WO1999026480A1 - Vecteurs de therapie genique anti-angiogenique et leurs applications dans le cadre du traitement d'affections liees a l'angiogenese - Google Patents

Vecteurs de therapie genique anti-angiogenique et leurs applications dans le cadre du traitement d'affections liees a l'angiogenese Download PDF

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Publication number
WO1999026480A1
WO1999026480A1 PCT/US1998/024950 US9824950W WO9926480A1 WO 1999026480 A1 WO1999026480 A1 WO 1999026480A1 US 9824950 W US9824950 W US 9824950W WO 9926480 A1 WO9926480 A1 WO 9926480A1
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Prior art keywords
polypeptide
human
cells
angiogenesis
angiostatin
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PCT/US1998/024950
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English (en)
Inventor
Philippe Leboulch
Robert James Pawliuk
Thomas Bachelot
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Genetix Pharmaceuticals, Inc.
Massachusetts Institute Of Technology
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Priority to AU15985/99A priority Critical patent/AU1598599A/en
Publication of WO1999026480A1 publication Critical patent/WO1999026480A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/515—Angiogenesic factors; Angiogenin
    • 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
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/484—Plasmin (3.4.21.7)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • This invention relates generally to gene therapy for, e.g., cancer.
  • Angiogenesis is the process by which new capillaries are formed from existing vasculature. It is a complex process which involves proliferation and migration of endothelial cells. It plays a fundamental role in reproduction, development and wound repair. Unregulated angiogenesis, however, can further the progression of many diseases, including tumor growth and metastasis, arthritis, diabetes, and some forms of blindness. For example, there is experimental evidence that limits of tumor size and growth are not the failure of the tumor cells to proliferate, but rather a failure of the tumor to provide sufficient nutrients and waste removal to its constituent cells by recruiting surrounding vasculature.
  • the invention features a method for inhibiting tumor growth in a human patient harboring a solid tumor, involving administering to the patient a nucleic acid molecule which expresses in the patient an anti-angiogenic polypeptide selected from the group consisting of human angiostatin, murine angiostatin, human endostatin, murine endostatin, and angiogenesis-inhibiting fragments thereof, wherein expression of the anti-angiogenic polypeptide in the patient inhibits angiogenesis in the vicinity of the tumor and/or systemically by diffusion of the recombinant protein to the vascular compartment from secreting transduced cells, thereby inhibiting its growth.
  • the invention features tumor inhibition, of the type just described, using nucleic acids molecules of the formula A-B, where A and B are polypeptide and/or export signal joined by a peptide bond; peptide A contains at least 100 amino acids and includes at least kringles 1, 2, and 3 of human or murine angiostatin; and peptide B contains at least 100 amino acids and includes at least 75% of the amino acid sequence of human or murine endostatin.
  • Expression of the fusion anti-angiogenic polypeptide in the patient inhibits angiogenesis in the vicinity of the tumor and/or systemically by diffusion of the recombinant protein to the vascular compartment from secreting transduced cells, thereby inhibiting its growth.
  • polypeptide and/or export signal A further includes kringle region 4 of angiostatin, and can also include kringle region 5 of plasminogen (the larger protein molecule of which angiostatin is a portion).
  • the nucleic acid molecule preferably constitutes a portion of a viral vector or a plasmid, which can either be administered to the patient so that cells of the patient in the vicinity of the tumor and/or systemically by diffusion of the recombinant protein to the vascular compartment from secreting transduced cells are infected or transfected with the nucleic acid encoding the angiogenesis-inhibiting polypeptide, or cells (of the patient, or another human donor, or an animal) are infected or transfected ex-vivo, and those infected or transfected cells are then infused into the patient so that the anti-angiogenic polypeptide is expressed in the vicinity of the tumor and/or systemically by diffusion of the recombinant protein to the vascular compartment from secreting transduced cells.
  • the nucleic acid molecule includes a nucleotide sequence encoding a preactivation polypeptide and/or export signal for effecting Golgi and/or endoplasmic reticulum export of the anti-angiogenic polypeptide.
  • the invention features a method for treating a human patient suffering from diabetic retinopathy, involving administering to the patient one of the nucleic acid molecules described above.
  • FIG. 1 depicts the structural relationship of angiostatin with plasminogen.
  • Fig. 2 depicts the structural relationship of endostatin with collagen type XVIII.
  • Fig. 3 depicts various viral (A. MSCV murine retrovirus; B. Adeno- associated virus; C. HIV based retrovirus; E. recombinant adeno- virus) and non- viral (D. plasmid) vectors used in the construction of gene therapy vectors for this invention.
  • MSCV Murine Stem Cell Virus
  • RSV Rous Sarcoma Virus
  • HTV Human Immunodeficiency Virus
  • GFP Green Fluorescence Protein
  • HBPRE Hepatitis B Export Element
  • RRE Rev Response Element poly A: polyadenylation site ⁇ +: , ,> iral packaging sequence
  • the inve ⁇ ed triangle shows the site at which the anti-angiogenic constructs will be inserted usins engineered Mlul and Xhol restriction sites.
  • * denotes specific mutations within the long terminal repeat and leader which bestows the abihty for expression in embryonic stem and hematopoietic stem cells.
  • the arrow denotes the direction of transcription.
  • Fis. 4 depicts in the left (A) panel nude mice which were implanted with human neuroblastoma cells (line SK-N-AS) transduced with a mock virus and in the right (B) panel, nude mice which were transplanted with human neuroblastoma cells transduced with a retroviral gene therapy vector encoding an angiostatin-endostatin fusion protein.
