WO1996036362A1 - Compositions containing nucleic acids and ligands for therapeutic treatment - Google Patents

Compositions containing nucleic acids and ligands for therapeutic treatment Download PDF

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Publication number
WO1996036362A1
WO1996036362A1 PCT/US1996/007164 US9607164W WO9636362A1 WO 1996036362 A1 WO1996036362 A1 WO 1996036362A1 US 9607164 W US9607164 W US 9607164W WO 9636362 A1 WO9636362 A1 WO 9636362A1
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Prior art keywords
receptor
nucleic acid
fgf
dna
binding domain
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PCT/US1996/007164
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French (fr)
Inventor
Barbara A. Sosnowski
J. Andrew Baird
Lois A. Chandler
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Prizm Pharmaceuticals, Inc.
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Publication date
Application filed by Prizm Pharmaceuticals, Inc. filed Critical Prizm Pharmaceuticals, Inc.
Priority to JP8535090A priority Critical patent/JPH11505805A/en
Priority to AU58628/96A priority patent/AU710309B2/en
Priority to EP96920274A priority patent/EP0833665A1/en
Publication of WO1996036362A1 publication Critical patent/WO1996036362A1/en

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    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0042—Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C07K14/50—Fibroblast growth factors [FGF]
    • C07K14/503—Fibroblast growth factors [FGF] basic FGF [bFGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/04—Fusion polypeptide containing a localisation/targetting motif containing an ER retention signal such as a C-terminal HDEL motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • the present invention relates generally to the treatment of diseases, and more specifically, to the preparation and use of complexes containing receptor-binding internalized ligands NABD and cytocide-encoding agents to alter the function, gene expression, or viability of a cell in a therapeutic manner.
  • a major goal of treatment of neoplastic diseases and hyperproliferative disorders is to ablate the abnormally growing cells while leaving normal cells untouched.
  • Various methods are under development for providing treatment, but none provide the requisite degree of specificity.
  • Immunotoxins and cytotoxins are protein conjugates of toxin molecules with either antibodies or factors which bind to receptors on target cells.
  • Three major problems may limit the usefulness of immunotoxins.
  • the antibodies may react with more than one cell surface molecule, thereby effecting delivery to multiple cell types, possibly including normal cells.
  • the antibody reactive molecule may be present on normal cells.
  • the toxin molecule may be toxic to cells prior to delivery and internalization. Cytotoxins suffer from similar disadvantages of specificity and toxicity.
  • Another limitation in the therapeutic use of immunotoxins and cytotoxins is the relatively low ratio of therapeutic to toxic dosage. Additionally, it may be difficult to direct sufficient concentrations of the toxin into the cytoplasm and intracellular compartments in which the agent can exert its desired activity.
  • cytotoxic therapy has been attempted using viral vectors to deliver DNA encoding the toxins into cells. If eukaryotic viruses are used, such as the retroviruses currently in use, they may recombine with host DNA to produce infectious virus. Moreover, because retro viral vectors are often inactivated by the complement system, use in vivo is limited. Retroviral vectors also lack specificity in delivery; receptors for most viral vectors are present on a large fraction, if not all, cells. Thus, infection with such a viral vector will infect normal as well as abnormal cells. Because of this general infection mechanism, it is not desirable for the viral vector to directly encode a cytotoxic molecule.
  • the present invention exploits the use of conjugates which have increased specificity and deliver higher amounts of nucleic acids to targeted cells, while providing other related advantages.
  • the present invention generally provides therapeutic compositions.
  • the composition has the formula: receptor-binding internalized ligand — nucleic acid binding domai — cytocide-encoding agent.
  • the receptor-binding internalized ligand is a polypeptide reactive with a cell surface receptor
  • the nucleic acid binding domain binds to a nucleic acid
  • the cytocide-encoding agent is a nucleic acid molecule encoding a cytocide and which binds to the nucleic acid binding domain
  • the composition binds to the cell surface receptor and internalizes the cytocide-encoding agent in cells bearing the receptor.
  • the composition has the formula: receptor-binding internalized ligand-nucleic acid binding comain-prodrug-encoding agent.
  • the receptor-binding internalized ligand is a polypeptide reactive with an FGF receptor, VEGF receptor, HBEGF receptor, or a cytokine.
  • the cytocide-encoding agent encodes a protein that inhibits protein synthesis and is preferably a ribosome inactivating protein, most preferably saporin.
  • the protein is gelonin or diphtheria toxin in other embodiments.
  • the prodrug-encoding agent encodes HSV-thymidine kinase.
  • the nucleic acid binding domain is poly-L-lysine in one embodiment.
  • the nucleic acid binding domain is a transcription factor selected from the group consisting of helix-turn-helix motif proteins, homeodomain proteins, zinc finger motif proteins, steroid receptor proteins, leucine zipper motif proteins, helix- loop-helix motif proteins, and ⁇ -sheet motif proteins.
  • the nucleic acid binding domain binds nonspecifically to nucleic acids and is selected from the group consisting of poly-L-lysine, protamine, histone and spermine.
  • the nucleic acid binding domain binds the coding region of a ribosome inactivating protein such as saporin.
  • FGF is conjugated to poly-L-lysine.
  • the cytocide-encoding agent contains a tissue-specific promoter, such as alpha-crystalline, gamma-crystalline, ⁇ -fetoprotein, CEA, prostate-specific antigen, erbB-2, tyrosinase, ⁇ -actin, c-myc, VEGF receptor, FGF receptor or cyclin D.
  • the composition also contains a linker.
  • the linker increases the flexibility of the conjugate and is (Gly m Ser p ) n , (Ala Ala Pro Ala) n , wherein n is 1 to 6, m is 1 to 6 and p is 1 to 4, or the linker is a disulfide bond.
  • the composition has the formula: receptor-binding internalized ligand-cytocide encoding agent-nucleic acid binding domain, wherein the receptor-binding internalized ligand is conjugated to the cytocide-encoding agent, which is bound to the nucleic acid binding domain to form a complex.
  • the invention provides methods for preventing excessive cell proliferation in the anterior eye following surgery, treating corneal clouding following excimer laser surgery, preventing closure of a trabeculectomy, preventing pterygii recurrence, treating hyperproliferative diseases in the back of the eye, such as macular degeneration, diabetic retinopathy and proliferative virtreal retinopathy, treating smooth muscle cell hyperplasia after a wound healing response to a procedure, e.g., vein grafting, endarterectomies and arterio venous shunts and treating cancer.
  • an effective amount of the compositions described above are administered.
  • Figure 1 is a photograph of an SDS-PAGE of FGF2-K152 under non- reducing (left) and reducing (right) conditions. Lane 1, FGF2-K152; lane 2, FGF2; lane 3, FGF2-K152: lane 4, FGF2.
  • the open arrow identifies material unable to enter the gel.
  • the closed arrow identifies a protein band corresponding to FGF2.
  • Figure 2 is a graph depicting the proliferation of bovine aortic endothelial cells in response to FGF2 (closed box) and FGF2-K152 (open circle) conjugate.
  • Figure 3 is a photograph of a gel showing the effects of various lengths of poly-L-lysine on the ability to interact with DNA.
  • Thirty-five ng of labeled DNA were added to increasing concentrations of either FGF2 or FGF2-K: lanes 1, 0 ng; lanes 2, 0.1 ng; lanes 3, 1 ng; lanes 4, 10 ng; lanes 5, 20 ng; lanes 6, 35 ng; lanes 7, 100 ng.
  • Panel A FGF2; panel B, FGF2-K152; panel C, FGF2-K13; panel D, FGF2- K84; panel E, EGF2-K267; panel F, FGF2-K39.
  • the lengths of the digested DNA are indicated.
  • Figure 4 is a chart depicting the activity of ⁇ -gal following transfection of FGF2/poly-L-lysine/DNA ⁇ -gal into COS cells.
  • the five bars, from left to right, are FGF2, FGF2-K13, FGF2- K39, FGF2-K84, and FGF2-K152.
  • Figure 5 are photographs of toroid format observed by electron microscopy. The upper panel shows an example of a toroid; the lower panel shows an incomplete toroid.
  • Figure 6 is a graph depicting proliferation of bovine aortic-endothelial cells.
  • cells were treated with FGF2-K152-DNA; in the lower panel, cells were treated with a mixture of FGF2, Kl 52, and DNA.
  • Figure 7A is a graph displaying ⁇ -gal activity after transfection of FGF2/poly-L-lysine/pSV ⁇ -gal into COS cells (lane 1), B16 cells (lane 2), NIH 3T3 cells (lane 3), and BHK cells (lane 4).
  • Figure 7B is a graph depicting ⁇ -gal expression in COS cells, pSV ⁇ -gal (lanes 1, 3) or pNASS ⁇ -gal (lanes 2, 4) were incubated with (lanes 1, 2) or without (lanes 3, 4) FGF2-K84 and the complexes incubated on COS cells for 48 hrs.
  • Figure 7C is a graph showing activity of ⁇ -gal activity at various times following transfection with either plasmid alone or with complexes of FGF2/K84/pSV ⁇ -gal. - ⁇ -, DNA alone; - ⁇ -, FGF2-K84-DNA.
  • Figure 7D is a graph showing ⁇ -gal activity after transfection of various concentrations of FGF2/K84/pSV ⁇ -gal. Lane 1, O ⁇ g; lane 2, 0,1 ⁇ g; lane 3, l ⁇ g; lane 4, 5 ⁇ g; lane 5, lO ⁇ g.
  • Figure 8A is a graph showing ⁇ -gal activity in COS cells following transfection of FGF2-K84- ⁇ SV ⁇ -gal (lane 1), FGF2+K84+pSV ⁇ -gal (lane 2), FGF2+pSV ⁇ -gal (lane 3), K84+pSV ⁇ -gal (lane 4); pSV ⁇ -gal (lane 5), FGF2-K84 (lane 6), FGF2 (lane 7) and K84 (lane 8).
  • Figure 8B is a graph showing completion for cell bindings.
  • Lane 1 FGF2-K84-pSV ⁇ -gal complex transfected into COS cells; lane 2, FGF2-K84-pSV ⁇ -gal plus 100 ⁇ g FGF2; lane 3, no complex.
  • Figure 8C is a graph showing the attenuation of ⁇ -gal activity upon the addition of heparin during transfection.
  • Figure 8D is a graph showing ligand targeting of DNA, pSV ⁇ -gal DNA alone (lane 1), FGF2-K84 (lane 2), histone H1-K84 (lane 3) and cytochrome C-K84 (lane 4) were condensed with pSV ⁇ -gal DNA and added to BHK cells, ⁇ -gal activity was measured 48 hr later.
  • Figure 9A is a graph showing the effect of chloroquine on ⁇ -gal expression, pSV ⁇ -gal and FGF2-K84 were mixed in the absence (lane 1) or presence (lane 2) of 100 ⁇ M chloroquine and incubated for 1 hr at room temperature prior to addition of the complexes to COS cells. Lane 3, chloroquine alone; lane 4, DNA alone.
  • Figure 9B is a graph showing the effect of endosome disruptive peptide on ⁇ -gal expression. Lane 1, control; lane 2, FGF2-K84-pSV ⁇ -gal; lane 3, FGF2-K84- pSV ⁇ -gal+EDP.
  • Figure 9C are photographs of cells stained for ⁇ -gal activity following transfection of COS cells with (right panel) or without (left panel) endosome disruptive peptide and FGF2-K84-pSV ⁇ -gal.
  • Figure 10 is a photograph of a fluorograph .analyzing cell-free translation products. Lane 1, no RNA; lane 2, saporin RNA; lane 3, luciferase RNA; lane 4, saporin RNA and luciferase RNA; lane 5, saporin RNA followed 30 min later with luciferase RNA.
  • Figure 11 is a graph depicting direct cytotoxicity of cells transfected by a CaPO 4 with an expression vector encoding saporin. Lane 1, mock transfection; lane 2, transfection with pSV ⁇ -gal; lane 3, transfection with saporin-containing vector.
  • Figure 12 is a pair of graphs showing cytotoxicity of cells transfected with FGF2-K84-pSVSAP. Left panel, BHK21 cells; right panel, NIH 3T3 cells. Lane 1, FGF2-K84-pSV ⁇ -gal; lane 2, FGF2-K84-pSVSAP.
  • Figure 13A is a graph showing ⁇ -gal activity with an endosome disruptive peptide in the complex.
  • Figure 13B is a graph showing ⁇ -gal activity with an endosome disruptive peptide in the complex.
  • Figure 13C is a graph showing ⁇ -gal activity with an endosome disruptive peptide in the complex.
  • amino acids which occur in the various amino acid sequences appearing herein, are identified according to their well known, three letter or one letter abbreviations.
  • nucleotides which occur in the various DNA fragments, are designated with the standard single letter designations used routinely in the art.
  • binding refers to the ability of a ligand to specifically recognize and detectably bind to such receptors, as assayed by standard in vitro assays.
  • binding measures the capacity of a VEGF conjugate, VEGF monomer, or VEGF dimer to recognize a VEGF receptor on a vascular endothelial cell, such as an aortic vascular endothelial cell line, using a procedure substantially as described in Moscatelli, J Cell Physiol. 757:123-130, 1987.
  • biological activity refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture.
  • Biological activity thus encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures.
  • Such biological activity may be defined with reference to particular in vitro activities as measured in a defined assay.
  • reference herein to the biological activity of FGF, or fragments of FGF refers to the ability of FGF to bind to cells bearing FGF receptors and internalize a linked agent.
  • Such activity is typically assessed in vitro by linking the FGF to a cytotoxic agent, such as saporin, contacting cells bearing FGF receptors, such as fibroblasts, with the conjugate and assessing cell proliferation or growth.
  • In vivo activity may be determined using recognized animal models, such as the mouse xenograft model for anti -tumor activity (see, e.g., Beitz et al., Cancer Research 52:227-230, 1992; Houghton et al., Cancer Res. ⁇ 2:535-539, 1982; Bogden et al., Cancer (Philadelphia) 4-5:10-20, 1981; Hoogenhout et al., Int. J. Radiat. Oncol, Biol. Phys. 9:871-879, 1983; Stastny et al., Cancer Res. 55:5740-5744, 1993).
  • mouse xenograft model for anti -tumor activity see, e.g., Beitz et al., Cancer Research 52:227-230, 1992; Houghton et al., Cancer Res. ⁇ 2:535-539, 1982; Bogden et al., Cancer (Philadelphia)
  • biological activity of a cytocide- encoding agent refers to the ability of such agent to interfere with the metabolism of the cell by inhibiting protein synthesis.
  • biological or cytotoxic activity may be assayed by any method known to those of skill in the art including, but not limited to, in vitro assays that measure protein synthesis and in vivo assays that assess cytotoxicity by measuring the effect of a test compound on cell proliferation or on protein synthesis. Assays that assess cytotoxicity in targeted cells are particularly preferred.
  • conjugate refers to a molecule that contains at least one receptor-internalized binding ligand and at least one nucleic acid binding domain that are linked directly or via a linker and that are produced by chemical coupling methods or by recombinant expression of chimeric DNA molecules to produce fusion proteins.
  • a "cytocide-encoding agent” is a nucleic acid molecule that encodes a protein that inhibits protein synthesis. Such a protein may act by cleaving rRNA or ribonucloprotein, inhibiting an elongation factor, cleaving mRNA, or other mechanism that reduces protein synthesis to a level such that the cell cannot survive.
  • the cytocide-encoding agent may contain additional elements besides the cytocide gene. Such elements include a promoter, enhancer, splice sites, transcription terminator, poly(A) signal sequence, bacterial or mammalian origins of replication, selection markers, and the like.
  • cytotoxic agent refers to a molecule capable of inhibiting cell function.
  • the agent may inhibit proliferation or may be toxic to cells.
  • a variety of cytotoxic agents can be used and include those that inhibit protein synthesis and those that inhibit expression of certain genes essential for cellular growth or survival. Cytotoxic agents include those that result in cell death and those that inhibit cell growth, proliferation and/or differentiation.
  • cytotoxic agents include, but are not limited to, saporin, the ricins, abrin and other ribosome inactivating proteins (RIPs), aquatic-derived cytotoxins, Pseudomonas exotoxin, inhibitors of DNA, RNA or protein synthesis, such as antisense nucleic acids, other metabolic inhibitors, such as DNA cleaving molecules, prodrugs, such as thymidine kinase from HSV and bacterial cytosine deaminase, and light activated porphyrin.
  • Suitable RIPs include ricin, ricin A chain, maize RIP, gelonin, diphtheria toxin, diphtheria toxin A chain, trichosanthin, tritin, pokeweed antiviral protein (PAP), mirabilis antiviral protein (MAP), Dianthins 32 and 30, abrin, monordin, bryodin, shiga, a catalytic inhibitor of protein biosynthesis from cucumber seeds (see, e.g., WO 93/24620), Pseudomonas exotoxin, biologically active fragments of cytotoxins and others known to those of skill in this art.
  • Suitable cytotoxic agents also include cytotoxic molecules that inhibit cellular metabolic processes, including transcription, translation, biosynthetic or degradative pathways, DNA synthesis, and other such processes that kill cells or inhibit cell proliferation.
  • Heparin-binding growth factor refers to any member of a family of heparin-binding growth factor proteins, in which at least one member of the family binds heparin.
  • Preferred growth factors in this regard include FGF, VEGF, and HBEGF.
  • Such growth factors encompass isoforms, peptide fragments derived from a family member, splice variants, and single or multiple exons, some forms of which may not bind heparin.
  • hybridize under conditions of a specified stringency is used to describe the stability of hybrids formed between two single-stranded nucleic acid molecules.
  • Stringency of hybridization is typically expressed in conditions of ionic strength and temperature at which such hybrids are annealed and washed.
  • high, medium and low stringency encompass the following conditions or equivalent conditions thereto:
  • high stringency 0.1 x SSPE or SSC, 0.1% SDS, 65°C
  • medium stringency 0.2 x SSPE or SSC, 0.1% SDS, 50°C
  • NABD nucleic acid binding domain
  • the NABD may bind to single or double strands of RNA or DNA or mixed RNA DNA hybrids.
  • the nucleic acid binding domain may bind to a specific sequence or bind irrespective of the sequence.
  • nucleic acids refer to RNA or DNA that are intended for internalization into a cell and includes, but are not limited to, DNA encoding a therapeutic protein, DNA encoding a cytotoxic protein, DNA encoding a prodrug, DNA encoding a cytocide, the complement of these DNAs, an antisense nucleic acid and other such molecules.
  • Reference to nucleic acids includes duplex DNA, single-stranded DNA, RNA in any form, including triplex, duplex or single-stranded RNA, anti-sense RNA, polynucleotides, oligonucleotides, single nucleotides, chimeras, and derivatives thereof.
  • Nucleic acids may be composed of the well-known deoxyribonucleotides and ribonucleotides composed of the bases adenosine, cytosine, guanine, thymidine, and uridine. As well, various other nucleotide derivatives and non-phosphate backbones or phosphate-derivative backbones may be used.
  • PO oligonucleotides normal phosphodiester oligonucleotides
  • PO oligonucleotides are sensitive to DNA- and RNA-specific nucleases
  • several resistant types of oligonucleotides have been developed in which the phosphate group has been altered to a phosphotriester, methylphosphonate, or phosphorothioate (see U.S. Patent No. 5,218,088).
  • operative linkage or operative association of heterologous DNA to regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, refers to the functional relationship between such DNA and such sequences of nucleotides.
  • operative linkage of heterologous DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA in reading frame.
  • polypeptide reactive with .an FGF receptor refers to any polypeptide that specifically interacts with an FGF receptor, preferably the high-affinity FGF receptor and that is transported into the cell by virtue of its interaction with the FGF receptor.
  • Polypeptides reactive with an FGF receptor are also called FGF proteins.
  • Such polypeptides include, but are not limited to, FGF-1 to FGF- 9.
  • bFGF FGF-2 should be generally understood to refer to polypeptides having substantially the same amino acid sequences and receptor-targeting activity as that of bovine bFGF or human bFGF. It is understood that differences in amino acid sequences can occur among FGFs of different species as well as among FGFs from individual organisms or species.
  • a prodrug is a compound that metabolizes or otherwise converts an inactive, nontoxic compound to a biologically, pharmaceutically, therapeutically, of toxic active form of the compound.
  • a prodrug may also be a pharmaceutically inactive compound that is modified upon administration to yield an active compound through metabolic or other processes.
  • the prodrug may alter the metabolic stability or the transport characteristics of a drug, mask side effects or toxicity, improve or alter other characteristics or properties of a drug.
  • pharmacodynamic processes and drug metabolism in vivo those of skill in this art, once a pharmaceutically active compound is known, can design inactive forms of the compound (see, e.g., Nogrady, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, 1985).
  • receptor-binding internalized ligand refers to any peptide, polypeptide, protein or non-protein, such as a peptidomimetic, that is capable of binding to a cell-surface molecule and is internalized.
  • the receptor-binding internalized ligand is conjugated to a nucleic acid binding domain, either as a fusion protein or through chemical conjugation, and is used to deliver a cytocide-encoding or pro-drug encoding agent to a cell.
  • the ligand is directly conjugated to a nucleic acid molecule, which may be further complexed with a nucleic acid binding domain.
  • Such ligands include growth factors, cytokines, antibodies or fragments thereof, hormones, and the like.
  • SAP serum-derived neurotrophic factor
  • SAP refers to polypeptides that are isolated from the leaves or seeds of Saponaria officinalis, as well as modified forms that have amino acid substitutions, deletions, insertions or additions, which still express substantial ribosome inactivating activity. Purified preparations of saporin are frequently observed to include several molecular isoforms of the protein. It is understood that differences in amino acid sequences can occur in saporin from different species as well as between saporin molecules from individual organisms of the same species. Saporin for use herein may be purified from leaves, chemically synthesized, or synthesized by expression of DNA encoding a saporin polypeptide.
  • a targeted agent is a nucleic acid molecule that is intended for internalization by complexing or linkage to a receptor-binding internalized ligand, and nucleic acid binding domain, and that upon internalization in some manner alters or affects cellular metabolism, growth, activity, viability or other property or characteristic of the cell.
  • a "therapeutic nucleic acid” describes any nucleic acid molecule used in the context of the invention that modifies gene transcription or translation. This term also includes nucleic acids that bind to sites on proteins. It includes, but is not limited to, the following types of nucleic acids: nucleic acids encoding a protein, antisense RNA, DNA intended to form triplex molecules, extracellular protein binding oligonucleotides, and small nucleotide molecules.
  • a therapeutic nucleic acid may be used to effect genetic therapy by serving as a replacement for a defective gene, by encoding a therapeutic product, such as TNF, or by encoding a cytotoxic molecule, especially an enzyme, such as saporin.
  • the therapeutic nucleic acid may encode all or a portion of a gene, and may function by recombining with DNA already present in a cell, thereby replacing a defective portion of a gene. It may also encode a portion of a protein and exert its effect by virtue of co-suppression of a gene product.
  • the present invention provides cytocide-encoding agents complexed with a conjugate of a receptor-binding internalized ligand and a nucleic acid binding domain. Upon binding to an appropriate receptor, the complex is internalized by the cell and is trafficked through the cell via the endosomal compartment, where at least a portion of the complex may be cleaved.
  • receptor-binding internalized ligands are used to deliver a cytocide-encoding agent to a cell expressing an appropriate receptor on its cell surface.
  • Numerous molecules that bind specific receptors have been identified and are suitable for use in the present invention. Such molecules include growth factors, cytokines, and antibodies. Many growth factors and families of growth factors share structural and functional features and may be used in the present invention. One such family of growth factors specifically binds to heparin. The ability of heparin-binding growth factors to interact with heparin appears in general to be a reflection of a physiologically more relevant interaction occurring in vivo between these factors and heparin sulfate proteoglycan molecules, which are found on the surface of cells and in extracellular matrix.
  • Heparin-binding growth factors include the fibroblast growth factors FGF-1 through FGF-9, vascular endothelial growth factor (VEGF), and heparin binding-epidermal growth factor (HBEGF).
  • Antibodies that are specific to cell surface molecules expressed by a selected cell type are readily generated as monoclonals or polyclonal antisera. Many such antibodies are available (e.g., American Type Culture Collection, Rockville, MD).
  • Other growth factors such as PDGF (platelet-derived growth factor), EGF (epidermal growth factor), TGF- ⁇ (tumor growth factor), TGF- ⁇ , IGF-I (insulin-like growth factor), and IGF-II also bind to specific identified receptors on cell surfaces and may be used in the present invention.
  • Cytokines including interleukins, CSFs (colony stimulating factors), and interferons, have specific receptors, which are mostly found on hematopoeitic cells, and may be used as described herein. These ligands are discussed in more detail below.
  • Fragments of these ligands may be used within the present invention, so long as the fragment retains the ability to bind to the appropriate cell surface molecule. Likewise, ligands with substitutions or other alterations, but which retain binding ability, may also be used.
  • Fibroblast growth factors One family of growth factors that has a broad spectrum of activities is the fibroblast growth factor (FGF) family. These proteins share the ability to bind to heparin, induce intracellular receptor-mediated tyrosine phosphorylation and the expression of the c-fos mRNA transcript, and stimulate DNA synthesis and cell proliferation. This family of proteins includes FGFs designated FGF-1 (acidic FGF (aFGF)), FGF-2 (basic FGF (bFGF)), FGF-3 (int-2) (see, e.g., Moore et al., EMBO J.
  • FGF-1 acidic FGF
  • bFGF basic FGF
  • FGF-3 int-2
  • FGF-4 hst-1/K-FGF
  • FGF-5 see, e.g., U.S. Patent No.
