combination with a therapy that promotes bone strength and/or growth, e.g., an anti-resorptive therapy.
  • a delivery vehicle e.g., a recombinant virus
  • a therapeutic agent are administered to the subject in combination with a bisphosphonate, testosterone, hyaluronic acid, vitamin D, calcium, testosterone, estrogen, progesterone, a selective estrogen modulator (SERM), calcitonin, a RANK or RANKL inhibitor (e.g., denosumab), pulsed ultrasound (e.g., low intensity pulsed ultrasound (LIPU)), electrical stimulation, extracorporeal shockwave therapy (ESWT), low energy laser therapy, and/or pulsed electromagnetic field (PEMF) therapy.
  • a bisphosphonate e.g., testosterone, hyaluronic acid
  • vitamin D calcium
  • testosterone, estrogen, progesterone e.g., progesterone
  • SERM selective estrogen modulator
  • calcitonin calcitonin
  • a RANK or RANKL inhibitor e.g., denosumab
  • pulsed ultrasound e.
  • a delivery vehicle e.g., a recombinant virus
  • a therapeutic agent are administered to the subject in combination with a bisphosphonate.
  • exemplary bisphosphonates include disodium pamidronate, alendronate, etidronate, tiludronate, risedronate, zoledronic acid, sodium clodronate, and ibandronic acid.
  • a delivery vehicle e.g., a recombinant virus
  • a therapeutic agent of the invention is administered in combination with a tyrosine kinase inhibitor, e.g., erlotinib.
  • a delivery vehicle e.g., a recombinant virus
  • a therapeutic agent of the invention is administered in combination with a checkpoint inhibitor, e.g., an anti-CTLA-4 antibody, an anti -PD- 1 antibody, or an anti-PD-Ll antibody.
  • a checkpoint inhibitor e.g., an anti-CTLA-4 antibody, an anti -PD- 1 antibody, or an anti-PD-Ll antibody.
  • Exemplary anti-PD-l antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech).
  • Exemplary anti-PD-Ll antibodies include, for example, atezolizumab (Tecentriq®, Genentech), duvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).
  • the term administered "in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the subject overlap at a point in time.
  • the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous" or “concurrent delivery.”
  • the delivery of one treatment ends before the delivery of the other treatment begins.
  • the treatment is more effective because of combined administration.
  • the second treatment is more effective, e.g.
  • delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
  • the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
  • the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
  • the effective amount of a delivery vehicle (e.g., a recombinant virus) and/or a therapeutic agent of the invention is identified by measuring an immune response to an antigen in the subject and/or the method of treating the subject further comprises measuring an immune response to an antigen in the subject.
  • a delivery vehicle e.g., a recombinant virus
  • a therapeutic agent of the invention is identified by measuring an immune response to an antigen in the subject and/or the method of treating the subject further comprises measuring an immune response to an antigen in the subject.
  • Hyperproliferative diseases e.g., cancers, may be characterized by immunosuppression, and measuring an immune response to an antigen in the subject may be indicative of the level of
  • measuring an immune response to an antigen in the subject may be indicative of the efficacy of the treatment and/or the effective amount of the recombinant virus.
  • the immune response to the antigen in the subject may be measured by any method known in the art.
  • the immune response to the antigen is measured by injecting the subject with the antigen at an injection site on the skin of the subject and measuring the size of an induration or amount of inflammation at the injection site.
  • the immune response to the antigen is measured by release of a cytokine from a cell of the subject (e.g., interferon gamma, IL-4 and/or IL-5) upon exposure to the antigen.
  • compositions, devices, and systems are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions, devices, and systems of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
  • viruses, compositions, systems, processes and methods, or features thereof are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges. By way of other examples, an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • This Example describes the construction of a recombinant adenovirus type 5 (Ad5) that expresses a protein that defines a binding site or a protein that mediates the production of a biomolecule that defines a binding site for a therapeutic agent, e.g., BMP -2 or BMP-7.
  • Ad5 a recombinant adenovirus type 5
  • a plasmid carrying the 5' portion of the adenovirus type 5 genomic sequence was modified to carry the deletion of a nucleotide region located from -304 to -255 upstream of the Ela initiation site, which renders Ela expression cancer-selective (as previously described in U.S. Patent No. 9,073,980).
  • the modified plasmid is hereafter referred to as the TAV plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV virus.
  • the TAV plasmid was further modified to carry a Sall site at the start of the Elb- l9k region and an Xhol site 200 base pairs 3' of the Sall site to facilitate insertion of therapeutic transgenes.
  • a Sall site at the start of the Elb- l9k region and an Xhol site 200 base pairs 3' of the Sall site to facilitate insertion of therapeutic transgenes.
  • the plasmid was cut with Sall and Xhol and self-ligated.
  • the nucleotide sequence of the modified Elb-l9k region is as follows, with the residual bases from the fused Sall and Xhol sites underlined:
  • the modified plasmid is hereafter referred to as the TAV-Al9k plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV-Al9k virus.
  • Exogenous nucleotide sequences encoding BMP -2 or BMP-7 were cloned into the modified Elb-l9k region.
  • Bone morphogenetic proteins (BMPs) are osteoinductive growth factors that belong to the transforming growth factor b (TGF-b) family.
  • TGF-b transforming growth factor b
  • the modified plasmid is hereafter referred to as the TAV-BMP-2 plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV-BMP-2 virus.
  • a nucleotide sequence encoding exemplary protein BMP-7 was cloned in to the modified Elb-l9k region of the TAV-Al9k plasmid.
  • the modified plasmid is hereafter referred to as the TAV-BMP-7 plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV-BMP-7 virus.
  • This Example describes the construction of a recombinant adenovirus type 5 (Ad5) that expresses a protein that defines a binding site or a protein that mediates the production of a biomolecule that defines a binding site for a therapeutic agent, e.g., BMP -4 or BMP-6.
  • Ad5 a recombinant adenovirus type 5
  • a plasmid carrying the 5' portion of the adenovirus type 5 genomic sequence is modified to carry the deletion of a nucleotide region located from -304 to -255 upstream of the Ela initiation site, which renders Ela expression cancer-selective (as previously described in U.S. Patent No. 9,073,980).
  • the modified plasmid is hereafter referred to as the TAV plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV virus.
  • the TAV plasmid is further modified to carry a Sall site at the start of the Elb- l9k region and an Xhol site 200 base pairs 3' of the Sall site to facilitate insertion of therapeutic transgenes.
  • a Sall site at the start of the Elb- l9k region and an Xhol site 200 base pairs 3' of the Sall site to facilitate insertion of therapeutic transgenes.
  • the plasmid is cut with Sall and Xhol and self-ligated.
  • the nucleotide sequence of the modified Elb-l9k region is as follows, with the residual bases from the fused Sall and Xhol sites underlined:
  • TAV-A 19k plasmid ATCTTGGTTACATCTGACCTCGTCGAGTCACCAGGCGCTTTTCCAA ( SEQ ID NO: 16).
  • TAV-D 19k virus any resulting viral particles produced therefrom are hereafter referred to as the TAV-D 19k virus.
  • BMPs Bone morphogenetic proteins
  • TGF-b transforming growth factor b
  • a nucleotide sequence encoding exemplary protein BMP-4 is cloned in to the modified Elb-l9k region of the TAV-Al9k plasmid.
  • the modified plasmid is hereafter referred to as the TAV-BMP-4 plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV-BMP-4 virus.
  • a nucleotide sequence encoding exemplary protein BMP-6 is cloned in to the modified Elb-l9k region of the TAV- Al9k plasmid.
  • the modified plasmid is hereafter referred to as the TAV-BMP-6 plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV-BMP-6 virus.
  • This Example describes the expression of BMPs from recombinant viruses.
  • A549 cells human lung cancer cells
  • (i) were infected with the TAV-BMP-2 virus, prepared as described in Example 1, at a multiplicity of infection (MOI) of 5,
  • (ii) were infected with the TAV-Al9k virus, without the BMP -2 gene, prepared as described in example 1, at an MOI of 5, or (ii) kept as non-infected controls.
  • BMP -2 levels in collected media were assayed by ELISA four days after infection. As depicted in FIGURE 1, BMP -2 expression from the TAV-BMP-2 virus resulted in approximately 10 ng/ml of BMP -2 in the collected media.
  • A549 cells were similarly (i) were infected with the TAV-BMP-7 virus, prepared as described in Example 1, at a multiplicity of infection (MOI) of 5, (ii) were infected with the TAV-Al9k virus, without the BMP-7 gene, prepared as described in example 1, at an MOI of 5, or (ii) kept as non-infected controls.
  • BMP-7 levels in conditioned media were measured by ELISA four days after infection. As shown in FIGURE 2, BMP-7 expression from the TAV-BMP-7 virus resulted in approximately 8 ng/ml of BMP-7 in the collected media.
  • Example 4 Cytotoxicity of BMP Expressing Viruses
  • This Example describes cytotoxicity resulting from BMP expressing recombinant viruses.
  • A549 cells human lung cancer cells were infected with the TAV-A19k.
  • TAV-A19k human lung cancer cells
  • TAV-BMP-7 viruses produced as described in Example 1.
  • Cells were stained with crystal violet, which stains viable cells blue, at the indicated time points after infection.
  • infection with the TAV-BMP-2 or TAV-BMP-7 results in cell death starting at approximately 6 days after infection.
  • This Example describes treatment of lung cancer in a mouse model with BMP expressing viruses.
  • mice were injected subcutaneously with ADS-12 (mouse lung cancer) cells and allowed to form tumors. After the tumors became large enough to treat, the mice were treated with TAV-Al9k, TAV-BMP-2, or TAV-BMP-7 (produced as described in Example 1), each at a dose of 1E9 PFU (plaque forming units). Viruses were injected intratumorally every four days for a total of three doses. Buffer without virus was used as a control. Tumor volumes of mice treated with buffer, TAV-Al9k, and TAV-BMP-7 are shown in FIGURE 4. Tumor volumes after treatment with TAV-BMP-2 were not smaller than with TAV-Al9k and are omitted for clarity.
  • ADS-12 mamouse lung cancer

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Abstract

The invention relates to compositions comprising (a) a delivery vehicle comprising an exogenous nucleotide sequence encoding a protein that defines a binding site (e.g., a bone morphogenetic protein (BMP) or prostate specific membrane antigen (PSMA)), or a protein that mediates the production of a biomolecule that defines a binding site (e.g., a BMP); and (b) an optional therapeutic agent that binds to the binding site, which may be used for treating cancer, treating bone loss, and/or hypercalcemia, or generating bone tissue in a subject.

Description

METHODS AND COMPOSITIONS FOR TARGETING
CANCER CELLS FOR TREATMENT
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62/696,450, filed July 11, 2018, the entire disclosures of which are incorporated by reference herein.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 11, 2019, is named RDX-037WO_ST25.txt and is 72,471 bytes in size.
FIELD OF THE INVENTION
[0003] The field of the invention is methods and compositions for treating cancer.
BACKGROUND
[0004] Despite extensive knowledge of the underlying molecular mechanisms that cause cancer, most advanced cancers remain incurable with current chemotherapy and radiation protocols. Oncolytic viruses have emerged as a platform technology that has the potential to significantly augment current standard treatment for a variety of malignancies (Kumar, S. et al. (2008) CURRENT OPINION IN MOLECULAR THERAPEUTICS l0(4):37l-379; Kim, D. (2001) EXPERT OPINION ON BIOLOGICAL THERAPY l(3):525-538; Kim D. (2000) ONCOGENE 19(56): 6660-6669). These viruses have shown promise as oncolytic agents that not only directly destroy malignant cells via an infection-to-reproduction-to-lysis chain reaction but also indirectly induce anti-tumor immunity. These immune stimulatory properties have been augmented with the insertion of therapeutic transgenes that are copied and expressed each time the virus replicates.
[0005] Previously developed oncolytic viruses include the oncolytic serotype 5 adenovirus (Ad5) referred to as TAV-255 that is transcriptionally attenuated in normal cells but transcriptionally active in cancer cells (see, PCT Publication No. W02010/101921). It is believed that the mechanism by which the TAV-255 vector achieves such tumor selectivity is through targeted deletion of three transcriptional factor (TF) binding sites for the transcription factors Pea3 and E2F, proteins that regulate adenovirus expression of Ela, the earliest gene to be transcribed after virus entry into the host cell, through binding to specific DNA sequences.
[0006] Despite the efforts to date, there is a need for improved oncolytic viruses for treating cancers and hyperproliferative disorders in human patients, and improved therapies for treating cancers and hyperproliferative disorders in human patients.
SUMMARY OF THE INVENTION
[0007] The invention is based, in part, upon the discovery that a delivery vehicle, e.g., a recombinant oncolytic virus, can be used to selectively express a protein defining a binding site or a protein that mediates the production of a biomolecule defining a binding site in a cancer cell, and thereby target the cancer cell for a treatment by a therapeutic agent that binds to the binding site. Furthermore, it has been discovered that a delivery vehicle, e.g., a recombinant oncolytic virus described herein, can be used to selectively express a bone morphogenetic protein (BMP) in a cancer cell and mediate the production of bone tissue in a tumor. Surprisingly, although certain cancers and metastases are associated with BMP expression, it has been discovered that selective recombinant expression of a BMP in a cancer cell allows for treatment of a cancer by targeting a therapeutic agent that binds to bone tissue, e.g., a bone targeting radionuclide, to a tumor. In addition, the delivery vehicle, e.g., the recombinant oncolytic virus, can also be used to treat bone loss and/or hypercalcemia, or promote the generation of bone tissue in a subject in need thereof.
[0008] Accordingly, in one aspect, the invention provides a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject: (a) an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and (b) an effective amount of a therapeutic agent that binds to the binding site.
[0009] In certain embodiments, the biomolecule defining a binding site is bone tissue, and/or the protein that mediates the production of the biomolecule defining a binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR), osteocalcin, osteopontin, bone sialoprotein, osteonectin, osteoprotegerin (OPG) and alkaline phosphatase. In certain embodiments, the protein defining the binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR) and prostate specific membrane antigen (PSMA). [0010] In certain embodiments, the therapeutic agent comprises a radionuclide or an antibody. In certain embodiments, the radionuclide is a bone targeting radionuclide, e.g., 89Sr (e.g., 89Sr chloride), 223Ra (e.g, 223Ra dichloride),153 Sm (e.g, 153Sm-EDTMP), 177Lu, 90Y, 186Re (e.g., 186Re HEDP), 117mSn (e.g, 117mSn DTP A), and 32P. In certain embodiments, the antibody is an antibody drug conjugate or an antibody radionuclide conjugate.
[0011] In another aspect, the invention provides a method of treating bone loss and/or hypercalcemia in a subject in need thereof, the method comprising administering to the subject an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding a protein that mediates the production of bone tissue. In certain embodiments, the subject has cancer.
[0012] In another aspect, the invention provides a method of generating bone tissue in a subject, the method comprising administering to the subject an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding a protein that mediates the production of bone tissue.
[0013] In certain embodiments, in any of the foregoing methods, the protein that mediates the production of bone is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR), osteocalcin, osteopontin, bone sialoprotein, osteonectin, osteoprotegerin (OPG) and alkaline phosphatase.
[0014] In certain embodiments, in any of the foregoing methods, the BMP is selected from BMP -2, BMP-4, BMP-6, and BMP-7. In certain embodiments, the BMP comprises SEQ ID NO: 1 or a functional fragment thereof, SEQ ID NO: 2 or a functional fragment thereof, SEQ ID NO: 3 or a functional fragment thereof, or SEQ ID NO: 4 or a functional fragment thereof, or an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
[0015] In certain embodiments, in any of the foregoing methods, the delivery vehicle is a recombinant oncolytic virus. In certain embodiments, the recombinant oncolytic virus is selected from an adenovirus, fowlpox virus, lentivirus, herpes virus, vesicular stomatitis virus (VSV), maraba virus, polio virus, and alpha virus. In certain embodiments, the recombinant virus is a recombinant oncolytic adenovirus, e.g., a type 5 adenovirus (Ad5).