  • Fis. 5 shows the nucleotide sequence (SEQ ID NO: 1 ) and amino acid sequence (SEQ ID NO: 2) of human plasminogen and the nucleotide sequence (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 6) of human angiostatin.
  • Fig. 6 shows the nucleotide sequence (SEQ ID NO: 9) and amino acid sequence (SEQ ID NO: 10) of murine endostatin.
  • Fig. 7 shows the nucleotide sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of murine plasminogen and the nucleotide (SEQ ID NO: 7) and amino acid sequence (SEQ ID NO: 8) of murine angiostatin.
  • This invention provides gene therapy using a vector having a nucleotide sequence encoding one of the above-identified anti-angiogenic polypeptides. Described below in more detail are some of the components of the vectors and methods of the invention.
  • a gene therapy vector is meant a vector useful for gene therapy.
  • Gene therapy vectors carry a gene of interest that is useful for gene therapy.
  • the gene therapy vectors are able to be transferred to the cells of an animal, e.g., a human, and are able to express the gene of interest in such cells so as to effect gene therapy.
  • the vector can be, e.g., chromosomal, non-chromosomal, or synthetic, and can be RNA or DNA.
  • the vector can be, e.g., a plasmid, a virus or a phage.
  • Preferred vectors include, e.g., retroviral vectors, adenoviral vectors, adeno-associated vectors, herpes virus vectors, Simliki Forest Virus- based vector, Human Immunodeficiency virus, Simian Immunodeficiency virus, and non- viral plasmids.
  • a preferred retroviral vector is Murine Stem Cell Virus (MSCV), which is a variant of Moloney Murine Leukemia Virus (MoMLV).
  • anti-angiogenic polypeptide is meant a polypeptide which inhibits angiogenesis.
  • angiogenesis is meant the process by which new vasculature, in particular, new capillaries, are formed from existing vasculature.
  • Angiogenesis is a complex process entailing numerous steps, including local dissolution of the basement membrane, migration of endothelial cells into the surrounding stroma, proliferation of the endothelial cells at the leading edge to form a migrating column of cells, branching and fusion of the newly formed vascular loops, and formation of a new basement membrane.
  • inhibiting angiogenesis is meant completely or partially inhibiting the formation of such new vasculature.
  • the anti-angiogenic polypeptide is an anti- angiogenic fragment of plasminogen (in particular, angiostatin), an anti- angiogenic fragment of collagen XVIII (endostatin) or a fusion of the two fragments.
  • Angiostatin is an internal fragment of plasminogen having a molecular weight of 38 or 45 kDa, depending on whether it contains kringles 1- 3 or 1-4. In the invention, either can be used, or a molecule including kringles 1-3 and a portion of kringle 4 can be used.
  • Angiostatin can be naturally produced in vivo in small amounts by tumor cells, e.g.
  • the gene therapy vectors of this invention encode angiostatin having kringles 1, 2 and 3, or angiostatin having kringles 1, 2, 3 and 4.
  • the anti-angiogenic polypeptide is endostatin or a biologically active analog or fragment thereof.
  • Endostatin can be naturally produced in vivo in small amounts by tumor cells, e.g., murine angiosarcoma cells, by proteolytic cleavage of endogenous collagen XVIII so as to eliminate the N-terminal portion including the signal polypeptide and/or export signal and the preactivation polypeptide and/or export signal, as well as the C-terminal portion following kringle 3 or 4. See Fig.2.
  • Mouse endostatin has been sequenced, and the human molecule (SEQ ID NOs: 17 and 18) forms a portion of collagen 18 (SEQ ID NOs: 19 and 20).
  • the human molecule position and sequence are apparent from an alignment of the active, Lys-terminated active region of human collagen 18 with murine endostatin, such that the C-terminal lysine residues align, bringing the active endostatin sequences into alignment.
  • the anti-angiogenic polypeptide is an in-frame fusion of angiostatin or a biologically active analog or fragment thereof and endostatin or a biologically active analog or fragment thereof.
  • the angiostatin or biologically active analog or fragment is 5' of the endostatin or biologically active analog or fragment.
  • the angiostatin-endostatin fusion proteins exhibit synergistic anti-angiogenic properties.
  • fragment is meant some portion of the naturally occurring anti- angiogenic polypeptide.
  • the fragment is at least 20 amino acid residues, more preferably at least 50 amino acid residues, and most preferably at least 100 amino acid residues in length.
  • Fragments include chimeric constructs composed of at least a portion of the relevant gene and another molecule.
  • the ability of a candidate fragment to exhibit a biological activity of the anti-angiogenic polypeptide can be assessed by methods known to those skilled in the art, e.g., by its ability to inhibit proliferation of bovine capillary cells, or by its ability to inhibit growth of primary mmor cells, e.g., as described herein. See, e.g.. Example 9.
  • Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide.
  • the gene therapy vector of this invention is capable of hybridizing to the native anti-angiogenesis polypepti de-encoding regions and has at least about 80%, preferably at least about 90%, and more preferably at least about 95%, sequence identity to the native nucleotide sequences, and encodes a polypeptide which has anti-angiogenic activity; or a biologically active fragment of any of the above nucleotide sequences wherein the encoded polypeptide has anti-angiogenic activity.
  • the nucleotide sequences of the present invention can be in the form of RNA or DNA, and the nucleotide sequence can be double-stranded or single stranded and, if single stranded, can be the coding strand or non-coding (antisense) strand.
  • the coding sequence which encodes the anti-angiogenic polypeptide can be identical to the native coding sequences, or can be a different coding sequence which, as a result of the degeneracy of the genetic code, encodes the same anti-angiogenic polypeptide.