  • FGF-6 hst-2
  • FGF-7 keratinocyte growth factor
  • FGF-8 see, e.g., Tanaka et al., Proc Natl. Acad. Sci. USA 59:8528-8532, 1992
  • FGF-9 see, Miyamoto et al., Mol. Cell. Biol. 75:4251-4259, 1993.
  • DNA encoding FGF peptides and/or the amino acid sequences of FGFs are known to those of skill in the art.
  • DNA encoding an FGF may be prepared synthetically based on a known amino acid or DNA sequence, isolated using methods known to those of skill in the art, or obtained from commercial or other sources.
  • DNA encoding virtually all of the FGF family of peptides is known.
  • DNA encoding human FGF-1 Jaye et al., Science 255:541-545, 1986; U.S. Patent No. 5,223,483
  • bovine FGF-2 Abraham et al., Science 255:545-548, 1986; Esch et al., Proc. Natl. Acad. Sci.
  • DNA encoding an FGF may be produced from any of the preceding DNA fragments by substitution of degenerate codons. It is understood that once the complete amino acid sequence of a peptide, such as an FGF peptide, and the DNA fragment encoding such peptide are available to those of skill in this art, it is routine to substitute degenerate codons and produce any of the possible DNA fragments that encode such peptide.
  • FGF refers to polypeptides having amino acid sequences of native FGF proteins, as well as modified sequences, having amino acid substitutions, deletions, insertions or additions in the native protein but retaining the ability to bind to FGF receptors and to be internalized. It is understood that differences in amino acid sequences can occur among FGFs of different species as well as among FGFs from individual organisms or species.
  • FGFs are intended to encompass proteins isolated from natural sources as well as those made synthetically, as by recombinant means or possibly by chemical synthesis.
  • FGF also encompasses muteins that possess the ability to bind to FGF-receptor expressing cells.
  • Such muteins include, but are not limited to, those produced by replacing one or more of the cysteines with serine as described herein or that have any other amino acids deleted or replaced as long as the resulting protein has the ability to bind to FGF-receptor bearing cells and internalize the linked targeted agent.
  • muteins will have conservative amino acid changes, such as those set forth below in Table 1.
  • DNA encoding such muteins will, unless modified by replacement of degenerate codons, hybridize under conditions of at least low stringency to native DNA sequence encoding the starting FGF.
  • Acidic and basic FGF are about 55% identical at the amino acid level and are highly conserved among species.
  • the other members of the FGF family have a high degree of amino acid sequence similarities and common physical and biological properties with FGF-1 and FGF-2, including the ability to bind to one or more FGF receptors.
  • Basic FGF, int-2, hst-1 K-FGF, FGF-5, hst-2/FGF-6 and FGF-8 may have oncogenic potential; bFGF is expressed in melanomas, int-2 is expressed in mammary tumor virus and hst-1/K-FGF is expressed in angiogenic tumors.
  • Acidic FGF, bFGF, KGF and FGF-9 are expressed in normal cells and tissues.
  • FGFs exhibit a mitogenic effect on a wide variety of mesenchymal, endocrine and neural cells and are also important in differentiation and development. Of particular interest is their stimulatory effect on collateral vascularization and angiogenesis. In some instances, FGF-induced mitogenic stimulation may be detrimental. For example, cell proliferation and angiogenesis are an integral aspect of tumor growth. Members of the FGF family, including bFGF, are thought to play a pathophysiological role, for example, in tumor development, rheumatoid arthritis, proliferative diabetic retinopathies and other complications of diabetes.
  • FGFs are mediated by high affinity receptor tyrosine kinases present on the cell surface of FGF-responsive cells (see, e.g., PCT WO 91/00916, WO 90/05522, PCT WO 92/12948; Imamura et al., Biochem. Biophys. Res. Comm. 755:583-590, 1988; Huang et al., J. Biol. Chem. 267:9568-9571, 1986; Partanen et al., EMBO J. 10:1347, 1991; and Moscatelli, J Cell. Physiol. 757:123, 1987). Lower affinity receptors also appear to play a role in mediating FGF activities.
  • the high affinity receptor proteins are single chain polypeptides with molecular weights ranging from 110 to 150 kD, depending on cell type that constitute a family of structurally related FGF receptors.
  • FGF receptor genes Four FGF receptor genes have been identified, and three of these genes generate multiple mRNA transcripts via alternative splicing of the primary transcript.
  • VEGFs Vascular endothelial growth factors
  • VEGF was originally isolated from a guinea pig heptocarcinoma cell line, line 10 (see, e.g., U.S. Patent No. 4,456,550), and has subsequently been identified in humans and in normal cells. It is expressed during normal development and in certain normal adult organs. Purified VEGF is a basic, heparin-binding, homodimeric glycoprotein that is heat-stable, acid-stable and may be inactivated by reducing agents.
  • DNA sequences encoding VEGF and methods to isolate these sequences may be found primarily in U.S. Patent No. 5,240,848, U.S. Patent No. 5,332,671, U.S. Patent No. 5,219,739, U.S. Patent No. 5,194,596, and Houch et al., Mol. Endocrin. 5:180, 1991.
  • "DNA encoding a VEGF peptide or polypeptide” refers to any of the DNA fragments set forth herein as coding such peptides, to any such DNA fragments known to those of skill in the art, any DNA fragment that encodes a VEGF that binds to a VEGF receptor and is internalized thereby.
  • VEGF DNA may be isolated from a human cell library, for example, using any of the preceding DNA fragments as a probe or any DNA fragment that encodes any of the VEGF peptides set forth in SEQ ID NOs. 1-4. It is understood that once the complete amino acid sequence of a peptide, such as a VEGF peptide, and the DNA fragment encoding such peptide are available to those of skill in this art, it is routine to substitute degenerate codons and produce any of the possible DNA fragments that encode such peptide. It is also generally possible to synthesize DNA encoding such peptide based on the amino acid sequence.
  • VEGF family members arise from a single gene organized as eight exons and spanning approximately 14 kb in the human genome.
  • Four molecular species of VEGF result from alternative splicing of mRNA and contain 121, 165, 189 and 206 amino acids. The four species have similar biological activities, but differ markedly in their secretion patterns.
  • the predominant isoform secreted by a variety of normal and transformed cells is VEGF 165 .
  • Transcripts encoding VEGF 121 and VEGF lg9 are detectable in most cells and tissues that express the VEGF gene.
  • VEGF 206 is less abundant and has been identified only in a human fetal liver cDNA library.
  • VEGF 121 is a weakly acidic polypeptide that lacks the heparin binding domain and, consequently, does not bind to heparin.
  • VEGF I89 and VEGF 206 are more basic than VEGF 165 and bind to heparin with greater affinity.
  • all isoforms are considered to be heparin-binding growth factors within the context of this invention.
  • the secreted isoforms, VEGF, 21 and VEGF 165 are preferred VEGF proteins.
  • VEGF vascular endothelial growth
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • VEGF protein or fragment thereof bind the VEGF receptor and be internalized into the cell bearing the receptor.
  • VEGF it may be desirable in certain contexts for VEGF to manifest certain of its biological activities.
  • VEGF it would be desirable that VEGF exhibit vessel permeability activity and promotion of fibroblast migration and angiogenesis. It will be apparent from the teachings provided within the subject application which of the activities of VEGF are desirable to maintain.
  • VEGF promotes an array of responses in endothelium, including blood vessel hyperpermeability, endothelial cell growth, angiogenesis, and enhanced glucose transport.
  • VEGF stimulates the growth of endothelial cells from a variety of sources (including brain capillaries, fetal and adult aortas, and umbilical veins) at low concentrations, but is reported to have no effect on the growth of vascular smooth muscle cells, adrenal cortex cells, keratinocytes, lens epithelial cells, or BHK-21 fibroblasts.
  • VEGF also is a potent polypeptide regulator of blood vessel function; it causes a rapid but transient increase in microvascular permeability without causing endothelial cell damage or mast cell degranulation, and its action is not blocked by antihistamines. VEGF has also been reported to induce monocyte migration and activation and has been implicated as a tumor angiogenesis factor in some human gliomas. Also, VEGF is a chemoattractant for monocytes and VEGF has been shown to enhance the activity of the inflammatory mediator tumor necrosis factor (TNF).
  • TNF tumor necrosis factor
  • VEGF receptors Two tyrosine kinases have been identified as VEGF receptors.
  • the first known as fins-like tyrosine kinase or FLT is a receptor tyrosine kinase that is specific for VEGF.
  • FLT fins-like tyrosine kinase
  • expression of FLT mRNA is localized to the endothelium and to populations of cells that give rise to endothelium.
  • the second receptor, KDR (human kinase insert domain- containing receptor), and its mouse homologue FLK-1 are closely related to FLT.
  • the KDR FLK-1 receptor is expressed in endothelium during the fetal growth stage, during earlier embryonic development, and in adult tissues.
  • messenger RNA encoding FLT and KDR have been identified in tumor blood vessels and specifically by endothelial cells of blood vessels supplying glioblastomas.
  • FLT and KDR mRNAs are upregulated in tumor blood vessels in invasive human colon adenocarcinoma, but not in the blood vessels of adjacent normal tissues.
  • HBEGF heparin-binding EGF-like growth factor
  • HBEGFs exhibit a mitogenic effect on a wide variety of cells including BALB/c 3T3 fibroblast cells and smooth muscle cells, but unlike VEGFs, are not mitogenic for endothelial cells (Higashiyama et al., Science 257:936-939, 1991). HBEGF also has a stimulatory effect on collateral vascularization and angiogenesis. Members of the HBEGF family are thought to play a pathophysiological role, for example, in a variety of tumors, such as bladder carcinomas, breast tumors and non- small cell lung tumors. Thus, these cell types are likely candidates for delivery of cytocide-encoded agents.
  • HBEGF isolated from U-937 cells is heterogeneous in structure and contains at least 86 amino acids and two sites of O-linked glycosyl groups (Higashiyama et al., J. Biol. Chem. 267:6205-6212, 1992).
  • the carboxyl-terminal half of the secreted HBEGF shares approximately 35% sequence identity with human EGF, including six cysteines spaced in the pattern characteristic of members of the EGF protein family.
  • the amino-terminal portion of the mature factor is characterized by stretches of hydrophilic residues and has no structural equivalent in EGF.
  • Site-directed mutagenesis of HBEGF and studies with peptide fragments have indicated that the heparin-binding sequences of HBEGF reside primarily in a 21 amino acid stretch upstream of and slightly overlapping the EGF-like domain.
  • HBEGFs The effects of HBEGFs are mediated by EGF receptor tyrosine kinases expressed on cell surfaces of HBEGF-responsive cells (see, e.g., U.S. Patent Nos. 5,183,884 and 5,218,090; and Ullrich et al., Nature 509:4113-425, 1984).
  • the EGF receptor proteins which are single chain polypeptides with molecular weights 170 kD, constitute a family of structurally related EGF receptors.
  • Cells known to express the EGF receptors include smooth muscle cells, fibroblasts, keratinocytes, and numerous human cancer cell lines, such as the: A431 (epidermoid); KB3-1 (epidermoid); COLO 205 (colon); CRL 1739 (gastric); HEP G2 (hepatoma); LNCAP (prostate); MCF-7 (breast); MDA-MB-468 (breast); NCI 417D (lung); MG63 (osteosarcoma); U-251 (glioblastoma); D-54MB (glioma); and SW-13 (adrenal).
  • A431 epidermoid
  • KB3-1 epidermoid
  • COLO 205 colon
  • CRL 1739 gastric
  • HEP G2 hepatoma
  • LNCAP prostate
  • MCF-7 breast
  • MDA-MB-468 breast
  • NCI 417D lung
  • MG63 osteo
  • HBEGF need only bind a specific HBEGF receptor and be internalized. Any member of the HBEGF family, whether or not it binds heparin, is useful within the context of this invention as long as it meets the requirements set forth above. Members of the HBEGF family are those that have sufficient nucleotide identity to hybridize under normal stringency conditions (typically greater than 75% nucleotide identity). Subfragments or subportions of a full-length HBEGF may also be desirable. One skilled in the art may find from the teachings provided within that certain biological activities are more or less desirable, depending upon the application.
  • DNA encoding an HBEGF peptide or polypeptide refers to any DNA fragment encoding an HBEGF, as defined above.
  • Exemplary DNA fragments include: any such DNA fragments known to those of skill in the art; any DNA fragment that encodes an HBEGF or fragment that binds to an HBEGF receptor and is internalized thereby; and any DNA fragment that encodes any of the HBEGF polypeptides set forth in SEQ ID NOs. 5-8.
  • Such DNA sequences encoding HBEGF fragments are available from publicly accessible databases, such as: EMBL, GenBank (Accession Nos.
  • any DNA fragment that may be produced from any of the preceding DNA fragments by substitution of degenerate codons is also contemplated for use herein. It is understood that since the complete amino acid sequence of HBEGF polypeptides, and DNA fragments encoding such peptides, are available to those of skill in this art, it is routine to substitute degenerate codons and produce any of the possible DNA fragments that encode such HBEGF polypeptides. It is also generally possible to synthesize DNA encoding such peptides based on the amino acid sequence.
  • receptor-binding ligands may be used in the present invention. Any protein, polypeptide, analogue, or fragment that binds to a cell-surface receptor and is internalized may be used. In general, in addition to the specific heparin-binding growth factors discussed above, other growth factors and cytokines are especially well suited for use. These ligands may be produced by recombinant or other means in preparation for conjugation to the nucleic acid binding domain. The DNA sequences and methods to obtain the sequences of these receptor-binding internalized ligands are well known. For example, these ligands include CSF-1 (GenBank Accession Nos.
  • IGF-Ia Insulin-like growth factor la
  • IGF-Ib GenBank Accession No. X56774 S61860; Sandberg-Nordqvist et al., Brain Res. Mol. Brain Res. 72:275-277, 1992; Sandberg-Nordqvist, A.C., Cancer Res. 55:2475-2478, 1993); IGF-I (GenBank Accession No. X03563, M29644; Dull et al., Nature 570:771-781, 1984; Rail et al., Meth. Enzymol. 746:239-248, 1987); IGF-II (GenBank Accession No. J03242; Shen et al., Proc. Natl. Acad. Sci.
  • IL-l- ⁇ interleukin 1 alpha
  • IL-l- ⁇ interleukin 1 beta
  • IL-1 GenBank Accession No. K02770, M54933, M38756; Auron et al, Proc. Natl. Acad. Sci. USA 57:7907-791 1, 1984; Webb et al., Adv. Gene Technol. 22:339-340, 1985
  • IL-2 GenBank Accession No. A14844, A21785, X00695, X00200, X00201, X00202; Lupker et al., Patent No. EP 0307285-A, March 15, 1989; Perez et al., Patent No.
  • IL-5 GenBank Accession No. X04688, J03478; Azuma et al., Nucleic Acids Res. 74:9149- 9158, 1986; Tanabe et al., J. Biol. Chem. 262:16580-16584, 1987
  • IL-6 GenBank Accession No. Y00081, X04602, M54894, M38669, M14584; Yasukawa et al., EMBO J. 6:2939-2945, 1987; Hirano et al., Nature 524:73-76, 1986; Wong et al., Behring Inst. Mitt.
  • IL-7 GenBank Accession No. J04156; Goodwin et al., Proc. Natl. Acad. Sci. USA 56:302- 306, 1989
  • IL-8 GenBank Accession No. Zl 1686; Kusner et al., Kidney Int. 59:1240- 1248, 1991
  • IL-10 GenBank Accession No. X78437, M57627; Vieira et al, Proc. Natl. Acad. Sci. USA 55:1172-1176, 1991
  • IL-11 GenBank Accession No.
  • DNA sequences of other suitable receptor-binding internalized ligands may be obtained from GenBank or EMBL DNA databases, reverse- synthesized from protein sequence obtained from PIR database or isolated by standard methods (Sambrook et al., supra) from cDNA or genomic libraries.
  • ligands may be customized for a particular application. Means for modifying proteins is provided below. Briefly, additions, substitutions and deletions of amino acids may be produced by any commonly employed recombinant DNA method.
  • An amino acid residue of FGF, VEGF, HBEGF or other receptor- binding internalized ligand is non-essential if the polypeptide that has been modified by deletion of the residue possesses substantially the same ability to bind to its receptor and internalize a linked agent as the unmodified polypeptide.
  • any polypeptide or peptide analogue including peptidomimetics, that is reactive with an FGF receptor, a VEGF receptor, an HBEGF receptor, other growth factor receptor (e.g., PDGF receptor), cytokine receptor or other cell surface molecule including members of the families and fragments thereof, or constrained analogs of such peptides that bind to the receptor and internalize a linked targeted agent may be used in the context of this invention.
  • Members of the FGF peptide family including FGF-1 to FGF-9, are preferred.
  • Modified peptides, especially those lacking proliferative function, and chimeric peptides, which retain the specific binding and internalizing activities are also contemplated for use herein.
  • a modification that is effected substantially near the N-terminus of a polypeptide is generally effected within the first about ten residues of the protein.
  • Such modifications include the addition or deletion of residues, such as the addition of a cysteine to facilitate conjugation and form conjugates that contain a defined molar ratio, preferably a ratio of 1 : 1 of the polypeptides.
  • DNA encoding one of the receptor-binding internalized ligands discussed above may be mutagenized using standard methodologies to delete or replace any cysteine residues that are responsible for aggregate formation. If necessary, the identity of cysteine residues that contribute to aggregate formation may be determined empirically, by deleting and/or replacing a cysteine residue and ascertaining whether the resulting protein aggregates in solutions containing physiologically acceptable buffers and salts. In addition, fragments of these receptor-binding internalized ligands may be constructed and used. The binding region of many of these ligands have been delineated. Fragments may also be shown to bind and internalize by any one of the tests described herein.
  • Modification of the polypeptide may be effected by any means known to those of skill in this art.
  • the preferred methods herein rely on modification of DNA encoding the polypeptide and expression of the modified DNA.
  • DNA encoding the FGF polypeptide may be isolated, synthesized or obtained from commercial sources (the amino acid sequences of FGF-1 - FGF-9 are set forth in SEQ ID NOs. 10-18; DNA sequences may be based on these amino acid sequences or may be obtained from public DNA databases and references (see, e.g., GenBank, see also U.S. Patent No. 4,956,455, U.S. Patent No. 5,126,323, U.S. Patent No. 5,155,217, U.S. Patent No. 4,868,113, PCT Application WO 90/08771, EP Application 0 488 196 A2, and Miyamoto et al., Mol. Cell. Biol. 75:4251-4259, 1993).
  • GenBank see also U.S. Patent No. 4,956,455, U.S. Patent No. 5,126,323, U.S. Patent No. 5,155,217, U.S. Patent No. 4,868,113, PCT Application WO 90/08771, EP Application 0 4
  • DNA encoding any of the other receptor-binding internalized ligands may also be isolated, synthesized, or obtained from commercial sources.
  • DNA sequences are available in public databases, such as GenBank. Based on these sequences, oligonucleotide primers may be designed and used to amplify the gene from cDNA or mRNA by polymerase chain reaction technique as one means of obtaining DNA.
  • Mutations may be made by any method known to those of skill in the art, including site-specific or site-directed mutagenesis of DNA encoding the protein and the use of DNA amplification methods using primers to introduce and amplify alterations in the DNA template, such as PCR splicing by overlap extension (SOE).
  • Site-directed mutagenesis is typically effected using a phage vector that has single- and double-stranded forms, such as Ml 3 phage vectors, which are well-known and commercially available.
  • Other suitable vectors that contain a single-stranded phage origin of replication may be used (see, e.g., Veira et al., Meth. Enzymol. 15:3, 1987).
  • site-directed mutagenesis is performed by preparing a single-stranded vector that encodes the protein of interest (i.e., a member of the FGF family or a cytotoxic molecule, such as a saporin).
  • An oligonucleotide primer that contains the desired mutation within a region of homology to the DNA in the single-stranded vector is annealed to the vector followed by addition of a DNA polymerase, such as E. coli DNA polymerase I (Klenow fragment), which uses the double stranded region as a primer to produce a heteroduplex in which one strand encodes the altered sequence and the other the original sequence.
  • the heteroduplex is introduced into appropriate bacterial cells and clones that include the desired mutation are selected.
  • the resulting altered DNA molecules may be expressed recombinantly in appropriate host cells to produce the modified protein.
  • nucleic acid binding domains interact with the target nucleic acid either in a sequence-specific manner or a sequence-nonspecific manner.
  • the nucleic acid binding domain binds nucleic acid regardless of the sequence.
  • poly-L-lysine is a basic polypeptide that binds to oppositely charged DNA.
  • HTH helix-turn-helix
  • HHLH helix-loop-helix
  • ⁇ -sheets Other classes or subclasses may eventually be delineated as more factors are discovered and defined. Proteins from those classes or proteins that do not fit within one of these classes but bind nucleic acid in a sequence-specific manner, such as SV40 T antigen and p53 may also be used.
  • Helix-turn-helix proteins include the well studied ⁇ Cro protein, ⁇ cl, and E. coli CAP proteins (see Steitz et al., Proc. Natl. Acad. Sci. USA 79:3097-3100, 1982; Ohlendorf et al., J. Mol. Biol. 769:757-769, 1983).
  • the lac repressor Kerin et al., J. Mol. Biol. 752:179-182, 1985
  • Trp repressor Scheritz et al., Nature 577:782-786, 1985
  • Members of the homeodomain family include the Drosophila protein Antennapaedia (Qian et al., Cell.
  • Zinc finger proteins include TFIIIA (Miller et al., EMBO J. 4: 1609- 1614, 1985), Sp- 1 , zif 268, and many others (see generally Krizek et al., J. Am. Chem. Soc. 775:4518-4523, 1991).
  • Steroid receptor proteins include receptors for steroid hormones, retinoids, vitamin D, thyroid hormones, as well as other compounds. Specific examples include retinoic acid, knirps, progesterone, androgen, glucocosteroid and estrogen receptor proteins.
  • the leucine zipper family was defined by a heptad repeat of leucines over a region of 30 to 40 residues. Specific members of this family include C/ ⁇ BP, c-fos, c-jun, GCN4, sis-A, and CR ⁇ B (see generally O'Shea et al., Science 254:539-544, 1991).
  • the helix-loop- helix (HLH) family of proteins appears to have some similarities to the leucine zipper family.
  • Well-known members of this family include myoD (Weintraub et al., Science 257:761-766, 1991); c-myc; and AP-2 (Williams and Tijan, Science 257:1067-1071, 1991).
  • the ⁇ -sheet family uses an antiparallel ⁇ -sheet for DNA binding, rather than the more common ⁇ -helix.
  • the family contains the MeU (Phillips, Curr. Opin. Struc. Biol. 7:89-98, 1991), Arc (Breg et al., Nature 546:586-589, 1990) and Mnt repressors.
  • Other motifs are used for DNA binding, such as the cysteine-rich motif in yeast GAL4 repressor, and the GATA factor.
  • Viruses also contain gene products that bind specific sequences.
  • One of the most-studied such viral genes is the rev gene from HIV.
  • the rev gene product binds a sequence called RR ⁇ (rev responsive element) found in the env gene.
  • the inserted DNA sequences may be randomly generated or variants of a known DNA-binding domain. Generally, the inserts encode from 6 to 20 amino acids.
  • the peptide encoded by the inserted sequence is displayed on the surface of the bacteriophage. Bacteriophage expressing a desired nucleic acid- binding domain are selected for by binding to the cytocide-encoding agent. This target molecule may be single stranded or double stranded DNA or RNA.
  • the appropriate target is single-stranded.
  • the target molecule is preferably double-stranded.
  • the entire coding region of the cytocide-encoding agent is used as the target.
  • elements necessary for transcription that are included for in vivo or in vitro delivery may be present in the target DNA molecule. Bacteriophage that bind the target are recovered and propagated. Subsequent rounds of selection may be performed. The final selected bacteriophage are propagated and the DNA sequence of the insert is determined.
  • sufficient peptide for use herein as an nucleic acid binding domain may be made either by recombinant means or synthetically. Recombinant means is used when the receptor-binding internalized ligand/nucleic acid binding domain is produced as a fusion protein.
  • the peptide may be generated as a tandem array of two or more peptides, in order to maximize affinity or binding of multiple DNA molecules to a single polypeptide.
  • DNA-binding domain/peptide that recognizes the coding region of saporin is isolated.
  • DNA fragments encoding saporin may be isolated from a plasmid containing these sequences.
  • the plasmid FPFS1 contains the entire coding region of saporin. Digestion of the plasmid with Ncol and EcoRI restriction enzymes liberates the saporin specific sequence as a single fragment of approximately 780 bp. This fragment may be purified by any one of a number of methods, such as agarose gel electrophoresis and subsequent elution from the gel.
  • the saporin fragment is fixed to a solid support, such as in the wells of a 96-well plate.
  • a coating such as a positively charged molecule, may be used to promote D ⁇ A adherence.
  • the phage library is added to the wells and an incubation period allows for binding of the phage to the D ⁇ A. Unbound phage are removed by a wash, typically containing 10 mM Tris, 1 mM ⁇ DTA, and without salt or with a low salt concentration. Bound phage are eluted starting at a 0.1 M ⁇ aCl containing buffer. The ⁇ aCl concentration is increased in a step-wise fashion until all the phage are eluted. Typically, phage binding with higher affinity will only be released by higher salt concentrations.
  • Eluted phage are propagated in the bacteria host. Further rounds of selection may be performed to select for a few phage binding with high affinity. The D ⁇ A sequence of the insert in the binding phage is then determined. In addition, peptides having a higher affinity may be isolated by making variants of the insert sequence and subjecting these variants to further rounds of selection.
  • a cytocide-encoding agent is a nucleic acid molecule (D ⁇ A or R ⁇ A) that, upon internalization by a cell, and subsequent transcription (if D ⁇ A) and[/or] translation into a cytocidal agent, is cytotoxic to a cell or inhibits cell growth by inhibiting protein synthesis.