[0016] In certain embodiments, in any of the foregoing methods, the exogenous nucleotide sequence is inserted into an Elb-l9K insertion site located between the start site of Elb-l9K and the start site of Elb-55K. In certain embodiments, the Elb-l9K insertion site is located between the start site of Elb-l9K and the stop site of Elb-l9K. In certain embodiments, the Elb-l9K insertion site comprises a deletion of from about 100 to about 305, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 305, about 150 to about 300, about 150 to about 250, or about 150 to about 200 nucleotides adjacent the start site of Elb-l9K. In certain embodiments, the Elb-l9K insertion site comprises a deletion of about 200 nucleotides, e.g., 203 nucleotides adjacent the start site of Elb-l9K. In certain embodiments, the Elb-l9K insertion site comprises a deletion corresponding to nucleotides 1714-1916 of the Ad5 genome (SEQ ID NO: 5), or, the exogenous nucleotide sequence is inserted between nucleotides corresponding to 1714 and 1916 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the exogenous nucleotide sequence is inserted between CTGACCTC (SEQ ID NO: 6) and TCACCAGG (SEQ ID NO: 7), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CTGACCTC (SEQ ID NO: 6), the exogenous nucleotide sequence, and
TCACCAGG (SEQ ID NO: 7).
[0017] In certain embodiments, in any of the foregoing methods, the exogenous nucleotide sequence is inserted into an E3 insertion site located between the stop site of pVIII and the start site of Fiber. In certain embodiments, the E3 insertion site comprises a deletion of from about 500 to about 3185, from about 500 to about 3000, from about 500 to about 2500, from about 500 to about 2000, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 3185, from about 1000 to about 3000, from about 1000 to about 2500, from about 1000 to about 2000, from about 1000 to about 1500, from about 1500 to about 3185, from about 1500 to about 3000, from about 1500 to about 2000, from about 2000 to about 3185, from about 2000 to about 3000, from about 2000 to about 2500, from about 2500 to about 3185, from about 2500 to about 3000, or from about 3000 to about 3185 nucleotides. In certain embodiments, the E3 insertion site is located between the stop site of E3-10.5K and the stop site of E3-14.7K. In certain embodiments, the E3 insertion site comprises a deletion of from about 500 to about 1551, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 1551, from about 1000 to about 1500, or from about 1500 to about 1551 nucleotides adjacent the stop site of E3-10.5K. In certain embodiments, the E3 insertion site comprises a deletion of about 1050 nucleotides adjacent the stop site of E3-10.5K, e.g., the E3 insertion site comprises a deletion of 1063 or 1064 nucleotides adjacent the stop site of E3-10.5K. In certain embodiments, the E3 insertion site comprises a deletion corresponding to the Ad5 dl309 E3 deletion. In certain embodiments, the E3 insertion site comprises a deletion corresponding to nucleotides 29773-30836 of the Ad5 genome (SEQ ID NO: 5), or, the exogenous nucleotide sequence is inserted between nucleotides corresponding to 29773 and 30836 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the exogenous nucleotide sequence is inserted between CAGTATGA (SEQ ID NO: 8) and TAATAAAAAA (SEQ ID NO: 9), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CAGTATGA (SEQ ID NO: 8), the exogenous nucleotide sequence, and TAATAAAAAA (SEQ ID NO: 9).
[0018] In certain embodiments, in any of the foregoing methods, the recombinant oncolytic adenovirus may comprise a deletion of at least one Pea3 binding site, or a functional portion thereof, e.g., the virus may comprise a deletion of nucleotides
corresponding to about -300 to about -250 upstream of the initiation site of E la or a deletion of nucleotides corresponding to -305 or -304 to -255 upstream of the initiation site of Ela. In certain embodiments, the recombinant adenovirus may comprise a deletion of nucleotides corresponding to 195-244 of the Ad5 genome (SEQ ID NO: 5), and/or the recombinant adenovirus may comprise the sequence GGTGTTTTGG (SEQ ID NO: 10). In certain embodiments, the recombinant oncolytic adenovirus may comprise a deletion of at least one Pea3 binding site, or a functional portion thereof, and not comprise a deletion of an E2F binding site.
[0019] In certain embodiments, in any of the foregoing methods, the recombinant oncolytic adenovirus may comprise a deletion of at least one E2F binding site, or a functional portion thereof. In certain embodiments, the recombinant oncolytic adenovirus may comprise a deletion of at least one E2F binding site, or a functional portion thereof, and not comprise a deletion of a Pea3 binding site.
[0020] In certain embodiments, in any of the foregoing methods, the recombinant oncolytic adenovirus may comprise an Ela promoter having a deletion of a functional TATA box, e.g., the deletion of an entire TATA box. For example, the virus may comprise a deletion of nucleotides corresponding to -29 to -26, -33 to -26, -44 to +52, or -148 to +52 of the Ela promoter. In certain embodiments, the deletion comprises a deletion of nucleotides corresponding to 353-552 of the Ad5 genome (SEQ ID NO: 5), and/or the Ela promoter comprises the sequence CTAGGACTG (SEQ ID NO: 11). [0021] In certain embodiments, in any of the foregoing methods, the exogenous nucleotide sequence is not operably linked to an exogenous promoter sequence. In certain embodiments, the recombinant oncolytic adenovirus may selectively replicate in a hyperproliferative cell. In certain embodiments, the recombinant oncolytic adenovirus may selectively express the protein defining a binding site or the protein that mediates the production of a biomolecule defining a binding site in a hyperproliferative cell. The hyperproliferative cell may be a cancer cell, e.g., a lung cancer cell, a colon cancer cell, and a pancreatic cancer cell.
[0022] In certain embodiments, in any of the foregoing methods, the recombinant oncolytic adenovirus comprises a nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
[0023] In certain embodiments, in any of the foregoing methods, the recombinant oncolytic virus further comprises an exogenous nucleotide sequence encoding a
costimulatory molecule, an antiangiogenic and/or antivascular molecule, an anti-fibrotic molecule, an anti-inflammatory or pro-inflammatory molecule, a monoclonal antibody or fragment thereof, a pro-apoptotic molecule, an enzyme, an immunomodulatory molecule, a cytokine, or a cytokine inhibitor (e.g., a cytokine trap).
[0024] In certain embodiments, in any of the foregoing methods, the delivery vehicle (e.g. , a recombinant oncolytic virus) is administered to the subject before, at the same time as, or after the therapeutic agent. In certain embodiments, the delivery vehicle (e.g., a recombinant oncolytic virus) and the therapeutic agent are administered in combination with one or more therapies selected from surgery, radiation, chemotherapy, immunotherapy, hormone therapy, and virotherapy.
[0025] In certain embodiments, the cancer is selected from melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, head and neck cancer, breast cancer, anal cancer, cervical cancer, non-small cell lung cancer, mesothelioma, small cell lung cancer, renal cell carcinoma, prostate cancer, gastroesophageal cancer, colorectal cancer, testicular cancer, bladder cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, brain and central nervous system cancer, thyroid cancer, parathyroid cancer (e.g., parathyroid carcinoma), endometrial cancer, neuroendocrine cancer, lymphoma (e.g., Hodgkin and non- Hodgkin), leukemia, merkel cell carcinoma, gastrointestinal stromal tumors, multiple myeloma, uterine cancer, a sarcoma, kidney cancer, ocular cancer, pancreatic cancer, and a germ cell cancer (e.g., ovarian germ cell cancer).
[0026] In certain embodiments, in any of the foregoing methods, the delivery vehicle and/or the therapeutic agent are administered in combination with a bisphosphonate, hyaluronic acid, vitamin D, calcium, testosterone, estrogen, progesterone, a selective estrogen modulator (SERM), calcitonin, a RANK or RANKL inhibitor (e.g., denosumab), pulsed ultrasound (e.g., low intensity pulsed ultrasound (LIPU)), electrical stimulation,
extracorporeal shockwave therapy (ESWT), low energy laser therapy, and/or pulsed electromagnetic field (PEMF) therapy.
[0027] In certain embodiments, in any of the foregoing methods, the effective amount of the recombinant oncolytic virus is 102-1015 plaque forming units (pfiis). In each of the foregoing methods, the subject can, e.g., be a human, e.g., a pediatric human, or an animal.
[0028] In another aspect, the invention provides a pharmaceutical composition comprising: (a) an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and (b) optionally, an effective amount of a therapeutic agent that binds to the binding site.
[0029] In certain embodiments, the biomolecule defining a binding site is bone tissue, and/or the protein that mediates the production of the biomolecule defining a binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR), osteocalcin, osteopontin, bone sialoprotein, osteonectin, osteoprotegerin (OPG) and alkaline phosphatase.
[0030] In certain embodiments, the protein defining the binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR) and prostate specific membrane antigen (PSMA).
[0031] In certain embodiments, in any of the foregoing compositions, the BMP comprises SEQ ID NO: 1 or a functional fragment thereof, SEQ ID NO: 2 or a functional fragment thereof, SEQ ID NO: 3 or a functional fragment thereof, or SEQ ID NO: 4 or a functional fragment thereof, or an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. [0032] In certain embodiments, in any of the foregoing compositions, the therapeutic agent comprises a radionuclide or an antibody. In certain embodiments, the radionuclide is a bone targeting radionuclide, e.g., 89Sr (e.g., 89Sr chloride), 223Ra (e.g., 223Ra dichloride), 153 Sm (e.g, 153Sm-EDTMP), 177Lu, 90Y, 186Re (e.g., 186Re HEDP), 117mSn (e.g, 117mSn DTP A), and 32P. In certain embodiments, the antibody is an antibody drug conjugate or an antibody radionuclide conjugate.
[0033] In certain embodiments, in any of the foregoing compositions, the delivery vehicle is a recombinant oncolytic virus. In certain embodiments, the recombinant oncolytic virus is selected from an adenovirus, fowlpox virus, lentivirus, herpes virus, vesicular stomatitis virus (VSV), maraba virus, polio virus, and alpha virus. In certain embodiments, the recombinant virus is a recombinant oncolytic adenovirus, e.g., a type 5 adenovirus (Ad5).
[0034] In certain embodiments, in any of the foregoing compositions, the exogenous nucleotide sequence is inserted into an Elb-l9K insertion site located between the start site of Elb-l9K and the start site of Elb-55K. In certain embodiments, the Elb-l9K insertion site is located between the start site of Elb-l9K and the stop site of Elb-l9K. In certain embodiments, the Elb-l9K insertion site comprises a deletion of from about 100 to about 305, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 305, about 150 to about 300, about 150 to about 250, or about 150 to about 200 nucleotides adjacent the start site of Elb-l9K. In certain embodiments, the Elb-l9K insertion site comprises a deletion of about 200 nucleotides, e.g., 203 nucleotides adjacent the start site of Elb-l9K. In certain embodiments, the Elb-l9K insertion site comprises a deletion corresponding to nucleotides 1714-1916 of the Ad5 genome (SEQ ID NO: 5), or, the exogenous nucleotide sequence is inserted between nucleotides corresponding to 1714 and 1916 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the exogenous nucleotide sequence is inserted between CTGACCTC (SEQ ID NO: 6) and TCACCAGG (SEQ ID NO: 7), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CTGACCTC (SEQ ID NO: 6), the exogenous nucleotide sequence, and
TCACCAGG (SEQ ID NO: 7).
[0035] In certain embodiments, in any of the foregoing compositions, the exogenous nucleotide sequence is inserted into an E3 insertion site located between the stop site of pVIII and the start site of Fiber. In certain embodiments, the E3 insertion site comprises a deletion of from about 500 to about 3185, from about 500 to about 3000, from about 500 to about 2500, from about 500 to about 2000, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 3185, from about 1000 to about 3000, from about 1000 to about 2500, from about 1000 to about 2000, from about 1000 to about 1500, from about 1500 to about 3185, from about 1500 to about 3000, from about 1500 to about 2000, from about 2000 to about 3185, from about 2000 to about 3000, from about 2000 to about 2500, from about 2500 to about 3185, from about 2500 to about 3000, or from about 3000 to about 3185 nucleotides. In certain embodiments, the E3 insertion site is located between the stop site of E3-10.5K and the stop site of E3-14.7K. In certain embodiments, the E3 insertion site comprises a deletion of from about 500 to about 1551, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 1551, from about 1000 to about 1500, or from about 1500 to about 1551 nucleotides adjacent the stop site of E3-10.5K. In certain embodiments, the E3 insertion site comprises a deletion of about 1050 nucleotides adjacent the stop site of E3-10.5K, e.g., the E3 insertion site comprises a deletion of 1063 or 1064 nucleotides adjacent the stop site of E3-10.5K. In certain embodiments, the E3 insertion site comprises a deletion corresponding to the Ad5 dl309 E3 deletion. In certain embodiments, the E3 insertion site comprises a deletion corresponding to nucleotides 29773-30836 of the Ad5 genome (SEQ ID NO: 5), or, the exogenous nucleotide sequence is inserted between nucleotides corresponding to 29773 and 30836 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the exogenous nucleotide sequence is inserted between CAGTATGA (SEQ ID NO: 8) and TAATAAAAAA (SEQ ID NO: 9), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CAGTATGA (SEQ ID NO: 8), the exogenous nucleotide sequence, and TAATAAAAAA (SEQ ID NO: 9).
[0036] In certain embodiments, in any of the foregoing compositions, the recombinant oncolytic adenovirus may comprise a deletion of at least one Pea3 binding site, or a functional portion thereof, e.g., the virus may comprise a deletion of nucleotides corresponding to about -300 to about -250 upstream of the initiation site of E la or a deletion of nucleotides corresponding to -305 or -304 to -255 upstream of the initiation site of Ela. In certain embodiments, the recombinant adenovirus may comprise a deletion of nucleotides corresponding to 195-244 of the Ad5 genome (SEQ ID NO: 5), and/or the recombinant adenovirus may comprise the sequence GGTGTTTTGG (SEQ ID NO: 10). In certain embodiments, the recombinant oncolytic adenovirus may comprise a deletion of at least one Pea3 binding site, or a functional portion thereof, and not comprise a deletion of an E2F binding site. [0037] In certain embodiments, in any of the foregoing compositions, the recombinant oncolytic adenovirus may comprise a deletion of at least one E2F binding site, or a functional portion thereof. In certain embodiments, the recombinant oncolytic adenovirus may comprise a deletion of at least one E2F binding site, or a functional portion thereof, and not comprise a deletion of a Pea3 binding site.
[0038] In certain embodiments, in any of the foregoing compositions, the recombinant oncolytic adenovirus may comprise an Ela promoter having a deletion of a functional TATA box, e.g., the deletion of an entire TATA box. For example, the virus may comprise a deletion of nucleotides corresponding to -29 to -26, -33 to -26, -44 to +52, or -148 to +52 of the Ela promoter. In certain embodiments, the deletion comprises a deletion of nucleotides corresponding to 353-552 of the Ad5 genome (SEQ ID NO: 5), and/or the Ela promoter comprises the sequence CTAGGACTG (SEQ ID NO: 11).
[0039] In certain embodiments, in any of the foregoing compositions, the exogenous nucleotide sequence is not operably linked to an exogenous promoter sequence. In certain embodiments, the recombinant oncolytic adenovirus may selectively replicate in a hyperproliferative cell. In certain embodiments, the recombinant oncolytic adenovirus may selectively express the protein defining a binding site or the protein that mediates the production of a biomolecule defining a binding site in a hyperproliferative cell. The hyperproliferative cell may be a cancer cell, e.g., a lung cancer cell, a colon cancer cell, and a pancreatic cancer cell.
[0040] In certain embodiments, in any of the foregoing compositions, the recombinant oncolytic adenovirus comprises a nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
[0041] In certain embodiments, in any of the foregoing compositions, the recombinant oncolytic virus further comprises an exogenous nucleotide sequence encoding a
costimulatory molecule, an antiangiogenic and/or antivascular molecule, an anti-fibrotic molecule, an anti-inflammatory or pro-inflammatory molecule, a monoclonal antibody or fragment thereof, a pro-apoptotic molecule, an enzyme, an immunomodulatory molecule, a cytokine, or a cytokine inhibitor (e.g., a cytokine trap). [0042] In certain embodiments, in any of the foregoing compositions, the composition further comprises a bisphosphonate, hyaluronic acid, vitamin D, calcium, testosterone, estrogen, progesterone, a selective estrogen modulator (SERM), calcitonin, or a RANK or RANKL inhibitor (e.g., denosumab).