  • the gene therapy vector also has a nucleotide sequence encoding a signal polypeptide and/or export signal (SP) for effecting secretion of the anti-angiogenic polypeptide.
  • signal polypeptide and/or export signal include plasminogen signal polypeptide and/or export signal.
  • the signal polypeptide and/or export signal is 5' (i.e., upstream) of the nucleotide sequence encoding the anti-angiogenic polypeptide.
  • the gene therapy vector has a nucleotide sequence encoding a preactivation polypeptide and/or export signal (PAP), which is a small polypeptide and/or export signal which effects folding and secretion of the anti- angiogenic polypeptide in vivo.
  • PAP preactivation polypeptide and/or export signal
  • Examples of preactivation polypeptide and/or export signal include plasminogen preactivation polypeptide and/or export signal, described herein, and PAP's of other proteins in the blood clotting cascade.
  • the preactivation polypeptide and/or export signal is positioned 5' of the nucleotide sequence encoding the anti-angiogenic polypeptide.
  • the preactivation polypeptide and/or export signal is 5' of the nucleotide sequence encoding the anti-angiogenic polypeptide, and 3' of the nucleotide sequence encoding the signal polypeptide and/or export signal.
  • PAP refers to a polypeptide and/or export signal which is naturally associated with a eukaryotic (preferably human) protein, the exportation of which is facilitated by its associated PAP.
  • eukaryotic preferably human
  • Examples of other human proteins whose Golgi/ER export is PAP-facilitated include other secreted proteins of the blood coagulation cascade, e.g., fibrinogen, prothrombin, Factor VIII, and Factor IX. Other secreted human proteins also are associated with potentially useful PAPs.
  • PAPs useful according to the invention preferably have 75% or greater amino acid sequence identity with a native PAP.
  • the gene therapy vector has a nucleotide sequence encoding a tag for identification of the anti-angiogenic polypeptide and/or export signal.
  • the tag is 5' of the nucleotide sequence encoding the anti-angiogenic polypeptide; in other embodiments, the tag is 3' of the nucleotide sequence encoding the anti-angiogenic polypeptide.
  • the anti-angiogenic polypeptide is endostatin or an angiostatin-endostatin fusion, it is preferred that the tag be 5' of the nucleotide sequence encoding endostatin.
  • the gene therapy vector includes a selectable marker, e.g., a Neomycin phosphotransferase gene, or a humanized red-shifted green fluorescent protein.
  • a selectable marker e.g., a Neomycin phosphotransferase gene, or a humanized red-shifted green fluorescent protein.
  • the invention also includes a cell infected or transfected with a gene therapy vector described herein.
  • the cell is an animal cell, more preferably an autologous or allogeneic human cell.
  • the gene therapy vectors described herein can be introduced into a cell, e.g., by transformation, transfection, transduction, infection, or ex vivo injection. They can be targeted to a particular cell type.
  • nucleic acid e.g., a gene therapy vector
  • administration of nucleic acid can be accomplished by any method which allows the nucleic acid to reach the target cells. These methods include, e.g., injection, deposition, implantation, suppositories, oral ingestion, inhalation, topical administration, or any other method of administration where access to the target cells by the nucleic acid is achieved. Injections can be, e.g., intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal.
  • Implantation includes inserting implantable drug delivery systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused or partially fused pellets.
  • Suppositories include glycerin suppositories.
  • Oral ingestion doses can be enterically coated.
  • Inhalation includes administering the nucleic acid with an aerosol in an inhalator, either alone or attached to a carrier that can be absorbed.
  • administration can be designed so as to result in sequential exposures to the nucleic acid over some time period, e.g., hours, days, weeks, months or years. This can be accomplished by repeated administrations of the nucleic acid, e.g., by one of the methods described above, or alternatively, by a controlled release delivery system in which the nucleic acid is delivered to the animal over a prolonged period without repeated administrations.
  • a controlled release delivery system is meant that total release of the nucleic acid does not occur immediately upon administration, but rather is delayed for some time. Release can occur in bursts or it can occur gradually and continuously.
  • Administration of such a system can be, e.g., by long acting oral dosage forms, bolus injections, transdermal patches or subcutaneous implants.
  • systems in which release occurs in bursts include, e.g., systems in which the nucleic acid is entrapped in liposomes which are encapsulated in a polymer matrix, the liposomes being sensitive to a specific stimulus, e.g., temperamre, pH, light, magnetic field, or a degrading enzyme, and systems in which the nucleic acid agent is encapsulated by an ionically-coated microcapsule with a microcapsule core-degrading enzyme.
  • nucleic acid examples include, e.g., erosional systems in which the nucleic acid is contained in a form within a matrix, and diffusional systems in which the nucleic acid permeates at a controlled rate, e.g., through a polymer.
  • sustained release systems can be, e.g., in the form of pellets or capsules.
  • the nucleic acid is administered to the patient in a therapeutically effective amount.
  • therapeutically effective amount is meant that amount which is capable of at least partially preventing or reversing the disease.
  • a therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on consideration of the patient's size, age, the efficacy of the particular nucleic acid used, the type of delivery system used, the time of administration relative to the onset of disease symptoms, and whether a single, multiple, or controlled release dose regimen is employed.
  • a therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
  • a therapeutically effective amount of an anti- angiogenic polypeptide is administered by providing to the animal a nucleic acid encoding the polypeptide and expressing the polypeptide in vivo.