  • Cytocides include saporin, the ricins, abrin and other ribosome inactivating proteins, Pseudomonas exotoxin, diphtheria toxin, angiogenin, tritin, dianthins 32 and 30, momordin, pokeweed antiviral protein, mirabilis antiviral protein, bryodin, angiogenin, and shiga exotoxin, as well as other cytocides that are known to those of skill in the art.
  • cytocide gene products may be noncytotoxic but activate a compound, which is endogenously produced or exogenously applied, from a nontoxic form to a toxic product that inhibits protein synthesis.
  • DNA molecules that encode an enzyme that results in cell death or renders a cell susceptible to cell death upon the addition of another product.
  • saporin is an enzyme that cleaves rRNA and inhibits protein synthesis.
  • Other enzymes that inhibit protein synthesis are especially well suited for use in the present invention.
  • enzymes may be used where the enzyme activates a compound with little or no cytotoxicity into a toxic product that inhibits protein synthesis.
  • Ribosome-inactivating proteins which include ricin, abrin, and saporin, are plant proteins that catalytically inactivate eukaryotic ribosomes. Ribosome- inactivating proteins inactivate ribosomes by interfering with the protein elongation step of protein synthesis.
  • the ribosome-inactivating protein saporin hereinafter also referred to as SAP
  • SAP ribosome-inactivating protein saporin
  • a 4324 in 28S rRNA corresponds to A 2660 in E. coli 23 S rRNA.
  • ribosome inactivating proteins also appear to interfere with protein synthesis in prokaryotes, such as E. coli.
  • Saporin is preferred as a cytocide, but other suitable ribosome inactivating proteins (RIPs) and toxins may be used.
  • suitable RIPs include, but are not limited to, ricin, ricin A chain, maize ribosome inactivating protein, gelonin, diphtheria toxin, diphtheria toxin A chain, trichosanthin, tritin, pokeweed antiviral protein (PAP), mirabilis antiviral protein (MAP), Dianthins 32 and 30, abrin, monordin, bryodin, shiga (see, e.g., WO 93/24620) and others (see, e.g., Barbieri et al., Cancer Surveys 7:489-520, 1982, and European patent application No.
  • ribosome inactivating proteins such as abrin and ricin
  • Some ribosome inactivating proteins contain two constituent chains: a cell- binding chain that mediates binding to cell surface receptors and internalization of the molecule and a chain responsible for toxicity.
  • Single chain ribosome inactivating proteins such as the saporins, do not have a cell-binding chain. As a result, unless internalized, they are substantially less toxic to whole cells than the ribosome inactivating proteins that have two chains.
  • Saponaria officinalis Several structurally related ribosome inactivating proteins have been isolated from seeds and leaves of the plant Saponaria officinalis (soapwort) (GB Patent 2,194,241 B; GP Patent 2,216,891; EP Patent 89306016).
  • Saporin proteins for use in this invention have amino acid sequences found in the natural plant host Saponaria officinalis or modified sequences, having amino acid substitutions, deletions, insertions or additions, but which still express substantial ribosome inactivating activity. Purified preparations of saporin are frequently observed to include several molecular isoforms of the protein. It is understood that differences in amino acid sequences can occur in saporin from different species as well as between saporin molecules from individual organisms of the same species.
  • SO-6 is the most active and abundant, representing 7% of total seed proteins.
  • Saporin is very stable, has a high isoelectric point, does not contain carbohydrates, and is resistant to denaturing agents, such as sodium dodecyl sulfate (SDS), and a variety of proteases.
  • SDS sodium dodecyl sulfate
  • the amino acid sequences of several saporin-6 isoforms from seeds are known, and there appear to be families of saporin ribosome inactivating proteins differing in few amino acid residues. Any of these saporin proteins or modified proteins that are cytotoxic may be used in the present invention.
  • saporin polypeptides include other members of the multi-gene family coding for isoforms of saporin-type ribosome inactivating proteins including SO-1 and SO-3 (Fordham- Skelton et al., Mol. Gen. Genet. 227:134-138, 1990), SO-2 (see, e.g., U.S. Application Serial No. 07/885,242; GB 2,216,891; see also Fordham-Skelton et al., Mol. Gen. Genet. 229:460-466, 1991), SO-4 (see, e.g., GB 2,194,241 B; see also Lappi et al., Biochem. Biophys. Res. Commun. 129:934-942, 1985) and SO-5 (see, e.g., GB 2,194,241 B; see also Montecucchi et al., 7 «t. J. Peptide Protein Res. 55:263-267, 1989).
  • SO-1 and SO-3 Forms
  • the saporin polypeptides for use in this invention include any of the isoforms of saporin that may be isolated from Saponaria officinalis or related species or modified forms that retain cytotoxic activity.
  • modified saporin may be produced by modifying the DNA encoding the protein (see, e.g., International PCT Application Serial No. PCT/US93/05702, and United States Application Serial No. 07/901,718; see also U.S. Patent Application No. 07/885,242, and Italian Patent No. 1,231,914) by altering one or more amino acids or deleting or inserting one or more amino acids.
  • Any such protein, or portion thereof, that exhibits cytotoxicity in standard in vitro or in vivo assays within at least about an order of magnitude of the saporin conjugates described herein is contemplated for use herein.
  • the saporin DNA sequence contains mammalian-preferred codons (SEQ. ID NO. 79). Preferred codon usage as exemplified in Current Protocols in Molecular Biology, infra, and Zhang et al. (Gene 705:61, 1991) for mammals, yeast, Drosophila, E. coli, and primates is established for saporin sequence.
  • the cytocide-encoding agent such as saporin DNA sequence
  • the cytocide-encoding agent is introduced into a plasmid in operative linkage to an appropriate promoter for expression of polypeptides in the organism.
  • the presently preferred saporin proteins are SO-6 and SO-4.
  • the DNA can optionally include sequences, such as origins of replication that allow for the extrachromosomal maintenance of the saporin-containing plasmid, or can be designed to integrate into the genome of the host (as an alternative means to ensure stable maintenance in the host).
  • cytocides that inhibit protein synthesis are useful in the present invention.
  • the gene sequences for these cytocides may be isolated by standard methods, such as PCR, probe hybridization of genomic or cDNA libraries, antibody screenings of expression libraries, or clones may be obtained from commercial or other sources.
  • the DNA sequences of many of these cytocides are well known, including ricin A chain (GenBank Accession No. X02388); maize ribosome inactivating protein (GenBank Accession No. L26305); gelonin (GenBank Accession No. LI 2243; PCT Application WO 92/03155; U.S. Patent No. 5,376,546; diphtheria toxin (GenBank Accession No.
  • DNA molecules encoding these proteins may be synthesized, and preferably contain mammalian- preferred codons.
  • a nucleic acid molecule encoding a prodrug may alternatively be used within the context of the present invention.
  • Prodrugs are inactive in the host cell until either a substrate is provided or an activating molecule is provided. Most typically, a prodrug activates a compound with little or no cytotoxicity into a toxic product.
  • Two of the more often used prodrug molecules, both of which may be used in the present invention, are HSV thymidine kinase and E. coli cytosine deaminase.
  • gene products which either directly or indirectly activate a compound with little or no cytotoxicity into a toxic product may be utilized within the context of the present invention.
  • Representative examples of such gene products include HSVTK (herpes simplex virus thymidine kinase) and VZVTK (varicella zoster virus thymidine kinase), which selectively phosphorylate certain purine arabinosides and substituted pyrimidine compounds. Phosphoryation converts these compounds to metabolites that are cytotoxic or cytostatic.
  • ganciclovir for example, exposure of the drugs ganciclovir, acyclovir, or any of their analogues (e.g., FIAU, FIAC, DHPG) to cells expressing HSVTK allows conversion of the drug into its corresponding active nucleotide triphosphate form.
  • analogues e.g., FIAU, FIAC, DHPG
  • E. coli guanine phosphoribosyl transferase which converts thioxanthine into toxic thioxanthine monophosphate (Besnard et al., Mol. Cell. Biol.
  • alkaline phosphatase which converts inactive phosphorylated compounds such as mitomycin phosphate and doxorubicin-phosphate to toxic dephosphorylated compounds
  • fungal e.g., Fusarium oxysporum
  • bacterial cytosine deaminase which converts 5-fluorocytosine to the toxic compound 5-fluorouracil
  • carboxypeptidase G2 which cleaves glutamic acid from para-N-bis (2-chloroethyl) aminobenzoyl glutamic acid, thereby creating a toxic benzoic acid mustard
  • Penicillin-V amidase which converts phenoxyacetabide derivatives of doxorubicin and melphalan to toxic compounds (see generally, Vrudhula et al., J.
  • Herpesviridae thymidine kinases including both primate and non-primate he ⁇ esviruses, are suitable.
  • Such herpesviruses include He ⁇ es Simplex Virus Type 1 (McKnight et al., Nuc. Acids Res 5:5949-5964, 1980), He ⁇ es Simplex Virus Type 2 (Swain and Galloway, J Virol. 46:1045-1050, 1983), Varicella Zoster Virus (Davison and Scott, J. Gen. Virol.
  • thymidine kinase can phosphorylate nucleosides (e.g., dT) and nucleoside analogues such as ganciclovir (9- ⁇ [2-hydroxy-l-(hydroxymethyl)ethoxyl methyl ⁇ guanosine), famciclovir, buciclovir, penciclovir, valciclovir, acyclovir (9-[2-hydroxy ethoxy)methyl] guanosine), trifluorothymidine, l-[2-deoxy, 2-fluoro, beta-D-arabino furanosyl]-5-iodouracil, ara-A (adenosine arabinoside, vivarabine), 1-beta-D-arabinofuranoxyl thymine, 5-ethyl-2'- deoxyuridine, 5-iodo-5'-amino
  • nucleosides e.g., dT
  • the conjugates provided herein may also be used to deliver other types of nucleic acids to targeted cells.
  • nucleic acids include antisense RNA, antisense DNA, ribozymes, triplex-forming oligonucleotides, and oligonucleotides that bind proteins.
  • the nucleic acids can also include RNA trafficking signals, such as viral packaging sequences (see, e.g., Sullenger et al. (1994) Science 262:1566-1569).
  • the nucleic acids also include DNA molecules that encode proteins that replace defective genes, such as the gene associated with cystic fibrosis (see, e.g., PCT Application WO 93/03709, U.S. Application Serial No.
  • DNA molecules may encode tumor-specific cytotoxic molecules, such as tumor necrosis factor, viral antigens and other proteins to render a cell susceptible to anti-cancer agents.
  • Nucleic acids and oligonucleotides for use as described herein can be synthesized by any method known to those of skill in this art (see, e.g., WO 93/01286, U.S. Application Serial No. 07/723,454; U.S.. Patent No. 5,218,088; U.S. Patent No. 5,175,269; U.S. Patent No. 5,109,124).
  • Identification of oligonucleotides and ribozymes for use as antisense agents and DNA encoding genes for targeted delivery for genetic therapy involve methods well known in the art. For example, the desirable properties, lengths and other characteristics of such oligonucleotides are well known.
  • Antisense oligonucleotides are typically designed to resist degradation by endogenous nucleolytic enzymes and include, but are not limited to: phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and other such linkages (see, e.g., Agrwal et al., Tetrehedron Lett. 25:3539-3542 (1987); Miller et al., J. Am. Chem. Soc. 95:6657-6665 (1971); Stec et al., Tetrehedron Lett. 26:2191-2194 (1985); Moody et al., Nucl.
  • Antisense nucleotides are oligonucleotides that bind in a sequence- specific manner to nucleic acids, such as mRNA or DNA. When bound to mRNA that has complementary sequences, antisense prevents translation of the mRNA (see, e.g., U.S. Patent No. 5,168,053 to Altman et al.; U.S. Patent No. 5,190,931 to Inouye, U.S. Patent No. 5,135,917 to Burch; U.S. Patent No. 5,087,617 to Smith and Clusel et al. (1993) Nucl. Acids Res. 27:3405-3411, which describes dumbbell antisense oligonucleotides).
  • Triplex molecules refer to single DNA strands that bind duplex DNA forming a colinear triplex molecule and thereby prevent transcription (see, e.g., U.S. Patent No. 5,176,996 to Hogan et al., which describes methods for making synthetic oligonucleotides that bind to target sites on duplex DNA).
  • Particularly useful antisense nucleotides and triplex molecules are molecules that are complementary or bind to the sense strand of DNA or mRNA that encodes an oncogene, such as bFGF, int-2, hst-1/K-FGF, FGF-5, hst-2/FGF-6, FGF-8.
  • an oncogene such as bFGF, int-2, hst-1/K-FGF, FGF-5, hst-2/FGF-6, FGF-8.
  • Other useful antisense oligonucleotides include those that are specific for IL-8 (see, e.g., U.S. Patent No.
  • a ribozyme is an RNA molecule that specifically cleaves RNA substrates, such mRNA, .and thus inhibits or interferes with cell growth or expression.
  • RNA substrates such mRNA
  • Ribozymes can be targeted to any RNA transcript and can catalytically cleave such transcript (see, e.g., U.S. Patent No. 5,272,262; U.S. Patent No. 5,144,019; and U.S. Patent Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246 to Cech et al., which described ribozymes and methods for production thereof).
  • Any such ribosome may be linked to the growth factor for delivery to a cell bearing a receptor for a receptor-internalized binding ligand.
  • the ribozymes may be delivered to the targeted cells by DNA encoding the ribozyme linked to a eukaryotic promoter, such as a eukaryotic viral promoter, such that upon introduction into the nucleus, the ribozyme will be directly transcribed.
  • a eukaryotic promoter such as a eukaryotic viral promoter
  • the construct will also include a nuclear translocation sequence, generally as part of the ligand or as part of a linker between the ligand and nucleic acid binding domain.
  • DNA that encodes a therapeutic product contemplated for use includes DNA encoding correct copies of defective genes, such as the defective gene (CFTR) associated with cystic fibrosis (see, e.g., International Application WO 93/03709, U.S. Application Serial No. 07/745,900; and Riordan et al. (1989) Science 245:1066-1073), and anticancer agents, such as tumor necrosis factors.
  • the conjugate preferably includes an NTS. If the conjugate is designed such that the ligand and nucleic acid binding domain are cleaved in the cytoplasm, then the NTS should be included in a portion of the conjugate or linker that remains bound to the DNA.
  • the nuclear translocation sequence (NTS) may be a heterologous sequence or a may be derived from the selected growth factor.
  • F. Construct containing cytocidal-encoding agent In the case of cytotocide molecules such as the ribosome inactivating proteins, very few molecules may need to be expressed to effect cell killing. Indeed, only a single molecule of diphtheria toxoid introduced into a cell was sufficient to kill the cell. With other cytocides or prodrugs, it may be that propagation or stable maintenance of the construct is necessary to attain a sufficient amount or concentration of the gene product for effective gene therapy. Examples of replicating and stable eukaryotic plasmids may be found in the scientific literature.
  • constructs will also contain elements necessary for transcription and translation. If the cytocide-encoding agent is DNA, then it must contain a promoter.
  • the choice of the promoter will depend upon the cell type to be transformed and the degree or type of control desired. Promoters can be constitutive or active in any cell type, tissue specific, cell specific, event specific temporal-specific or inducible. Cell-type specific promoters and event type specific promoters are preferred. Examples of constitutive or nonspecific promoters include the SV40 early promoter (U.S. Patent No. 5,118,627), the SV40 late promoter (U.S. Patent No. 5,118,627), CMV early gene promoter (U.S. Patent No. 5,168,062), and adenovirus promoter.
  • cellular promoters are also amenable within the context of this invention.
  • cellular promoters for the so-called housekeeping genes are useful.
  • Viral promoters are preferred, because generally they are stronger promoters than cellular promoters.
  • Tissue specific promoters are particularly useful when a certain tissue type is to be targeted for transformation. By using one of this class of promoters, an extra margin of specificity can be attained.
  • the indication to be treated is ophthalmological (e.g., secondary lens clouding)
  • either the alpha-crystalline promoter or gamma-crystalline promoter is preferred.
  • cellular promoters for specific tumor markers or promoters more active in tumor cells should be chosen.
  • the prostate-specific antigen promoter is especially useful.
  • the tyrosinase promoter or tyrosinase- related protein promoter is a preferred promoter for melanoma treatment.
  • the VEGF receptor promoter is preferred.
  • the VEGF receptor is expressed in developing capillaries.
  • the promoter from heat shock protein 27 is preferred; for treatment of colon or lung cancer, the promoter from carcinoembryonic antigen is preferred; for treatment of restenosis or other diseases involving smooth muscle cells, the promoter from ⁇ -actin or myosin heavy chain is preferred.
  • tissue specific promoters For B lymphocytes, the immunoglobulin variable region gene promoter; for T lymphocytes, the TCR receptor variable region promoter; for helper T lymphocytes, the CD4 promoter; for liver, the albumin or ⁇ -fetoprotein promoter, are a few additional examples of tissue specific promoters. Many other examples of tissue specific promoters are readily available to one skilled in the art. Some of these promoters are temporally regulated, such as c-myc and cyclin D. Inducible promoters may also be used. These promoters include the
  • MMTV LTR PCT WO 91/13160
  • dexamethasone metallothionein
  • promoters with cAMP response elements which are inducible by cAMP.
  • Event-type specific promoters are active or up-regulated only upon the occurrence of an event, such as tumorigenecity or viral infection.
  • the HIV LTR is a well known example of an event-specific promoter.
  • the promoter is inactive unless the tat gene product is present, which occurs upon viral infection.
  • Another promoter is c-myc.
  • promoters that are coordinately regulated with a particular cellular gene may be used.
  • promoters of genes that are coordinately expressed when a particular FGF receptor gene is expressed may be used. Then, the nucleic acid will be transcribed when the FGF receptor, such as FGFRl, is expressed, and not when FGFR2 is expressed.
  • This type of promoter is especially useful when one knows the pattern of FGF receptor expression in a particular tissue, so that specific cells within that tissue may be killed upon transcription of a cytotoxic agent gene without affecting the surrounding tissues. If the domain binds in a sequence specific manner, the construct must contain the sequence that binds to the nucleic acid binding domain.
  • the target nucleotide sequence may be contained within the coding region of the cytocide, in which case, no additional sequence need be inco ⁇ orated. Additionally, it may be desirable to have multiple copies of target sequence. If the target sequence is coding sequence, the additional copies must be located in non-coding regions of the cytocide-encoding agent.
  • the target sequences of the nucleic acid binding domains are typically generally known. If unknown, the target sequence may be readily determined. Techniques are generally available for establishing the target sequence (e.g., see PCT Application WO 92/05285 and U.S. Serial No. 586,769).
  • NTS nuclear translocation or targeting sequence
  • Table 2 Examples of NTSs are set forth in Table 2 below. Comparison with known NTSs, and if necessary testing of candidate sequences, should permit those of skill in the art to readily identify other amino acid sequences that function as NTSs.
  • a heterologous NTS refers to an NTS that is different from the NTS that occurs in the wild-type peptide, polypeptide, or protein.
  • the NTS may be derived from another polypeptide, it may be synthesized, or it may be derived from another region in the same polypeptide.
  • the conjugate In order to deliver the nucleic acid to the nucleus, the conjugate should include an NTS. If the conjugate is designed such that the receptor-binding internalized ligand and linked nucleic acid binding domain is cleaved or dissociated in the cytoplasm, then the NTS should be included in a portion of the complex that remains bound to the nucleic acid, so that, upon internalization, the conjugate will be trafficked to the nucleus. Thus, the NTS is preferably included in the nucleic acid binding domain, but may additionally be included in the ligand. An NTS is preferred if the cytocide-encoding agent is DNA. If the cytocide-encoding agent is mRNA, an NTS may be omitted.
  • the nuclear translocation sequence may be a heterologous sequence or a may be derived from the selected growth factor. All presently identified members of the FGF family of peptides contain an NTS (see, e.g., International Application WO 91/15229 and Table 2).
  • a typical consensus NTS sequence contains an amino-terminal proline or glycine followed by at least three basic residues in a array of seven to nine amino acids (see, e.g., Dang et al., J. Biol. Chem. 264:18019-18023, 1989; Dang et al., Mol. Cell. Biol 5:4049-4058, 1988, and Table 2).
  • Cytoplasm-translocation signal sequence is a sequence of amino acids in a protein that cause retention of proteins in the lumen of the endoplasmic reticulum and/or translocate proteins to the cytosol.
  • the signal sequence in mammalian cells is KDEL (Lys-Asp-Glu-Leu) (SEQ ID NO. 42) (Munro and Pelham, Cell 45:899-907, 1987). Some modifications of this sequence have been made without loss of activity. For example, the sequences RDEL (Arg-Asp-Glu-Leu) (SEQ ID NO. 43) and KEEL (Lys-Glu-Glu-Leu) (SEQ ID NO. 44) confer efficient or partial retention, respectively, in plants (Denecke et al., Embo. J. 77:2345-2355, 1992).
  • a cytoplasm-translocation signal sequence may be included in either the receptor-internalized binding ligand or the nucleic acid binding domain part or both. If cleavable linkers are used to link the ligand with the nucleic acid binding domain, the cytoplasm-translocation signal is preferably included in the nucleic acid binding domain, which will stay bound to the cytocide-encoding agent. Additionally, a cytoplasmic-translocation signal sequence may be included in the receptor-internalized binding ligand, as long as it does not interfere with receptor binding. Similarly, the signal sequence placed in the nucleic acid binding domain should not interfere with binding to the cytocide-encoding agent.
  • membrane-disruptive peptides may be inco ⁇ orated into the complexes.
  • adenoviruses are known to enhance disruption of endosomes.
  • Virus-free viral proteins such as influenza virus hemagglutinin HA-2, also disrupt endosomes and are useful in the present invention.
  • Other proteins may be tested in the assays described herein to find specific endosome disrupting agents that enhance gene delivery. In general, these proteins and peptides are amphipathic (see Wagner et al., Adv. Drug. Del. Rev. 14: 113-135, 1994).
  • Endosome-disruptive peptides may be inco ⁇ orated into the complex of receptor-internalized binding ligand, nucleic acid binding domain, and cytocide-encoding agent.
  • Two such peptides derived from influenza virus are: GLFEAIEGFIENGWEGMIDGGGC (SEQ. ID NO. 45) and GLFEAIEGFIENGWEGMIDGWYGC (SEQ. ID NO. 46).
  • Other peptides useful for disrupting endosomes may be identified by general characteristics: 25-30 residues in length, contain an alternating pattern of hydrophobic domains and acidic domains, and at low pH (e.g., pH 5) from amphipathic ⁇ -helices.
  • a candidate endosome-disrupting peptide is tested by inco ⁇ orating it into the complex and determining whether it increases the total number of cells expressing the target gene.
  • the peptides are added to a complex having excess negative charge.
  • a DNA construct is complexed with an FGF-poly-L-lysine chemical conjugate so that only a portion of the negative charge of the DNA is neutralized.
  • Poly-L-lysine is added to further bind the DNA and a fusogenic peptide is then added.
  • Optional ratios of DNA, poly-L-lysine and fusogenic peptide are determined using assays, such as gene expression and cell viability.
  • the fusogenic peptides may alternatively be inco ⁇ orated into the complex as a fusion protein with either the ligand or the nucleic acid binding domain or both.
  • the endosome-disruptive peptide may be present as single or multiple copies at the N- or C- terminus of the ligand.
  • a single fusion protein of the endosome-disruptive peptide, nucleic acid binding domain, and receptor-internalized binding ligand may be constructed and expressed.
  • DNA encoding the endosome-disruptive peptide may be synthesized by PCR using overlapping oligonucleotides and inco ⁇ orating a restriction site at the 5' and 3' end to facilitate cloning. The sequence may be verified by sequence analysis. 4.
  • a "linker” is an extension that links the receptor-binding internalized ligand or fragment thereof and the nucleic acid binding domain. In certain instances, the linker is used to conjugate the ligand directly to the nucleic acid.
  • the linkers provided herein confer specificity, enhance intracellular availability, serum stability and/or solubility on the conjugate and may serve to promote condensation of the nucleic acid.
  • the linkers provided herein confer specificity and serum stability on the cytotoxic conjugate, for example, by conferring specificity for certain proteases, particularly proteases that are present in only certain subcellular compartments or that are present at higher levels in tumor cells than normal cells. Specificity for proteases present in intracellular compartments and absent in blood is particularly preferred.
  • the linkers may also include sorting signals that direct the conjugate to particular intracellular loci or compartments. Additionally, the linkers may reduce steric hindrance between the growth factor and other protein or linked nucleic acid by distancing the components of the conjugate. Linkers may also condense the nucleic acid. For this pu ⁇ ose, the linker comprises highly basic amino acids (e.g., Lys, Arg) and may even by poly-L-lysine.
  • linkers In order to increase the serum stability, solubility and/or intracellular concentration or condense the targeted agent, one or more linkers (are) inserted between the receptor-binding internalized ligand and the nucleic acid binding domain.
  • linkers include peptide linkers, such as intracellular protease substrates, and chemical linkers, such as acid labile linkers, ribozyme substrate linkers and others.
  • Peptides linkers may be inserted using heterobifunctional reagents, described below, or, preferably, are linked to FGF, other growth factors, including heparin-binding growth factors, or cytokines by linking DNA encoding the ligand to the DNA encoding the nucleic acid binding domain.
  • Chemical linkers may be inserted by covalently coupling the linker to the
  • FGF FGF
  • other growth factor protein or cytokine and the nucleic acid binding domain.
  • the linker may be bound via the N- or C-terminus or an internal residue.
  • the heterobifunctional agents, described below, may be used to effect such covalent coupling.