[0043] These and other aspects and advantages of the invention are illustrated by the following figures, detailed description and claims.
DESCRIPTION OF THE DRAWINGS
[0044] The invention can be more completely understood with reference to the following drawings.
[0045] FIGURE 1 is a bar graph showing the expression of BMP-2 by the TAV-BMP-2 virus in A549 cells as determined by ELISA.
[0046] FIGURE 2 is a bar graph showing the expression of BMP-7 by the TAV-BMP-7 virus in A549 cells as determined by ELISA.
[0047] FIGURE 3 depicts crystal violet staining of A549 cells at the indicated time points following infection with the TAV- A 19k, TAV-BMP-2, and TAV-BMP-7 viruses.
[0048] FIGURE 4 depicts tumor volumes of mice carrying subcutaneous ADS-12 tumors treated with vehicle, TAVA19k. or TAV-BMP-7 viruses. Lines show the mean tumor volume of 10 mice in each treatment group and error bars show SEM.
DETAILED DESCRIPTION
[0049] The invention is based, in part, upon the discovery that a delivery vehicle, e.g., a recombinant oncolytic virus, can be used to selectively express a protein defining a binding site or a protein that mediates the production of a biomolecule defining a binding site in a cancer cell, and thereby target the cancer cell for a treatment by a therapeutic agent that binds to the binding site. Furthermore, it has been discovered that a delivery vehicle, e.g., a recombinant oncolytic virus described herein, can be used to selectively express a bone morphogenetic protein (BMP) in a cancer cell, and cause a cancer to acquire a bone cell phenotype, e.g., an osteoblast-like cell phenotype, and/or mediate the production of bone tissue in a tumor. Certain cancers and metastases are associated with increased levels of BMP expression, and cancer cells that acquire a bone cell phenotype have a greater likelihood of surviving and proliferating in bone. Surprisingly, in spite of this fact, it has been discovered that selective recombinant expression of a BMP in a cancer cell, and acquisition of a bone cell phenotype, e.g., an osteoblast-like cell, and/or meditation of the production of bone tissue in a tumor, can facilitate the treatment of a cancer by providing a target for a therapeutic agent that binds to bone tissue, e.g., a bone targeting radionuclide, to a tumor.
[0050] Accordingly, in one aspect, the invention provides a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject: (a) an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and (b) an effective amount of a therapeutic agent that binds to the binding site. In another aspect, the invention provides a pharmaceutical composition comprising: (a) an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and (b) optionally, an effective amount of a therapeutic agent that binds to the binding site.
[0051] In other aspects, the invention provides methods for treating bone loss and/or hypercalcemia or promoting the growth of bone in a subject in need thereof using a delivery vehicle (e.g., a recombinant oncolytic virus) described herein.
[0052] Various features and aspects of the invention are discussed in more detail below.
I. Delivery Vehicles
[0053] Delivery vehicles suitable for use in the invention include, e.g., viruses, bacteria (e.g., Listeria monocytogenes, Salmonella enterica or Serovar typhi murium), nanoparticles, liposomes, exosomes, or microemulsions.
[0054] The term "virus" is used herein to refer any of the obligate intracellular parasites having no protein-synthesizing or energy-generating mechanism. The viral genome may be RNA or DNA. The viruses useful in the practice of the present invention include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picomoviridiae, herpesviridiae, poxyiridae, or adenoviridiae. A recombinantly modified virus is referred to herein as a“recombinant virus.” A recombinant virus may, e.g., be modified by recombinant DNA techniques to be replication deficient, conditionally replicating, or replication competent, and/or be modified by recombinant DNA techniques to include expression of exogenous transgenes. Chimeric viral vectors which exploit advantageous elements of each of the parent vector properties (See, e.g., Feng et al. (1997) NATURE BIOTECHNOLOGY 15:866-870) may also be useful in the practice of the present invention. Although it is generally favored to employ a virus from the species to be treated, in some instances it may be advantageous to use vectors derived from different species that possess favorable pathogenic features. For example, equine herpes virus vectors for human gene therapy are described in PCT Publication No. WO 98/27216. The vectors are described as useful for the treatment of humans as the equine virus is not pathogenic to humans. Similarly, ovine adenoviral vectors may be used in human gene therapy as they are claimed to avoid the antibodies against the human adenoviral vectors. Such vectors are described in PCT Publication No. WO 97/06826.
[0055] Exemplary viruses useful in the practice of the invention include adenovirus, fowlpox virus, lentivirus, herpes virus, vesicular stomatitis virus (VSV), maraba virus, polio virus, and alpha virus. Preferably, the recombinant virus is an adenovirus. Adenoviruses are medium-sized (90-100 nm), non-enveloped (naked), icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. Adenoviruses replicate in the nucleus of mammalian cells using the host's replication machinery. The term "adenovirus" refers to any virus in the genus Adenoviridiae including, but not limited to, human, bovine, ovine, equine, canine, porcine, murine, and simian adenovirus subgenera. In particular, human adenoviruses includes the A-F subgenera as well as the individual serotypes thereof, the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Adl la and Adl lp), 12, 13, 14, 15, 16, 17, 18, 19, l9a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91. Preferred are recombinant viruses derived from human adenovirus types 2 and 5. Unless stated otherwise, all adenovirus type 5 nucleotide numbers are relative to the NCBI reference sequence AC_000008.1, which is depicted herein in SEQ ID NO: 5.
[0056] The adenovirus replication cycle has two phases: an early phase, during which 4 transcription units (El, E2, E3, and E4) are expressed, and a late phase which occurs after the onset of viral DNA synthesis, and during which late transcripts are expressed primarily from the major late promoter (MLP). The late messages encode most of the virus's structural proteins. The gene products of El, E2 and E4 are responsible for transcriptional activation, cell transformation, viral DNA replication, as well as other viral functions, and are necessary tor viral growth.
[0057] The term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a gene if it affects the transcription of the gene. Operably linked nucleotide sequences are typically contiguous. However, as enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome.
[0058] in certain embodiments, the virus has one or more modifications to a regulatory sequence or promoter. A modification to a regulatory sequence or promoter comprises a deletion, substitution, or addition of one or more nucleotides compared to the wild-type sequence of the regulatory sequence or promoter.
[0059] In certain embodiments, the modification of a regulatory sequence or promoter comprises a modification of sequence of a transcription factor binding site to reduce affinity for the transcription factor, for example, by deleting a portion thereof, or by inserting a single point mutation into the binding site. In certain embodiments, the additional modified regulatory sequence enhances expression in neoplastic cells, but attenuates expression in normal cells.
[0060] In certain embodiments, the modified regulatory sequence is operably linked to a sequence encoding a protein. In certain embodiments, at least one of the adenoviral Ela and Elb genes (coding regions) is operably linked to a modified regulatory sequence. In certain embodiments, the Ela gene is operably linked to the modified regulatory sequence.
[0061] The Ela regulatory sequence contains five binding sites for the transcription factor Pea3, designated Pea3 I, Pea3 II, Pea3 III, Pea3 IV, and Pea3 V, where Pea3 I is the Pea3 binding site most proximal to the Ela start site, and Pea3 V is most distal. The Ela regulatory sequence also contains binding sites for the transcription factor E2F, hereby designated E2F I and E2F II, where E2F I is the E2F binding site most proximal to the Ela start site, and E2F II is more distal. From the Ela start site, the binding sites are arranged: Pea3 I, E2F I, Pea3 II, E2F II, Pea3 III, Pea3 IV, and Pea3 V.
[0062] In certain embodiments, at least one of these seven binding sites, or a functional portion thereof, is deleted. A "functional portion" is a portion of the binding site that, when deleted, decreases or even eliminates the functionality, e.g. binding affinity, of the binding site to its respective transcription factor (Pea3 or E2F) by, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to the complete sequence. In certain embodiments, one or more entire binding sites are deleted. In certain embodiments, a functional portion of one or more binding sites is deleted. A "deleted binding site" encompasses both the deletion of an entire binding site and the deletion of a functional portion thereof. When two or more binding sites are deleted, any combination of entire binding site deletion and functional portion deletion may be used.
[0063] In certain embodiments, at least one Pea3 binding site, or a functional portion thereof, is deleted. The deleted Pea3 binding site can be Pea3 I, Pea3 II, Pea3 III, Pea3 IV, and/or Pea3 V. In certain embodiments, the deleted Pea3 binding site is Pea3 II, Pea3 III, Pea3 IV, and/or Pea3 V. In certain embodiments, the deleted Pea3 binding site is Pea3 IV and/or Pea3 V. In certain embodiments, the deleted Pea3 binding site is Pea3 II and/or Pea3
III. In certain embodiments, the deleted Pea3 binding site is both Pea3 II and Pea3 III. In certain embodiments, the Pea3 I binding site, or a functional portion thereof, is retained.
[0064] In certain embodiments, at least one E2F binding site, or a functional portion thereof, is deleted. In certain embodiments, at least one E2F binding site, or a functional portion thereof, is retained. In certain embodiments, the retained E2F binding site is E2F I and/or E2F II. In certain embodiments, the retained E2F binding site is E2F II. In certain embodiments, the total deletion consists essentially of one or more of Pea3 II, Pea3 III, Pea3
IV, and/or Pea3 V, or functional portions thereof.
[0065] In certain embodiments, the virus has a deletion of a 50 base pair region located from -304 to -255 upstream of the Ela initiation site, e.g., corresponding to 195-244 of the Ad5 genome (SEQ ID NO: 5), hereafter referred to as the TAV-255 deletion. In certain embodiments, the TAV-255 deletion results in an Ela promoter that comprises the sequence GGTGTTTTGG (SEQ ID NO: 10).
[0066] In certain embodiments, the virus comprises an Ela promoter having a deletion of a functional TATA box, e.g., the deletion of an entire TATA box. As used herein, a “functional TATA box” refers to a TATA box that is capable of binding to a TATA box binding protein (TBP), e.g., a TATA box that has at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or at least 40%, of the TBP binding activity of a corresponding wild-type TATA box sequence. As used herein, a“non-functional TATA box” refers to a TATA box that, e.g., has less than 30%, less than 20%, less than 10%, or 0% of the TBP binding activity of a corresponding wild-type TATA box sequence. Assays for determining whether a TBP binds to a TATA box are known in the art. Exemplary binding assays include electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and DNAse footprinting assays.
[0067] For example, in certain embodiments, the virus comprises a deletion of nucleotides corresponding to -27 to -24, -31 to -24, -44 to +54, or -146 to +54 of the adenovirus type 5 Ela promoter, which correspond, respectively, to nucleotides 472 to 475, 468 to 475, 455 to 552, and 353 to 552 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the virus comprises a deletion of nucleotides corresponding to -29 to -26, -33 to -26, -44 to +52, or -148 to +52 of the adenovirus type 5 Ela promoter. In certain embodiments, the virus comprises a deletion of nucleotides corresponding to 353 to 552 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the virus comprises a polynucleotide deletion that results in an adenovirus comprising the sequence CTAGGACTG (SEQ ID NO: 11), AGTGCCCG (SEQ ID NO: 19), or TATTCCCG (SEQ ID NO: 20), which result from joining the two polynucleotide sequences that would otherwise flank the deleted polynucleotide sequence. In certain embodiments, the virus comprises a polynucleotide deletion that results in an adenovirus comprising the sequence CTAGGACTG (SEQ ID NO: 11) .
[0068] In certain embodiments, the virus comprises an Ela promoter having a deletion of a functional CAAT box, e.g., the deletion of an entire CAAT box. As used herein, a “functional CAAT box” refers to a CAAT box that is capable of binding to a C/EBP or NF-Y protein, e.g., a CAAT box that has at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or at least 40%, of the a C/EBP or NF-Y binding activity of a corresponding wild-type CAAT box sequence. As used herein, a“non-functional CAAT box” refers to a CAAT box that, e.g., has less than 30%, less than 20%, less than 10%, or 0% of the a C/EBP or NF-Y binding activity of a corresponding wild-type CAAT box sequence. Assays for determining whether a C/EBP or NF-Y protein binds to a CAAT box are known in the art. Exemplary binding assays include electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and DNAse footprinting assays.
[0069] For example, in certain embodiments, the virus comprises a deletion of nucleotides corresponding to -76 to -68 of the adenovirus type 5 Ela promoter, which corresponds to nucleotides 423 to 431 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the virus comprises a polynucleotide deletion that results in an adenovirus comprising the sequence TTCCGTGGCG (SEQ ID NO: 21), which results from joining the two polynucleotide sequences that would otherwise flank the deleted polynucleotide sequence.
[0070] The adenoviral Elb-l9k gene functions primarily as an anti-apoptotic gene and is a homolog of the cellular anti-apoptotic gene, BCL-2. Since host cell death prior to maturation of the progeny viral particles would restrict viral replication, Elb-l9k is expressed as part of the El cassette to prevent premature cell death thereby allowing the infection to proceed and yield mature virions. Accordingly, in certain embodiments, a recombinant virus is provided that includes an Elb-l9K insertion site, e.g., the adenovirus has an exogenous nucleotide sequence inserted into an Elb-l9K insertion site.
[0071] In certain embodiments, the Elb-l9K insertion site is located between the start site of Elb-l9K (i.e., the nucleotide sequence encoding the start codon of Elb-l9k, e.g., corresponding to nucleotides 1714-1716 of SEQ ID NO: 5) and the start site of Elb-55K (i.e., the nucleotide sequence encoding the start codon of Elb-55k, e.g., corresponding to nucleotides 2019-2021 of SEQ ID NO: 5). Throughout the description and claims, an insertion between two sites, for example, an insertion between (i) a start site of a first gene (e.g., Elb-l9k) and a start site of a second gene, (e.g., Elb-55K), (ii) a start site of a first gene and a stop site of a second gene, (iii) a stop site of a first gene and start site of a second gene, or (iv) a stop site of first gene and a stop site of a second gene, is understood to mean that all or a portion of the nucleotides constituting a given start site or a stop site surrounding the insertion may be present or absent in the final virus. Similarly, an insertion between two nucleotides is understood to mean that the nucleotides surrounding the insertion may be present or absent in the final virus.
[0072] In certain embodiments, the Elb-l9K insertion site is located between the start site of Elb-l9K (i.e., the nucleotide sequence encoding the start codon of Elb-l9k, e.g., corresponding to nucleotides 1714-1716 of SEQ ID NO: 5) and the stop site of Elb-l9K (i.e., the nucleotide sequence encoding the stop codon of Elb-l9k, e.g., corresponding to nucleotides 2242-2244 of SEQ ID NO: 5). In certain embodiments, the Elb-l9K insertion site comprises a deletion of from about 100 to about 305, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 305, about 150 to about 300, about 150 to about 250, or about 150 to about 200 nucleotides adjacent the start site of Elb-l9K. In certain embodiments, the Elb-l9K insertion site comprises a deletion of about 200 nucleotides, e.g., 203 nucleotides adjacent the start site of Elb-l9K. In certain embodiments, the Elb-l9K insertion site comprises a deletion corresponding to nucleotides 1714-1916 of the Ad5 genome (SEQ ID NO: 5), or the exogenous nucleotide sequence is inserted between nucleotides corresponding to 1714 and 1916 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the exogenous nucleotide sequence is inserted between CTGACCTC (SEQ ID NO: 6) and TCACCAGG (SEQ ID NO: 7), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CTGACCTC (SEQ ID NO: 6), the exogenous nucleotide sequence, and TCACCAGG (SEQ ID NO: 7). CTGACCTC (SEQ ID NO: 6) and TCACCAGG (SEQ ID NO: 7) define unique boundary sequences for the Elb- 19K insertion site within the Ad5 genome (SEQ ID NO: 5). Throughout the description and claims, a deletion adjacent to a site, for example, a deletion adjacent to a start site of a gene or a deletion adjacent to a stop site of a gene, is understood to mean that the deletion may include a deletion of all, a portion, or none of the nucleotides constituting a given start site or a stop site.