  • Nucleic acids encoding the polypeptide, or mutants thereof can be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the nucleotide sequence for the anti-angiogenic polypeptide to cells in vivo.
  • Approaches include, e.g., insertion of the nucleic acid into viral vectors.
  • Viral vectors can be delivered to the cells, e.g., by infection or transduction using the virus. Viral vectors can also be delivered to the cells, e.g., by physical means, e.g., by electroporation, lipids, cationic lipids, liposomes, DNA gun, Ca 3 (P0 4 ) 2 precipitation, or delivery of naked DNA.
  • the virus is administered by injection, e.g., intramuscular injection, in a dose range of about 10 3 to about 10 10 infectious particles per injection, more preferably in a dose range of about 10 5 to about 10 8 infectious particles per injection.
  • injection e.g., intramuscular injection
  • Single or multiple doses can be administered over a given period of time, depending, e.g., upon the disease.
  • Plasmid DNA can be delivered to cells with the help of, e.g., cationic liposomes (lipofectinTM; Life Technologies, Inc., Gaithersburg, MD) or derivatized (e.g., antibody conjugated) polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or Ca 3 (P0 4 ) 2 precipitation carried out in vivo, or by use of a gene gun.
  • lipofectinTM lipofectinTM
  • derivatized e.g., antibody conjugated
  • Targets for delivery of the nucleic acid can be, e.g., specific target cells which are diseased.
  • the target can be, e.g., the peritoneal cavity, gastro-intestinal tract, bone manow cavity, liver, lungs, muscles, vasculature, pericardial cavity, pleural cavity, skin, sub-cutaneous or deep connective tissues, central nervous system, spinal fluid, eye, or specific sites of tumor growth.
  • the nucleic acid can be, e.g., coupled to an antibody, to a ligand to a cell surface receptor, or to a toxin component, or can be contained in a particle which is selectively internalized into cells, e.g., liposomes, or a virus where the viral receptor binds specifically to a certain cell type, or a viral particle lacking the viral nucleic acid, or can be administered by local injection.
  • the nucleic acid is administered to the patient by introducing ex vivo the nucleic acid into cells of the patient, or into syngeneic or allogeneic or xenogeneic cells, and then administering the cells having the nucleic acid to the animal. Any cell type can be used.
  • the cells having the introduced nucleic acid are expanded and/or selected after the nucleic acid transfer.
  • the cells having the transfened nucleic acid are subsequently administered to the patient.
  • the cells are administered in a dose range of about 1 x 10 6 to about 1 x 10 9 cells/dosage/day, and most preferably at about 1 x 10 7 to about 1 x 10 8 cells/dosage/day.
  • the cells can be administered by any method which results in delivering the transfened nucleic acid in the cells to the desired target.
  • the cells can be implanted directly into a specific tissue of the patient, or implanted after encapsulation within an artificial polymer matrix.
  • sites of implantation include, e.g., the peritoneal cavity, gastro-intestinal tract, bone manow cavity, liver, lungs, muscles, vasculature, pericardial cavity, pleural cavity, skin, sub-cutaneous or deep connective tissues, central nervous system, spinal fluid, eye, or specific sites of tumor growth.
  • Systemic delivery can be achieved, e.g., by introducing the nucleic acid into cells which circulate in the peripheral blood of the patient, or which give rise to cells which circulate in the peripheral blood.
  • the nucleic acid is introduced into such cells ex vivo, and these cells are then administered to the patient, resulting in systemic delivery within the peripheral blood.
  • These cells can be the cells of the patient or allogeneic cells.
  • Prefened cells in which the nucleic acid can be introduced are hematopoietic cells.
  • other therapy is additionally administered.
  • other tumor therapy e.g., another therapeutic agent, chemotherapy, radiation or surgery, is additionally administered to the patient, either simultaneously or at different times.
  • Treating is meant to include, e.g., preventing, treating, reducing the symptoms of, or curing the disease.
  • treating a tumor includes preventing growth of the tumor, causing shrinkage of the tumor, or preventing development of micro-metasteses.
  • the recombinant nucleic acid is a gene therapy vector, e.g., as described herein.
  • the anti-angiogenic polypeptide is angiostatin, endostatin, an angiostatin-endostatin fusion protein, or biologically active analogs or fragments thereof.
  • the angiostatin has kringles 1, 2 and 3; in other embodiments, the angiostatin has kringles 1, 2, 3 and 4, and, in some embodiments, kringle 5 of human or murine plasminogen.
  • Angiostatin is described in O'Reilly and Folkman U.S. Patent No. 5,639,725, hereby incorporated by reference. Endostatin is described in O'Reilly and Folkman PCT Appln. No. WO 97/15666, published May 1, 1997, hereby incorporated by reference.
  • the recombinant nucleic acid has been introduced ex vivo into cells so as to express the anti-angiogenic polypeptide in the cells, and the recombinant nucleic acid is administered to the patient by administering to the patient the cells containing the recombinant nucleic acid.
  • the cells are derived from the patient; in other embodiments the cells are allogeneic cells relative to the cells of the patient.
  • the cells are preferably hematopoietic cells, but can also be mesenchymal cells, stem cells, epithelial cells (e.g., from the gut), or dendritic cells.
  • the gene therapy vectors of the invention can be provided in a pharmaceutical composition comprising a therapeutically effective amount of the recombinant nucleic acid together with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, e.g., water, saline, dextrose, glycerol, ethanol, liposomes and lipid emulsions.