  • protease-specific substrates may be introduced between the ligand and the nucleic acid binding domain.
  • the peptides may be inserted using heterobifunctional reagents, as described below, or preferably inserted by recombinant means and expression of the resulting chimera.
  • Any protease specific substrate may be introduced as a linker as long as the substrate is cleaved in an intracellular compartment.
  • Preferred substrates include those that are specific for proteases that are expressed at higher levels in tumor cells, that are preferentially expressed in the endosome, or that are absent in blood. The following substrates are among those contemplated for use in accord with the methods herein: cathepsin B substrate, cathepsin D substrate, trypsin substrate, thrombin substrate, and recombinant subtilisin substrate.
  • linkers which reduce steric hindrance, and linkers that increase solubility of the conjugates are contemplated for use, either alone or with other linkers, such as the protease specific substrate linkers.
  • these linkers are simple polymers of small amino acids (i.e., small side groups) with uncharged polar side groups. These amino acids (Gly, Ser, Thr, Cys, Tyr, Asn, Gin) are more soluble in water. Of these amino acids, Gly and Ser are preferred.
  • Such linkers include, but are not limited to, (Gly 4 Ser) n , (Ser 4 Gly) n and (AlaAlaProAla) n in which n is 1 to 6, preferably 1-4, such as: a .
  • heterobifunctional cross-linking reagents Numerous heterobifunctional cross-linking reagents that are used to form covalent bonds between amino groups and thiol groups and to introduce thiol groups into proteins, are known to those of skill in this art (see, e.g., the PIERCE CATALOG, ImmunoTechnology Catalog & Handbook, 1992-1993, which describes the preparation of and use of such reagents and provides a commercial source for such reagents; see also, e.g., Cumber et al., Bioconjugate Chem. 5:397-401, 1992; Tho ⁇ e et al., Cancer Res. 47:5924-5931, 1987; Gordon et al., Proc. Natl.
  • reagents include, but are not limited to: N-succu ⁇ imidyl-3-(2- pyridyldithio)propionate (SPDP; disulfide linker); sulfosuccinimidyl 6-[3-(2- pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP); succinimidyloxycarbonyl- ⁇ - methyl benzyl thiosulfate (SMBT, hindered disulfate linker); succinimidyl 6-[3-(2- pyridyldithio) propionamido]hexanoate (LC-SPDP); sulfosuccmimidyl
  • sulfo-SMCC 4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate
  • SPDB succinimidyl 3-(2-pyridyldithio)butyrate
  • SPED hindered disulfide bond linker
  • SAED sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide) ethyl- l,3'-dithiopropionate
  • SAED sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate
  • SAMCA sulfosuccinimidyl 6-[alpha-methyl-alpha-(2-pvridyldithio)toluamido]hexanoate
  • sulfo-LC-SMPT 4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate
  • SPDB succinimidyl 3-(
  • Acid cleavable linkers include, but are not limited to, bismaleimideothoxy propane, adipic acid dihydrazide linkers (see, e.g., Fattom et al., Infection & Immun. 60:584-589, 1992) and acid labile transferrin conjugates that contain a sufficient portion of transferrin to permit entry into the intracellular transferrin cycling pathway (see, e.g., Welh ⁇ ner et al., J. Biol. Chem. 266:4309-4314, 1991). Conjugates linked via acid cleavable linkers should be preferentially cleaved in acidic intracellular compartments, such as the endosome.
  • Photocleavable linkers are linkers that are cleaved upon exposure to light (see, e.g., Goldmacher et al, Bioconj. Chem. 5:104-107, 1992), thereby releasing the targeted agent upon exposure to light.
  • Goldmacher et al, Bioconj. Chem. 5:104-107, 1992 thereby releasing the targeted agent upon exposure to light.
  • nitrobenzyl group as a photocleavable protective group for cysteine; Yen et al., Makromol.
  • the eye or skin or other body part can be exposed to light, resulting in release of the targeted moiety from the conjugate.
  • Heat sensitive linkers would also have similar applicability.
  • Host organisms include those organisms in which recombinant production of heterologous proteins have been carried out, such as bacteria (for example, E. coli), yeast (for example, Saccharomyces cerevisiae and Pichia pastoris), mammalian cells, and insect cells.
  • bacteria for example, E. coli
  • yeast for example, Saccharomyces cerevisiae and Pichia pastoris
  • mammalian cells for example, E. coli bacterial strains.
  • Presently preferred host organisms are E. coli bacterial strains.
  • the DNA construct encoding the desired protein is introduced into a plasmid for expression in an appropriate host.
  • the host is a bacterial host.
  • the sequence encoding the ligand or nucleic acid binding domain is preferably codon-optimized for expression in the particular host.
  • the codons would be optimized for bacterial usage.
  • the gene can be synthesized as a single oligonucleotide.
  • splicing of multiple oligonucleotides, mutagenesis, or other techniques known to those in the art may be used.
  • sequence of a bacterial-codon preferred FGF-SAP fusion is shown in SEQ. ID NO. 80.
  • the sequences of nucleotides in the plasmids that are regulatory regions, such as promoters and operators, are operationally associated with one another for transcription.
  • the sequence of nucleotides encoding the growth factor or growth factor-chimera may also include DNA encoding a secretion signal, whereby the resulting peptide is a precursor protein.
  • the resulting processed protein may be recovered from the periplasmic space or the fermentation medium.
  • the DNA plasmids also include a transcription terminator sequence.
  • a "transcription terminator region" has either (a) a subsegment that encodes a polyadenylation signal and polyadenylation site in the transcript, and or (b) a subsegment that provides a transcription termination signal that terminates transcription by the polymerase that recognizes the selected promoter.
  • the entire transcription terminator may be obtained from a protein-encoding gene, which may be the same or different from the inserted gene or the source of the promoter. Transcription terminators are optional components of the expression systems herein, but are employed in preferred embodiments.
  • the plasmids used herein include a promoter in operable association with the DNA encoding the protein or polypeptide of interest and are designed for expression of proteins in a bacterial host. It has been found that tightly regulatable promoters are preferred for expression of saporin. Suitable promoters for expression of proteins and polypeptides herein are widely available and are well known in the art. Inducible promoters or constitutive promoters that are linked to regulatory regions are preferred. Such promoters include, but are not limited to, the T7 phage promoter and other T7-like phage promoters, such as the T3, T5 and SP6 promoters, the t ⁇ , lpp, and lac promoters, such as the lacUV5, from E.
  • coli the PI 0 or polyhedron gene promoter of baculovirus/insect cell expression systems (see, e.g., U.S. Patent Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784) and inducible promoters from other eukaryotic expression systems.
  • promoters are inserted in a plasmid in operative linkage with a control region such as the lac operon.
  • Preferred promoter regions are those that are inducible and functional in
  • E. coli E. coli.
  • suitable inducible promoters and promoter regions include, but are not limited to: the E. coli lac operator responsive to isopropyl ⁇
  • IPTG -D-thiogalactopyranoside
  • the metallothionein promoter metal-regulatory-elements responsive to heavy-metal (e.g., zinc) induction see, e.g., U.S. Patent No. 4,870,009 to Evans et al.
  • the phage T71ac promoter responsive to IPTG see, e.g., U.S. Patent No. 4,952,496; and Studier et al., Meth. Enzymol. 755:60-89, 1990
  • the TAC promoter see, et al. Nakamura et al., Cell 75:1109-1117, 1979
  • heavy-metal e.g., zinc
  • the phage T71ac promoter responsive to IPTG see, e.g., U.S. Patent No. 4,952,496; and Studier et al., Meth. Enzymol. 755:60-89, 1990
  • TAC promoter see, e
  • the plasmids also preferably include a selectable marker gene or genes that are functional in the host.
  • a selectable marker gene includes any gene that confers a phenotype on bacteria that allows transformed bacterial cells to be identified and selectively grown from among a vast majority of untransformed cells.
  • Suitable selectable marker genes for bacterial hosts include the ampicillin resistance gene (Amp r ), tetracycline resistance gene (Tc r ) and the kanamycin resistance gene (Kan r ). The kanamycin resistance gene is presently preferred.
  • the plasmids may also include DNA encoding a signal for secretion of the operably linked protein.
  • Secretion signals suitable for use are widely available and are well known in the art. Prokaryotic and eukaryotic secretion signals functional in E. coli may be employed. The presently preferred secretion signals include, but are not limited to, those encoded by the following E. coli genes: ompA, ompT, ompF, ompC, beta-lactamase, and alkaline phosphatase, and the like (von Heijne, J. Mol. Biol. 754:99-105, 1985).
  • the bacterial pelB gene secretion signal (Lei et al., J.
  • Bacteriol 169:4379, 1987), the phoA secretion signal, and the cek2 functional in insect cell may be employed.
  • the most preferred secretion signal is the E. coli ompA secretion signal.
  • Other prokaryotic and eukaryotic secretion signals known to those of skill in the art may also be employed (see, e.g., von Heijne, J. Mol. Biol. 754:99-105, 1985).
  • one of skill in the art can substitute secretion signals that are functional in either yeast, insect or mammalian cells to secrete proteins from those cells.
  • coli cells include the p ⁇ T expression vectors (see U.S patent 4,952,496; available from Novagen, Madison, WI; see also literature published by Novagen describing the system).
  • Such plasmids include p ⁇ T l la, which contains the T71ac promoter, T7 terminator, the inducible E. coli lac operator, and the lac repressor gene; p ⁇ T 12a-c, which contains the T7 promoter, T7 terminator, and the E.
  • coli ompT secretion signal and p ⁇ T 15b (Novagen, Madison, WI), which contains a His-TagTM leader sequence for use in purification with a His column and a thrombin cleavage site that permits cleavage following purification over the column, the T7-lac promoter region and the T7 terminator.
  • Other preferred plasmids include the pKK plasmids, particularly pKK 223-3, which contains the tac promoter, (available from Pharmacia; see also Brosius et al., Proc. Natl. Acad. Sci. 57:6929, 1984; Ausubel et al., Current Protocols in Molecular Biology; U.S. Patent Nos.
  • Plasmid pKK has been modified by replacement of the ampicillin resistance marker gene, by digestion with EcoRI, with a kanamycin resistance cassette with EcoRI sticky ends (purchased from Pharmacia; obtained from pUC4K, see, e.g., Vieira et al. (Gene 79:259-268, 1982; and U.S. Patent No. 4,719,179).
  • Baculovirus vectors such as pBlueBac (also called pJV ⁇ TL and derivatives thereof), particularly pBlueBac III, (see, e.g., U.S. Patent Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784; available from Invitrogen, San Diego) may also be used for expression of the polypeptides in insect cells.
  • pBlueBac also called pJV ⁇ TL and derivatives thereof
  • pBlueBac III see, e.g., U.S. Patent Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784; available from Invitrogen, San Diego
  • the pBlueBacIII vector is a dual promoter vector and provides for the selection of recombinants by blue/white screening as this plasmid contains the ⁇ -galactosidase gene (lacZ) under the control of the insect recognizable ⁇ TL promoter and is inducible with IPTG.
  • a DNA construct may be made in baculovirus vector pBluebac III and then co-transfected with wild type virus into insect cells Spodoptera frugiperda (sf9 cells; see, e.g., Luckow et al., Bio/technology 6:47-55, 1988, and U.S. Patent No. 4,745,051).
  • Other plasmids include the pIN-IIIompA plasmids (see U.S. Patent
  • the pIN-IIIompA plasmids include an insertion site for heterologous DNA linked in transcriptional reading frame with four functional fragments derived from the lipoprotein gene of E. coli.
  • the plasmids also include a DNA fragment coding for the signal peptide of the ompA protein of E. coli, positioned such that the desired polypeptide is expressed with the ompA signal peptide at its amino terminus, thereby allowing efficient secretion across the cytoplasmic membrane.
  • the plasmids further include DNA encoding a specific segment of the E. coli lac promoter-operator, which is positioned in the proper orientation for transcriptional expression of the desired polypeptide, as well as a separate functional E. coli lad gene encoding the associated repressor molecule that, in the absence of lac operon inducer, interacts with the lac promoter-operator to prevent transcription therefrom.
  • Expression of the desired polypeptide is under the control of the lipoprotein (lpp) promoter and the lac promoter-operator, although transcription from either promoter is normally blocked by the repressor molecule.
  • the repressor is selectively inactivated by means of an inducer molecule thereby inducing transcriptional expression of the desired polypeptide from both promoters.
  • the DNA fragment is replicated in bacterial cells, preferably in E. coli.
  • the preferred DNA fragment also includes a bacterial origin of replication, to ensure the maintenance of the DNA fragment from generation to generation of the bacteria. In this way, large quantities of the DNA fragment can be produced by replication in bacteria.
  • Preferred bacterial origins of replication include, but are not limited to, the fl-ori and col El origins of replication.
  • Preferred hosts contain chromosomal copies of DNA encoding T7 RNA polymerase operably linked to an inducible promoter, such as the lacUV promoter (see U.S. Patent No. 4,952,496). Such hosts include, but are not limited to, lysogens E.
  • the pLys strains provide low levels of T7 lysozyme, a natural inhibitor of T7 RNA polymerase.
  • the DNA fragments provided may also contain a gene coding for a repressor protein.
  • the repressor protein is capable of repressing the transcription of a promoter that contains sequences of nucleotides to which the repressor protein binds. The promoter can be derepressed by altering the physiological conditions of the cell.
  • the alteration can be accomplished by adding to the growth medium a molecule that inhibits the ability to interact with the operator or with regulatory proteins or other regions of the DNA or by altering the temperature of the growth media.
  • Preferred repressor proteins include, but are not limited to the E. coli lad repressor responsive to IPTG induction, the temperature sensitive ⁇ cI857 repressor, and the like.
  • the E. coli lad repressor is preferred.
  • DNA encoding full-length FGF-2 or FGF-2 mutein is linked to DNA encoding an nucleic acid binding domain, such as protamine, and introduced into the pET vectors, including pET-l la and pET-12a expression vectors (Novagen, Madison,
  • WI for intracellular and periplasmic expression, respectively, of FGF-protamine fusion proteins.
  • a nucleic acid binding domain is coupled to a receptor- binding internalized ligand, either by chemical conjugation or as a fusion protein.
  • the ligand may alternatively be coupled directly to the nucleic acid and then complexed with a nucleic acid binding protein, such as poly-lysine, which serves to condense the nucleic acid.
  • Linkers as described above may optionally be used.
  • the receptor-binding internalized ligand confers specificity of delivery in a cell-specific manner. The choice of the receptor-binding internalized ligand to use will depend upon the receptor expressed by the target cells.
  • the receptor type of the target cell population may be determined by conventional techniques such as antibody staining, PCR of cDNA using receptor-specific primers, and biochemical or functional receptor binding assays. It is preferable that the receptor be cell type-specific or have increased expression or activity (i.e., higher rate of internalization) within the target cell population.
  • the nucleic acid binding domain can be of two types, non-specific in its ability to bind nucleic acid, or highly specific so that the amino acid residues bind only the desired nucleic acid sequence.
  • Nonspecific binding proteins, polypeptides, or compounds are generally polycationic or highly basic. Lys and Arg are the most basic of the 20 common amino acids; proteins enriched for these residues are candidates for nucleic acid binding domains. Examples of basic proteins include histones, protamines, and repeating units of lysine and arginine.
  • Poly-L-lysine is an often-used nucleic acid binding domain (see U.S. Patent Nos. 5,166,320 and 5,354,844). Poly-L-lysine and protamine are preferred.
  • nucleic acids such as spermine and spermidine
  • sequence-specific proteins including gal4, Sp-1, AP-1, myoD and the rev gene product from HIV, may be used.
  • Specific nucleic acid binding domains can be cloned in tandem, individually, or multiply to a desired region of the receptor-binding internalized ligand of interest. Alternatively, the ligand and binding domain can be chemically conjugated to each other.
  • the corresponding sequence that binds a sequence-specific domain is inco ⁇ orated into the construct to be delivered.
  • Complexing the cytocidal-encoding agent to the receptor-binding internalized ligand/nucleic acid binding domain allows specific binding to the nucleic acid binding domain. Even greater specificity of binding may be achieved by identifying and using the minimal amino acid sequence that binds to the cytocidal-encoding agent of interest.
  • phage display methods can be used to identify amino acids residues of varying length that will bind to specific nucleic acid sequences with high affinity. (See U.S. Patent No. 5,223,409.)
  • the peptide sequence can then be cloned into the receptor-binding internalized ligand as a single copy or multiple copies.
  • the peptide may be chemically conjugated to the receptor-binding internalized ligand. Incubation of the cytocide-encoding agent with the conjugated proteins will result in a specific binding between the two.
  • cytocide gene is cloned downstream of a mammalian promoter such as c-myc, SV40 early or late gene, CMV-IE, TK or adenovirus promoter.
  • promoters of interest may be active in any cell type, active only in a tissue-specific manner, such as ⁇ -crystalline or tyrosinase, event specific, or inducible, such as the MMTV LTR.
  • Receptor-binding internalized ligands are prepared as discussed by any suitable method, including recombinant DNA technology, isolation from a suitable source, purchase from a commercial source, or chemical synthesis.
  • the selected linker or linkers is (are) linked to the receptor-binding internalized ligands by chemical reaction, generally relying on an available thiol or amine group on the receptor-binding internalized ligands.
  • Heterobifunctional linkers are particularly suited for chemical conjugation.
  • the linker is a peptide linker, then the receptor-binding internalized ligands, linker and nucleic acid binding domain can be expressed recombinantly as a fusion protein.
  • any protein that binds and internalizes through a receptor interaction may be used herein.
  • any member of the FGF family of peptides or portion thereof that binds to an FGF receptor and internalizes a linked agent may be used herein.
  • the protein may be produced recombinantly, produced synthetically or obtained from commercial or other sources.
  • the DNA encoding the FGF may be obtained from any known source or synthesized according to its DNA or amino acid sequences (see discussion above). Although any of the growth factors may be conjugated in this manner,
  • FGF FGF, VEGF, and HBEGF conjugation are discussed merely by way of example and not by way of limitation.
  • the heterogeneity of preparations of ligand (e.g., FGF) containing chemical conjugates and fusion proteins can be reduced by modifying the ligand by deleting or replacing a site(s) that causes the heterogeneity.
  • sites in FGF are typically cysteine residues that upon folding of the protein remain available for interaction with other cysteines or for interaction with more than one cytotoxic molecule per molecule of FGF peptide.
  • cysteine residues do not include any cysteine residue that is required for proper folding of the FGF peptide or for binding to an FGF receptor and internalization.
  • one cysteine residue that in physiological conditions is available for interaction is not replaced but is used as the site for linking the cytotoxic moiety.
  • the resulting modified FGF is thus conjugated with a single species of nucleic acid binding domain (or nucleic acid).
  • the polypeptide reactive with an FGF receptor may be modified by removing one or more reactive cysteines that are not required for receptor binding, but that are available for reaction with appropriately derivatized cytotoxic agent, so that the resulting FGF protein has only one cysteine residue available for conjugation with the cytotoxic agent. If necessary, the contribution of each cysteine to the ability to bind to FGF receptors may be determined empirically. Each cysteine residue may be systematically replaced with a conservative amino acid change (see Table 1, above) or deleted. The resulting mutein is tested for the requisite biological activity, the ability to bind to FGF receptors and internalize linked cytotoxic moieties. If the mutein retains at least 50% of wild-type activity, then the cysteine residue is not required.
  • cysteines are systematically deleted and replaced and the resulting muteins are tested for activity. In this manner the minimum number and identity of the cysteines needed to retain the ability to bind to an FGF receptor and internalize may be determined.
  • the resulting mutant FGF is then tested for retention of the ability to target a cytotoxic agent to a cell that expresses an FGF receptor and to internalize the cytotoxic agent into such cells. Retention of proliferative activity is indicative, though not definitive, of the retention of such activities.
  • Proliferative activity may be measured by any suitable proliferation assay, such as the assay, exemplified below, that measures the increase in cell number of bovine aortic endothelial cells.
  • modified or mutant FGFs may exhibit reduced or no proliferative activity, but may be suitable for use herein, if they retain the ability to target cytocide-encoding agent to cells bearing FGF receptors and result in internalization.
  • Certain residues of FGF-2 have been associated with proliferative activity. Modification of these residues arg 116, lys 119, tyr 120, t ⁇ 123 to ile 116, glu 119, ala 120, ala 123 may be made individually (see SEQ ID NOs. 81-84) to remove this function. The resulting protein is tested for proliferative activity by a standard assay. Any of FGF-1 - FGF-9 may be used.
  • FGF-1 - FGF- 9 The complete amino acid sequence of each of FGF-1 - FGF- 9 is known (see, e.g., SEQ ID NO. 10 (FGF-1) and SEQ ID NOs. 12-18 (FGF-3 - FGF-9, respectively)). Comparison among the amino acid sequences of FGF-1 -FGF-9 reveals that one Cys is conserved among FGF family of peptides (see Table 3). These cysteine residues may be required for secondary structure and are not preferred residues to be altered. Each of the remaining cysteine residues may be systematically deleted and/or replaced by a serine residue or other residue that would not be expected to alter the structure of the protein. The resulting peptide is tested for biological activity. If the cysteine residue is necessary for retention of biological activity it is not deleted; if it not necessary, then it is preferably replaced with a serine or other residue that should not alter the secondary structure of the resulting protein.
  • cysteine residues from each of FGF-1 - FGF-9 that appear to be essential for retention of biological activity and that are not preferred residues for deletion or replacement are as follows:
  • FGF-1 has cysteines at positions 31, 98 and 132; FGF-2 has cysteines at positions 34, 78, 96 and 101; FGF-3 has cysteines at positions 50 and 115; FGF-4 has cysteines at positions 88 and 155; FGF-5 has cysteines at positions 19, 93, 160 and 202; FGF-6 has cysteines at positions 80 and 147; FGF-7 has cysteines at positions 18, 23, 32, 46, 71, 133 and 137; FGF-8 has cysteines at positions 10, 19, 109 and 127; and FGF-9 has cysteines at positions 68 and 134.
  • FGF-3, FGF-4 and FGF-6 have only two cysteines, for pu ⁇ oses of chemical conjugation, preferably neither cysteine is deleted or replaced, unless another residue, preferably one near either terminus, is replaced with a cysteine.
  • cysteine With respect to the other FGF family members, at least one cysteine must remain available for conjugation with the cytotoxic conjugate and probably two cysteines, but at least the cysteine residues set forth in Table 3.
  • a second cysteine may be required to form a disulfide bond.
  • any FGF peptide that has more than three cysteines is be modified for chemical conjugation by deleting or replacing the other cysteine residues.
  • FGF peptides that have three cysteine residues are modified by elimination of one cysteine, conjugated to a cytotoxic moiety and tested for the ability to bind to FGF receptors and internalize the cytotoxic moiety.
  • DNA obtained from pFC80 (see PCT Application Serial No. PCT/US93/05702; United States Application Serial No. 07/901,718; see also SEQ ID NO. 52) encoding basic FGF has been mutagenized.
  • cysteine 78 of basic FGF (FGF-2) to serine ([C78S]FGF) or cysteine 96 to serine ([C96SJFGF) produced two mutants that retain virtually complete proliferative activity of native basic FGF as judged by the ability to stimulate endothelial cell proliferation in culture.
  • the activities of the two mutants and the native protein do not significantly differ as assessed by efficacy or maximal response.
  • Sequence analysis of the modified DNA verified that each of the mutants has one codon for cysteine converted to that for serine.
  • the construction and biological activity of FGF-1 with cysteine substitutions of one, two or all three cysteines has been disclosed (U.S. Patent No. 5,223,483).
  • the mitogenic activity of the mutants was similar to or increased over the native protein.
  • any of the cysteines may be mutated and FGF-1 will still bind and internalize.
  • the resulting mutein FGF or unmodified FGF is reacted with a nucleic acid binding domain.
  • the bFGF muteins may react with a single species of derivatized nucleic acid binding domain (mono-derivatized nucleic acid binding domain), thereby resulting in monogenous preparations of FGF-nucleic acid binding domain conjugates and homogeneous compositions of FGF-nucleic acid binding domain chemical conjugates.
  • the resulting chemical conjugates do not aggregate and retain the requisite biological activities.
  • VEGF or HBEGF may be isolated from a suitable source or may be produced using recombinant DNA methodology, discussed below.
  • the growth factor protein is conjugated generally via a reactive amine group or thiol group to the nucleic acid binding domain directly or through a linker to the nucleic acid binding domain.
  • the growth factor protein is conjugated either via its N-terminus, C-terminus, or elsewhere in the polypeptide.
  • the growth factor protein is conjugated via a reactive cysteine residue to the linker or to the nucleic acid binding domain.
  • the growth factor can also be modified by addition of a cysteine residue, either by replacing a residue or by inserting the cysteine, at or near the amino or carboxyl terminus, within about 20, preferably 10 residues from either end, and preferably at or near the amino terminus.
  • the heterogeneity of preparations may be reduced by mutagenizing the growth factor protein to replace reactive cysteines, leaving, preferably, only one available cysteine for reaction.
  • the growth factor protein is modified by deleting or replacing a site(s) on the growth factor that causes the heterogeneity.
  • sites are typically cysteine residues that, upon folding of the protein, remain available for interaction with other cysteines or for interaction with more than one cytotoxic molecule per molecule of heparin-binding growth factor peptide.
  • cysteine residues do not include any cysteine residue that are required for proper folding of the growth factor or for retention of the ability to bind to a growth factor receptor and internalize.
  • cysteine residue that, in physiological conditions, is available for interaction, is not replaced because it is used as the site for linking the cytotoxic moiety.
  • the resulting modified heparin-binding growth factor is conjugated with a single species of cytotoxic conjugate.
  • each cysteine residue may be systematically replaced with a conservative amino acid change (see Table 1 , above) or deleted.