[0073] In certain embodiments, an exogenous nucleotide sequence is inserted into an E3 insertion site located between the stop site of pVIII (i.e., the nucleotide sequence encoding the stop codon of pVIII, e.g., corresponding to nucleotides 27855-27857 of SEQ ID NO: 5) and the start site of Fiber (i.e., the nucleotide sequence encoding the start codon of Fiber, e.g., corresponding to nucleotides 31042-31044 of SEQ ID NO: 5). In certain embodiments, the E3 insertion site comprises a deletion of from about 500 to about 3185, from about 500 to about 3000, from about 500 to about 2500, from about 500 to about 2000, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 3185, from about 1000 to about 3000, from about 1000 to about 2500, from about 1000 to about 2000, from about 1000 to about 1500, from about 1500 to about 3185, from about 1500 to about 3000, from about 1500 to about 2000, from about 2000 to about 3185, from about 2000 to about 3000, from about 2000 to about 2500, from about 2500 to about 3185, from about 2500 to about 3000, or from about 3000 to about 3185 nucleotides. In certain embodiments, the E3 insertion site is located between the stop site of E3-10.5K (i.e., the nucleotide sequence encoding the stop codon of E3-10.5K, e.g., corresponding to nucleotides 29770-29772 of SEQ ID NO: 5) and the stop site of E3-14.7K (i.e., the nucleotide sequence encoding the stop codon of E3-14.7K, e.g., corresponding to nucleotides 30837-30839 of SEQ ID NO: 5). In certain embodiments, the E3 insertion site comprises a deletion of from about 500 to about 1551, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 1551, from about 1000 to about 1500, or from about 1500 to about 1551 nucleotides adjacent the stop site of E3-10.5K. In certain embodiments, the E3 insertion site comprises a deletion of about 1050 nucleotides adjacent the stop site of E3-10.5K, e.g., the E3 insertion site comprises a deletion of 1063 or 1064 nucleotides adjacent the stop site of E3-10.5K. In certain embodiments, the E3 insertion site comprises a deletion corresponding to the Ad5 dl309 E3 deletion. In certain embodiments, the E3 insertion site comprises a deletion corresponding to nucleotides 29773-30836 of the Ad5 genome (SEQ ID NO: 5), or the exogenous nucleotide sequence is inserted between nucleotides corresponding to 29773 and 30836 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the exogenous nucleotide sequence is inserted between CAGTATGA (SEQ ID NO: 8) and TAATAAAAAA (SEQ ID NO: 9), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, CAGTATGA (SEQ ID NO: 8), the exogenous nucleotide sequence, and TAATAAAAAA (SEQ ID NO: 9). CAGTATGA (SEQ ID NO: 8) and TAATAAAAAA (SEQ ID NO: 9) define unique boundary sequences for an E3 insertion site within the Ad5 genome (SEQ ID NO: 5).
[0074] In certain embodiments, the E3 insertion site is located between stop site of E3- gpl9K (i.e.. the nucleotide sequence encoding the stop codon of E3-gpl9K, e.g., corresponding to nucleotides 29215-29217 of SEQ ID NO: 5) and the stop site of E3-14.7K (i.e., the nucleotide sequence encoding the stop codon of E3-14.7K, e.g., corresponding to nucleotides 30837-30839 of SEQ ID NO: 5). In certain embodiments, the E3 insertion site comprises a deletion of from about 500 to about 1824, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 1824, from about 1000 to about 1500, or from about 1500 to about 1824 nucleotides adjacent the stop site of E3-gpl9K. In certain embodiments, the E3 insertion site comprises a deletion of about 1600 nucleotides adjacent the stop site of E3-gpl9K. e.g., the E3 insertion site comprises a deletion of 1622 nucleotides adjacent the stop site of E3-gpl9K. In certain embodiments, the E3 insertion site comprises a deletion corresponding to nucleotides 29218-30839 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the exogenous nucleotide sequence is inserted between nucleotides corresponding to 29218 and 30839 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the exogenous nucleotide sequence is inserted between TGCCTTAA (SEQ ID NO: 17) and TAAAAAAAAAT (SEQ ID NO: 18), e.g., the recombinant adenovirus comprises, in a 5’ to 3’ orientation, TGCCTTAA (SEQ ID NO: 17), the exogenous nucleotide sequence, and TAAAAAAAAAT (SEQ ID NO: 18). TGCCTTAA (SEQ ID NO: 17) and TAAAAAAAAAT (SEQ ID NO: 18) define unique boundary sequences for an E3 insertion site within the Ad5 genome (SEQ ID NO: 5). [0075] In certain embodiments, the vims comprises an E4 deletion. In certain embodiments, the E4 deletion is located between the start site of E4-ORF6/7 (i.e., the nucleotide sequence encoding the start codon of E4-ORF6/7, e.g., corresponding to nucleotides 34075-34077 of SEQ ID NO: 5) and the right inverted terminal repeat (ITR; e.g., corresponding to nucleotides 35836-35938 of SEQ ID NO: 5). In certain embodiments, the E4 deletion is located between the start site of E4-ORF6/7 and the start site of E4-ORF1 (i.e.. the nucleotide sequence encoding the start codon of E4-ORF1, e.g., corresponding to nucleotides 35524-35526 of SEQ ID NO: 5). In certain embodiments, the E4 deletion comprises a deletion of a nucleotide sequence between the start site of E4-ORF6/7 and the start site of E4-ORF1. In certain embodiments, the E4 deletion comprises a deletion of from about 500 to about 2500, from about 500 to about 2000, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 2500, from about 1000 to about 2000, from about 1000 to about 1500, from about 1500 to about 2500, from about 1500 to about 2000, or from about 2000 to about 2500 nucleotides. In certain embodiments, the E4 deletion comprises a deletion of from about 250 to about 1500, from about 250 to about 1250, from about 250 to about 1000, from about 250 to about 750, from about 250 to about 500, from 500 to about 1500, from about 500 to about 1250, from about 500 to about 1000, from about 500 to about 750, from 750 to about 1500, from about 750 to about 1250, from about 750 to about 1000, from about 1000 to about 1500, or from about 1000 to about 1250 nucleotides adjacent the start site of E4-ORF6/7. In certain embodiments, the E4 deletion comprises a deletion of about 1450 nucleotides adjacent the start site of E4-ORF6/7, e.g., the E4 deletion comprises a deletion of about 1449 nucleotides adjacent the start site of E4-ORF6/7. In certain embodiments, the E4 deletion comprises a deletion corresponding to nucleotides 34078-35526 of the Ad5 genome (SEQ ID NO: 5).
[0076] In certain embodiments, the virus includes an IX-E2 insertion site, e.g., the virus has an exogenous nucleotide sequence inserted into an IX-E2 insertion site. In certain embodiments, the IX-E2 insertion site is located between the nucleotide sequence encoding the stop codon of IX and the nucleotide sequence encoding the stop codon of IVa2. In certain embodiments, the nucleotide sequence is inserted between nucleotides corresponding to 4029 and 4093 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the nucleotide sequence is inserted between nucleotides corresponding to 4029 and 4050, nucleotides corresponding to 4051 and 4070, or nucleotides corresponding to 4071 and 4093 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the IX-E2 insertion site comprises a deletion of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides.
[0077] In certain embodiments, the virus includes an L5-E4 insertion site, e.g., the virus has an exogenous nucleotide sequence inserted into an L5-E4 insertion site. In certain embodiments, the L5-E4 insertion site is located between the nucleotide sequence encoding the stop codon of Fiber and the nucleotide sequence encoding the stop codon of E4-ORF6 or E40RF6/7. In certain embodiments, the nucleotide sequence is inserted between nucleotides corresponding to 32785 to 32916 of the Ad5 genome (SEQ ID NO: 5). In certain
embodiments, the nucleotide sequence is inserted between nucleotides corresponding to 32785 and 32800, nucleotides corresponding to 32801 and 32820, nucleotides corresponding to 32821 and 32840, nucleotides corresponding to 32841 and 32860, nucleotides
corresponding to 32861 and 32880, nucleotides corresponding to 32881 and 32900, or nucleotides corresponding to 32901 and 32916 of the Ad5 genome (SEQ ID NO: 5). In certain embodiments, the L5-E4 insertion site comprises a deletion of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or 130 nucleotides.
[0078] In certain embodiments, the recombinant virus comprises a nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
[0079] Sequence identity may be determined in various ways that are within the skill in the art, e.g., using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al, ( 1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES. 25 :3389-3402, incorporated by reference) are tailored for sequence similarity searching. For a discussion of basic issues in searching sequence databases see Altschul et al., (1994) NATURE GENETICS 6: 1 19-129, which is fully incorporated by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The search parameters for histogram, descriptions, alignments, expect (i.e.. the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the
BLOSUM62 matrix (Henikoff et al, (1992) PROC. NATL. ACAD. SCI. USA 89: 10915-10919, fully incorporated by reference). Four blastn parameters may be adjusted as follows: Q=l0 (gap creation penalty); R=l0 (gap extension penalty); wink=l (generates word hits at every wink.sup.th position along the query); and gapw=l6 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings may be Q=9; R=2; wink=l; and gapw=32. Searches may also be conducted using the NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameter (e.g. : -G, Cost to open gap [Integer]: default = 5 for nucleotides/ 11 for proteins; -E, Cost to extend gap [Integer] : default = 2 for nucleotides/ 1 for proteins; -q, Penalty for nucleotide mismatch [Integer] : default = -3; -r, reward for nucleotide match [Integer]: default = 1; -e, expect value [Real]: default = 10; -W, wordsize [Integer]: default = 11 for nucleotides/ 28 for megablast/ 3 for proteins; -y, Dropoff (X) for blast extensions in bits: default = 20 for blastn/ 7 for others; - X, X dropoff value for gapped alignment (in bits): default = 15 for all programs, not applicable to blastn; and -Z, final X dropoff value for gapped alignment (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, e.g., Blosum62 matrix and Gap Opening Penalty = 10 and Gap Extension Penalty = 0.1). A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.
[0080] In certain embodiments, a recombinant virus is an oncolytic virus, e.g., a virus that exhibits tumor-selective replication and/or viral mediated lysis. In certain embodiments, a recombinant virus of the invention exhibits selective expression of a protein, e.g., a protein defining a binding site or the protein that mediates the production of a biomolecule defining a binding site therapeutic transgene in a hyperproliferative cell, e.g., a cancer cell, relative to a non-hyperprobferative cell. In certain embodiments, the expression of the protein in a non- hyperproliferative cell is about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10% , or about 5% of the expression of in a
hyperproliferative cell. In certain embodiments, the virus exhibits no detectable expression of the protein in a non-hyperproliferative cell. Therapeutic transgene expression may be determined by any appropriate method known in the art, e.g., Western blot or ELISA. [0081] The hyperproliferative cell may be a cancer cell, e.g., a carcinoma, sarcoma, leukemia, lymphoma, prostate cancer, lung cancer, gastrointestinal tract cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, stomach cancer, thyroid cancer, mesothelioma, liver cancer, kidney cancer, skin cancer, head and neck cancer, or brain cancer cell.
II. Methods of Viral Production
[0082] Methods for producing recombinant viruses of the invention are known in the art. Typically, a disclosed virus is produced in a suitable host cell line using conventional techniques including culturing a transfected or infected host cell under suitable conditions so as to allow the production of infectious viral particles. Nucleic acids encoding viral genes can be incorporated into plasmids and introduced into host cells through conventional transfection or transformation techniques. Exemplary suitable host cells for production of disclosed viruses include human cell lines such as HeLa, Hela-S3, HEK293, 911, A549, HER96, or PER-C6 cells. Specific production and purification conditions will vary depending upon the virus and the production system employed. For adenovirus, the traditional method for the generation of viral particles is co-transfection followed by subsequent in vivo recombination of a shuttle plasmid (usually containing a small subset of tire adenoviral genome and optionally containing a potential transgene an expression cassette) and an adenoviral helper plasmid (containing most of the entire adenoviral genome).
[0083] Alternative technologies for the generation of adenovirus include utilization of the bacterial artificial chromosome (BAG) system, in vivo bacterial recombination in a recAT bacterial strain utilizing two plasmids containing complementary adenoviral sequences, and the yeast artificial chromosome (YAC) system.
[0084] Following production, infectious viral particles are recovered from the culture and optionally purified. Typical purification steps may include plaque purification, centrifugation, e.g., cesium chloride gradient centrifugation, clarification, enzymatic treatment, e.g., benzonase or protease treatment, chromatographic steps, e.g., ion exchange chromatography or filtration steps.
III. Therapeutic Agents and Therapeutic Agent Binding Sites
[0085] The invention provides methods and compositions comprising: (a) an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and (b) an effective amount of a therapeutic agent that binds to the binding site.
[0086] As used herein, the term“therapeutic agent that binds to a binding site” refers to any therapeutic agent (/. e. , any agent that imparts a therapeutic effect in a target cell, body fluid, tissue, organ, physiological system, or subject) that reacts or associates more frequently, more rapidly, with greater duration and/or with stronger affinity with a particular binding site (e.g., a binding site on a protein encoded by a delivery vehicle or a binding site on a biomolecule the production of which is mediated by a protein encoded by a delivery vehicle, e.g., bone tissue) than it does with an alternative binding site. The therapeutic agent may, e.g., have affinity for the binding site stronger than 100 nM, 50 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM, as determined by surface plasmon resonance. As used herein, the term“a biomolecule defining a binding site” is understood to contemplate an individual biomolecule, e.g., a protein, and/or a collection of biomolecules, e.g., a tissue, e.g., bone tissue.
[0087] In certain embodiments, the protein that defines a binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR) and prostate specific membrane antigen (PSMA), and/or the therapeutic agent that binds to the protein is selected from an anti-BMP antibody, an anti-BMPR antibody, and an anti-PSMA antibody.
[0088] The BMP may, for example, be selected from BMP-2, BMP-4, BMP-6 and BMP- 7, and accordingly, the anti-BMP antibody may, for example, be selected from an anti-BMP - 2 antibody, an anti-BMP -4 antibody, an anti-BMP-6 antibody, and an anti-BMP-7 antibody.
[0089] In certain embodiments, the therapeutic agent is an antibody. In general, antibodies are multimeric proteins that contain four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains), and two of the polypeptide chains are called immunoglobulin light chains (L chains). The immunoglobulin heavy and light chains are connected by an interchain disulfide bond. The immunoglobulin heavy chains are connected by interchain disulfide bonds. A light chain consists of one variable region (VL) and one constant region (CL). The heavy chain consists of one variable region (VH) and at least three constant regions (CHi, CH2 and CH3). The variable regions determine the binding specificity of the antibody. [0090] Each variable region contains three hypervariable regions known as
complementarity determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs). The extent of the FRs and CDRs has been defined (Rabat, E.A., et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, FIFTH EDITION, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. MOL. BIOL. 196:901-917). The three CDRs, referred to as CDRi, CDR2, and CDR3, contribute to the antibody binding specificity. Naturally occurring antibodies have been used as starting material for engineered antibodies, such as chimeric antibodies and humanized antibodies.
[0091] As used herein, unless otherwise indicated, the term“antibody” means an intact antibody (e.g., an intact monoclonal antibody) or antigen-binding fragment of an antibody, including an intact antibody or antigen-binding fragment that has been modified, engineered or chemically conjugated. Examples of antibodies that have been modified or engineered are chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab’, F(ab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, and diabodies. An antibody conjugated to a toxin, radioisotope, cytokine, or enzyme is an example of a chemically conjugated antibody. In certain embodiments, the antibody the antibody is selected from an antibody drug conjugate and an antibody radionuclide conjugate.
[0092] In certain embodiments, an antibody may mediate an“effector functions,” e.g., a biological activity attributable to the Fc region of an antibody that varies with the antibody isotype. Examples of antibody effector functions include Clq binding and complement dependent cytotoxicity, Fc receptor binding, antibody-dependent cell mediated cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.