  • Example 1 Construction of Inserts for Gene Therapy Vectors Containing cDNA for Angiostatin. Endostatin or Angiostatin-Endostatin Fusion Proteins
  • the following genetic constructs are inserted into retroviral gene therapy vectors; the genetic constmcts contain human or murine cDNA for angiostatin, endostatin or an angiostatin-endostatin fusion, and DNA encoding a signal polypeptide and/or export signal (SP), a tag (FLAG), and, preferably, a preactivation polypeptide and/or export signal (PAP).
  • SP signal polypeptide and/or export signal
  • FLAG tag
  • PAP preactivation polypeptide and/or export signal
  • the constructs are all made using standard genetic engineering techniques, and their insertion into retroviral gene therapy vectors is carried out using known methods.
  • the constructs have the following components:
  • SP-PAP-Kl-K2-K3-K4-Flag SEQ ID NO: 1 1 and 12
  • SP-K1-K2-K3-K4- Flag-Endo SEQ ID NO: 15 and 16
  • the entire coding region of the human plasminogen cDNA from the start (ATG) to the stop (TAA) codon is 2433bp in size.
  • This sequence encodes a signal peptide (bp 1-57), a preactivation peptide (bp 58- 288), and 5 distinct structural regions known as kringles (K1-K3 from bp 289-1092; K4 from bp 1093- 1380; K5 from bp 1381-1740).
  • K1-K3 from bp 289-1092
  • K4 from bp 1093- 1380
  • K5 from bp 1381-1740
  • a DNA fragment encoding a portion of the human plasminogen protein from bp 1 to 1377 was obtained by PCR of a widely available human liver cD A library using synthetic DNA oligonucleotides complementary to sequences immediately preceding the sisnal peptide and immediatiy following kringle 4.
  • This fragment contains the sigal pepdde fbpl-57), the preactivation peptide (bp 58-288), kringles f (bp289-549), 2 (bp 550-804), 3 (bp 805-1092) and 4 (bp 1093-1380).
  • the synthetic oligonucXtides used for this reaction contained engineered recognition sites for the restricdon enzymes EcoRI and Xhol.
  • the amplified fragment was cloned into the EcoRI/XhoI sites of BluescriptSK(-) (Stratgene) using standard techniques (Maniatis). Following cloning the integrity of the amplified sequence was verified by sequencing both strands using die Sanger method (Sanger). Various derivatives of the cloned fragment were subsequently constructed using BluescriptSK(-) (Stratagene) as a backbone. A full list of the derivadves are described in Table 1. Briefly, the variations are composed of constructs containing various combinations of kringles with or without the signal and/or preactivation peptide sequences.
  • the coding sequence for murine plasminogen is 2439bp in size and, similar to the human plasminogen cDNA encompasses a sequence encoding signal and preactivation ceptides (bp 1-57 and 58-288 respectively) in addition to 5 kringle regions; kringle 1-3 (bp 289- 1092), kringle 4 (bp 1093-1380) and kringle 5 (bp 1381-1743).
  • kringle 1-3 bp 289- 1092
  • kringle 4 bp 1093-1380
  • kringle 5 bp 1381-1743
  • the murine plasminogen cDNA has previously been cloned and was mace available to us. Derivatives of murine plasminogen were constructed using sequences derived from bp 1-1743 of the coding sequence. Various combinations of kringle regions with or without signal and preacivation peptide regions were made using BluescriptSK(-) ⁇ Stratagene, La Jolla, CA) as the vector backbone.
  • Retroviral Gene Therapy Vectors This example illustrates the construction of retroviral gene therapy vectors comprising cDNA for angiostatin, endostatin or angiostatin-endostatin fusion proteins.
  • the DNA inserts from Example 1 were inserted into two retroviral vectors. Both vectors were derived from the Murine Stem Cell Virus (MSCV), which is a variant of Moloney Murine Leukemia Virus (MoMLV) having several mutations allowing high, sustained expression in hematopoietic stem cells and their progeny. In both cases, the angiostatin, endostatin, or angiostatin-endostatin fusion DNA inserts were under the transcriptional control of the retroviral left Long Terminal Repeat (LTR).
  • MSCV Murine Stem Cell Virus
  • MoMLV Moloney Murine Leukemia Virus
  • the dominant selectable marker was the Neomycin phosphofransferase gene (NeoR), which confers resistance to G418, and is driven by an internal phosphoglycerate kinase (PGK) promoter.
  • the dominant selectable marker was the humanized, red-shifted green fluorescent protein (EGFP), which is co-translationally expressed by means of an Internal Ribosome Entry Site (IRES) from the Encephalomyocarditis virus (EMCV).
  • IRS Internal Ribosome Entry Site
  • EMCV Encephalomyocarditis virus
  • Viral supernatants were collected two days thereafter and filtered through 0.45 mm filters. Filtered viral supernatants were subsequently used to infect GENETIX's stable amphotropic retroviral packaging cell-line AM 12 (Genetix Pharmaceuticals, Inc., Cambridge, MA). After another two days, viral supernatants from transduced AM 12 were filtered and used to infect GENETIX's stable ecotropic retroviral packaging cell-line
  • NeoR NeoR
  • FACS Fluorescent Activated Cell Sorter
  • Retrovirus (RCR) was detected in standard assays.
  • Example 3 Transduction of Target Cells Using Retroviral Gene Therapy Vectors
  • This example illustrates the stability of retroviral gene therapy vector transmission and the lack of toxicity in non-endothelial target cells.
  • viral supernatant was removed and filtered (0.45 ⁇ m filter, Gelman Sciences, Ann Arbor, MI).
  • Viral supernatant containing 7 ⁇ g/ml polybrene (Sigma, St. Louis, MO), was added to target cells 24 hours after plating the target cells.