  • the resulting mutein is tested for the requisite biological activity: the ability to bind to growth factor receptors and internalize linked nucleic acid binding domain and agents. If the mutein retains this activity, then the cysteine residue is not required. Additional cysteines are systematically deleted and replaced and the resulting muteins are tested for activity.
  • Each of the remaining cysteine residues may be systematically deleted and/or replaced by a serine residue or other residue that would not be expected to alter the structure of the protein.
  • the resulting peptide is tested for biological activity. If the cysteine residue is necessary for retention of biological activity it is not deleted; if it not necessary, then it is preferably replaced with a serine or other residue that should not alter the secondary structure of the resulting protein. In this manner the minimum number and identity of the cysteines needed to retain the ability to bind to a heparin-binding growth factor receptor and internalize may be determined.
  • modified or mutant heparin-binding growth factors may exhibit reduced or no proliferative activity, but may be suitable for use herein, if they retain the ability to target a linked cytotoxic agent to cells bearing receptors to which the unmodified heparin-binding growth factor binds and result in internalization of the cytotoxic moiety.
  • VEGF 121 contains 9 cysteines and each of VEGF ]65 , VEGF 189 and VEGF 206 contain 7 additional residues in the region not present in VEGF 121 . Any of the 7 are likely to be non-essential for targeting and internalization of linked cytotoxic agents.
  • the VEGF monomers are preferably linked via non-essential cysteine residues to the linkers or to the targeted agent.
  • VEGF that has been modified by introduction of a Cys residue at or near one terminus, preferably the N-terminus is preferred for use in chemical conjugation.
  • the VEGF is dimerized prior to linkage to the linker and/or targeted agent.
  • cysteines in the HBEGF polypeptide that are not required for biological activity can be deleted or replaced.
  • all except one of these cysteines which will be used for chemical conjugation to the cytotoxic agent, can be deleted or replaced.
  • Each of the HBEGF polypeptides described herein have six cysteine residues.
  • Each of the six cysteines may independently be replaced and the resulting mutein tested for the ability to bind to HBEGF receptors and to be internalized.
  • the resulting mutein-encoding DNA is used as part of a construct containing DNA encoding the nucleic acid binding domain linked to the HBEGF-encoding DNA. The construct is expressed in a suitable host cell and the resulting protein tested for the ability to bind to HBEGF receptors and internalize. As long as this ability is retained the mutein is suitable for use herein.
  • Methods for chemical conjugation of proteins are known to those of skill in the art.
  • the preferred methods for chemical conjugation depend on the selected components, but preferably rely on disulfide bond formation.
  • the targeted agent is SPDP-derivatized saporin
  • VEGF is modified to include a cysteine residue at or near the N-, preferably, or C- terminus, then dimerization should follow coupling to the nucleic acid binding domain.
  • the HBEGF polypeptide is linked via one or more selected linkers or directly to the nucleic acid binding domain.
  • a nucleic acid binding domain is prepared for chemical conjugation.
  • a nucleic acid binding domain may be derivatized with SPDP or other suitable chemicals. If the binding domain does not have a Cys residue available for reaction, one can be either inserted or substituted for another amino acid. If desired, mono-derivatized species may be isolated, essentially as described.
  • nucleic acid binding domain may be derivatized or modified such that it includes a cysteine residue for conjugation to the receptor-binding internalized ligand.
  • derivatization proceeds by reaction with SPDP. This results in a heterogeneous population.
  • nucleic acid binding domain that is derivatized by SPDP to a level of 0.9 moles pyridine-disulfide per mole of nucleic acid binding domain includes a population of non-derivatized, mono-derivatized and di-derivatized SAP.
  • nucleic acid binding domain proteins which are overly derivatized with SPDP, may lose ability to bind nucleic acid because of reaction with sensitive lysines (Lambert et al., Cancer Treat. Res. 57:175-209, 1988).
  • the quantity of non-derivatized nucleic acid binding domain in the preparation of the non-purified material can be difficult to judge and this may lead to errors in being able to estimate the correct proportion of derivatized nucleic acid binding domain to add to the reaction mixture.
  • the methods herein rely on this charge difference for purification of mono-derivatized nucleic acid binding domain by Mono-S cation exchange chromatography.
  • the use of purified mono- derivatized nucleic acid binding domain has distinct advantages over the non-purified material.
  • the amount of receptor-binding internalized ligand that can react with nucleic acid binding domain is limited to one molecule with the mono-derivatized material, and it is seen in the results presented herein that a more homogeneous conjugate is produced.
  • nucleic acid binding domain may react with the succinimidyl moiety, it is possible that more than one amino group on the surface of the protein is reactive. This creates potential for heterogeneity in the mono-derivatized nucleic acid binding domain.
  • the nucleic acid binding domain can be modified by the introduction of a cysteine residue.
  • Preferred loci for introduction of a cysteine residue include the N-terminus region, preferably within about one to twenty residues from the N-terminus of the nucleic acid binding domain.
  • compositions containing the conjugates also appear to be free of aggregates.
  • Fusion protein of receptor-binding internalized ligands and nucleic acid binding domain As a preferred alternative, heterogeneity can be avoided by producing a fusion protein of receptor-binding internalized ligand and nucleic acid binding domain, as described below. Expression of DNA encoding a fusion of a receptor-binding internalized ligand polypeptide linked to the nucleic acid binding domain results in a more homogeneous preparation of cytotoxic conjugates. Aggregate formation can be reduced in preparations containing the fusion proteins by modifying the receptor- binding internalized ligand, such as by removal of nonessential cysteines, and/or the nucleic acid binding domain to prevent interactions between conjugates via free cysteines.
  • one or more coding regions for endosome-disruptive peptide may be constructed as part of the fusion protein.
  • DNA encoding the polypeptides may be isolated, synthesized or obtained from commercial sources or prepared as described herein. Expression of recombinant polypeptides may be performed as described herein; and DNA encoding these polypeptides may be used as the starting materials for the methods herein.
  • DNA encoding FGF, VEGF, HBEGF hepatocyte growth factor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-11, IL-13, TNF, GM- CSF, IFN and IGF polypeptides and/or the amino acid sequences of these factors are described above.
  • DNA may be prepared synthetically based on the amino acid or DNA sequence or may be isolated using methods known to those of skill in the art, such as PCR, probe hybridization of libraries, and the like or obtained from commercial or other sources. For example, suitable methods are described in the Examples for amplifying FGF encoding cDNA from plasmids containing FGF encoding cDNA.
  • cysteine residues may be mutagenized using standard methodologies to delete or replace any cysteine residues that are responsible for aggregate formation.
  • identity of cysteine residues that contribute to aggregate formation may be determined empirically, by deleting and/or replacing a cysteine residue and ascertaining whether the resulting growth factor with the deleted cysteine forms aggregates in solutions containing physiologically acceptable buffers and salts.
  • Loci for insertion of cysteine residues may also be determined empirically. Generally, regions at or near (within 20, preferably 10 amino acids) the C- or, preferably, the N-terminus are preferred.
  • the DNA construct encoding the fusion protein can be inserted into a plasmid and expressed in a selected host, as described above, to produce a recombinant receptor-binding internalized Kenya — nucleic acid binding domain conjugate. Multiple copies of the chimera can be inserted into a single plasmid in operative linkage with one promoter. When expressed, the resulting protein will then be a multimer. Typically, two to six copies of the chimera are inserted, preferably in a head to tail fashion, into one plasmid.
  • FGF peptides such as FGF-3, FGF-4 and FGF-6, that have two cysteines can be modified by replacing the second cysteine, which is not listed in Table 3, and the resulting mutein used as part of a construct containing DNA encoding the cytotoxic agent linked to the FGF-encoding DNA.
  • the construct is expressed in a suitable host cell and the resulting protein tested for the ability to bind to FGF receptors and internalize the cytotoxic agent.
  • conjugates containing bFGF muteins in which Cys 78 and Cys 96 have been replaced with serine residues have been prepared.
  • DNA encoding the FGF protein or other receptor-binding internalized ligand is modified so that, upon expression, the resulting FGF portion of the fusion protein does not include any cysteines available for reaction.
  • DNA encoding an FGF polypeptide is linked to DNA encoding a nucleic acid binding domain.
  • the DNA encoding the FGF polypeptide or other receptor-binding internalized ligand is modified in order to remove the translation stop codon and other transcriptional or translational stop signals that may be present and to remove or replace DNA encoding the available cysteines.
  • the DNA is then ligated to the DNA encoding the nucleic acid binding domain polypeptide directly or via a linker region of one or more codons between the first codon of the nucleic acid binding domain and the last codon of the FGF.
  • the size of the linker region may be any length as long as the resulting conjugate binds and is internalized by a target cell. Presently, spacer regions of from about one to about seventy-five to ninety codons are preferred.
  • the order of the receptor-binding internalized ligand and nucleic acid binding domain in the fusion protein may be reversed. If the nucleic acid binding domain is N-terminal, then it is modified to remove the stop codon and any stop signals.
  • any heparin-binding protein including FGF, VEGF,
  • HBEGF, cytokine, growth factor and the like may be modified and expressed in accord with the methods herein. Binding to an FGF receptor followed by internalization are the only activities required for an FGF protein to be suitable for use herein. All of the FGF proteins induce mitogenic activity in a wide variety of normal diploid mesoderm- derived and neural crest-derived cells and this activity is mediated by binding to an FGF cell surface receptor followed by internalization.
  • a test of such "FGF mitogenic activity” which reflects the ability to bind to FGF receptors and to be internalized, is the ability to stimulate proliferation of cultured bovine aortic endothelial cells (see, e.g., Gospodarowicz et al., J. Biol. Chem. 257:12266-12278, 1982; Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 75:4120-4124, 1976).
  • FGF FGF may be fluorescently labeled with FITC or radiolabeled with 125 I. Fluorescein-conjugated FGF is incubated with cells and examined microscopically by fluorescence microscopy or confocal microscopy for internalization. When FGF is labeled with 125 I, the labeled FGF is incubated with cells at 4°C.
  • the ligand can be conjugated with an nucleic acid binding domain by any of the methods described herein and complexed with a plasmid encoding saporin. As discussed below, the complex may be used to transfect cells and cytotoxicity measured.
  • the DNA encoding the resulting receptor-binding internalized ligand — ucleic acid binding domain can be inserted into a plasmid and expressed in a selected host, as described above, to produce a monogenous preparation. Fusion proteins of FGF-2 and protamine are especially suitable for use in the present invention.
  • modified receptor-binding internalized ligand/nucleic acid binding domain chimera can be inserted into a single plasmid in operative linkage with one promoter. When expressed, the resulting protein will be a multimer. Typically two to six copies of the chimera are inserted, preferably in a head to tail fashion, into one plasmid.
  • SEQ ID NO. 52 has been mutagenized as described in the Examples using splicing by overlap extension (SOE).
  • SOE overlap extension
  • Another preferred coding region is set forth in SEQ ID NO. 53.
  • the DNA is modified by replacing the cysteines at positions 78 and 96 with serine.
  • the codons encoding cysteine residues at positions 78 and 96 of FGF were converted to serine codons by SOE.
  • Each application of the SOE method uses two amplified oligonucleotide products, which have complementary ends as primers and which include an altered codon at the locus at which the mutation is desired, to produce a hybrid product.
  • a second amplification reaction that uses two primers that anneal at the non-overlapping ends amplify the hybrid to produce DNA that has the desired alteration.
  • the receptor-binding internalized ligand/nucleic acid binding domain is incubated with the cytocide-encoding agent, preferably a linear DNA molecule, to be delivered under conditions that allow binding of the nucleic acid binding domain to the agent.
  • Conditions will vary somewhat depending on the nature of the nucleic acid binding domain, but will typically occur in 0.1M NaCl and 20 mM HEPES or other similar buffer. Alternatively, salt conditions can be varied to increase the packing or condensation of DNA. The extent of binding is preferably tested for each preparation. After complexing, additional nucleic acid binding domain, such as poly-L-lysine, may be added to further condense the nucleic acid.
  • test constructs have been made and tested.
  • One construct is a chemical conjugate of bFGF and poly-L-lysine.
  • the bFGF molecule is a variant in which the Cys residue at position 96 has been changed to a serine; thus, only the Cys at position 78 is available for conjugation.
  • This bFGF is called FGF2-3.
  • the poly-L-lysine was derivatized with SPDP and coupled to FGF2-3.
  • This FGF2- 3/poly-L-lysine conjugate was used to deliver a plasmid able to express the ⁇ -galactosidase gene.
  • the ability of a construct to bind nucleic acid molecules may be conveniently assessed by agarose gel electrophoresis.
  • a plasmid such as pSV ⁇
  • restriction enzymes to yield a variety of fragment sizes.
  • the fragments may be labeled with 32 P either by filling in of the ends with DNA polymerase I or by phosphorylation of the 5 '-end with polynucleotide kinase following dephosphorylation by alkaline phosphatase.
  • the plasmid fragments are then incubated with the receptor-binding internalized ligand/nucleic acid binding domain in this case, FGF2-3/poly-L-lysine in a buffered saline solution, such as 20 mM HEPES, pH 7.3, 0.1M NaCl.
  • the reaction mixture is electrophoresed on an agarose gel alongside similarly digested, but nonreacted fragments. If a radioactive label was inco ⁇ orated, the gel may be dried and autoradiographed. If no radioactive label is present, the gel may be stained with ethidium bromide and the DNA visualized through appropriate red filters after excitation with UV. Binding has occurred if the mobility of the fragments is retarded compared to the control. In the example case, the mobility of the fragments was retarded after binding with the FGF2-3/poly-L-lysine conjugate. If there is insufficient binding, poly-L-lysine may be additionally added until binding is observed.
  • the conjugate binds to the cell surface receptor and is internalized into the cell. It is not necessary that the receptor-binding internalized ligand part of the conjugate retain complete biological activity. For example, FGF is mitogenic on certain cell types. As discussed above, this activity may not always be desirable. If this activity is present, a proliferation assay is performed. Likewise, for each desirable activity, an appropriate assay may be performed. However, for application of the subject invention, the only criteria that need be met are receptor binding and internalization. Receptor binding and internalization may be measured by the following three assays. (1) A competitive inhibition assay of the complex to cells expressing the appropriate receptor demonstrates receptor binding.
  • Receptor binding and internalization may be assayed by measuring expression of a reporter gene, such as ⁇ -gal (e.g., enzymatic activity), in cells that have been transformed with a complex of a plasmid encoding a reporter gene and a conjugate of a receptor-binding internalized ligand and nucleic acid binding domain.
  • a reporter gene such as ⁇ -gal (e.g., enzymatic activity)
  • ⁇ -gal e.g., enzymatic activity
  • This assay is particularly useful for optimizing conditions to give maximal transformation.
  • the optimum ratio of receptor- binding internalized ligand/nucleic acid binding domain to nucleic acid and the amount of DNA per cell may readily be determined by assaying and comparing the enzymatic activity of ⁇ -gal.
  • the preferred assay is a cytotoxicity assay performed on cells transformed with a cytocide-encoding agent bound by receptor-binding internalized ligand/nucleic acid binding domain. While, in general, any cytocidal molecule may be used, ribosome inactivating proteins are preferred and saporin, or another type I ribosome inactivating protein, is particularly preferred. A statistically significant reduction in cell number demonstrates the ability of the receptor-binding internalized ligand/nucleic acid binding domain conjugate or fusion to deliver nucleic acids into a cell.
  • the receptor-internalized binding ligand may be conjugated to the nucleic acid, either directly or through a linker.
  • Methods for conjugating nucleic acids, at the 5' ends, 3' ends and elsewhere, to the amino and carboxyl termini and other sites in proteins are known to those of skill in the art (for a review see, e.g., Goodchild, (1993) In: Perspectives in Bioconjugate Chemistry, Mears, Ed., American Chemical Society, Washington, D.C. pp. 77-99).
  • proteins have been linked to nucleic acids using ultraviolet irradiation (Sperling et al. (1978) Nucleic Acids Res. 5:2755-2773; Fiser et al.
  • the reagents N-acetyl-N'-(p-glyoxylylbenzolyl)cystamine and 2-iminothiolane have been used to couple DNA to proteins, such as ⁇ -macroglobulin (C M) via mixed disulfide formation (see Cheng et al., Nucleic Acids Res. 77:659-669, 1983).
  • N-acetyl-N'-(p-glyoxylylbenzolyl)cystamine reacts specifically with nonpaired guaninine residues and, upon reduction, generates a free sulfhydryl group.
  • 2-iminothiolane reacts with proteins to generate sulfhydryl groups that are then conjugated to the derivatized DNA by an intermolecular disulfide interchange reaction.
  • Any linkage may be used provided that the targeted nucleic acid is active upon internalization of the conjugate.
  • cleavage of the linkage may be necessary, although it is contemplated that for some reagents, such as DNA encoding ribozymes linked to promoters or DNA encoding therapeutic agents for delivery to the nucleus, such cleavage may not be necessary.
  • Thiol linkages which are preferred, can be readily formed using heterbiofunctional reagents.
  • Amines have also been attached to the terminal 5' phosphate of unprotected oligonucleotides or nucleic acids in aqueous solutions by reacting the nucleic acid with a water-soluble carbodiimide, such as l-ethyl-3'[3- dimethylaminopropyljcarbodiimide (EDC) or N-ethyl-N'(3-dimethylaminopropylcar- bodiimidehydrochloride (EDCI), in imidazole buffer at pH 6 to produce the 5'phosphorimidazolide.
  • a water-soluble carbodiimide such as l-ethyl-3'[3- dimethylaminopropyljcarbodiimide (EDC) or N-ethyl-N'(3-dimethylaminopropylcar- bodiimidehydrochloride (EDCI)
  • the unreacted protein may be removed from the mixture by column chromatography using, for example, Sephadex G75 (Pharmacia) using 0.1 M ammonium carbonate solution, pH 7.0 as an eluting buffer.
  • the isolated conjugate may be lyophilized and stored until used.
  • U.S. Patent No. 5,237,016 provides methods for preparing nucleotides that are bromacetylated at their 5' termini and reacting the resulting oligonucleotides with thiol groups. Oligonucleotides derivatized at their 5'-termini bromoacetyl groups can be prepared by reacting 5'-aminohexyl-phosphoramidate oligonucleotides with bromoacetic acid-N-hydroxysuccinimide ester as described in U.S. Patent No. 5,237,016. This patent also describes methods for preparing thiol-derivatized nucleotides, which can then be reacted with thiol groups on the selected growth factor.
  • thiol-derivatized nucleotides are prepared using a 5 '-phosphorylated nucleotide in two steps: (1) reaction of the phosphate group with imidazole in the presence of a diimide and displacement of the imidazole leaving group with cystamine in one reaction step; and reduction of the disulfide bond of the cystamine linker with dithiothreitol (see, also, Orgel et al. ((1986) Nucl. Acids Res. 74:651, which describes a similar procedure).
  • the 5'-phosphorylated starting oligonucleotides can be prepared by methods known to those of skill in the art (see, e.g., Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, p. 122).
  • the nucleic acid such as a methylphosphonate oligonucleotide (MP- oligomer)
  • MP- oligomer may be derivatized by reaction with SPDP or SMPB.
  • the resulting MP- oligomer may be purified by HPLC and then coupled to an FGF, such as an FGF or FGF mutein, modified by replacement of one or more cysteine residues, as described above.
  • the MP-oligomer (about 0.1 ⁇ M) is dissolved in about 40-50 ⁇ l of 1 :1 acetonitrile/water to which phosphate buffer (pH 7.5, final concentration 0.1 M) and a 1 mg MP-oligomer in about 1 ml phosphate buffered saline is added.
  • FGF-oligonucleotide conjugates can be purified on heparin sepharose Hi Trap columns (1 ml, Pharmacia) and eluted with a linear or step gradient. The conjugate should elute in 0.6 M NaCl.
  • the ligand may be conjugated to the nucleic acid construct encoding the cytocide or cytotoxic agent or may be conjugated to a mixture of oligonucleotides complementary to one strand of the construct.
  • the oligonucleotides are then added to single stranded construct produced by melting a double-stranded construct or grown and isolated as single-stranded.
  • the oligonucleotides should hybridize at a higher temperature than the construct alone, if a double-stranded construct is used as the starting material.
  • the gaps are filled in by DNA polymerase I to generate a construct with one strand conjugated to ligand and one strand unconjugated.
  • Oligonucleotides conjugated to ligand and complementary to the other strand may be used in addition to generate a mixture of constructs with different strands linked to ligand.
  • Any remaining single stranded plasmid may be digested with a single strand specific endonuclease.
  • the ligand-conjugated constructs are then mixed with a nucleic acid binding domain, such as protamine or polylysine, to effect condensation of the construct for delivery.
  • Optimal ratios of ligand to DNA may be determined experimentally by receptor-mediated transfection of a construct containing a reporter gene.
  • treatment means any manner in which the symptoms of a conditions, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein.
  • amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.
  • these conjugates and complexes may be used to treat complications of the eye following laser surgery, glaucoma surgery, and removal of pterygii.
  • the conjugates and complexes inhibit the proliferation of these cells.
  • the conjugates and complexes may be used in general to treat pathophysiological conditions, especially FGF-, VEGF-, or HBEGF-mediated pathophysiological conditions by specifically targeting to cells having corresponding receptors.
  • FGF-mediated pathophysiological condition refers to a deleterious condition characterized by or caused by proliferation of cells that are sensitive to FGF mitogenic stimulation.
  • Basic FGF-mediated pathophysiological conditions include, but are not limited to, melanoma, other tumors, rheumatoid arthritis, restenosis, Dupuytren's Contracture and certain complications of diabetes, such as proliferative retinopathy.
  • HBEGF-mediated pathophysiological condition refers to a deleterious condition characterized by or caused by proliferation of cells that are sensitive to HBEGF mitogenic stimulation.
  • HBEGF-mediated pathophysiological conditions include conditions involving pathophysiological proliferation of smooth muscle cells, such as restenosis, certain tumors, such as solid tumors including breast and bladder tumors, tumors involving pathophysiological expression of EGF receptors, dermatological disorders, such as psoriasis, and ophthalmic disorders involving epithelial cells, such as recurrence of pterygii and secondary lens clouding.
  • cytokine receptors or growth factor receptors may be eliminated.
  • diseases include restenosis, Dupuytren's Contracture, diabetic retinopathies, rheumatoid arthritis, Kaposi's sarcoma, lymphomas, leukemias, tumors such as renal cell carcinoma, colon carcinoma, breast cancer, bladder cancer, disorders with underlying vascular proliferation, such as diseases in the back of the eye (e.g., proliferative vitreoritinopathy, inacular degeneration and diabetic retinopathy).
  • diseases in the back of the eye e.g., proliferative vitreoritinopathy, inacular degeneration and diabetic retinopathy.
  • use of the VEGF-receptor promoter to control expression of the cytocide or cytotoxic agent is preferred.
  • the conjugates may be used to prevent corneal haze or clouding that results from exposure of the cornea to laser radiation during eye surgery, particularly LRK.
  • the haze or clouding appears to result from fibroblastic keratocyte proliferation in the subepithelial zone following photoablation of the cornea.
  • the conjugates may be used to treat a "hype ⁇ roliferative skin disorder.”
  • a "hype ⁇ roliferative skin disorder” is a disorder that is manifested by a proliferation of endothelial cells of the skin coupled with an underlying vascular proliferation, resulting in a localized patch of scaly or horny or thickened skin or a tumor of endothelial origin.
  • disorders include actinic and atopic dermatitis, toxic eczema, allergic eczema, psoriasis, skin cancers and other tumors, such as Kaposi's sarcoma, angiosarcoma, hemangiomas, and other highly vascularized tumors, and vascular proliferative responses, such as varicose veins.
  • the conjugates may be used to treat or prevent restenosis, a process and the resulting condition that occurs following angioplasty in which the arteries become reclogged.
  • denudation of the interior wall of the vessel occurs, including removal of the endothelial cells that constitute the lining of the blood vessels.
  • smooth muscle cells SMCs
  • This process and the resulting condition is restenosis.
  • compositions suitable for administration of the conjugates and complexes provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the conjugates and complexes may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • the conjugates and complexes can be administered by any appropriate route, for example, orally, parenterally, including intravenously, intradermally, subcutaneously, or topically, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration.
  • Preferred modes of administration depend upon the indication treated. Dermatological and ophthalmologic indications will typically be treated locally; whereas, tumors and restenosis, will typically be treated by systemic, intradermal, or intramuscular modes of administration.
  • conjugates and complexes herein may be formulated into pharmaceutical compositions suitable for topical, local, intravenous and systemic application.
  • local administration either by topical administration or by injection is preferred.
  • Time release formulations are also desirable.
  • an "effective amount" of a compound for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Repeated administration may be required to achieve the desired amelioration of symptoms.
  • an ophthalmically effective amount is that amount which, in the composition administered and by the technique administered, provides an amount of therapeutic agent to the involved eye tissues sufficient to prevent or reduce corneal haze following excimer laser surgery, prevent closure of a trabeculectomy, prevent or substantially slow the recurrence of pterygii, and other conditions.
  • concentrations or amounts of the conjugates and complexes that are effective requires delivery of an amount, upon administration, that ameliorates the symptoms or treats the disease.
  • the compositions are formulated for single dosage administration.
  • Therapeutically effective concentrations and amounts may be determined empirically by testing the conjugates and complexes in known in vitro and in vivo systems, such as those described here; dosages for humans or other animals may then be extrapolated therefrom.
  • the conjugate is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the conjugates may be delivered as pharmaceutically acceptable salts, esters or other derivatives of the conjugates include any salts, esters or derivatives that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects. It is understood that number and degree of side effects depends upon the condition for which the conjugates and complexes are administered. For example, certain toxic and undesirable side effects are tolerated when treating life-threatening illnesses, such as tumors, that would not be tolerated when treating disorders of lesser consequence.