[0093] In certain embodiments, the therapeutic agent is a cell, e.g., a T-cell. The T-cell can be any T-cell, such as a cultured T-cell, e.g., a primary T-cell, or a T-cell from a cultured T-cell line, e.g., Jurkat, SupTi, etc., or a T-cell obtained from a mammal. If obtained from a mammal, the T-cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T-cells can also be enriched for or purified. Preferably, the T-cell is a human T-cell, which can be an autologous or heterologous cell. The T-cell can be any type of T-cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T-cells, CD4+ helper T-cells, e.g., Thl and Th2 cells, CD4+ T-cells, CD8+ T-cells (e.g., cytotoxic T-cells), tumor infiltrating lymphocytes (TILs), memory T-cells (e.g., central memory T-cells and effector memory T-cells), naive T-cells, and the like. The cells can include autologous cells derived from a subject to be treated, or alternatively allogenic cells derived from a donor.
[0094] In certain embodiments, a T-cell binds to a binding site through a T-cell receptor. The T-cell receptor may be an endogenous or a recombinant T-cell receptor. T-cell receptors comprise two chains referred to as the a- and b-chains, that form a pair on the surface of a T- cell to form a heterodimeric receptor. The T-cell receptor is involved in recognition of MHC- restricted antigens. Each of a- and b- chain comprises two regions, a constant region and a variable region. Each variable region of the a- and b- chains defines three loops, referred to as complementary determining regions (CDRs) known as CDRi, CDR2, and CDR3 that confer the T-cell receptor with antigen binding activity and binding specificity.
[0095] In certain embodiments, a T-cell binds to a binding site through a chimeric antigen receptor (CAR). As used herein, chimeric antigen receptor, or CAR, refers to any artificial receptor including an antigen-specific binding moiety and one or more signaling chains derived from an immune receptor. Commonly, CARs comprise a single chain fragment variable (scFv) of an antibody specific for an antigen coupled via hinge and transmembrane regions to cytoplasmic domains of T-cell signaling molecules, e.g. a T-cell costimulatory domain (e.g., from CD28, CD 137, 0X40, ICOS, and CD27) in tandem with a T-cell triggering domain (e.g. from CD3z). A T-cell expressing a chimeric antigen receptor is referred to as a CAR T-cell.
[0096] In certain embodiments, the protein that mediates the production of a biomolecule defining a binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR), osteocalcin, osteopontin, bone sialoprotein, osteonectin, osteoprotegerin (OPG) and alkaline phosphatase and/or the biomolecule defining a binding site is bone tissue, e.g., expression of a BMP, a BMPR, osteocalcin, osteopontin, bone sialoprotein, osteonectin, OPG or alkaline phosphatase by the delivery vehicle mediates the production of bone tissue. The BMP may, for example, be selected from BMP-2, BMP-4, BMP-6 or BMP-7. In certain embodiments, expression of a BMP, a BMPR, osteocalcin, osteopontin, bone sialoprotein, osteonectin, OPG or alkaline phosphatase by the delivery vehicle in a cancer cell results in the cancer cell acquiring an osteoblast-like cell phenotype. [0097] In certain embodiments, the therapeutic agent that binds to a biomolecule, e.g., bone, is a radionuclide, i.e., an atom that emits radiation and, e.g., kills surrounding cancer cells. Radionuclides may, e.g., be g-emitting radionuclides, b-emitting radionuclides, a- emitting radionuclides, and positron-emitting radionuclides. Exemplary radionuclides include 32P, 47Sc, 64Cu, 67Cu, 89Sr, 86Y, 87Y, 90Y, 105Rh, mAg, mIn, 117mSn, 149Pm, 153Sm, 166Ho, 177Lu, I S6RC. 188Re, 211At, 212Bi, and 223Ra. In certain embodiments, the radionuclide is a bone targeting (i.e., a bone-homing) radionuclide. In certain embodiments, a bone-targeting radionuclide is a calcium mimetic (e.g., Sr and Ra). In certain embodiments, a bone homing radionuclide is conjugated to a ligand (e.g., ethylenediaminetetramethylene phosphonate (EDTMP), e.g., 153Sm-EDTMP), which binds to calcium. In certain embodiments, the radionuclide is selected from 89Sr (e.g., 89Sr chloride, i.e., Metastron™),
223 Ra (e.g., 223Ra dichloride, i.e., Xofigo®),153Sm (e.g., 153Sm-EDTMP, i.e., 153Sm
lexidronam, i.e., Quadramet®), 177Lu, 90Y, 186Re (e.g, 186Re HEDP), 117mSn (e.g, 117mSn DTP A), and 32P.
[0098] In any disclosed method or composition, a bone morphogenetic protein (BMP) may, for example, be selected from BMP-2, BMP -4, BMP-6 or BMP-7.
[0099] An exemplary amino acid sequence of BMP-2, corresponding to NCBI reference sequence NP_001 191.1, is as follows:
MVAGTRCLLALLLPQVLLGGAAGLVPELGRRKFAAAS SGRP SSQP SDEVLSEFELRLLSMFG LKQRPTP SRDAWPPYMLDLYRRHSGQP GSPAPDHRLERAASRANTVRSFHHEE SLEELPET SGKTTRRFFFNLSS IP TEEF I TSAELQVFREQMQDALGNNS SFHHRINIYE I IKPATANSKF PVTRLLDTRLVNQNASRWESFDVTPAVMRWTAQGHANHGFVVEVAHLEEKQGVSKRHVRI SR SLHQDEHSWSQ IRP LLVTFGHDGKGHPLHKREKRQAKHKQRKRLKSSCKRHP LYVDF SDVGW NDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSKIPKACCVPTELSAI SML YLDENEKWLKNYQDMWEGCGCR (SEQ ID NO: 1).
[00100] An exemplary nucleotide sequence of BMP -2 cloned into an exemplary recombinant adenovirus delivery vehicle is as follows, where the flanking adenoviral sequence including restriction sites is underlined:
ATCTGACCTCGTCGACATGGTGGCCGGGACCCGCTGTCTTCTAGCGTTGCTGCTTCCCCAGG
TCCTCCTGGGCGGCGCGGCTGGCCTCGTTCCGGAGCTGGGCCGCAGGAAGTTCGCGGCGGCG
TCGTCGGGCCGCCCCTCATCCCAGCCCTCTGACGAGGTCCTGAGCGAGTTCGAGTTGCGGCT
GCTCAGCATGTTCGGCCTGAAACAGAGACCCACCCCCAGCAGGGACGCCGTGGTGCCCCCCT ACATGCTAGACCTGTATCGCAGGCACTCAGGTCAGCCGGGCTCACCCGCCCCAGACCACCGG
TTGGAGAGGGCAGCCAGCCGAGCCAACACTGTGCGCAGCTTCCACCATGAAGAATCTTTGGA AGAACTACCAGAAACGAGTGGGAAAACAACCCGGAGATTCTTCTTTAATTTAAGTTCTATCC CCACGGAGGAGTTTATCACCTCAGCAGAGCTTCAGGTTTTCCGAGAACAGATGCAAGATGCT TTAGGAAACAATAGCAGTTTCCATCACCGAATTAATATTTATGAAATCATAAAACCTGCAAC AGCCAACTCGAAATTCCCCGTGACCAGACTTTTGGACACCAGGTTGGTGAATCAGAATGCAA GCAGGTGGGAAAGTTTTGATGTCACCCCCGCTGTGATGCGGTGGACTGCACAGGGACACGCC AACCATGGATTCGTGGTGGAAGTGGCCCACTTGGAGGAGAAACAAGGTGTCTCCAAGAGACA TGTTAGGATAAGCAGGTCTTTGCACCAAGATGAACACAGCTGGTCACAGATAAGGCCATTGC TAGTAACTTTTGGCCATGATGGAAAAGGGCATCCTCTCCACAAAAGAGAAAAACGTCAAGCC AAACACAAACAGCGGAAACGCCTTAAGTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTT CAGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCCCCGGGGTATCACGCCTTTTACTGCC ACGGAGAATGCCCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATCATGCCATTGTTCAG ACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAG TGCTATCTCGATGCTGTACCTTGACGAGAATGAAAAGGTTGTATTAAAGAACTATCAGGACA TGGTTGTGGAGGGTTGTGGGTGTCGCTAGCTCGAGTCACCAGGCG ( SEQ ID NO: 12)
[00101] An exemplary amino acid sequence of BMP-4, corresponding to NCBI reference sequence NP_00l334843. l, is as follows:
MIPGNRMLMWLLCQVLLGGASHASLIPETGKKKVAEIQGHAGGRRSGQSHELLRDFEATLL QMFGLRRRPQPSKSAVIPDYMRDLYRLQSGEEEEEQIHSTGLEYPERPASRANTVRSFHHEE HLENIPGTSENSAFRFLFNLSSIPENEVISSAELRLFREQVDQGPDWERGFHRINIYEVMKP PAEVVPGHLITRLLDTRLVHHNVTRWETFDVSPAVLRWTREKQPNYGLAIEVTHLHQTRTHQ GQHVRISRSLPQGSGNWAQLRPLLVTFGHDGRGHALTRRRRAKRSPKHHSQRARKKNKNCRR HSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCPFPLADHLNSTNHAIVQTLVNSVNSSIPKAC CVPTELSAISMLYLDEYDKWLKNYQEMWEGCGCR (SEQ ID NO: 2).
[00102] An exemplary nucleotide sequence of BMP -4 cloned into an exemplary recombinant adenovirus delivery vehicle is as follows, where the flanking adenoviral sequence including restriction sites is underlined:
ATCTGACCTCGTCGACATGATTCCTGGTAACCGAATGCTGATGGTCGTTTTATTATGCCAAG
TCCTGCTAGGAGGCGCGAGCCATGCTAGTTTGATACCTGAGACGGGGAAGAAAAAAGTCGCC
GAGATTCAGGGCCACGCGGGAGGACGCCGCTCAGGGCAGAGCCATGAGCTCCTGCGGGACTT
CGAGGCGACACTTCTGCAGATGTTTGGGCTGCGCCGCCGCCCGCAGCCTAGCAAGAGTGCCG TCATTCCGGACTACATGCGGGATCTTTACCGGCTTCAGTCTGGGGAGGAGGAGGAAGAGCAG
ATCCACAGCACTGGTCTTGAGTATCCTGAGCGCCCGGCCAGCCGGGCCAACACCGTGAGGAG CTTCCACCACGAAGAACATCTGGAGAACATCCCAGGGACCAGTGAAAACTCTGCTTTTCGTT TCCTCTTTAACCTCAGCAGCATCCCTGAGAACGAGGTGATCTCCTCTGCAGAGCTTCGGCTC TTCCGGGAGCAGGTGGACCAGGGCCCTGATTGGGAAAGGGGCTTCCACCGTATAAACATTTA TGAGGTTATGAAGCCCCCAGCAGAAGTGGTGCCTGGGCACCTCATCACACGACTACTGGACA CGAGACTGGTCCACCACAATGTGACACGGTGGGAAACTTTTGATGTGAGCCCTGCGGTCCTT CGCTGGACCCGGGAGAAGCAGCCAAACTATGGGCTAGCCATTGAGGTGACTCACCTCCATCA GACTCGGACCCACCAGGGCCAGCATGTCAGGATTAGCCGATCGTTACCTCAAGGGAGTGGGA ATTGGGCCCAGCTCCGGCCCCTCCTGGTCACCTTTGGCCATGATGGCCGGGGCCATGCCTTG ACCCGACGCCGGAGGGCCAAGCGTAGCCCTAAGCATCACTCACAGCGGGCCAGGAAGAAGAA TAAGAACTGCCGGCGCCACTCGCTCTATGTGGACTTCAGCGATGTGGGCTGGAATGACTGGA TTGTGGCCCCACCAGGCTACCAGGCCTTCTACTGCCATGGGGACTGCCCCTTTCCACTGGCT GACCACCTCAACTCAACCAACCATGCCATTGTGCAGACCCTGGTCAATTCTGTCAATTCCAG TATCCCCAAAGCCTGTTGTGTGCCCACTGAACTGAGTGCCATCTCCATGCTGTACCTGGATG AGTATGATAAGGTGGTACTGAAAAATTATCAGGAGATGGTAGTAGAGGGATGTGGGTGCCGC TGACTCGAGTCACCAGGCG ( SEQ ID NO: l3)
[00103] An exemplary amino acid sequence of BMP-6, corresponding to NCBI reference sequence NP_00l709. l, is as follows:
MPGLGRRAQWLCWWWGLLCSCCGPPPLRPPLPAAAAAAAGGQLLGDGGSPGRTEQPPPSPQS SSGFLYRRLKTQEKREMQKEILSVLGLPHRPRPLHGLQQPQPPALRQQEEQQQQQQLPRGEP PPGRLKSAPLFMLDLYNALSADNDEDGASEGERQQSWPHEAASSSQRRQPPPGAAHPLNRKS LLAPGSGSGGASPLTSAQDSAFLNDADMVMSFVNLVEYDKEFSPRQRHHKEFKFNLSQIPEG EVVTAAEFRIYKDCVMGSFKNQTFLISIYQVLQEHQHRDSDLFLLDTRWWASEEGWLEFDI TATSNLWWTPQHNMGLQLSVVTRDGVHVHPRAAGLVGRDGPYDKQPFMVAFFKVSEVHVRT TRSASSRRRQQSRNRSTQSQDVARVSSASDYNSSELKTACRKHELYVSFQDLGWQDWIIAPK GYAANYCDGECSFPLNAHMNATNHAIVQTLVHLMNPEYVPKPCCAPTKLNAISVLYFDDNSN VILKKYRNMWRACGCH (SEQ ID NO: 3).
[00104] An exemplary nucleotide sequence of BMP-6 cloned into an exemplary recombinant adenovirus delivery vehicle is as follows, where the flanking adenoviral sequence including restriction sites is underlined: ATCTGACCTCGTCGACATGCCGGGGCTGGGGCGGAGGGCGCAGTGGCTGTGCTGGTGGTGGG GGCTGCTGTGCAGCTGCTGCGGGCCCCCGCCGCTGCGGCCGCCCTTGCCCGCTGCCGCGGCC GCCGCCGCCGGGGGGCAGCTGCTGGGGGACGGCGGGAGCCCCGGCCGCACGGAGCAGCCGCC GCCGTCGCCGCAGTCCTCCTCGGGCTTCCTGTACCGGCGGCTCAAGACGCAGGAGAAGCGGG AGATGCAGAAGGAGATCTTGTCGGTGCTGGGGCTCCCGCACCGGCCCCGGCCCCTGCACGGC CTCCAACAGCCGCAGCCCCCGGCGCTCCGGCAGCAGGAGGAGCAGCAGCAGCAGCAGCAGCT GCCTCGCGGAGAGCCCCCTCCCGGGCGACTGAAGTCCGCGCCCCTCTTCATGCTGGATCTGT ACAACGCCCTGTCCGCCGACAACGACGAGGACGGGGCGTCGGAGGGGGAGAGGCAGCAGTCC TGGCCCCACGAAGCAGCCAGCTCGTCCCAGCGTCGGCAGCCGCCCCCGGGCGCCGCGCACCC GCTCAACCGCAAGAGCCTTCTGGCCCCCGGATCTGGCAGCGGCGGCGCGTCCCCACTGACCA GCGCGCAGGACAGCGCCTTCCTCAACGACGCGGACATGGTCATGAGCTTTGTGAACCTGGTG GAGTACGACAAGGAGTTCTCCCCTCGTCAGCGACACCACAAAGAGTTCAAGTTCAACTTATC CCAGATTCCTGAGGGTGAGGTGGTGACGGCTGCAGAATTCCGCATCTACAAGGACTGTGTTA TGGGGAGTTTTAAAAACCAAACTTTTCTTATCAGCATTTATCAAGTCTTACAGGAGCATCAG CACAGAGACTCTGACCTGTTTTTGTTGGACACCCGTGTAGTATGGGCCTCAGAAGAAGGCTG GCTGGAATTTGACATCACGGCCACTAGCAATCTGTGGGTTGTGACTCCACAGCATAACATGG GGCTTCAGCTGAGCGTGGTGACAAGGGATGGAGTCCACGTCCACCCCCGAGCCGCAGGCCTG GTGGGCAGAGACGGCCCTTACGACAAGCAGCCCTTCATGGTGGCTTTCTTCAAAGTGAGTGA GGTGCACGTGCGCACCACCAGGTCAGCCTCCAGCCGGCGCCGACAACAGAGTCGTAATCGCT CTACCCAGTCCCAGGACGTGGCGCGGGTCTCCAGTGCTTCAGATTACAACAGCAGTGAATTG AAAACAGCCTGCAGGAAGCATGAGCTGTATGTGAGTTTCCAAGACCTGGGATGGCAGGACTG GATCATTGCACCCAAGGGCTATGCTGCCAATTACTGTGATGGAGAATGCTCCTTCCCACTCA ACGCACACATGAATGCAACCAACCACGCGATTGTGCAGACCTTGGTTCACCTTATGAACCCC GAGTATGTCCCCAAACCGTGCTGTGCGCCAACTAAGCTAAATGCCATCTCGGTTCTTTACTT TGATGACAACTCCAATGTCATTCTGAAAAAATACAGGAATATGGTTGTAAGAGCTTGTGGAT GCCACTAACTCGAGTCACCAGGCG ( SEQ ID NO: 14).