  • Fresh medium was added after 4-12 hours, and, after an additional 48 hours, cells were selected for retroviral infection by exposure to medium containing 1 mg/ml G418 (Gibco BRL, Grand Island, NY) or by FACS sorting (FACStar cell sorter, Becton Dickinson, San Jose, CA).
  • Example 2 The stability of transmission of the retroviral gene therapy vectors described in Example 2 was examined by Southern blot analysis of transduced NIH3T3 cells, using specific probes (EGFP) and restriction enzyme digestion of genomic DNA with Sacl, which cuts only once in each LTR. Stable chromosomal integration of intact proviruses of appropriate length was observed with all constructs.
  • non-endothelial cells The lack of non-specific toxicity on non-endothelial cells was established by using filtered viral supernatants to transduce various tumor cell- types and cell-lines (NIH3T3 cells, K562 cells (ATCC), and human SK-N-AS neuroblastoma cells; Cohen, P.S., Cancer Research, 55:2380 (1995). Transduced cell populations were subsequently selected with G418 or sorted for EGFP expression by FACS. No obvious effects on cell viability, growth or other phenotypical characteristics were detected.
  • This example illustrates that recombinant angiostatin, endostatin, and angiostatin-endostatin fusion proteins were readily detected in retrovirally transduced cells and their supernatant, indicating efficient expression and secretion.
  • MSCV virus based vectors containing sequences encoding murine Kringle 1 (Kl), K1K5, K1K2K3, K1K2K3K4, and K1K2K3K4K5 were used to transduce NIH3T3 cells.
  • murine recombinant proteins Western blot analysis of transduced cells and their supernatant was performed by means of a monoclonal antibody that recognizes the FLAG polypeptide and or export signal. Because this antibody is not mono-specific, significant cross-reactivity with murine proteins was apparent. However, by comparing the pattern obtained with mock cells, it was clear that the antibody revealed an additional band of appropriate size in all transduced cells.
  • the recombinant proteins were detected in cell supernatants at levels above 50 ng/ml, using a protein concentration/semi-purification procedure (Centricon columns, Amicon, Beverly, MA).
  • a protein concentration/semi-purification procedure (Centricon columns, Amicon, Beverly, MA).
  • no FLAG tag was added, so a monoclonal antibody that recognizes specifically the first three kringles of human plasminogen in its native, non- dena red form was used; O'Reilly et al., Cell 79:315 (1994). Because of this constraint, Western blot analysis using denaturing gels could not be performed.
  • An ELISA assay was performed which indicated that human recombinant angiostatin was detected at levels likely to be therapeutic according to previous findings in the model of Lewis Lung Carcinoma Id.
  • Human SK-N-AS neuroblastoma cells (Cohen, 1995) were transduced with the retroviral gene therapy vector containing the angiostatin-endostatin fusion protein, described in Example 2. These cells (1,000,000) were suspended in 1 mL Dulbecco's phosphate buffered saline and injected into the right mid-quadrant of nude immuno-compromised mice. While no impairment of the in vitro growth of transduced cells was observed, a dramatic decrease in tumor growth in nude mice cells following subcutaneous implantation of the transduced cells was evident as compared to "mock virus"-transduced control cells.
  • This example illustrates infection of primary hematopoietic cells from donor mice with retroviral gene therapy vectors encoding angiostatin, endostatin, or an angiostatin-endostatin fusion protein, and a selectable GFP marker, and subsequent transplantation of the transduced hematopoietic cells into recipient mice.
  • Femoral bone manow cells are harvested from male donor C57BL6/J-
  • Ly5.1 mice (Jackson Labs, Bar Harbor, ME), intravenously injected four days previously with 150 mg/kg of 5-fluorouracil (5-FU).
  • Bone manow cells are cultured for two days in medium composed of DMEM, 15% fetal calf serum, 10 ng/ml human IL-6, 6 ng/ml murine IL-3 and 100 ng/ml murine Steel factor prior to two days of culmre atop a confluent monolayer of inadiated ( 1 ,500 cGy, 137 Cs ⁇ -inadiation) viral producer cells in the above medium including 6 ug/ml of prolamine sulfate.
  • the viral producer cells are transfected with a retroviral gene therapy vector, as described above.
  • Retrovirally transduced cells expressing the transfened GFP gene are subsequently identified and selected for, using a FACStar+ cell sorter (Becton Dickinson, San Jose, CA).
  • the GFP+ cells are intravenously injected into congenic female C57BL6/J-Ly5.2 recipient mice (National Cancer Institute, Washington, DC) previously given 950 cGy (83cGy/min, 137 Cs ⁇ -rays) of whole body inadiation.
  • This example illustrates engraftment of the recipient mice with the donor-derived transfected hematopoietic cells from Example 6.
  • the donor and recipient mice are phenotypically distinguishable on the basis of Y chromosome specific sequences, as well as on the basis of allelic differences at the murine CD45 cell surface antigen locus.
  • Male donor mice are homozygous for the CD45.2 allele, while female recipient mice are homozygous for CD45.1.
  • the engraftment of recipient mice with donor- derived (CD45.2+) cells is assessed at both short (5 weeks) and long (34 months) time points post-transplant by flow cytometric analysis of peripheral blood samples stained with a phycoerythrin labeled antibody specific for the CD45.2 antigen (Pharmingen, San Diego, C A). The results indicate that engraftment occurs.
  • Example 8 Proviral Marking and GFP Expression in Recipient Mice
  • This example illustrates the presence of recombinant pro virus and expression of the transfened GFP gene in the recipient mice from Example 6.