  • concentration of conjugate in the composition will depend on abso ⁇ tion, inactivation and excretion rates thereof, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • the conjugate and complex are substantially pure.
  • substantially pure means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.
  • TLC thin layer chromatography
  • HPLC high performance liquid chromatography
  • a therapeutically effective dosage should produce a serum concentration of active ingredient of from about 0.1 ng/ml to about 50-100 ⁇ g/ml.
  • the pharmaceutical compositions typically should provide a dosage of from about 0.01 mg to about 100 - 2000 mg of conjugate, depending upon the conjugate selected, per kilogram of body weight per day. For example, for treatment of restenosis a daily dosage of about between 0.05 and 0.5 mg/kg (based on FGF-SAP chemical conjugate or an amount of conjugate provided herein equivalent on a molar basis thereto) should be sufficient.
  • Local application for ophthalmic disorders and dermatological disorders should provide about 1 ng up to 100 ⁇ g, preferably about 1 ng to about 10 ⁇ g, per single dosage administration.
  • the amount to administer will be a function of the conjugate selected, the indication treated, and possibly the side effects that will be tolerated.
  • Therapeutically effective concentrations and amounts may be determined for each application herein empirically by testing the conjugates and complexes in known in vitro and in vivo systems (e.g., murine, rat, rabbit, or baboon models), such as those described herein; dosages for humans or other animals may then be extrapolated therefrom.
  • Demonstration that the conjugates and complexes prevent or inhibit proliferation of serum stimulated corneal keratocytes or fibroblasts explanted from eyes, as shown herein, and demonstration of any inhibition of proliferation of such tissues in rabbits should establish human efficacy.
  • the rabbit eye model is a recognized model for studying the effects of topically and locally applied drugs (see, e.g., U.S. Patent Nos. 5,288,735, 5,263,992, 5,262,178, 5,256,408, 5,252,319, 5,238,925, 5,165,952; see also Mirate et al., Curr. Eye Res. 7:491-493, 1981).
  • the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
  • the conjugates and complexes may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracistemal or intraspinal application.
  • solutions particularly those intended for ophthalmic use, may be formulated as 0.01% -10% isotonic solutions, pH about 5-7, with appropriate salts.
  • the ophthalmic compositions may also include additional components, such as hyaluronic acid.
  • the conjugates and complexes may be formulated as aerosols for topical application (see, e.g., U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923).
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of toxicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent
  • antimicrobial agents such as benzyl alcohol and methyl parabens
  • antioxidants such as ascorbic acid and sodium bisulfit
  • parental preparations can be enclosed in ampules, disposable syringes or multiple dose vials made of glass, plastic or other suitable material.
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • PBS physiological saline or phosphate buffered saline
  • Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art.
  • the resulting mixture may be a solution, suspension, emulsion or the like.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the conjugate in the selected carrier or vehicle.
  • the effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined based upon in vitro and/or in vivo data, such as the data from the mouse xenograft model for tumors or rabbit ophthalmic model. If necessary, pharmaceutically acceptable salts or other derivatives of the conjugates and complexes may be prepared.
  • the active materials can also be mixed with other active materials, that do not impair the desired action, or with materials that supplement the desired action, including viscoelastic materials, such as hyaluronic acid, which is sold under the trademark HEALON (solution of a high molecular weight (MW of about 3 millions) fraction of sodium hyaluronate; manufactured by Pharmacia, Inc. see, e.g., U.S. Patent Nos.
  • HEALON solution of a high molecular weight (MW of about 3 millions) fraction of sodium hyaluronate
  • VISCOAT fluorine-containing (meth)acrylates, such as, lH,lH,2H,2H-hepta- decafluorodecylmethacrylate; see, e.g., U.S. Patent Nos. 5,278,126, 5,273,751 and 5,214,080; commercially available from Alcon Surgical, Inc.), ORCOLON (see, e.g., U.S. Patent Nos.
  • the viscoelastic materials are present generally in amounts ranging from about 0.5 to 5.0%, preferably 1 to 3% by weight of the conjugate material and serve to coat and protect the treated tissues.
  • the compositions may also include a dye, such as methylene blue or other inert dye, so that the composition can be seen when injected into the eye or contacted with the surgical site during surgery.
  • the conjugates and complexes may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye.
  • Such solutions particularly those intended for ophthalmic use, may be formulated as 0.01%- 10% isotonic solutions, pH about 5-7, with appropriate salts.
  • Suitable ophthalmic solutions are known (see, e.g., U.S. Patent No. 5,116,868, which describes typical compositions of ophthalmic irrigation solutions and solutions for topical application).
  • Such solutions which have a pH adjusted to about 7.4, contain, for example, 90-100 mM sodium chloride, 4-6 mM dibasic potassium phosphate, 4-6 mM dibasic sodium phosphate, 8-12 mM sodium citrate, 0.5-1.5 mM magnesium chloride, 1.5-2.5 mM calcium chloride, 15-25 mM sodium acetate, 10-20 mM D.L.-sodium ⁇ - hydroxybutyrate and 5-5.5 mM glucose.
  • the conjugates and complexes may be prepared with carriers that protect them against rapid elimination from the body, such as time release formulations or coatings.
  • Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others.
  • the composition may be applied during surgery using a sponge, such as a commercially available surgical sponges (see, e.g., U.S. Patent Nos. 3,956,044 and 4,045,238; available from Week, Alcon, and Mentor), that has been soaked in the composition and that releases the composition upon contact with the eye.
  • a sponge such as a commercially available surgical sponges (see, e.g., U.S. Patent Nos. 3,956,044 and 4,045,238; available from Week, Alcon, and Mentor), that has been soaked in the composition and that releases the composition upon contact with the eye.
  • compositions may also be applied in pellets (such as Elvax pellets(ethylene- vinyl acetate copolymer resin); about 1- 5 ⁇ g of conjugate per 1 mg resin) that can be implanted in the eye during surgery.
  • Ophthalmologically effective concentrations or amounts of one or more of the conjugates and complexes are mixed with a suitable pharmaceutical carrier or vehicle.
  • concentrations or amounts of the conjugates and complexes that are effective requires delivery of an amount, upon administration, that prevents or substantially reduces corneal clouding, trabeculectomy closure, or pterygii recurrence.
  • the conjugates and complexes herein are formulated into ophthalmologically acceptable compositions and are applied to the affected area of the eye during or immediately after surgery.
  • the composition is applied to the cornea; following trabeculectomy the composition is applied to the fistula; and following removal of pterygii the composition is applied to the cornea.
  • the compositions may also be used to treat pterygii.
  • the conjugates and complexes are applied during and immediately following surgery and may, if possible be applied post-operatively, until healing is complete.
  • the compositions are applied as drops for topical and subconjunctival application or are injected into the eye for intraocular application.
  • the compositions may also be absorbed to a biocompatible support, such as a cellulosic sponge or other polymer delivery device, and contacted with the affected area.
  • the ophthalmologic indications herein are typically be treated locally either by the application of drops to the affected tissue(s), contacting with a biocompatible sponge that has absorbed a solution of the conjugates and complexes or by injection of a composition.
  • the composition will be applied during or immediately after surgery in order to prevent closure of the trabeculectomy, prevent a proliferation of keratocytes following excimer laser surgery, or to prevent a recurrence of pterygii.
  • the composition may also be injected into the affected tissue following surgery and applied in drops following surgery until healing is completed. For example, to administer the formulations to the eye, it can be slowly injected into the bulbar conjunctiva of the eye.
  • Conjugates and complexes with photocleavable linkers are among those preferred for use in the methods herein.
  • the eye Upon administration of such composition to the affected area of the eye, the eye is exposed to light of a wavelength, typically visible or UV that cleaves the linker, thereby releasing the cytotoxic agent.
  • the conjugate should be provided in a composition that protects it from the acidic environment of the stomach.
  • the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine.
  • the composition may also be formulated in combination with an antacid or other such ingredient.
  • Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules.
  • the active compound or compounds can be inco ⁇ orated with excipients and used in the form of tablets, capsules or troches.
  • compositions can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth and gelatin; an excipient such as starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, and fruit flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth and gelatin
  • an excipient such as starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch
  • a lubricant such as, but not limited to, magnesium stearate
  • the dosage unit form When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil.
  • dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.
  • the conjugates and complexes can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as cis-platin for treatment of tumors.
  • the compounds may be packaged as articles of manufacture containing packaging material, one or more conjugates and complexes or compositions as provided herein within the packaging material, and a label that indicates the indication for which the conjugate is provided.
  • Many methods have been developed to deliver nucleic acid into cells including retroviral vectors, electroporation, CaPO 4 precipitation and microinjection, but each of these methods has distinct disadvantages. Microinjecting nucleic acid into cells is very time consuming because each cell must be manipulated individually. Retroviral vectors can only hold a limited length of nucleic acid and can activate oncogenes depending upon the insertion site in the target chromosome. Conditions for electroporation and CaP0 4 -mediated transfection are harsh and cause much cell death.
  • receptor mediated gene delivery as described herein is a more desirable method of selectively targeting toxic genes into cells that have "more active" receptors or that overexpress the specific receptor on the cell surface.
  • a receptor may be more active because it has a higher rate of internalization or higher cycling rate through the endosome to the cell surface.
  • Advantages of this method over other gene delivery methods include increased specificity of delivery, the absence of nucleic acid length limitations, reduced toxicity, and reduced immunogenicity of the conjugate. These characteristics allow for repeated administration of the material with minimal harm to cells and may allow increased level of expression of the toxic protein.
  • primary cultures can also be treated using this method.
  • E. coli strain JA221 (lpp- hdsM+ t ⁇ E5 leuB6 lacY recAl F'[lacl ⁇ e lac + pro + ]) is publicly available from the American Type Culture Collection (ATCC), Rockville, MD 20852, under the accession number ATCC 33875. (JA221 is also available from the Northern Regional Research Center (NRRL), Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604, under the accession number NRRL B-15211; see also U.S. Patent No. 4,757,013 to Inouye; and Nakamura et al., Cell 75:1109-1117, 1979). Strain INVl ⁇ is commercially available from Invitrogen, San Diego, CA.
  • restriction and modification enzymes employed herein are commercially available in the U.S. Native saporin and rabbit polyclonal antiserum to saporin were obtained as previously described in Lappi et al., Biochem. Biophys. Res.
  • Western blotting was accomplished by transfer of the electrophoresed protein to nitrocellulose using the PhastTransfer system, as described by the manufacturer.
  • the antiserum to SAP was used at a dilution of 1:1000.
  • Horseradish peroxidase labeled anti-IgG was used as the second antibody (see Davis et al., Basic Methods In Molecular Biology, New York, Elsevier Science Publishing Co., pp 1-338, 1986).
  • One cycle included a denaturation step (94°C for 1 min), an annealing step (60°C for 2 min), and an elongation step (72°C for 3 min). After 30 cycles, a 10 ⁇ l aliquot of each reaction was run on a 1.5% agarose gel to verify the structure of the amplified product.
  • the amplified DNA was digested with EcoRI and subcloned into EcoRI- restricted M13mpl8 (New England Biolabs, Beverly, MA; see also Yanisch-Perron et al. (1985), "Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mpl8 and pUC19 vectors", Gene 55:103).
  • Single-stranded DNA from recombinant phages was sequenced using oligonucleotides based on internal points in the coding sequence of saporin (see Bennati et al., Eur. J. Biochem. 755:465- 470, 1989).
  • Nine of the M13mpl8 derivatives were sequenced and compared.
  • the clones were designated M13mpl8-G4, -Gl, -G2, -G7, and -G9. Each of these clones contains all of the saporin coding sequence and 45 nucleotides of DNA encoding the native saporin N-terminal leader peptide. Saporin DNA sequence was also cloned in the pETl la vector. Briefly, the DNA encoding SAP-6 was amplified by polymerase chain reaction (PCR) from the parental plasmid pZlBl .
  • PCR polymerase chain reaction
  • the plasmid pZlBl contains the DNA sequence for human FGF-2 linked to SAP-6 by a two-amino-acid linker (Ala-Met). PZlBl also includes the T7 promoter, lac operator, ribosomal binding site, and T7 terminator present in the pET- l la vector.
  • the 5' primer 5'
  • the digested SAP-6 DNA fragment was subcloned into the Nc /if ⁇ mHI-digested pZlBl. This digestion removed FGF-2 and the 5' portion of SAP-6 (up to nucleotide position 650) from the parental rFGF2- SAP vector (pZlBl) and replaced this portion with a SAP-6 molecule containing a Cys at position -1 relative to the start site of the native mature SAP-6 protein. The resultant plasmid was designated as pZ50B.
  • pZ50B was transformed into E. coli strain ⁇ ovaBlue for restriction and sequencing analysis. The appropriate clone was then transformed into E. coli strain BL21(DE3) for expression and large-scale production.
  • SAP plant protein saporin
  • restriction enzyme sites were added to the ends of each fragment, and added or removed within each fragment without changing the corresponding amino acid sequence.
  • the 5' end of the cD ⁇ A was modified to include a Kozak sequence for optimal expression in mammalian cells.
  • Fragments A, B, and D were each synthesized by annealing 4 oligos (2 sense, 2 antisense) with 20 base overlaps and using PCR to fill-in and amplify the fragments.
  • PCR products were then purified using GeneClean (Biol 01), digested with restriction enzymes recognizing the sites in the primers, and subcloned into pBluescript (SK+) (Stratagene). The sequence of the inserts was verified using Sequenase Version 2.0 (United States Biochemical/Amersham). Fragment C was synthesized in two steps: The 5' and 3' halves of the fragment were independently synthesized by PCR using 2 overlapping oligos. The products of these using 2 reactions were then purified and combined and the full-length fragment C was generated by PCR using the outermost oligos as primers. Full-length fragment C was subcloned into pBluescript for sequencing.
  • Fragments A and B were ligated together in pBluescript at an overlapping Kspl site.
  • Fragments C and D were ligated together in pBluescript at an overlapping RvuII site.
  • Fragments A-B and C-D were then joined in pBluescript at an overlapping Aval site to give the full-length mammalian optimized SAP cDNA.
  • ⁇ -gal sequences were excised from the plasmids pNASS- ⁇ and pSV- ⁇ (Clontech) by digestion with NotI and replaced with the synthetic SAP gene, which has NotI ends. Orientation of the insert was confirmed by restriction enzyme digestion. Large scale plasmid preparations were performed using Qiagen Maxi 500 columns. The oligos used to synthesize each SAP fragment are (5 '-3'):
  • Ml 3 mpl8-G4 was digested with EcoR I, and the resulting fragment was ligated into the EcoR I site of the vector pIN-IIIompA2 (see, e.g., see, U.S. Patent No. 4,575,013 to Inouye; and Duffaud et al., Meth. Enz. 153:492-507, 1987) using the methods described herein.
  • the ligation was accomplished such that the DNA encoding saporin, including the N-terminal extension, was fused to the leader peptide segment of the bacterial ompA gene.
  • the resulting plasmid pOMPAG4 contains the Ipp promoter (Nakamura et al., Cell 75:1109-1117, 1987), the E. coli lac promoter operator sequence (lac O) and the E. coli ompA gene secretion signal in operative association with each other and with the saporin and native N-terminal leader-encoding DNA listed in S ⁇ Q ID NO. 19.
  • the plasmid also includes the E. coli lac repressor gene (lac I).
  • the Ml 3 mpl8-Gl, -G2, -G7, and -G9 clones, containing S ⁇ Q ID NOs. 20-23, respectively, are digested with EcoR I and ligated into EcoR I digested pIN- IIIompA2 as described for Ml 3 mpl8-G4 above in this example.
  • the resulting plasmids, labeled pOMPAGl, ⁇ OMPAG2, pOMPAG7, pOMPA9, are screened, expressed, purified, and characterized as described for the plasmid pOMPAG4.
  • INVl ⁇ competent cells were transformed with pOMPAG4 and cultures containing the desired plasmid structure were grown further in order to obtain a large preparation of isolated pOMPAG4 plasmid using methods described herein.
  • the pOMPAG4 transformed E. coli cells were grown under conditions in which the expression of the saporin-containing protein is repressed by the lac repressor until the end of the log phase of growth, at which time IPTG was added to induce expression of the saporin-encoding DNA.
  • IPTG IPTG was added to induce expression of the saporin-encoding DNA.
  • an overnight culture approximately 16 hours growth of JA221 E.
  • coli cells transformed with the plasmid pOMPAG4 in LB broth (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) containing 125 mg/ml ampicillin was diluted 1 :100 into a flask containing 750 ml LB broth with 125 mg/ml ampicillin. Cells were grown at logarithmic phase with shaking at 37°C until the optical density at 550 nm reached 0.9 measured in a spectrophotometer.
  • the E. coli cells containing C-SAP construct in p ⁇ Tl la were grown in a high-cell density fed-batch fermentation with the temperature and pH controlled at 30°C and 6.9, respectively.
  • a glycerol stock (1 ml) was grown in 50 ml Luria broth until the A 600 reached 0.6 Inoculum (10 ml) was injected into a 7-1-Applikon (Foster City CA) fermentor containing 21 complex batch medium consisting of 5 g/1 of glucose, 1.25 g/1 each of yeast extract and tryptone (Difco Laboratories), 7 g/1 of K 2 HPO 4 , 8 g/1 of KH 2 P0 4 , 1.66 g/1 of (NH 4 ) 2 SO 4 , 1 g/1 of MgS0 4 • 7H 2 O, 2 ml/1 of a trace metal solution (74 g/1 of trisodium citrate, 27 g/1 of FeCl 3 • 6H 2 O, 2.0 g/1 of CoCl 2
  • the culture was fed with a solution containing 100 g/1 of glucose, 100 g/1 of yeast extract, and 200 g/1 of tryptone.
  • the cells were harvested by centrifugation (8000xg, 10 min) and frozen at -80°C until further processed.
  • the cell pellet ( «400 g wet mass) containing C-SAP was resuspended in
  • Buffer B (10 mM sodium phosphate pH 7.0, 5 mM EDTA, 5 mM EGTA, and 1 mM dithiothreitol). The suspension was passed through a microfluidizer three times at
  • the diluted lysate was loaded onto an expanded bed of Streamline SP cation-exchange resin (300 ml) equilibrated with buffer C (20 mM sodium phosphate pH 7.0, 1 mM EDTA) at 100 ml/min upwards flow.
  • the resin was washed with buffer C until it appeared clear.
  • the plunger was then lowered at 2 cm/min while washing continued at 70 ml/min. Upwards flow was stopped when the plunger was approximately 8 cm away from the bed and the plunger was allowed to move to within 0.5 cm of the packed bed.
  • the resin was further washed at 70 ml/min downwards flow until A 28o reached baseline. Buffer C plus 0.25 M NaCl was then used to elute proteins containing C-SAP at the same flow rate.
  • the eluate was buffer exchanged into buffer D (50 mM sodium borate pH 8.5, 1 mM EDTA) using the Sartocon Mini crossflow filtration system with a 10000
  • the ribosome inactivating protein activity of recombinant saporin was compared to the ribosome inactivating protein activity of native SAP in an in vitro assay measuring cell-free protein synthesis in a nuclease-treated rabbit reticulocyte lysate (Promega).
  • Samples of immunoaffinity-purified saporin were diluted in PBS and 5 ⁇ l of sample was added on ice to 35 ⁇ l of rabbit reticulocyte lysate and 10 ⁇ l of a reaction mixture containing 0.5 ⁇ l of Brome Mosaic Virus RNA, 1 mM amino acid mixture minus leucine, 5 ⁇ Ci of tritiated leucine and 3 ⁇ l of water.
  • Assay tubes were incubated 1 hour in a 30°C water bath. The reaction was stopped by transferring the tubes to ice and adding 5 ⁇ l of the assay mixture, in triplicate, to 75 ⁇ l of 1 N sodium hydroxide, 2.5% hydrogen peroxide in the wells of a Millititer HA 96-well filtration plate (Millipore). When the red color had bleached from the samples, 300 ⁇ l of ice cold 25% trichloroacetic acid (TCA) were added to each well and the plate left on ice for another 30 min. Vacuum filtration was performed with a Millipore vacuum holder. The wells were washed three times with 300 ⁇ l of ice cold 8% TCA. After drying, the filter paper circles were punched out of the 96-well plate and counted by liquid scintillation techniques.
  • TCA trichloroacetic acid
  • the IC 50 for the recombinant and native saporin were approximately 20 pM. Therefore, recombinant saporin-containing protein has full protein synthesis inhibition activity when compared to native saporin.
  • Restriction and modification enzymes were purchased from BRL (Gaithersburg, MD), Stratagene (La Jolla, CA) and New England Biolabs (Beverly, MA).
  • Plasmid pFC80 containing the basic FGF coding sequence, was a gift of
  • Plasmid pFC80 has been described in the PCT Application Serial No. WO 90/02800 and PCT Application Serial No. PCT/US93/05702, which are herein inco ⁇ orated in their entirety by reference.
  • the sequence of DNA encoding bFGF in pFC80 is that set forth in PCT Application Serial No. PCT/US 93/05702 and in SEQ ID NO. 52.
  • SDS gel electrophoresis was performed on a PhastSystem utilizing 20% gels (Pharmacia). Western blotting was accomplished by transfer of electrophoresed protein to nitrocellulose using the PhastTransfer system (Pharmacia), as described by the manufacturer. The antisera to SAP and basic FGF were used at a dilution of 1 : 1000. Horseradish peroxidase labeled anti-IgG was used as the second antibody as described (Davis, L., Dibner et al. (1986) Basic Methods in Molecular Biology, p. 1, Elsevier Science Publishing Co., New York).
  • Cysteine to serine substitutions were made by oligonucleotide-directed mutagenesis using the Amersham (Arlington Heights, IL) in v tro-mutagenesis system 2.1. Oligonucleotides encoding the new amino acid were synthesized using a 380B automatic DNA synthesizer (Applied Biosystems, Foster City, CA).
  • the oligonucleotide used for in vitro mutagenesis of cysteine 78 was
  • the oligonucleotide for mutagenesis of cysteine 96 was TTCTAAATCGGTTACCGATGACTG (SEQ ID NO. 57), which spans nucleotides
  • the mutated replicative form DNA was transformed into E. coli strain JM109 and single plaques were picked and sequenced for verification of the mutation.
  • the FGF mutated gene was then cut out of Ml 3, ligated into the expression vector pFC80, which had the non-mutated form of the gene removed, and transformed into E. coli strain JM109. Single colonies were picked and the plasmids sequenced to verify the mutation was present. Plasmids with correct mutation were then transformed into the E. coli strain FIC ⁇ 2 and single colonies from these transformations were used to obtain the mutant basic FGFs. Approximately 20 mg protein per liter of fermentation broth was obtained.
  • Cells were grown overnight in 20 ml of LB broth containing 100 ⁇ g/ml ampicillin. The next morning the cells were pelleted and transferred to 500 ml of M9 medium with 100 ⁇ g/ml ampicillin and grown for 7 hours. The cells were pelleted and resuspended in lysis solution (10 mM TRIS, pH 7.4, 150 mM NaCl, lysozyme, 10 ⁇ g/mL, aprotinin, 10 ⁇ g/mL, leupeptin, 10 ⁇ g/mL, pepstatin A, 10 ⁇ g/mL and 1 mM PMSF; 45-60 ml per 16 g of pellet) and incubated while stirring for 1 hour at room temperature.
  • lysis solution 10 mM TRIS, pH 7.4, 150 mM NaCl, lysozyme, 10 ⁇ g/mL, aprotinin, 10 ⁇ g/mL, leupeptin, 10 ⁇ g/mL,
  • Extract volumes (40 ml) were diluted to 50 ml with lO mM TRIS, pH 7.4 (buffer A). Pools were loaded onto a 5 ml Hi-Trap heparin- Sepharose column (Pharmacia, Uppsala, Sweden) equilibrated in 150 mM sodium chloride in buffer A.
  • MyoD at a concentration of 4.1 mg/ml is dialyzed against 0.1 M sodium phosphate, 0.1 M sodium chloride, pH 7.5.
  • a 1.1 molar excess (563 ⁇ g in 156 ⁇ l of anhydrous ethanol) of SPDP (Pharmacia, Uppsala, Sweden) is added and the reaction mixture immediately agitated and put on a rocker platform for 30 minutes.
  • the solution is then dialyzed against the same buffer.
  • An aliquot of the dialyzed solution is examined for extent of derivatization according to the Pharmacia instruction sheet.
  • the extent of derivatization is typically 0.79 to 0.86 moles of SPDP per mole of nucleic acid binding domain.
  • Derivatized myoD (32.3 mg) is dialyzed in 0.1 M sodium borate, pH 9.0 and applied to a Mono S 16/10 column equilibrated with 25 mM sodium chloride in dialysis buffer. A gradient of 25 mM to 125 mM sodium chloride in dialysis buffer elutes free and derivatized nucleic acid binding domain. The flow rate is 4.0 ml/min, 4 ml fractions are collected. Aliquots of fractions were assayed for protein concentration (BCA Protein Assay, Pierce Chemical, Chicago, IL) and for pyridylthione released by reducing agent. Individual fractions (25 to 37) are analyzed for protein concentration and pyridyl-disulfide concentration.
  • the data indicate a separation according to the level of derivatization by SPDP.
  • the initial eluting peak is composed of myoD that is approximately di-derivatized; the second peak is mono-derivatized and the third peak shows no derivatization.
  • the di-derivatized material accounts for approximately 20% of the three peaks; the second accounts for approximately 48% and the third peak contains approximately 32%.