[00105] An exemplary amino acid sequence of BMP-7, corresponding to NCBI reference sequence NR_001710.1, is as follows:
MHVRSLRAAAPHSFVALWAPLFLLRSALADFSLDNEVHSSFIHRRLRSQERREMQREILSIL
GLPHRPRPHLQGKHNSAPMFMLDLYNAMAVEEGGGPGGQGFSYPYKAVFSTQGPPLASLQDS
HFLTDADMVMSFVNLVEHDKEFFHPRYHHREFRFDLSKIPEGEAVTAAEFRIYKDYIRERFD
NETFRISVYQVLQEHLGRESDLFLLDSRTLWASEEGWLVFDITATSNHWVVNPRHNLGLQLS
VETLDGQSINPKLAGLIGRHGPQNKQPFMVAFFKATEVHFRSIRSTGSKQRSQNRSKTPKNQ EALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNSYMN
ATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACGCH
(SEQ ID NO: 4).
[00106] An exemplary nucleotide sequence of BMP-7 cloned into an exemplary recombinant adenovirus delivery vehicle is as follows, where the flanking adenoviral sequence including restriction sites is underlined:
ATCTGACCTCGTCGACATGCACGTGCGCTCACTGCGAGCTGCGGCGCCGCACAGCTTCGTGG CGCTCTGGGCACCCCTGTTCCTGCTGCGCTCCGCCCTGGCCGACTTCAGCCTGGACAACGAG GTGCACTCGAGCTTCATCCACCGGCGCCTCCGCAGCCAGGAGCGGCGGGAGATGCAGCGCGA GATCCTCTCCATTTTGGGCTTGCCCCACCGCCCGCGCCCGCACCTCCAGGGCAAGCACAACT CGGCACCCATGTTCATGCTGGACCTGTACAACGCCATGGCGGTGGAGGAGGGCGGCGGGCCC GGCGGCCAGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAGGGCCCCCCTCTGGC CAGCCTGCAAGATAGCCATTTCCTCACCGACGCCGACATGGTCATGAGCTTCGTCAACCTCG TGGAACATGACAAGGAATTCTTCCACCCACGCTACCACCATCGAGAGTTCCGGTTTGATCTT TCCAAGATCCCAGAAGGGGAAGCTGTCACGGCAGCCGAATTCCGGATCTACAAGGACTACAT CCGGGAACGCTTCGACAATGAGACGTTCCGGATCAGCGTTTATCAGGTGCTCCAGGAGCACT TGGGCAGGGAATCGGATCTCTTCCTGCTCGACAGCCGTACCCTCTGGGCCTCGGAGGAGGGC TGGCTGGTGTTTGACATCACAGCCACCAGCAACCACTGGGTGGTCAATCCGCGGCACAACCT GGGCCTGCAGCTCTCGGTGGAGACGCTGGATGGGCAGAGCATCAACCCCAAGTTGGCGGGCC TGATTGGGCGGCACGGGCCCCAGAACAAGCAGCCCTTCATGGTGGCTTTCTTCAAGGCCACG GAGGTCCACTTCCGCAGCATCCGGTCCACGGGGAGCAAACAGCGCAGCCAGAACCGCTCCAA GACGCCCAAGAACCAGGAAGCCCTGCGGATGGCCAACGTGGCAGAGAACAGCAGCAGCGACC AGAGGCAGGCCTGTAAGAAGCACGAGCTGTATGTCAGCTTCCGAGACCTGGGCTGGCAGGAC TGGATCATCGCGCCTGAAGGCTACGCCGCCTACTACTGTGAGGGGGAGTGTGCCTTCCCTCT GAACTCCTACATGAACGCCACCAACCACGCCATCGTGCAGACGCTGGTCCACTTCATCAACC CGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGCAGCTCAATGCCATCTCCGTCCTCTAC TTCGATGACAGCTCCAACGTCATCCTGAAGAAATACAGAAACATGGTGGTCCGGGCCTGTGG CTGCCACTAGCTCGAGTCACCAGGCG ( SEQ ID NO: 15).
[00107] In certain embodiments, a BMP comprises SEQ ID NO: 1 or a functional fragment thereof, SEQ ID NO: 2 or a functional fragment thereof, SEQ ID NO: 3 or a functional fragment thereof, or SEQ ID NO: 4 or a functional fragment thereof, or an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. As used herein, a functional fragment of a BMP refers to a fragment that exhibits at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity, e.g., BMP receptor binding activity, of a corresponding full length BMP protein.
IV. Therapeutic Transgenes
[00108] A disclosed delivery vehicle (e.g., a recombinant virus) may further comprise an exogenous nucleotide sequence that encodes for a therapeutic transgene. A therapeutic transgene may encode a therapeutic nucleic acid, e.g., an antisense RNA or ribozyme RNA. The therapeutic transgene may encode a therapeutic peptide or polypeptide, e.g., a costimulatory molecule, an antiangiogenic and/or antivascular molecule, an anti-fibrotic molecule, an anti-inflammatory or pro-inflammatory molecule, a monoclonal antibody or fragment thereof, a pro-apoptotic molecule, an enzyme, an immunomodulatory molecule, a cytokine, a cytokine inhibitor (e.g., a cytokine trap), an oncoprotein, a tumor suppressor, a lytic peptide, a vaccine antigen, and a molecule which complements genetic defects in somatic cells
[00109] In certain embodiments, the therapeutic transgene encodes a therapeutic polypeptide, or a fragment thereof, selected from acetylcholine, an androgen-receptor, an anti-PD-l antibody heavy chain and/or light chain, an anti-PD-Ll antibody heavy chain and/or light chain, BORIS/CTCFL, BRAF, CD19, CD20, CD30, CD80, CD86, CD137, CD137L, CD154, CEA, DKKl/Wnt, EGFRvIII, FGF, gplOO, Her-2/neu, ICAM, IL-l, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-l 7, IL-23A/pl9, p40, IL-24, IL-27, IL-27A/p28, IL- 27B/EBI3, IL-35, interferon-gamma, KRAS, MAGE, MAGE-A3, MART1, melan-A, mesothelin, MUC-l, NY-ESO-l, Podocalyxin (Podxl), p53, TGF-b, a TGF-b trap, thymidine kinase, and tyrosinase.
[00110] In certain embodiments, the therapeutic transgene encodes a cancer antigen derived from 9D7, androgen receptor, a BAGE family protein, b-catenin, BING-4, BRAF, BRCA1/2, a CAGE family protein, calcium-activated chloride channel 2, CD 19, CD20, CD30, CDK4, CEA, CML66, CT9, CT10, cyclin-Bl, EGFRvIII, Ep-CAM, EphA3, fibronectin, a GAGE family protein, gpl00/pmell7, Her-2/neu, HPV E6, HPV E7, Ig, immature laminin receptor, a MAGE family protein (e.g., MAGE-A3), MART- 1 /melan-A, MART2, MC1R, mesothelin, a mucin family protein (e.g., MUC-l), NY-ESO-l/LAGE-l, P.polypeptide, p53, podocalyxin (Podxl), PRAME, a ras family proteins (e.g., KRAS), prostate specific antigen, a SAGE family protein, SAP-l, SSX-2, survivin, TAG-72, TCR, telomerase, TGF- RII, TRP-l, TRP-2, tyrosinase, or a XAGE family protein.
V. Pharmaceutical Compositions
[00111] For therapeutic use, a delivery vehicle (e.g., a recombinant virus) and/or a therapeutic agent preferably is combined with a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” means buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The carrier(s) should be“acceptable” in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
[00112] Pharmaceutical compositions containing recombinant viruses disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, intraocular, intranasal, transdermal, topical, transmucosal, rectal, oral, parenteral, subcutaneous, intramuscular, ophthalmic, epidural, intratracheal, sublingual, buccal, vaginal, and nasal administration.
[00113] An exemplary route of administration is IV infusion. Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences, 18th ed. (Mack Publishing Company, 1990). Formulation components suitable for parenteral administration include 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 EDTA;
buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. [00114] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol), and suitable mixtures thereof.
[00115] Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
[00116] The term“effective amount” as used herein refers to the amount of an active component (e.g., the amount of a recombinant virus of the present invention) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
[00117] In certain embodiments, a therapeutically effective amount of active component is in the range of 0.1 mg/kg to 100 mg/kg, e.g., 1 mg/kg to 100 mg/kg, 1 mg/kg to 10 mg/kg, 1 mg/kg to 5 mg/kg, 10 mg/kg, 7.5 mg/kg, 5 mg/kg, or 2.5 mg/kg. In certain embodiments, a therapeutically effective amount of a recombinant virus is in the range of 102 to 1015 plaque forming units (pfiis), e.g., l02to 1010 , l02to 105, l05to 1015, l05to 1010, or l010to l015 plaque forming units. In certain embodiments, a therapeutically effective amount of a radionuclide virus is in the range of 1-500000 kBq/kg, e.g., 1-200000 kBq/kg, 1-150000 kBq/kg, 1-100000 kBq/kg, 1-50000 kBq/kg, 1-20000 kBq/kg, 1-10000 kBq/kg, 1-5000 kBq/kg, 1-2000 kBq/kg, 1-1000 kBq/kg, 1-500 kBq/kg, 1-100 kBq/kg, 1-50 kBq/kg, or 1-20 kBq/kg. The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the subject, the in vivo potency of the virus, the
pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue- level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study designed to run from 0.5 mg/kg to 20 mg/kg. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, the half-life of the recombinant virus, and the disease being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks. A preferred route of administration is parenteral, e.g., intravenous infusion.
VI. Therapeutic Uses
[00118] The methods and compositions disclosed herein can be used to treat various medical indications. For example, the methods and compositions disclosed herein can be used to treat cancers. The cancer cells are exposed to a therapeutically effective amount of a delivery vehicle (e.g., a recombinant virus) and/or a therapeutic agent so as to inhibit or reduce proliferation of the cancer cells. The invention provides a method of treating a cancer in a subject. The method comprises administering to the subject an effective amount of a delivery vehicle and/or a therapeutic agent to treat the cancer in the subject. In certain embodiments, administering an effective amount of a delivery vehicle and/or a therapeutic agent to a subject reduces tumor load in that subject by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The methods and compositions disclosed herein can be used to treat bone loss and/or hypercalcemia, e.g., bone loss and/or hypercalcemia associated with a cancer. The invention provides a method of bone loss and/or hypercalcemia in a subject, e.g., a subject with cancer. The method comprises administering to the subject an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding a protein that mediates the production of bone tissue to treat the bone loss and/or hypercalcemia in a subject.
[00119] As used herein,“treat”,“treating” and“treatment” mean the treatment of a disease in a subject, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state. As used herein, the terms“subject” and“patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.
[00120] Examples of cancers include solid tumors, soft tissue tumors, hematopoietic tumors and metastatic lesions. Examples of hematopoietic tumors include, leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), e.g., transformed CLL, diffuse large B-cell lymphomas (DLBCL), follicular lymphoma, hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, or Richter’s Syndrome (Richter’s Transformation). Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting head and neck (including pharynx), thyroid, lung (small cell or non-small cell lung carcinoma (NSCLC)), breast, lymphoid, gastrointestinal (e.g., oral, esophageal, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genitals and genitourinary tract (e.g., renal, urothelial, bladder, ovarian, uterine, cervical, endometrial, prostate, testicular), CNS (e.g., neural or glial cells, e.g., neuroblastoma or glioma), or skin (e.g, melanoma).
[00121] In certain embodiments, the cancer is selected from melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, head and neck cancer, breast cancer, anal cancer, cervical cancer, non-small cell lung cancer, mesothelioma, small cell lung cancer, renal cell carcinoma, prostate cancer, gastroesophageal cancer, colorectal cancer, testicular cancer, bladder cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, brain and central nervous system cancer, thyroid cancer, parathyroid cancer (e.g, parathyroid carcinoma), endometrial cancer, neuroendocrine cancer, lymphoma (e.g., Hodgkin and non- Hodgkin), leukemia, merkel cell carcinoma, gastrointestinal stromal tumors, multiple myeloma, uterine cancer, a sarcoma, kidney cancer, ocular cancer, pancreatic cancer, and a germ cell cancer (e.g., ovarian germ cell cancer).
[00122] The invention also provides a method of generating bone tissue in a subject in need thereof. The method comprises administering to the subject an effective amount of a delivery vehicle described herein to generate bone tissue in the subject. The generation of bone tissue may, e.g., treat a non-union or fracture in the subject, or be used for a spinal fusion or dental implant in the subject.
[00123] In certain embodiments, a delivery vehicle (e.g., a recombinant virus) and/or a therapeutic agent are administered to the subject in combination with one or more therapies, e.g., surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or virotherapy.
[00124] In certain embodiments, a delivery vehicle (e.g., a recombinant virus) and/or a therapeutic agent are administered to the subject in combination with a therapy that promotes bone strength and/or growth, e.g., an anti-resorptive therapy. In certain embodiments, a delivery vehicle (e.g., a recombinant virus) and/or a therapeutic agent are administered to the subject in combination with a bisphosphonate, testosterone, hyaluronic acid, vitamin D, calcium, testosterone, estrogen, progesterone, a selective estrogen modulator (SERM), calcitonin, a RANK or RANKL inhibitor (e.g., denosumab), pulsed ultrasound (e.g., low intensity pulsed ultrasound (LIPU)), electrical stimulation, extracorporeal shockwave therapy (ESWT), low energy laser therapy, and/or pulsed electromagnetic field (PEMF) therapy.
[00125] In certain embodiments, a delivery vehicle (e.g., a recombinant virus) and/or a therapeutic agent are administered to the subject in combination with a bisphosphonate. Exemplary bisphosphonates include disodium pamidronate, alendronate, etidronate, tiludronate, risedronate, zoledronic acid, sodium clodronate, and ibandronic acid.
[00126] In certain embodiments, a delivery vehicle (e.g., a recombinant virus) and/or a therapeutic agent of the invention is administered in combination with a tyrosine kinase inhibitor, e.g., erlotinib.
[00127] In certain embodiments, a delivery vehicle (e.g., a recombinant virus) and/or a therapeutic agent of the invention is administered in combination with a checkpoint inhibitor, e.g., an anti-CTLA-4 antibody, an anti -PD- 1 antibody, or an anti-PD-Ll antibody.
Exemplary anti-PD-l antibodies include, for example, nivolumab (Opdivo®, Bristol-Myers Squibb Co.), pembrolizumab (Keytruda®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-Ll antibodies include, for example, atezolizumab (Tecentriq®, Genentech), duvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).