  • the level of proviral marking in reconstituted animals is initially determined by Southern blot and semi-quantitative PCR analysis of DNA obtained from peripheral blood leukocytes.
  • the large majority of donor- derived (CD45.2+) cells in recipient mice contain a minimum of one copy of recombinant pro virus.
  • flow cytometric analysis of peripheral blood leukocytes is performed to ascertain the proportion of cells expressing the transfened GFP cDNA.
  • This example illustrates the presence of anti-angiogenic polypeptide in the sera of the recipient mice from Example 6, using both physical and functional assays.
  • Serum obtained from the transplanted animals described in Example 6 is used for ELISA using an antibody specific for the synthetic FLAG epitope (IBI, Eastman Kodak, New Haven, CT) and compared against known standards of purified protein. Results indicate the presence of the anti-angiogenic polypeptide in the serum.
  • IBI Eastman Kodak, New Haven, CT
  • cells are plated in 24 well dishes at 25,000 cells/ml and maintained in DMEM with 5% bovine calf serum for 24 hours. The medium is then replaced with fresh medium containing various dilutions of the test serum. After 20 minutes of incubation, fresh medium including b-FGF (final concentration 1 ng/ml) is added and the cells are culmred for 72 hours. Cells are then dispersed using trypsin and the cell number determined by Coulter counter. Results indicate that functional anti-angiogenic polypeptide is present in the sera of the recipient mice.
  • mice are subcutaneously injected with one million Lewis lung carcinoma (LLC) cells (O'Reilly, (1994)) at the proximal midline of their dorsal skin. The mice are closely monitored for survival, tumor size and growth, and overall health. Results indicate that the anti-angiogenic polypeptides from the sera of the recipient mice inhibit growth of the LLC tumor cells.
  • LLC Lewis lung carcinoma
  • bone manow cells is re-transplanted into secondary recipients to generate individual day 12 spleen colonies, as well as plated in methylcellulose to assess in vitro clonogenic progenitors.
  • Individual clones are analyzed for proviral DNA by PCR or Southern blot, and for gene expression by flow cytometry and ELISA. Results of these tests also indicate the presence of proviral DNA and expression of the anti-angiogenic polypeptides and marker proteins.
  • Example 10 Evaluating the Efficacy of Retroviral Gene Therapy Vectors Encoding Anti-Angiogenic Polypetides on Various Human Cancers Implanted in SCID Mice Using Ex Vivo Gene Therapy
  • This example illustrates a method for rapidly screening various forms of human cancer to determine susceptibility to treatment by the systemic delivery of anti-angiogenic polypeptides.
  • SCID mice are more sensitive to ⁇ -inadiation than C57BL6/J mice, the female SCID recipients receive a lower dose of 400cGy of whole body inadiation in contrast to the 950cGy required for C57BL6/J.
  • SCID mice do not possess allelic differences at the CD45 cell surface antigen locus, donor and recipient cells are phenotypically distinguished on the basis of Y chromosome specific sequences using Southern blot analysis.
  • This example illustrates the feasibility of using retroviral gene therapy vectors encoding anti-angiogenic polypeptides to achieve efficient gene transfer to established mmors in vivo using a well-established murine model of human ovarian cancer. Following injections, mice are closely monitored for tumor growth and survival.
  • Viral supernatant for in vivo injection is prepared as follows: Viral producer cells are grown to confluence in DMEM with 10% bovine calf serum, and the medium is then changed. After 24 hours of incubation, the viral conditioned supernatant is filtered though a 0.45 um low protein binding filter, protamine sulfate is added to a final concentration of 6ug/ml, the solution is aliquoted into 2 ml volumes, and frozen at -80°C.
  • Recipient mice receive three intraperitoneal injections of viral supernatant (2 mis per injection) in addition to the poly cation, over a period of 36 hours. Control mice are injected with medium collected from confluent dishes of NIH3T3 cells. Following injection of the viral conditioned supernatant, the mice are analyzed for survival as well as tumor growth over time as compared to mock injected controls. Results indicate that treatment of the ovarian cancer occurs. At death, the mmors are removed, weighed, and the cells dissociated for DNA extraction for Southern blot analysis to detect recombinant pro virus.
  • the invention provides methods and compositions for treating diseases and processes that are mediated by angiogenesis including, but not limited to, hemangioma, solid mmors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, plaque neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic neovascularization, macular degeneration, wound healing, peptic ulcer, Helicobacter related diseases, fractures, keloids, vasculogenesis, hematopoiesis, ovulation, menstmation, placentation, and cat scratch fever.
  • angiogenesis including, but not limited to, hemangioma, solid

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Abstract

Cette méthode, visant à inhiber la croissance tumorale chez un patient porteur d'une tumeur solide, consiste à lui administrer une molécule d'acide nucléique exprimant chez ce patient un polypeptide anti-angiogénique issu du groupe constitué par l'angiostatine humaine, l'angiostatine murine, l'endostatine humaine et l'endostatine murine ainsi que par leurs fragments inhibiteurs de l'angiogenèse. L'expression de ce polypeptide anti-angiogénique entrave l'angiogenèse localement, au voisinage de la tumeur, et/ou de manière générale, par diffusion vers le compartiment vasculaire de la protéine de recombinaison émanant de cellules sécrétrices transduites, ce qui, partant, a pour effet d'inhiber la croissance de celle-ci.