  • Material from the second peak is pooled and gives an average ratio of pyridyl-disulfide to myoD of 0.95. Fraction 33, which showed a divergent ratio of pyridine-2-thione to protein, was excluded from the pool.
  • Fractions that showed a ratio of SPDP to myoD greater than 0.85 but less than 1.05 are pooled, dialyzed against 0.1 M sodium chloride, 0.1 M sodium phosphate, pH 7.5 and used for derivatization with basic FGF.
  • myoD is modified by addition of a cysteine residue at or near the N-terminus-encoding portion of the DNA.
  • the resulting myoD can then react with an available cysteine on an FGF or react with a linker or a linker attached to an FGF to produce conjugates that are linked via the added Cys.
  • Modified myoD is prepared by modifying DNA encoding the myoD (GenBank Accession No. X56677). DNA encoding Cys is inserted at position -1 or at a codon within 10 or fewer residues of the N-terminus. The resulting DNA is inserted into pETl 1 a and pETl 5b and expressed in BL21 cells (NOVAGEN, Madison, WI).
  • Primer #2 is an antisense primer complementing the coding sequence of nucleic acid binding domain spanning nucleotides 1054-1077 and contains a BamHI site. 5 * -GGATCCGAGCACCTGGTATATCGGTGGGGG-3' (SEQ ID NO. 59)
  • MyoD DNA is amplified by PCR as follows using the above primers.
  • a clone containing a full-length DNA (or cDNA) for myoD (1 ⁇ l) is mixed in a final volume of 100 ⁇ l containing lO mM Tris-HCl (pH 8.3), 50 mM KCl, 0.01% gelatin, 2 mM MgCl 2 , 0.2 mM dNTPs, 0.8 ⁇ g of each primer.
  • 2.5 U TaqI DNA polymerase (Boehringer Mannheim) is added and the mixture is overlaid with 30 ⁇ l of mineral oil (Sigma). Incubations are done in a DNA Thermal Cycler.
  • Cycles include a denaturation step (94°C for 1 min), an annealing step (60°C for 2 min), and an elongation step (72°C for 3 min). After 35 cycles, a 10 ⁇ l aliquot of each reaction is run on a 1.5% agarose gel to verify the correct structure of the amplified product.
  • the amplified DNA is gel purified and digested with Ndel .and BamHI and subcloned into Ndel and Tf ⁇ mHI-digested plasmid containing FGF/myoD. This digestion and subcloning step removes the FGF-encoding DNA and 5' portion of SAP up to the BamHI site at nucleotides 555-560 (SEQ ID NO. 52) and replaces this portion with DNA encoding a myoD molecule that contains a cysteine residue at position -1 relative to the start site of the native mature SAP protein.
  • MyoD is amplified by polymerase chain reaction (PCR) from the parental plasmid encoding the FGF-nucleic acid binding domain fusion protein using primers that inco ⁇ orate a TGT or TGC codon at position 4 or 10.
  • PCR polymerase chain reaction
  • the PCR conditions are performed as described above, using the following cycles: denaturation step 94°C for 1 minute, annealing for 2 minutes at 60°C, and extension for 2 minutes at 72°C for 35 cycles.
  • the amplified DNA is gel purified, digested with Ndel and BamHI, and subcloned into Ndel and BamHI digested pETl la. This digestion removes the FGF and 5' portion of nucleic acid binding domain (up to the newly added BamHI) from the parental FGF- myoD vector and replaces this portion with a myoD molecule containing a Cys at position 4 or 10 relative to the start site of the native protein.
  • the resulting plasmid is digested with NdellBamHl and inserted into pET15b (NOVAGEN, Madison, WI), which has a His-TagTM leader sequence (SEQ ID NO. 60), that has also been digested NdellBamHl.
  • DNA encoding unmodified myoD can be similarly inserted into a pET5b or pETl 1 A and expressed as described below for the modified SAP-encoding DNA.
  • BL21(DE3) cells are transformed with the resulting plasmids and cultured as described in Example 2, except that all incubations were conducted at 30°C instead of 37°C. Briefly, a single colony is grown in LB AMP 100 to and OD 600 of 1.0-1.5 and then induced with IPTG (final concentration 0.1 mM) for 2 h. The bacteria are spun down.
  • Lysis buffer (20 mM NaPO 4 , pH 7.0, 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 0.5 ⁇ g/ml leupeptin, 1 ⁇ g/ml aprotinin, 0.7 ⁇ g/ml pepstatin) was added to the myoD cell paste (produced from pZ50Bl in BL21 cells, as described above) in a ratio of 1.5 ml buffer/g cells. This mixture is evenly suspended via a Polytron homogenizer and passed through a microfluidizer twice.
  • the resulting lysate is centrifuged at 50,000 rpm for 45 min.
  • the supernatant is diluted with SP Buffer. A (20 mM NaPO 4 , 1 mM EDTA, pH 7.0) so that the conductivity is below 2.5 mS/cm.
  • the diluted lysate supernatant is then loaded onto a SP-Sepharose column, and a linear gradient of 0 to 30% SP Buffer B (1 M NaCl, 20 mM NaP0 4 , 1 mM EDTA, pH 7.0) in SP Buffer A with a total of 6 column volumes is applied. Fractions containing myoD are combined and the resulting rnucleic acid binding domain had a purity of greater than 90%.
  • a buffer exchange step is used to get the SP eluate into a buffer containing 50 mM NaB0 3 , 1 mM EDTA, pH 8.5 (S Buffer A). This sample is then applied to a Resource S column (Pharmacia, Sweden) pre- equilibrated with S Buffer A. Pure nucleic acid binding domain is eluted off the column by 10 column volumes of a linear gradient of 0 to 300 mM NaCl in SP Buffer A.
  • ultracentrifugation is used clarify the lysate; other methods, such as filtration and using floculents also can be used.
  • Streamline S (PHARMACIA, Sweden) may also be used for large scale preparations.
  • FC78S FGF-nucleic acid binding domain rCCFN2 and l " C96SlFGF-nucleic acid binding domain CCCFN3 [C78SJTGF or [C96SJFGF (1 mg; 56 nmol) that had been dialyzed against phosphate-buffered saline is added to 2.5 mg mono-derivatized nucleic acid binding domain (a 1.5 molar excess over the basic FGF mutants) and left on a rocker platform overnight. The next morning the ultraviolet-visible wavelength spectrum is taken to determine the extent of reaction by the release of pyridylthione, which adsorbs at 343 nm with a known extinction coefficient.
  • reaction mixtures are treated identically for purification in the following manner: reaction mixture is passed over a HiTrap heparin- Sepharose column (1 ml) equilibrated with 0.15 M sodium chloride in buffer A at a flow rate of 0.5 ml/min. The column is washed with 0.6 M NaCl and 1.0 M NaCl in buffer A and the product eluted with 2.0 M NaCl in buffer A. Fractions (0.5 ml) are analyzed by gel electrophoresis and absorbance at 280 nm.
  • Peak tubes are pooled and dialyzed versus 10 mM sodium phosphate, pH 7.5 and applied to a Mono-S 5/5 column equilibrated with the same buffer. A 10 ml gradient between 0 and 1.0 M sodium chloride in equilibration buffer is used to elute the product. Purity is determined by gel electrophoresis and peak fractions were pooled. Under these conditions, virtually 100%) of the mutant FGFs reacts with mono-derivatized myoD. Because the free surface cysteine of each mutant acts as a free sulfhydryl, it is unnecessary to reduce cysteines after purification from the bacteria.
  • the resulting product is purified by heparin-Sepharose (data not shown), thus establish- ing that heparin binding activity of the conjugate is retained.
  • a two-stage method is used to produce recombinant FGF[C78/96S]- myoD protein (hereinafter FPFN4).
  • FPFN4 recombinant FGF[C78/96S]- myoD protein
  • E. coli strains BL21(DE3), BL21(DE3)pLysS, HMS174(DE3) and HMS174(DE3)pLysS were purchased from NOVAGEN, Madison, WI.
  • Plasmid pFC80, described below, has been described in the WIPO International Patent Application No. WO 90/02800, except that the bFGF coding sequence in the plasmid designated pFC80 herein has the sequence set forth as SEQ ID NO. 52, nucleotides 1-465.
  • the plasmids described herein may be prepared using pFC80 as a starting material or, alternatively, by starting with a fragment containing the ell ribosome binding site (SEQ ID NO. 61) linked to the FGF-encoding DNA (SEQ ID NO. 52).
  • E. coli strain JA221 (lpp- hdsM+ tipE5 leuB6 lacY recAl F'[lacl ⁇ - lac + pro + ]) is publicly available from the American Type Culture Collection (ATCC), Rockville, MD 20852, under the accession number ATCC 33875. (JA221 is also available from the Northern Regional Research Center (NRRL), Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604, under the accession number NRRL B-15211; see also U.S. Patent No. 4,757,013 to Inouye; and Nakamura et al., Cell 75:1109-1117, 1979). Strain INVl ⁇ is commercially available from Invitrogen, San Diego, CA.
  • a Nco I restriction site is introduced into the nucleic acid binding domain-encoding DNA by site-directed mutagenesis using the Amersham in vitro- mutagenesis system 2.1.
  • the oligonucleotide employed to create the Nco I restriction site is synthesized using a 380B automatic DNA synthesizer (Applied Biosystems). This oligonucleotide containing the Nco I site replaces the original nucleic acid binding domain-containing coding sequence.
  • Plasmid pFC80 is a derivative of pDS20 (see, e.g., Duester et al., Cell 50:855-864, 1982; see also U.S. Patent Nos. 4,914,027, 5,037,744, 5,100,784, and 5,187,261; see also PCT International Application No. WO 90/02800; and European Patent Application No.
  • Plasmid pKG1800 includes the 2880 bp EcoR l-Pvu II of pBR322 that contains the contains the ampicillin resistance gene and an origin of replication.
  • Plasmid pFC80 is prepared from pDS20 by replacing the entire galK gene with the FGF-encoding DNA of S ⁇ Q ID NO. 52, inserting the t ⁇ promoter (S ⁇ Q ID NO. 62) and the bacteriophage lambda ell ribosome binding site (S ⁇ Q. ID No. 61; see, e.g., Schwarz et al., Nature 272:410, 1978) upstream of and operatively linked to the FGF-encoding DNA.
  • the T ⁇ promoter can be obtained from plasmid pDR720 (Pharmacia PL Biochemicals) or synthesized according to S ⁇ Q ID NO. 62.
  • Plasmid pFC80 contains the 2880 bp EcoR 1-BamH I fragment of plasmid pSD20, a synthetic Sal l-Nde I fragment that encodes the T ⁇ promoter region:
  • the FGF-encoding DNA is removed from pFC80 by treating it as follows.
  • the pFC80 plasmid was digested by Hga l and Sai l, which produces a fragment containing the CII ribosome binding site linked to the FGF-encoding DNA.
  • the resulting fragment is blunt ended with Klenow's reagent and inserted into M13mpl8 that has been opened by Sma l and treated with alkaline phosphatase for blunt-end ligation.
  • an insert in the ORI minus direction is mutagenized using the Amersham kit, as described above, using the following oligonucleotide (S ⁇ Q ID NO.
  • GCTAAGAGCGCCATGGAGA which contains one nucleotide between the FGF carboxy terminal serine codon and a Nco I restriction site; it replaces the following wild type FGF encoding DNA having SEQ ID NO. 64:
  • FGFM13 The resulting mutant derivative of M13mpl8, lacking a native stop codon after the carboxy terminal serine codon of bFGF, was designated FGFM13.
  • the mutagenized region of FGFM13 contained the correct sequence (SEQ ID NO. 65).
  • Plasmid FGFM13 is cut with Nco I and S c I to yield a fragment containing the CII ribosome binding site linked to the bFGF coding sequence with the stop codon replaced.
  • An M13mpl8 derivative containing the myoD coding sequence is also cut with restriction endonucleases Nco I and Sac I, and the bFGF coding fragment from FGFM13 was inserted by ligation to D ⁇ A encoding the fusion protein bFGF- myoD into the M13mpl8 derivative to produce mpFGF- myoD, which contains the CII ribosome binding site linked to the FGF-nucleic acid binding domain fusion gene.
  • Plasmid mpFGF- myoD is digested with Xba I and EcoR I and the resulting fragment containing the bFGF- myoD coding sequence is isolated and ligated into plasmid p ⁇ T-l la (available from ⁇ OVAG ⁇ , Madison, WI; for a description of the plasmids see U.S. Patent No. 4,952,496; see also Studier et al., Meth. Enz. 755:60- 89, 1990; Studier et al., J. Mol. Biol. 759:113-130, 1986; Rosenberg et al., Gene 56: 125- 135, 1987) that has also been treated with EcoR I and Xba I.
  • E. coli strain BL21(D ⁇ 3)pLysS (NOVAGEN, Madison WI) may be transformed with the plasmid containing the fusion gene.
  • Plasmid FGF/myoD may be digested with EcoR I, the ends repaired by adding nucleoside triphosphates and Klenow DNA polymerase, and then digested with Nde I to release the FGF-encoding DNA without the CII ribosome binding site.
  • This fragment is ligated into p ⁇ T 1 la, which is BamH I digested, treated to repair the ends, and digested with Nde I.
  • the resulting plasmid includes the T7 transcription terminator and the pET-1 la ribosome binding site.
  • Plasmid FGF/myoD may be digested with EcoR I and Nde I to release the FGF-encoding DNA without the CII ribosome binding site and ends are repaired as described above. This fragment may be ligated into p ⁇ T 12a, which had been BamH I digested and treated to repair the ends. The resulting plasmid includes DNA encoding the OMP T secretion signal operatively linked to DNA encoding the fusion protein.
  • Protamines are small basic DNA binding proteins, approximately 6.8 kD in molecular weight with a isoelectric point of 12.175. Twenty-four of the fifty one amino acids are strongly basic. Human protamine has been shown to condense genomic DNA for packaging into the sperm head. The positive charges of the protamine react with the negative charges of the phosphate backbone of the DNA.
  • a FGF-protamine fusion protein that has the ability to bind to the FGF receptor and bind DNA with high affinity is constructed for expression in E. coli.
  • the sequence for the human protamine gene is obtained from GenBank (accession no. Y00443). Four overlapping oligonucleotides (60mers) are generated and used to amplify the protamine gene.
  • the amplified product is purified and ligated into the bacterial expression vector p ⁇ Tl la (Novagen). To facilitate subcloning, a Ncol and BamHI site are inco ⁇ orated into the primers.
  • the fragment is synthesized by annealing the 4 oligos (2 sense and 2 antisense) with 20 base overlaps and using PCR to fill-in and amplify the fragments.
  • the PCR products are digested with Ncol and BamHI, and subcloned into pBluescript SK+. The insert sequence is verified. The sequenced product is then cloned downstream and in-frame with FGF2, which has been previously cloned into the p ⁇ Tl la expression plasmid.
  • the oligos used to generate fragment A are (5'-3'):
  • Competent bacterial cells BL21 (DE3) are transformed with the pETl 1- FGF2-protamine construct.
  • the cells are initially plated on LB agar plates containing 100 ⁇ g/ml ampicillin.
  • a glycerol stock made from an individual colony added to 1 ml fresh LB broth and then to 250 ml of LB broth.
  • the cells are grown to an OD 600 of 0.7 and induced with IPTG.
  • the culture is harvested 4 hours after induction. The suspension is centrifuged; the supernatant is saved and the pellet is resuspended in lysis buffer, centrifuged again and the supernatants pooled.
  • Soluble protein is purified. Briefly, the cells are pelleted and resuspended in buffer A (10 mM sodium phosphate, pH 6.0, containing 10 mM EDTA, 10 mM EGTA and 50 mM NaCl) and passed through a microfluidizer (Microfluidics Co ⁇ ., Newton, MA) to break open the bacteria and shear DNA. The resultant mixture is diluted and loaded onto an expanded bed Streamline SP cation-exchange resin. The column is washed with step gradients of increasing concentrations of NaCl.
  • buffer A 10 mM sodium phosphate, pH 6.0, containing 10 mM EDTA, 10 mM EGTA and 50 mM NaCl
  • a microfluidizer Microfluidics Co ⁇ ., Newton, MA
  • the eluted material is analyzed by Western analysis for fractions containing the fusion protein. These fractions are pooled, diluted, and loaded onto a Heparin-Sepharose affinity column. After washing, the bound proteins are eluted in a batch-wise manner in buffer containing 1 M NaCl and then in buffer containing 2 M NaCl. Peak fractions of the 2M elution, as determined by optical density at 280 nm, are pooled and the purity determined by gel electrophoresis and Western analysis. The final pool of material will be loaded onto a column of Sephacryl S-100 equilibrated with 20 mM HEPES pH 7.4, 150 mM NaCl.
  • Fusion protein located in the pellet is isolated, solubilized and refolded. Briefly, each culture pellet is thawed completely and resuspended in buffer A (10 mM Tris, 1 mM EDTA, pH 8.0 + 0.1 mg/ml lyzozyme). The mixture is sonicated on ice, centrifuged at 16,000 X g, and the supernatant discarded.
  • buffer A 10 mM Tris, 1 mM EDTA, pH 8.0 + 0.1 mg/ml lyzozyme
  • Inclusion bodies are solubilized with solubilization buffer: (6 M guanidine-HCl, 100 mM Tris, 150 mM NaCl, 50 mM EDTA, 50 mM EGTA, pH 9.5,), vortexed, incubated for 30 minutes at room temperature, and centrifuged at 35,000 X g for 15 minutes. The supernatant is saved and diluted 1:10 in dilution buffer (100 mM Tris, 10 mM EDTA, 1% monothioglycerol, 0.25 M L-arginine, pH 9.5). The material is stirred, covered, at 4°C for 2 hours and then centrifuged at 35,000 X g for 20 minutes.
  • solubilization buffer 6 M guanidine-HCl, 100 mM Tris, 150 mM NaCl, 50 mM EDTA, 50 mM EGTA, pH 9.5,
  • the supernatant is dialyzed in against 5 liters PBS, pH 8.8, for 24 hours at 4°C with 3 changes of fresh PBS.
  • the material is concentrated approximately 10-fold using size-exclusion spin columns.
  • the soluble refolded material is then analyzed by gel electrophoresis.
  • FGF-protamine fusion protein can be achieved in mammalian cells by excising the insert with restriction enzymes Ndel and BamHI and ligating into a mammalian expression vector.
  • bFGF-nucleic acid binding domain fusion protein (hereinafter bFGF-nucleic acid binding domain fusion protein).
  • pFS92 plasmid-containing bacterial cells (strain BL21(DE3)pLysS) from an overnight culture (1 :100 dilution). Cells are grown at 37°C in an incubator shaker to an OD 600 of 0.7. IPTG (Sigma Chemical, St. Louis, MO) is added to a final concentration of 0.2 mM and growth was continued for 1.5 hours at which time cells were centrifuged.
  • the pellet is resuspended in lysis solution (45-60 ml per 16 g of pellet; 20 mM TRIS, pH 7.4, 5 mM EDTA, 10% sucrose, 150 mM NaCl, lysozyme, 100 ⁇ g/ml, aprotinin, 10 ⁇ g/ml, leupeptin, 10 ⁇ g/ml, pepstatin A, 10 ⁇ g/ml and 1 mM PMSF) and incubated with stirring for 1 hour at room temperature. The solution is frozen and thawed three times and sonicated for 2.5 minutes.
  • the suspension is centrifuged at 12,000 X g for 1 hour; the resulting first-supernatant saved and the pellet is resuspended in another volume of lysis solution without lysozyme.
  • the resuspended material is centrifuged again to produce a second-supernatant, and the two supernatants are pooled and dialyzed against borate buffered saline, pH 8.3.
  • bFGF-nucleic acid binding domain fusion protein elutes from the heparin-Sepharose column at the same concentration (2 M NaCl) as native and recombinantly-produced bFGF, indicating that the heparin affinity is retained in the bFGF-SAP fusion protein.
  • SDS gel electrophoresis is performed on a Phastsystem utilizing 20% acrylamide gels (Pharmacia).
  • Western blotting is accomplished by transfer of the electrophoresed protein to nitrocellulose using the PhastTransfer system (Pharmacia), as described by the manufacturer.
  • Antisera to bFGF is used at a dilution of 1 :1000.
  • Horseradish peroxidase labeled anti-IgG is used as the second antibody (Davis et al.,
  • Anti-FGF antisera should bind to a protein with an approximate molecular weight of 53,000, which corresponds to the sum of the independent molecular weights of nucleic acid binding domain (35,000) and bFGF (18,000).
  • oligos encoding protease substrates Complementary single-stranded oligos in which the sense strand encodes a protease substrate, have been synthesized either using a cyclone machine (Millipore, MA) according the instructions provided by the manufacturer, or were made by Midland Certified Reagent Co. (Midland, TX) or by National BioscienceSj, Inc. (MN). The following oligos have been synthesized.
  • the complementary oligos are annealed by heating at 95 °C for 15 min., cooled to room temperature, and then incubated at 4°C for a minute to about an hour.
  • the oligos are digested with Ncol and ligated overnight at a 3:1 (insert: vector) ratio at 15°C to Ncol-digested plasmid which has been treated with alkaline phosphatase (Boehringer Mannheim).
  • Bacteria ( ⁇ ovablue ( ⁇ OVAGE ⁇ , Madison, WI)) are transformed with the ligation mixture (1 ⁇ l) and plated on LB-amp or LB-Kan, depending upon the plasmid). Colonies are selected, clones isolated and sequenced to determine orientation of the insert. Clones with correct orientation are used to transform strain expression strain BL21(DE3) (NOVAGEN, Madison, WI). Glycerol stocks are generated from single transformed colonies. The transformed strains are cultured as described in Example 2 and fusion proteins with linkers were expressed.
  • exemplary fusion proteins containing cathepsin B substrate (FPFS9), cathepsin D substrate (FPFS5), Gly 4 Ser (FPFS7), (Gly 4 Ser) 2 (FPFS8), trypsin substrate (FPFS6), (Ser 4 Gly) 4 (FPFS12) and (Ser 4 Gly) 2 (FPFS11) linkers, respectively, are set forth in SEQ ID NOs. 72-78.
  • Polylysine polymer with average lengths of 13, 39, 89, 152, and 265 (K 13 , K 39 , K ⁇ , K 152 , K 265 ) are purchased from a commercial vendor (Sigma, St. Louis, MO) and dissolved in 0.1 M NaPO4, 0.1 M NaCl, 1 mM EDTA, pH 7.5 (buffer A) at 3-5 mg/ml.
  • poly-L-lysine solution Approximately 30 mg of poly-L-lysine solution is mixed with 0.187 ml of 3 mg/ml N-succinimidyl-3(pyridyldithio)proprionate (SPDP) in anhydrous ethanol resulting in a molar ratio of SPDP/poly-L-lysine of 1.5 and incubated at room temperature for 30 minutes. The reaction mixture is then dialyzed against 4 liters of buffer A for 4 hours at room temperature.
  • SPDP N-succinimidyl-3(pyridyldithio)proprionate
  • a solution containing 28.5 mg of poly-L-lysine-SPDP is added to 12.9 mg of FGF2-3 ([C96SJ-FGF2) in buffer A and incubated overnight at 4°C.
  • the molar ratio of poly-L-lysine-SPDP/FGF2-3 is approximately 1.5.
  • the conjugation reaction mixture is applied to a 6 ml Resource S (Pharmacia, Uppsala, Sweden) column.
  • a gradient of 0.15 M to 2.1 M NaCl in 20 mM NaPO4, 1 mM EDTA, pH 8.0 (Buffer B) over 24 column volumes is used for elution.
  • the FGF2- 3/poly-L-lysine conjugate is eluted off the column at approximately 1.8-2 M NaCl concentration. Unreacted FGF2-3 is eluted off by 0.5-0.6 M NaCl.
  • the fractions containing FGF2-K are concentrated and loaded onto a gel- filtration column (Sephacryl SI 00) for buffer exchange into 20 mM HEPES, 0.1 M NaCl, pH 7.3.
  • the molecular weight of FGF-K152 as determined by size exclusion HPLC is approximately 42 kD.
  • the chemical conjugate FGF2-K is loaded onto a heparin column and eluted off the column at 1.8- 2.0 M NaCl.
  • unconjugated FGF2-3 is eluted off heparin at 1.4 - 1.6 M NaCl.
  • poly-L-lysine contributes to FGF2-3 ability to bind heparin.
  • the ability of poly-L- lysine 152 to bind heparin is not determined; poly-L-lysine 84 elutes at approximately 1.6 M NaCl.
  • Histone Hl-polylysine was purchased and cytochrome C was conjugated to polylysine as described herein.
  • FGF2-K A sample of FGF2-K is electrophoresed on SDS-PAGE under non- reducing and reducing conditions.
  • the protein migrates at the same molecular weight as FGF.
  • the conjugate does not enter the gel because of its high charge density ( Figure 1, lanes 1, 2, non-reducing; lanes 3, 4, reducing).
  • Optimal conditions for complex formation are established. Varying quantities (0.2 to 200 ⁇ g) of ⁇ -galactosidase encoding plasmid nucleic acid pSV ⁇ or pNASS- ⁇ (lacking a promoter) are slowly mixed with 100 ⁇ g of FGF2-K in 20 mM HEPES pH 7.3, 0.15 M NaCl. The reaction is incubated for 1 hour at room temperature. Nucleic acid binding to the FGF-lysine conjugate is confirmed by gel mobility shift assay using 32 P-labeled SV40- ⁇ -gal nucleic acid cut with Hindi restriction endonuclease.
  • SV40 ⁇ -gal nucleic acid is digested with Hindi restriction endonucleases; ends are labeled by T 4 PNK following dephosphorylation with calf intestinal alkaline phosphatase.
  • To each sample of 35 ng of 32 P-labeled nucleic acid increasing amounts of FGF-polylysine conjugate is added to the mixture.