[00128] The term administered "in combination," as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the subject overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g. , an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
[00129] In certain embodiments, the effective amount of a delivery vehicle (e.g., a recombinant virus) and/or a therapeutic agent of the invention is identified by measuring an immune response to an antigen in the subject and/or the method of treating the subject further comprises measuring an immune response to an antigen in the subject. Hyperproliferative diseases, e.g., cancers, may be characterized by immunosuppression, and measuring an immune response to an antigen in the subject may be indicative of the level of
immunosuppression in the subject. Accordingly, measuring an immune response to an antigen in the subject may be indicative of the efficacy of the treatment and/or the effective amount of the recombinant virus. The immune response to the antigen in the subject may be measured by any method known in the art. In certain embodiments, the immune response to the antigen is measured by injecting the subject with the antigen at an injection site on the skin of the subject and measuring the size of an induration or amount of inflammation at the injection site. In certain embodiments, the immune response to the antigen is measured by release of a cytokine from a cell of the subject (e.g., interferon gamma, IL-4 and/or IL-5) upon exposure to the antigen.
[00130] Throughout the description, where compositions, devices, and systems are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions, devices, and systems of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.
[00131] In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. [00132] Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular virus, that virus can be used in various embodiments of compositions of the present invention and/or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.
[00133] It should be understood that the expression“at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and/or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.
[00134] The use of the term“include,”“includes,”“including,”“have,”“has,”“having,” “contain,”“contains,” or“containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.
[00135] At various places in the present specification, viruses, compositions, systems, processes and methods, or features thereof, are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges. By way of other examples, an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, and 20.
[00136] Where the use of the term“about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term“about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. [00137] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.
[00138] The use of any and all examples, or exemplary language herein, for example,
“such as” or“including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.
EXAMPLES
[00139] The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.
Example 1: Adenovirus Construction
[00140] This Example describes the construction of a recombinant adenovirus type 5 (Ad5) that expresses a protein that defines a binding site or a protein that mediates the production of a biomolecule that defines a binding site for a therapeutic agent, e.g., BMP -2 or BMP-7.
[00141] A plasmid carrying the 5' portion of the adenovirus type 5 genomic sequence was modified to carry the deletion of a nucleotide region located from -304 to -255 upstream of the Ela initiation site, which renders Ela expression cancer-selective (as previously described in U.S. Patent No. 9,073,980). The modified plasmid is hereafter referred to as the TAV plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV virus.
[00142] The TAV plasmid was further modified to carry a Sall site at the start of the Elb- l9k region and an Xhol site 200 base pairs 3' of the Sall site to facilitate insertion of therapeutic transgenes. To delete the 200 base pair Elb-l9k region the plasmid was cut with Sall and Xhol and self-ligated. The nucleotide sequence of the modified Elb-l9k region is as follows, with the residual bases from the fused Sall and Xhol sites underlined:
ATCTTGGTTACATCTGACCTCGTCGAGTCACCAGGCGCTTTTCCAA ( SEQ ID NO: 16).
[00143] The modified plasmid is hereafter referred to as the TAV-Al9k plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV-Al9k virus. [00144] Exogenous nucleotide sequences encoding BMP -2 or BMP-7 were cloned into the modified Elb-l9k region. Bone morphogenetic proteins (BMPs) are osteoinductive growth factors that belong to the transforming growth factor b (TGF-b) family. A nucleotide sequence encoding exemplary protein BMP -2 was cloned in to the modified Elb-l9k region of the TAV-Al9k plasmid. The modified plasmid is hereafter referred to as the TAV-BMP-2 plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV-BMP-2 virus. Additionally, a nucleotide sequence encoding exemplary protein BMP-7 was cloned in to the modified Elb-l9k region of the TAV-Al9k plasmid. The modified plasmid is hereafter referred to as the TAV-BMP-7 plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV-BMP-7 virus.
[00145] The various plasmids described were used along with other plasmids carrying the remainder of the adenovirus type 5 genomic sequence (based on strain dl309) to generate recombinant adenoviruses.
Example 2: Adenovirus Construction
[00146] This Example describes the construction of a recombinant adenovirus type 5 (Ad5) that expresses a protein that defines a binding site or a protein that mediates the production of a biomolecule that defines a binding site for a therapeutic agent, e.g., BMP -4 or BMP-6.
[00147] A plasmid carrying the 5' portion of the adenovirus type 5 genomic sequence is modified to carry the deletion of a nucleotide region located from -304 to -255 upstream of the Ela initiation site, which renders Ela expression cancer-selective (as previously described in U.S. Patent No. 9,073,980). The modified plasmid is hereafter referred to as the TAV plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV virus.
[00148] The TAV plasmid is further modified to carry a Sall site at the start of the Elb- l9k region and an Xhol site 200 base pairs 3' of the Sall site to facilitate insertion of therapeutic transgenes. To delete the 200 base pair Elb-l9k region the plasmid is cut with Sall and Xhol and self-ligated. The nucleotide sequence of the modified Elb-l9k region is as follows, with the residual bases from the fused Sall and Xhol sites underlined:
ATCTTGGTTACATCTGACCTCGTCGAGTCACCAGGCGCTTTTCCAA ( SEQ ID NO: 16). [00149] The modified plasmid is hereafter referred to as the TAV-A 19k plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV-D 19k virus.
[00150] Exogenous nucleotide sequences encoding BMP -4 or BMP-6 are cloned into the modified Elb-l9k region. Bone morphogenetic proteins (BMPs) are osteoinductive growth factors that belong to the transforming growth factor b (TGF-b) family.
[00151] A nucleotide sequence encoding exemplary protein BMP-4 is cloned in to the modified Elb-l9k region of the TAV-Al9k plasmid. The modified plasmid is hereafter referred to as the TAV-BMP-4 plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV-BMP-4 virus. Additionally, a nucleotide sequence encoding exemplary protein BMP-6 is cloned in to the modified Elb-l9k region of the TAV- Al9k plasmid. The modified plasmid is hereafter referred to as the TAV-BMP-6 plasmid, and any resulting viral particles produced therefrom are hereafter referred to as the TAV-BMP-6 virus.
[00152] The various plasmids described are used along with other plasmids carrying the remainder of the adenovirus type 5 genomic sequence (based on strain dl309) to generate recombinant adenoviruses.
Example 3: BMP Expression
[00153] This Example describes the expression of BMPs from recombinant viruses.
[00154] A549 cells (human lung cancer cells) (i) were infected with the TAV-BMP-2 virus, prepared as described in Example 1, at a multiplicity of infection (MOI) of 5, (ii) were infected with the TAV-Al9k virus, without the BMP -2 gene, prepared as described in example 1, at an MOI of 5, or (ii) kept as non-infected controls. BMP -2 levels in collected media were assayed by ELISA four days after infection. As depicted in FIGURE 1, BMP -2 expression from the TAV-BMP-2 virus resulted in approximately 10 ng/ml of BMP -2 in the collected media.
[00155] A549 cells were similarly (i) were infected with the TAV-BMP-7 virus, prepared as described in Example 1, at a multiplicity of infection (MOI) of 5, (ii) were infected with the TAV-Al9k virus, without the BMP-7 gene, prepared as described in example 1, at an MOI of 5, or (ii) kept as non-infected controls. BMP-7 levels in conditioned media were measured by ELISA four days after infection. As shown in FIGURE 2, BMP-7 expression from the TAV-BMP-7 virus resulted in approximately 8 ng/ml of BMP-7 in the collected media. Example 4: Cytotoxicity of BMP Expressing Viruses
[00156] This Example describes cytotoxicity resulting from BMP expressing recombinant viruses.
[00157] A549 cells (human lung cancer cells) were infected with the TAV-A19k. TAV-
BMP-2, or TAV-BMP-7 viruses produced as described in Example 1. Cells were stained with crystal violet, which stains viable cells blue, at the indicated time points after infection. As depicted in FIGURE 2, infection with the TAV-BMP-2 or TAV-BMP-7 results in cell death starting at approximately 6 days after infection.
Example 5: Treatment of Cancer with BMP Expressing Viruses
[00158] This Example describes treatment of lung cancer in a mouse model with BMP expressing viruses.
[00159] Adult 129S4 mice were injected subcutaneously with ADS-12 (mouse lung cancer) cells and allowed to form tumors. After the tumors became large enough to treat, the mice were treated with TAV-Al9k, TAV-BMP-2, or TAV-BMP-7 (produced as described in Example 1), each at a dose of 1E9 PFU (plaque forming units). Viruses were injected intratumorally every four days for a total of three doses. Buffer without virus was used as a control. Tumor volumes of mice treated with buffer, TAV-Al9k, and TAV-BMP-7 are shown in FIGURE 4. Tumor volumes after treatment with TAV-BMP-2 were not smaller than with TAV-Al9k and are omitted for clarity.
INCORPORATION BY REFERENCE
[00160] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.
EQUIVAUENTS
[00161] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and the range of equivalency of the claims are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:
1. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject:
(a) an effective amount of a delivery vehicle comprising an exogenous nucleotide
sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and
(b) an effective amount of a therapeutic agent that binds to the binding site.
2. The method of claim 1, wherein the protein that mediates the production of the biomolecule is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR), osteocalcin, osteopontin, bone sialoprotein, osteonectin, osteoprotegerin (OPG) and alkaline phosphatase.
3. The method of claims 1 or 2, wherein the biomolecule defining the binding site is bone tissue.
4. The method of claim 1, wherein the protein defining the binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR) and prostate specific membrane antigen (PSMA).
5. The method of any one of claims 1-4, wherein the therapeutic agent comprises a radionuclide, or an antibody.
6. The method of claim 5, wherein the radionuclide is a bone targeting radionuclide.
7. The method of claims 5 or 6, wherein the radionuclide is selected from 89Sr (e.g., 89Sr chloride), 223Ra
Figure imgf000045_0001
dichloride), 153 Sm (e.g., 153Sm-EDTMP), 177Lu, 90Y, 186Re (e.g., 186Re HEDP), 117mSn (e.g., 117mSn DTPA), and 32P.
8. The method of claim 5, wherein the antibody is selected from an antibody drug conjugate and an antibody radionuclide conjugate.
9. A method of treating bone loss and/or hypercalcemia in a subject in need thereof, the method comprising administering to the subject an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding a protein that mediates the production of bone tissue.
10. The method of claim 9, wherein the subject has cancer.
11. A method of generating bone tissue in a subject, comprising administering to the subject an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding a protein that mediates the production of bone tissue.
12. The method of any one of claims 9-11, wherein the protein that mediates the production of bone is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR), osteocalcin, osteopontin, bone sialoprotein, osteonectin, osteoprotegerin (OPG) and alkaline phosphatase.
13. The method of any one of claims 2-12, wherein the BMP is selected from BMP-2, BMP-4, BMP-6, and BMP-7.
14. The method of claim 13, wherein the BMP comprises SEQ ID NO: 1 or a functional fragment thereof, SEQ ID NO: 2 or a functional fragment thereof, SEQ ID NO: 3 or a functional fragment thereof, or SEQ ID NO: 4 or a functional fragment thereof, or an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
15. The method of any one of claims 1-14, wherein the delivery vehicle is a recombinant oncolytic virus.
16. The method of claim 15, wherein the recombinant oncolytic virus is selected from an adenovirus, fowlpox virus, lentivirus, herpes virus, vesicular stomatitis virus (VSV), maraba virus, polio virus, and alpha virus.
17. The method of claim 16, wherein the recombinant oncolytic virus is a recombinant oncolytic adenovirus.
18. The method of claim 17, wherein the recombinant oncolytic adenovirus is a type 5 adenovirus (Ad5).
19. The method of claims 17 or 18, wherein the exogenous nucleotide sequence is inserted into an Elb-l9K insertion site located between the start site of Elb-l9K and the start site of Elb-55K.
20. The method of claim 19, wherein the Elb-l9K insertion site is located between the start site of Elb-l9K and the stop site of Elb-l9K.
21. The method of claims 19 or 20, wherein the Elb-l9K insertion site comprises a deletion of from about 100 to about 305, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 305, about 150 to about 300, about 150 to about 250, or about 150 to about 200 nucleotides adjacent the start site of Elb-l9K.
22. The method of any one of claims 19-21, wherein the Elb-l9K insertion site comprises a deletion of about 200 nucleotides adjacent the start site of Elb-l9K.
23. The method of any one of claims 19-22, wherein the Elb-l9K insertion site comprises a deletion of 203 nucleotides adjacent the start site of Elb-l9K.
24. The method of any one of claims 19-23, wherein the Elb-l9K insertion site comprises a deletion corresponding to nucleotides 1714-1916 of the Ad5 genome (SEQ ID NO: 5).
25. The method of any one of claims 19-24, wherein the exogenous nucleotide sequence is inserted between nucleotides corresponding to 1714 and 1916 of the Ad5 genome (SEQ ID NO: 5).
26. The method of any one of claims 19-25, wherein the exogenous nucleotide sequence is inserted between CTGACCTC (SEQ ID NO: 6) and TCACCAGG (SEQ ID NO: 7).
27. The method of any one of claims 19-26, wherein the recombinant oncolytic adenovirus comprises, in a 5’ to 3’ orientation, CTGACCTC (SEQ ID NO: 6), the exogenous nucleotide sequence, and TCACCAGG (SEQ ID NO: 7).
28. The method of claims 17 or 18, wherein the exogenous nucleotide sequence is inserted into an E3 insertion site located between the stop site of pVIII and the start site of Fiber.
29. The method of claim 28, wherein the E3 insertion site is located between the stop site of E3-l0.5K and the stop site of E3-l4.7K.
30. The method of claims 28 or 29, wherein the E3 insertion site comprises a deletion of from about 500 to about 3185, from about 500 to about 3000, from about 500 to about 2500, from about 500 to about 2000, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 3185, from about 1000 to about 3000, from about 1000 to about 2500, from about 1000 to about 2000, from about 1000 to about 1500, from about 1500 to about 3185, from about 1500 to about 3000, from about 1500 to about 2000, from about 2000 to about 3185, from about 2000 to about 3000, from about 2000 to about 2500, from about 2500 to about 3185, from about 2500 to about 3000, or from about 3000 to about 3185 nucleotides.
31. The method of any one of claims 28-30, wherein the E3 insertion site comprises a deletion of from about 500 to about 1551, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 1551, from about 1000 to about 1500, or from about 1500 to about 1551 nucleotides adjacent the stop site of E3-10.5K.
32. The method of any one of claims 28-31, wherein the E3 insertion site comprises a deletion of about 1050 nucleotides adjacent the stop site of E3-10.5K.
33. The method of any one of claims 28-32, wherein the E3 insertion site comprises a deletion of 1064 nucleotides adjacent the stop site of E3-10.5K.
34. The method of any one of claims 28-33, wherein the E3 insertion site comprises a deletion corresponding to the Ad5 dl309 E3 deletion.
35. The method of any one of claims 28-34, wherein the E3 insertion site comprises a deletion corresponding to nucleotides 29773-30836 of the Ad5 genome (SEQ ID NO: 5).
36. The method of any one of claims 28-35, wherein the exogenous nucleotide sequence is inserted between nucleotides corresponding to 29773 and 30836 of the Ad5 genome (SEQ ID NO: 5).
37. The method of any one of claims 28-36, wherein the exogenous nucleotide sequence is inserted between CAGTATGA (SEQ ID NO: 8) and TAATAAAAAA (SEQ ID NO: 9).
38. The method of any one of claims 28-37, wherein the recombinant oncolytic adenovirus comprises, in a 5’ to 3’ orientation, CAGTATGA (SEQ ID NO: 8), the exogenous nucleotide sequence, and TAATAAAAAA (SEQ ID NO: 9).
39. The method of any one of claims 17-38, wherein the recombinant oncolytic adenovirus further comprises a deletion of a Pea3 binding site, or a functional fragment thereof.
40. The method of claim 39, wherein the recombinant oncolytic adenovirus comprises a deletion of nucleotides corresponding to about -300 to about -250 upstream of the initiation site of El a.
41. The method of claims 39 or 40, wherein the recombinant oncolytic adenovirus comprises a deletion of nucleotides corresponding to -304 or -305 to -255 upstream of the initiation site of El a.