PCT/US1998/024950 1997-11-20 1998-11-20 Vecteurs de therapie genique anti-angiogenique et leurs applications dans le cadre du traitement d'affections liees a l'angiogenese WO1999026480A1 (fr)

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WO1999029878A2 (fr) * 1997-12-08 1999-06-17 Beth Israel Deaconess Medical Center Procede de production de proteines anti-angiogeniques, dont l'endostatine, l'angiostatine ou la restine, au moyen d'un systeme d'expression de la levure pichia
WO2001002434A1 (fr) * 1999-07-02 2001-01-11 Bayer Ag Modulation de l'angiogenese au moyen d'angiootensine-7 antiangiogenique et de polynucleotides codant pour cette substance
WO2001012830A1 (fr) * 1999-08-13 2001-02-22 Novartis Ag Vecteurs adenoviraux renfermant des sequences d'adn codant des inhibiteurs de l'angiogenese
WO2001019989A2 (fr) * 1999-09-14 2001-03-22 Entremed, Inc. Procede de preparation et de purification d'une proteine d'endostatine¿tm?
WO2001058494A2 (fr) * 2000-02-11 2001-08-16 Genvec, Inc. Matieres et procedes destines au traitement de troubles oculaires
WO2002067971A2 (fr) * 2001-02-22 2002-09-06 Novartis Ag Traitement de la neovasculatisation oculaire
US6797488B1 (en) 1997-12-08 2004-09-28 Beth Israel Deaconess Medical Center Methods of producing anti-angiogenic proteins
US6821775B1 (en) 2000-02-11 2004-11-23 Genvec, Inc. Viral vector encoding pigment epithelium-derived factor
US7008921B2 (en) * 2000-09-05 2006-03-07 Karolinska Innovations Ab Materials and methods relating to endothelial cell growth inhibitors
US7723293B2 (en) 1998-10-28 2010-05-25 Cornell Research Foundation, Inc. Methods for increasing capillary density and maintaining viability of microvascular cardiac endothelial cells using trk receptor ligands
US7989426B2 (en) 2002-02-15 2011-08-02 Johns Hopkins University School Of Medicine Selective induction of apoptosis to treat ocular disease by expression of PEDF

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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6797488B1 (en) 1997-12-08 2004-09-28 Beth Israel Deaconess Medical Center Methods of producing anti-angiogenic proteins
WO1999029878A3 (fr) * 1997-12-08 1999-09-16 Beth Israel Hospital Procede de production de proteines anti-angiogeniques, dont l'endostatine, l'angiostatine ou la restine, au moyen d'un systeme d'expression de la levure pichia
WO1999029878A2 (fr) * 1997-12-08 1999-06-17 Beth Israel Deaconess Medical Center Procede de production de proteines anti-angiogeniques, dont l'endostatine, l'angiostatine ou la restine, au moyen d'un systeme d'expression de la levure pichia
US8853163B2 (en) 1998-10-28 2014-10-07 Cornell Research Foundation, Inc. Methods for increasing vascular density and maintaining viability of microvascular endothelial cells using trk receptor ligands
US7723293B2 (en) 1998-10-28 2010-05-25 Cornell Research Foundation, Inc. Methods for increasing capillary density and maintaining viability of microvascular cardiac endothelial cells using trk receptor ligands
WO2001002434A1 (fr) * 1999-07-02 2001-01-11 Bayer Ag Modulation de l'angiogenese au moyen d'angiootensine-7 antiangiogenique et de polynucleotides codant pour cette substance
WO2001012830A1 (fr) * 1999-08-13 2001-02-22 Novartis Ag Vecteurs adenoviraux renfermant des sequences d'adn codant des inhibiteurs de l'angiogenese
WO2001019989A3 (fr) * 1999-09-14 2001-08-09 Entremed Inc Procede de preparation et de purification d'une proteine d'endostatine¿tm?
US7476726B1 (en) 1999-09-14 2009-01-13 The Children's Medical Center Corp. Method of producing and purifying endostatin protein
WO2001019989A2 (fr) * 1999-09-14 2001-03-22 Entremed, Inc. Procede de preparation et de purification d'une proteine d'endostatine¿tm?
WO2001058494A3 (fr) * 2000-02-11 2002-04-04 Genvec Inc Matieres et procedes destines au traitement de troubles oculaires
US6821775B1 (en) 2000-02-11 2004-11-23 Genvec, Inc. Viral vector encoding pigment epithelium-derived factor
WO2001058494A2 (fr) * 2000-02-11 2001-08-16 Genvec, Inc. Matieres et procedes destines au traitement de troubles oculaires
US7008921B2 (en) * 2000-09-05 2006-03-07 Karolinska Innovations Ab Materials and methods relating to endothelial cell growth inhibitors
WO2002067971A2 (fr) * 2001-02-22 2002-09-06 Novartis Ag Traitement de la neovasculatisation oculaire
JP2004527494A (ja) * 2001-02-22 2004-09-09 ノバルティス アクチエンゲゼルシャフト 眼球血管新生を処置するための方法
JP4689144B2 (ja) * 2001-02-22 2011-05-25 ノバルティス アーゲー 眼球血管新生を処置するための方法
US8338384B2 (en) 2001-02-22 2012-12-25 Novartis Ag Method for treating ocular neovascularization
WO2002067971A3 (fr) * 2001-02-22 2003-12-31 Novartis Ag Traitement de la neovasculatisation oculaire
US9238057B2 (en) 2001-02-22 2016-01-19 Novartis Ag Method for treating ocular neovascularization
US7989426B2 (en) 2002-02-15 2011-08-02 Johns Hopkins University School Of Medicine Selective induction of apoptosis to treat ocular disease by expression of PEDF

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