42. The method of any one of claims 39-41, wherein the recombinant oncolytic adenovirus comprises a deletion of nucleotides corresponding to 195-244 of the Ad5 genome (SEQ ID NO: 5).
43. The method of any one of claims 39-42, wherein the recombinant oncolytic adenovirus comprises the sequence GGTGTTTTGG (SEQ ID NO: 10).
44. The method of any one of claims 39-43, wherein the recombinant oncolytic adenovirus does not comprise a deletion of an E2F binding site.
45. The method of any one of claims 17-39, wherein the recombinant oncolytic adenovirus further comprises a deletion of a E2F binding site, or a functional fragment thereof.
46. The method of claim 45, wherein the recombinant oncolytic adenovirus does not comprise a deletion of a Pea3 binding site, or a functional fragment thereof.
47. The method of any one of claims 17-46, wherein the recombinant oncolytic adenovirus comprises an Ela promoter having a deletion of a functional TATA box.
48. The method of claim 47, wherein the deletion comprises a deletion of the entire TATA box.
49. The method of claims 47 or 48, wherein the deletion comprises a deletion of nucleotides corresponding to -29 to -26 of the Ela promoter.
50. The method of any one of claims 47-49, wherein the deletion comprises a deletion of nucleotides corresponding to -33 to -26 of the Ela promoter.
51. The method of any one of claims 47-50, wherein the deletion comprises a deletion of nucleotides corresponding to -44 to +52 of the Ela promoter.
52. The method of any one of claims 47-51, wherein the deletion comprises a deletion of nucleotides corresponding to -148 to +52 of the Ela promoter.
53. The method of any one of claims 47-52, wherein the deletion comprises a deletion of nucleotides corresponding to 353-552 of the Ad5 genome (SEQ ID NO: 5).
54. The method of any one of claims 47-53, wherein the Ela promoter comprises the sequence CTAGGACTG (SEQ ID NO: 11).
55. The method of any one of claims 17-54, wherein the recombinant oncolytic adenovirus comprises the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
56. The method of any one of claims 15-55, wherein the recombinant oncolytic virus further comprises an exogenous nucleotide sequence encoding a costimulatory molecule, an antiangiogenic and/or antivascular molecule, an anti-fibrotic molecule, an anti-inflammatory or pro-inflammatory molecule, a monoclonal antibody or fragment thereof, a pro-apoptotic molecule, an enzyme, an immunomodulatory molecule, a cytokine, or a cytokine inhibitor (e.g., a cytokine trap).
57. The method of any one of claims 15-56, wherein the recombinant oncolytic virus selectively replicates in a hyperproliferative cell.
58. The method of any one of claims 15-57, wherein the recombinant oncolytic virus selectively expresses the protein that defines a binding site or the protein that mediates the production of a biomolecule that defines a binding a site in a hyperproliferative cell.
59. The method of claims 57 or 58, wherein the hyperproliferative cell is a cancer cell.
60. The method of any one claims 1-59, wherein the delivery vehicle is administered to the subject before, at the same time as, or after the therapeutic agent.
61. The method of any one claims 1-60, wherein the cancer is selected from melanoma, squamous cell carcinoma of the skin, basal cell carcinoma, head and neck cancer, breast cancer, anal cancer, cervical cancer, non-small cell lung cancer, mesothelioma, small cell lung cancer, renal cell carcinoma, prostate cancer, gastroesophageal cancer, colorectal cancer, testicular cancer, bladder cancer, ovarian cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, brain and central nervous system cancer, thyroid cancer, parathyroid cancer (e.g., parathyroid carcinoma), endometrial cancer, neuroendocrine cancer, lymphoma (e.g, Hodgkin and non-Hodgkin), leukemia, merkel cell carcinoma, gastrointestinal stromal tumors, multiple myeloma, uterine cancer, a sarcoma, kidney cancer, ocular cancer, pancreatic cancer, and a germ cell cancer (e.g, ovarian germ cell cancer).
62. The method of any one claims 1-61, wherein the delivery vehicle and the therapeutic agent are administered in combination with one or more therapies selected from surgery, radiation, chemotherapy, immunotherapy, hormone therapy, and virotherapy.
63. The method of any one claims 1-62, wherein the delivery vehicle and the therapeutic agent are administered in combination with a bisphosphonate, hyaluronic acid, vitamin D, calcium, testosterone, estrogen, progesterone, a selective estrogen modulator (SERM), calcitonin, a RANK or RANKL inhibitor (e.g., denosumab), pulsed ultrasound (e.g., low intensity pulsed ultrasound (LIPU)), electrical stimulation, extracorporeal shockwave therapy (ESWT), low energy laser therapy, and/or pulsed electromagnetic field (PEMF) therapy.
64. The method of any one of claims 1-63, wherein the effective amount of the recombinant oncolytic virus is 102-1015 plaque forming units (pfus).
65. The method of any one of claims 1-64, wherein the subject is a human or animal.
66. The method of claim 65, wherein the subject is a pediatric human.
67. A pharmaceutical composition comprising
(a) an effective amount of a delivery vehicle comprising an exogenous nucleotide sequence encoding: (i) a protein defining a binding site; or (ii) a protein that mediates the production of a biomolecule defining a binding site; and
(b) optionally, an effective amount of a therapeutic agent that binds to the binding site.
68. The pharmaceutical composition claim 67, wherein the protein that mediates the production of the biomolecule is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR), osteocalcin, osteopontin, bone sialoprotein, osteonectin, osteoprotegerin (OPG) and alkaline phosphatase.
69. The pharmaceutical composition of claims 67 or 68, wherein the biomolecule defining the binding site is bone tissue.
70. The pharmaceutical composition of claim 67, wherein the protein defining the binding site is selected from a bone morphogenetic protein (BMP), a bone morphogenetic protein receptor (BMPR) and prostate specific membrane antigen (PSMA).
71. The pharmaceutical composition of any one of claims 68-70, wherein the BMP is selected from BMP-2, BMP -4, BMP-6, and BMP-7.
72. The pharmaceutical composition of claim 71, wherein the BMP comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or an amino acid sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.
73. The pharmaceutical composition of any one of claims 67-72, wherein the therapeutic agent comprises a radionuclide or an antibody.
74. The pharmaceutical composition of claim 73, wherein the radionuclide is a bone targeting radionuclide.
75. The pharmaceutical composition of claims 73 or 74, wherein the radionuclide is selected from 89Sr (e.g., 89Sr chloride), 223Ra (e.g., 223Ra dichloride), 153Sm (e.g., 153Sm-EDTMP),
177LU, 90Y, 186Re (e.g., 186Re HEDP), 117mSn (e.g., 117mSn DTPA), and 32P.
76. The pharmaceutical composition of claim 73, wherein the antibody is selected from an antibody drug conjugate and an antibody radionuclide conjugate.
77. The pharmaceutical composition of any one of claims 67-76, wherein the delivery vehicle is a recombinant oncolytic virus.
78. The pharmaceutical composition of claim 77, wherein the recombinant oncolytic virus is selected from an adenovirus, fowlpox virus, lentivirus, herpes virus, vesicular stomatitis virus (VSV), maraba virus, polio virus, and alpha virus.
79. The pharmaceutical composition of claim 78, wherein the recombinant oncolytic virus is a recombinant oncolytic adenovirus.
80. The pharmaceutical composition of claim 79, wherein the recombinant oncolytic adenovirus is a type 5 adenovirus (Ad5).
81. The pharmaceutical composition of claims 79 or 80, wherein the exogenous nucleotide sequence is inserted into an Elb-l9K insertion site located between the start site of Elb-l9K and the start site of Elb-55K.
82. The pharmaceutical composition of claim 81, wherein the Elb-l9K insertion site is located between the start site of Elb-l9K and the stop site of Elb-l9K.
83. The pharmaceutical composition of claims 81 or 82, wherein the Elb-l9K insertion site comprises a deletion of from about 100 to about 305, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 305, about 150 to about 300, about 150 to about 250, or about 150 to about 200 nucleotides adjacent the start site of Elb-l9K.
84. The pharmaceutical composition of any one of claims 81-83, wherein the Elb-l9K insertion site comprises a deletion of about 200 nucleotides adjacent the start site of Elb- 19K.
85. The pharmaceutical composition of any one of claims 81-84, wherein the Elb-l9K insertion site comprises a deletion of 203 nucleotides adjacent the start site of Elb-l9K.
86. The pharmaceutical composition of any one of claims 81-85, wherein the Elb-l9K insertion site comprises a deletion corresponding to nucleotides 1714-1916 of the Ad5 genome (SEQ ID NO: 5).
87. The pharmaceutical composition of any one of claims 81-86, wherein the exogenous nucleotide sequence is inserted between nucleotides corresponding to 1714 and 1916 of the Ad5 genome (SEQ ID NO: 5).
88. The pharmaceutical composition of any one of claims 81-87, wherein the exogenous nucleotide sequence is inserted between CTGACCTC (SEQ ID NO: 6) and TCACCAGG (SEQ ID NO: 7).
89. The pharmaceutical composition of any one of claims 81-88, wherein the recombinant oncolytic adenovirus comprises, in a 5’ to 3’ orientation, CTGACCTC (SEQ ID NO: 6), the exogenous nucleotide sequence, and TCACCAGG (SEQ ID NO: 7).
90. The pharmaceutical composition of claims 79 or 80, wherein the exogenous nucleotide sequence is inserted into an E3 insertion site located between the stop site of pVIII and the start site of Fiber.
91. The pharmaceutical composition of claim 90, wherein the E3 insertion site is located between the stop site of E3-10.5K and the stop site of E3-14.7K.
92. The pharmaceutical composition of claims 90 or 91, wherein the E3 insertion site comprises a deletion of from about 500 to about 3185, from about 500 to about 3000, from about 500 to about 2500, from about 500 to about 2000, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 3185, from about 1000 to about 3000, from about 1000 to about 2500, from about 1000 to about 2000, from about 1000 to about 1500, from about 1500 to about 3185, from about 1500 to about 3000, from about 1500 to about 2000, from about 2000 to about 3185, from about 2000 to about 3000, from about 2000 to about 2500, from about 2500 to about 3185, from about 2500 to about 3000, or from about 3000 to about 3185 nucleotides.
93. The pharmaceutical composition of any one of claims 90-92, wherein the E3 insertion site comprises a deletion of from about 500 to about 1551, from about 500 to about 1500, from about 500 to about 1000, from about 1000 to about 1551, from about 1000 to about 1500, or from about 1500 to about 1551 nucleotides adjacent the stop site of E3-10.5K.
94. The pharmaceutical composition of any one of claims 90-93, wherein the E3 insertion site comprises a deletion of about 1050 nucleotides adjacent the stop site of E3-10.5K.
95. The pharmaceutical composition of any one of claims 90-94, wherein the E3 insertion site comprises a deletion of 1064 nucleotides adjacent the stop site of E3-10.5K.
96. The pharmaceutical composition of any one of claims 90-95, wherein the E3 insertion site comprises a deletion corresponding to the Ad5 dl309 E3 deletion.
97. The pharmaceutical composition of any one of claims 90-96, wherein the E3 insertion site comprises a deletion corresponding to nucleotides 29773-30836 of the Ad5 genome (SEQ ID NO: 5).
98. The pharmaceutical composition of any one of claims 90-97, wherein the exogenous nucleotide sequence is inserted between nucleotides corresponding to 29773 and 30836 of the Ad5 genome (SEQ ID NO: 5).
99. The pharmaceutical composition of any one of claims 90-98, wherein the exogenous nucleotide sequence is inserted between CAGTATGA (SEQ ID NO: 8) and TAATAAAAAA (SEQ ID NO: 9).
100. The pharmaceutical composition of any one of claims 90-99, wherein the recombinant oncolytic adenovirus comprises, in a 5’ to 3’ orientation, CAGTATGA (SEQ ID NO: 8), the exogenous nucleotide sequence, and TAATAAAAAA (SEQ ID NO: 9).
101. The pharmaceutical composition of any one of claims 90-100, wherein the recombinant oncolytic adenovirus further comprises a deletion of a Pea3 binding site, or a functional fragment thereof.
102. The pharmaceutical composition of claim 101, wherein the recombinant oncolytic adenovirus comprises a deletion of nucleotides corresponding to about -300 to about -250 upstream of the initiation site of El a.
103. The pharmaceutical composition of claims 101 or 102, wherein the recombinant oncolytic adenovirus comprises a deletion of nucleotides corresponding to -304 or -305 to - 255 upstream of the initiation site of Ela.
104. The pharmaceutical composition of any one of claims 101-103, wherein the recombinant oncolytic adenovirus comprises a deletion of nucleotides corresponding to 195- 244 of the Ad5 genome (SEQ ID NO: 5).
105. The pharmaceutical composition of any one of claims 101-104, wherein the recombinant oncolytic adenovirus comprises the sequence GGTGTTTTGG (SEQ ID NO:
10).
106. The pharmaceutical composition of any one of claims 101-105, wherein the recombinant oncolytic adenovirus does not comprise a deletion of an E2F binding site.
107. The pharmaceutical composition of any one of claims 79-100, wherein the recombinant oncolytic adenovirus further comprises a deletion of a E2F binding site, or a functional fragment thereof.
108. The pharmaceutical composition of claim 107, wherein the recombinant oncolytic adenovirus does not comprise a deletion of a Pea3 binding site, or a functional fragment thereof.
109. The pharmaceutical composition of any one of claims 79-108, wherein the recombinant oncolytic adenovirus comprises an Ela promoter having a deletion of a functional TATA box.
110. The pharmaceutical composition of claim 109, wherein the deletion comprises a deletion of the entire TATA box.
111. The pharmaceutical composition of claims 109 or 110, wherein the deletion comprises a deletion of nucleotides corresponding to -29 to -26 of the Ela promoter.
112. The pharmaceutical composition of any one of claims 109-111, wherein the deletion comprises a deletion of nucleotides corresponding to -33 to -26 of the Ela promoter.
113. The pharmaceutical composition of any one of claims 109-112, wherein the deletion comprises a deletion of nucleotides corresponding to -44 to +52 of the Ela promoter.
114. The pharmaceutical composition of any one of claims 109-113, wherein the deletion comprises a deletion of nucleotides corresponding to -148 to +52 of the Ela promoter.
115. The pharmaceutical composition of any one of claims 109-114, wherein the deletion comprises a deletion of nucleotides corresponding to 353-552 of the Ad5 genome (SEQ ID NO: 5).
116. The pharmaceutical composition of any one of claims 109-115, wherein the Ela promoter comprises the sequence CTAGGACTG (SEQ ID NO: 11).
117. The pharmaceutical composition of any one of claims 79-116, wherein the recombinant oncolytic adenovirus comprises the nucleotide sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a nucleotide sequence having 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
118. The pharmaceutical composition of any of claims 77-117, wherein the recombinant oncolytic virus further comprises an exogenous nucleotide sequence encoding a
costimulatory molecule, an antiangiogenic and/or antivascular molecule, an anti-fibrotic molecule, an anti-inflammatory or pro-inflammatory molecule, a monoclonal antibody or fragment thereof, a pro-apoptotic molecule, an enzyme, an immunomodulatory molecule, a cytokine, or a cytokine inhibitor (e.g., a cytokine trap).
119. The pharmaceutical composition of any of claims 77-118, wherein the recombinant oncolytic virus selectively replicates in a hyperproliferative cell.
120. The pharmaceutical composition of any of claims 77-119, wherein the recombinant oncolytic virus selectively expresses the protein that defines a binding site or the protein that mediates the production of a biomolecule that defines a binding a site in a hyperproliferative cell.
121. The pharmaceutical composition of claims 119 or 120, wherein the hyperproliferative cell is a cancer cell.
122. The pharmaceutical composition of any one of claims 67 - 121, wherein the composition further comprises a bisphosphonate, hyaluronic acid, vitamin D, calcium, testosterone, estrogen, progesterone, a selective estrogen modulator (SERM), calcitonin, and/or a RANK or RANKL inhibitor (e.g., denosumab).
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