  • the protein/nucleic acid mixture is electrophoresed in an agarose gel with 1 X TAE buffer. Binding of the conjugate to the radiolabeled DNA is shown by a shift in the complex to the top of the well. (Figure 3.)
  • As seen in Figure 3D as little as 10 ng of K 84 causes a complete shift of restriction fragments indicating binding.
  • With K 13 100 ng of poly-L-lysine was required ( Figure 3C).
  • With K 265 10 ng was required ( Figure 3E).
  • the optimal length of poly-L-lysine and weight ratios is determined by conjugation of FGF2-3 to poly-lysine of different lengths.
  • DNA encoding ⁇ -galactosidase was complexed with the conjugates at 10:1, 5:1, 2:1, 1:1, and 0.5:1 ( Figure 4, lanes 1-5, respectively) (w/w) ratios.
  • the ability of these FGF2-K complexes to bind DNA was determined by measuring the ability of FGF to promote the uptake of plasmid DNA into cells.
  • FGF2-K conjugates were evaluated at various protein to DNA ratios for their ability to deliver pSV ⁇ -gal DNA into cells ( Figure 4).
  • toroid formation which correlates with increased gene expression, was assessed by electron microscopy.
  • a representative toroid at a protein to DNA ratio of 2:1 is shown in Figure 5, upper panel. Toroidal structures are absent, or only partially formed, at low ratios (e.g., 0.5:1) ( Figure 5, lower panel).
  • a proliferation assay is performed to determine if the condensed nucleic acid had an effect on the ability of FGF2-K to bind to cognate receptor and stimulate mitogenesis. The proliferation assay shows that only the highest dose of nucleic acid (200 ⁇ g) has a slightly inhibitory effect on proliferation as compared to FGF2-3 plus poly-L-lysine + DNA (Figure 6).
  • a FGF2-K84-DNA at a protein:DNA ratio of 2:1 is introduced into COS cells and an endothelial cell line, ABAE, both of which express FGF receptors.
  • the cells are subsequently assayed for ⁇ -galactosidase enzyme activity.
  • COS and ABAE cells are grown on coverslips and incubated with the different ratios of FGF2-K:DNA for 48 hours. The cells are then fixed and stained with X-gal. Maximal ⁇ -galactosidase enzyme activity is seen when 50 ⁇ g of pSV ⁇ per 100 ⁇ g of FGF2-3 -polylysine conjugate is used.
  • FGF2-K84- ⁇ SV ⁇ -gal at a protein to DNA ratio of 2:1 was added to various cell lines and incubated for 48 hr.
  • Cell extracts were prepared, assayed for ⁇ -gal activity and total protein.
  • COS, B16, NIH3T3, and BHK cell lines were all able to take up complex and express ⁇ -gal.
  • ⁇ -gal requires FGF2 for targeting into cells.
  • pS V ⁇ or pNASS ⁇ plasmid DNA was incubated with ( Figure 7B, lanes 1, 2) or without (lanes 3, 4) FGF2-K84 for 1 hr at room temperature. Complexes were added to COS cells for 48 hr. Cell extracts were assayed for ⁇ -gal activity and normalized to total protein. Only background ⁇ -gal activity was seen unless the plasmid was complexed with FGF2/K84. Expression of ⁇ -gal is seen to be both time and dose-dependent ( Figures 7C and 7D).
  • Sensitivity of the receptor mediated gene delivery system is determined using the optimized FGF2-K/DNA ratio for complex formation.
  • Increasing amounts of the FGF2-K DNA complex is added to cells.
  • 100 ⁇ g of FGF2-K was mixed with 50 ug of pS V ⁇ for 1 hour at room temperature.
  • the COS and endothelial cells are incubated with increasing amounts of condensed material (0 ng, 1 ng, 10 ng, 100 ng, 1000 ng and 10,000 ng).
  • the cells are incubated for 48 hours and then were assayed for ⁇ -galactosidase activity.
  • cells grown on cover slips are treated with 1000 ng of FGF2-K-DNA for 48 hours, then fixed and stained using X-gal.
  • the ⁇ -gal enzyme assay reveals that with increasing amounts of material there is an increase in enzyme activity.
  • Figure 7D Cells incubated with X-gal show blue staining throughout the cytoplasm in approximately 3% of the cells on the coverslip.
  • FGF2-K84-pSV ⁇ -gal resulted in enzyme activity (lane 1), while only background levels of activity were seen with FGF2+K84+DNA (lane 2), FGF2+DNA (lane 3), K84+DNA (lane 4), DNA (lane 5), FGF2-K84 (lane 6), FGF2 alone (lane 7) and K84 alone (lane 8).
  • the expression of ⁇ -gal is specifically inhibited if free FGF2 is added during transfection (Figure 8B).
  • the addition of heparin attenuates the expression of ⁇ -gal ( Figure 8C).
  • histone HI and cytochrome C were ineffective in delivering pSV ⁇ -gal ( Figure 8C).
  • the peptide INF7 GLF EAIEGFIEN GWEGMIDGWYGC, derived from influenza virus, was synthesized.
  • the INF7 peptide was added 30 minutes later.
  • the complex is added to COS cells and ⁇ -gal activity is assayed 48 or 72 hr later.
  • the amount of free polylysine necessary to neutralize the DNA and allow INF7 to complex was determined. Polylysine was added at 4, 10, or 25 ⁇ g to the FGF2-K84/pSV ⁇ -gal complex. To each of these complexes four different concentrations of INF7 were added. Maximal ⁇ -gal expression was seen with 4 ⁇ g of K84 and 12 ⁇ g of INF7 ( Figure 13 A). When higher amounts of poly-lysine were used, more cell death resulted. The optimal amount of INF7 was determined using 4 ⁇ g of polylysine. As seen in Figure 13B, 24 ⁇ g of INF7 gave maximal ⁇ -gal activity. At 72 hr, 48 ⁇ g of INF7 gave maximal ⁇ -gal activity (approximately 20-32 fold enhancement) (Figure 13C).
  • the cytotoxicity assay measures viable cells after transfection with a cytocide-encoding agent.
  • FGF-2 is the receptor-binding internalized ligand
  • COS7 cells which express FGFR, may be used as targets, and T47D, which does not express a receptor for FGF-2 at detectable levels, may be used as negative control cells.
  • FGF2- K pZ200M a plasmid which expresses saporin
  • Controls include FGF2-K alone, pZ200M alone, and FGF-2 plus poly-L-lysine plus pZ200M.
  • cells are rinsed in PBS lacking Mg 4-1" and Ca "1-1" . Trypsin at 0.1% is added for 10 min and cells are harvested and washed. Cell number from each well is determined by a Coulter particle counter (or equivalent method). A statistically significant decrease in cell number for cells incubated with FGF2-K/pZ200M compared to FGF2-K or pZ200M alone indicates sufficient cytotoxicity.
  • FGF2-polylysine-DNASAP complexes show selective cytotoxicity.
  • saporin in mammalian cells, a synthetic saporin gene using preferred mammalian codons and introduced a "Kozak" sequence for translation initiation. The synthetic gene was then cloned into SV40 promoter and promoterless expression vectors. Because the expression of SAP from SAP-encoding DNA would only be feasible if the mammalian ribosome can synthesize the protein (SAP) prior to its inactivation by the SAP synthesized, the enzymatic activity of saporin encoded by the synthetic gene was tested.
  • SAP mammalian ribosome can synthesize the protein
  • SAP was cloned into a T7/SP6 promoter plasmid and sense RNA was generated using T7 RNA polymerase. The RNA was then added to a mammalian in vitro translation assay. The results from this cell-free in vitro translation assay clearly show that the saporin expressed in a mammalian system can inhibit the expression of protein mutagenesis ( Figure 10).
  • SAP mRNA When added above to the lysate, SAP mRNA is translated into a protein that has the anticipated molecular weight of the saporin protein (lane 2).
  • luciferase mRNA is added to the lysate, a molecule consistent with the luciferase protein is detected (lane 3).
  • FGF2-K was condensed with the pSV40-SAP plasmid DNA at a ratio of 2:1 (w:w).
  • BHK 21 .and NIH 3T3 cells were used as the target cells.
  • the cells (24,000 cells/well) were incubated with either FGF2-K-DNASAP or an FGF2-K-DNA ⁇ -gal complex. After 72 hours of incubation, cell number was determined. As shown in Figure 12, there is a significant decrease in cell number when cells are incubated with the FGF2-K-DNASAP complex compared to cells incubated with the FGF2-K-DNA ⁇ -gal complex.

Abstract

Preparations of conjugates of a receptor-binding internalized ligand and a cytocide-encoding agent and compositions containing such preparations are provided. The conjugates contain a polypeptide that is reactive with an FGF receptor, such as bFGF, or another heparin-binding growth factor, cytokine, or growth factor coupled to a nucleic acid binding domain. One or more linkers may be used in the conjugation. The linker is selected to increase the specificity, toxicity, solubility, serum stability, or intracellular availability, and promote nucleic acid condensation of the targeted moiety. The conjugates are complexed with a cytocide-encoding agent, such as DNA encoding saporin. Conjugates of a receptor-binding internalized ligand to a nucleic acid molecule are also provided.

Description

Description
COMPOSITIONS CONTAINING NUCLEIC
ACIDS AND LIGANDS FOR THERAPEUTIC TREATMENT
Technical Field
The present invention relates generally to the treatment of diseases, and more specifically, to the preparation and use of complexes containing receptor-binding internalized ligands NABD and cytocide-encoding agents to alter the function, gene expression, or viability of a cell in a therapeutic manner.
Background of the Invention
A major goal of treatment of neoplastic diseases and hyperproliferative disorders is to ablate the abnormally growing cells while leaving normal cells untouched. Various methods are under development for providing treatment, but none provide the requisite degree of specificity.
One method of treatment is to provide toxins. Immunotoxins and cytotoxins are protein conjugates of toxin molecules with either antibodies or factors which bind to receptors on target cells. Three major problems may limit the usefulness of immunotoxins. First, the antibodies may react with more than one cell surface molecule, thereby effecting delivery to multiple cell types, possibly including normal cells. Second, even if the antibody is specific, the antibody reactive molecule may be present on normal cells. Third, the toxin molecule may be toxic to cells prior to delivery and internalization. Cytotoxins suffer from similar disadvantages of specificity and toxicity. Another limitation in the therapeutic use of immunotoxins and cytotoxins is the relatively low ratio of therapeutic to toxic dosage. Additionally, it may be difficult to direct sufficient concentrations of the toxin into the cytoplasm and intracellular compartments in which the agent can exert its desired activity.
Given these limitations, cytotoxic therapy has been attempted using viral vectors to deliver DNA encoding the toxins into cells. If eukaryotic viruses are used, such as the retroviruses currently in use, they may recombine with host DNA to produce infectious virus. Moreover, because retro viral vectors are often inactivated by the complement system, use in vivo is limited. Retroviral vectors also lack specificity in delivery; receptors for most viral vectors are present on a large fraction, if not all, cells. Thus, infection with such a viral vector will infect normal as well as abnormal cells. Because of this general infection mechanism, it is not desirable for the viral vector to directly encode a cytotoxic molecule.
While delivery of nucleic acids offers advantages over delivery of cytotoxic proteins such as reduced toxicity prior to internalization, there is a need for high specificity of delivery, which is currently unavailable with the present systems.
In view of the problems associated with gene therapy, there is a compelling need for improved treatments which are more effective and are not associated with such disadvantages. The present invention exploits the use of conjugates which have increased specificity and deliver higher amounts of nucleic acids to targeted cells, while providing other related advantages.
Summary of the Invention
The present invention generally provides therapeutic compositions. In one aspect, the composition has the formula: receptor-binding internalized ligand — nucleic acid binding domai — cytocide-encoding agent. The receptor-binding internalized ligand is a polypeptide reactive with a cell surface receptor, the nucleic acid binding domain binds to a nucleic acid, the cytocide-encoding agent is a nucleic acid molecule encoding a cytocide and which binds to the nucleic acid binding domain, and the composition binds to the cell surface receptor and internalizes the cytocide-encoding agent in cells bearing the receptor. In another aspect, the composition has the formula: receptor-binding internalized ligand-nucleic acid binding comain-prodrug-encoding agent.
In certain embodiments, the receptor-binding internalized ligand is a polypeptide reactive with an FGF receptor, VEGF receptor, HBEGF receptor, or a cytokine. In other embodiments, the cytocide-encoding agent encodes a protein that inhibits protein synthesis and is preferably a ribosome inactivating protein, most preferably saporin. The protein is gelonin or diphtheria toxin in other embodiments. In other embodiments, the prodrug-encoding agent encodes HSV-thymidine kinase. The nucleic acid binding domain is poly-L-lysine in one embodiment. In other embodiments, the nucleic acid binding domain is a transcription factor selected from the group consisting of helix-turn-helix motif proteins, homeodomain proteins, zinc finger motif proteins, steroid receptor proteins, leucine zipper motif proteins, helix- loop-helix motif proteins, and β-sheet motif proteins. In other embodiments, the nucleic acid binding domain binds nonspecifically to nucleic acids and is selected from the group consisting of poly-L-lysine, protamine, histone and spermine. In a preferred embodiment, the nucleic acid binding domain binds the coding region of a ribosome inactivating protein such as saporin. In another preferred embodiment, FGF is conjugated to poly-L-lysine.
In yet other embodiments, the cytocide-encoding agent contains a tissue- specific promoter, such as alpha-crystalline, gamma-crystalline, α-fetoprotein, CEA, prostate-specific antigen, erbB-2, tyrosinase, α-actin, c-myc, VEGF receptor, FGF receptor or cyclin D. In another aspect, the composition also contains a linker. In various embodiments, the linker increases the flexibility of the conjugate and is (GlymSerp)n, (Ala Ala Pro Ala)n, wherein n is 1 to 6, m is 1 to 6 and p is 1 to 4, or the linker is a disulfide bond.
In yet another aspect, the composition has the formula: receptor-binding internalized ligand-cytocide encoding agent-nucleic acid binding domain, wherein the receptor-binding internalized ligand is conjugated to the cytocide-encoding agent, which is bound to the nucleic acid binding domain to form a complex.
In other aspects, the invention provides methods for preventing excessive cell proliferation in the anterior eye following surgery, treating corneal clouding following excimer laser surgery, preventing closure of a trabeculectomy, preventing pterygii recurrence, treating hyperproliferative diseases in the back of the eye, such as macular degeneration, diabetic retinopathy and proliferative virtreal retinopathy, treating smooth muscle cell hyperplasia after a wound healing response to a procedure, e.g., vein grafting, endarterectomies and arterio venous shunts and treating cancer. In these aspects, an effective amount of the compositions described above are administered.
Brief Description of the Drawings
Figure 1 is a photograph of an SDS-PAGE of FGF2-K152 under non- reducing (left) and reducing (right) conditions. Lane 1, FGF2-K152; lane 2, FGF2; lane 3, FGF2-K152: lane 4, FGF2. The open arrow identifies material unable to enter the gel. The closed arrow identifies a protein band corresponding to FGF2. Figure 2 is a graph depicting the proliferation of bovine aortic endothelial cells in response to FGF2 (closed box) and FGF2-K152 (open circle) conjugate.
Figure 3 is a photograph of a gel showing the effects of various lengths of poly-L-lysine on the ability to interact with DNA. Thirty-five ng of labeled DNA were added to increasing concentrations of either FGF2 or FGF2-K: lanes 1, 0 ng; lanes 2, 0.1 ng; lanes 3, 1 ng; lanes 4, 10 ng; lanes 5, 20 ng; lanes 6, 35 ng; lanes 7, 100 ng. Panel A: FGF2; panel B, FGF2-K152; panel C, FGF2-K13; panel D, FGF2- K84; panel E, EGF2-K267; panel F, FGF2-K39. The lengths of the digested DNA are indicated. Figure 4 is a chart depicting the activity of β-gal following transfection of FGF2/poly-L-lysine/DNAβ-gal into COS cells. Lane 1, 10:1 (w/w) ratio of FGF2/poly-L-lysine conjugate to DNA; lane 2, 5:1 ratio; lane 3, 2:1 ratio; lane 4, 1 :1 ratio; lane 5, 0.5:1 ratio. The five bars, from left to right, are FGF2, FGF2-K13, FGF2- K39, FGF2-K84, and FGF2-K152. Figure 5 are photographs of toroid format observed by electron microscopy. The upper panel shows an example of a toroid; the lower panel shows an incomplete toroid.
Figure 6 is a graph depicting proliferation of bovine aortic-endothelial cells. In the upper panel, cells were treated with FGF2-K152-DNA; in the lower panel, cells were treated with a mixture of FGF2, Kl 52, and DNA. Figure 7A is a graph displaying β-gal activity after transfection of FGF2/poly-L-lysine/pSVβ-gal into COS cells (lane 1), B16 cells (lane 2), NIH 3T3 cells (lane 3), and BHK cells (lane 4).
Figure 7B is a graph depicting β-gal expression in COS cells, pSVβ-gal (lanes 1, 3) or pNASSβ-gal (lanes 2, 4) were incubated with (lanes 1, 2) or without (lanes 3, 4) FGF2-K84 and the complexes incubated on COS cells for 48 hrs.
Figure 7C is a graph showing activity of β-gal activity at various times following transfection with either plasmid alone or with complexes of FGF2/K84/pSV β-gal. -Δ-, DNA alone; -■-, FGF2-K84-DNA. Figure 7D is a graph showing β-gal activity after transfection of various concentrations of FGF2/K84/pSVβ-gal. Lane 1, Oμg; lane 2, 0,1 μg; lane 3, lμg; lane 4, 5μg; lane 5, lOμg.
Figure 8A is a graph showing β-gal activity in COS cells following transfection of FGF2-K84-ρSVβ-gal (lane 1), FGF2+K84+pSVβ-gal (lane 2), FGF2+pSVβ-gal (lane 3), K84+pSVβ-gal (lane 4); pSVβ-gal (lane 5), FGF2-K84 (lane 6), FGF2 (lane 7) and K84 (lane 8).
Figure 8B is a graph showing completion for cell bindings. Lane 1, FGF2-K84-pSVβ-gal complex transfected into COS cells; lane 2, FGF2-K84-pSVβ-gal plus 100 μg FGF2; lane 3, no complex. Figure 8C is a graph showing the attenuation of β-gal activity upon the addition of heparin during transfection. Lane 1, FGF2-K84-pSVβ-gal+10μg heparin; lane 2, FGF2-K84-pSVβ-gal; lane 3, heparin alone; lane 4, pSVβ-gal alone.
Figure 8D is a graph showing ligand targeting of DNA, pSVβ-gal DNA alone (lane 1), FGF2-K84 (lane 2), histone H1-K84 (lane 3) and cytochrome C-K84 (lane 4) were condensed with pSVβ-gal DNA and added to BHK cells, β-gal activity was measured 48 hr later.
Figure 9A is a graph showing the effect of chloroquine on β-gal expression, pSVβ-gal and FGF2-K84 were mixed in the absence (lane 1) or presence (lane 2) of 100 μM chloroquine and incubated for 1 hr at room temperature prior to addition of the complexes to COS cells. Lane 3, chloroquine alone; lane 4, DNA alone. Figure 9B is a graph showing the effect of endosome disruptive peptide on β-gal expression. Lane 1, control; lane 2, FGF2-K84-pSVβ-gal; lane 3, FGF2-K84- pSVβ-gal+EDP.
Figure 9C are photographs of cells stained for β-gal activity following transfection of COS cells with (right panel) or without (left panel) endosome disruptive peptide and FGF2-K84-pSVβ-gal.
Figure 10 is a photograph of a fluorograph .analyzing cell-free translation products. Lane 1, no RNA; lane 2, saporin RNA; lane 3, luciferase RNA; lane 4, saporin RNA and luciferase RNA; lane 5, saporin RNA followed 30 min later with luciferase RNA.
Figure 11 is a graph depicting direct cytotoxicity of cells transfected by a CaPO4 with an expression vector encoding saporin. Lane 1, mock transfection; lane 2, transfection with pSVβ-gal; lane 3, transfection with saporin-containing vector.
Figure 12 is a pair of graphs showing cytotoxicity of cells transfected with FGF2-K84-pSVSAP. Left panel, BHK21 cells; right panel, NIH 3T3 cells. Lane 1, FGF2-K84-pSVβ-gal; lane 2, FGF2-K84-pSVSAP.
Figure 13A is a graph showing β-gal activity with an endosome disruptive peptide in the complex.
Figure 13B is a graph showing β-gal activity with an endosome disruptive peptide in the complex.
Figure 13C is a graph showing β-gal activity with an endosome disruptive peptide in the complex.
Detailed Description of the Invention Definitions
All U.S. patents and all publications mentioned herein are incorporated in their entirety by reference thereto.
The "amino acids," which occur in the various amino acid sequences appearing herein, are identified according to their well known, three letter or one letter abbreviations. The nucleotides, which occur in the various DNA fragments, are designated with the standard single letter designations used routinely in the art.
As used herein, to "bind to a receptor" refers to the ability of a ligand to specifically recognize and detectably bind to such receptors, as assayed by standard in vitro assays. For example, as used herein, binding measures the capacity of a VEGF conjugate, VEGF monomer, or VEGF dimer to recognize a VEGF receptor on a vascular endothelial cell, such as an aortic vascular endothelial cell line, using a procedure substantially as described in Moscatelli, J Cell Physiol. 757:123-130, 1987. As used herein, "biological activity" refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity thus encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Such biological activity may be defined with reference to particular in vitro activities as measured in a defined assay. For example, reference herein to the biological activity of FGF, or fragments of FGF, refers to the ability of FGF to bind to cells bearing FGF receptors and internalize a linked agent. Such activity is typically assessed in vitro by linking the FGF to a cytotoxic agent, such as saporin, contacting cells bearing FGF receptors, such as fibroblasts, with the conjugate and assessing cell proliferation or growth. In vivo activity may be determined using recognized animal models, such as the mouse xenograft model for anti -tumor activity (see, e.g., Beitz et al., Cancer Research 52:227-230, 1992; Houghton et al., Cancer Res. ¥2:535-539, 1982; Bogden et al., Cancer (Philadelphia) 4-5:10-20, 1981; Hoogenhout et al., Int. J. Radiat. Oncol, Biol. Phys. 9:871-879, 1983; Stastny et al., Cancer Res. 55:5740-5744, 1993). As used herein, reference to the "biological activity of a cytocide- encoding agent," such as DNA encoding saporin, refers to the ability of such agent to interfere with the metabolism of the cell by inhibiting protein synthesis. Such biological or cytotoxic activity may be assayed by any method known to those of skill in the art including, but not limited to, in vitro assays that measure protein synthesis and in vivo assays that assess cytotoxicity by measuring the effect of a test compound on cell proliferation or on protein synthesis. Assays that assess cytotoxicity in targeted cells are particularly preferred.
As used herein, a "conjugate" refers to a molecule that contains at least one receptor-internalized binding ligand and at least one nucleic acid binding domain that are linked directly or via a linker and that are produced by chemical coupling methods or by recombinant expression of chimeric DNA molecules to produce fusion proteins.
A "cytocide-encoding agent" is a nucleic acid molecule that encodes a protein that inhibits protein synthesis. Such a protein may act by cleaving rRNA or ribonucloprotein, inhibiting an elongation factor, cleaving mRNA, or other mechanism that reduces protein synthesis to a level such that the cell cannot survive. The cytocide- encoding agent may contain additional elements besides the cytocide gene. Such elements include a promoter, enhancer, splice sites, transcription terminator, poly(A) signal sequence, bacterial or mammalian origins of replication, selection markers, and the like.
As used herein, the term "cytotoxic agent" refers to a molecule capable of inhibiting cell function. The agent may inhibit proliferation or may be toxic to cells. A variety of cytotoxic agents can be used and include those that inhibit protein synthesis and those that inhibit expression of certain genes essential for cellular growth or survival. Cytotoxic agents include those that result in cell death and those that inhibit cell growth, proliferation and/or differentiation.
As used herein, cytotoxic agents include, but are not limited to, saporin, the ricins, abrin and other ribosome inactivating proteins (RIPs), aquatic-derived cytotoxins, Pseudomonas exotoxin, inhibitors of DNA, RNA or protein synthesis, such as antisense nucleic acids, other metabolic inhibitors, such as DNA cleaving molecules, prodrugs, such as thymidine kinase from HSV and bacterial cytosine deaminase, and light activated porphyrin. While saporin is the preferred RIP, other suitable RIPs include ricin, ricin A chain, maize RIP, gelonin, diphtheria toxin, diphtheria toxin A chain, trichosanthin, tritin, pokeweed antiviral protein (PAP), mirabilis antiviral protein (MAP), Dianthins 32 and 30, abrin, monordin, bryodin, shiga, a catalytic inhibitor of protein biosynthesis from cucumber seeds (see, e.g., WO 93/24620), Pseudomonas exotoxin, biologically active fragments of cytotoxins and others known to those of skill in this art. Suitable cytotoxic agents also include cytotoxic molecules that inhibit cellular metabolic processes, including transcription, translation, biosynthetic or degradative pathways, DNA synthesis, and other such processes that kill cells or inhibit cell proliferation.
"Heparin-binding growth factor" refers to any member of a family of heparin-binding growth factor proteins, in which at least one member of the family binds heparin. Preferred growth factors in this regard include FGF, VEGF, and HBEGF. Such growth factors encompass isoforms, peptide fragments derived from a family member, splice variants, and single or multiple exons, some forms of which may not bind heparin.
As used herein, to "hybridize" under conditions of a specified stringency is used to describe the stability of hybrids formed between two single-stranded nucleic acid molecules. Stringency of hybridization is typically expressed in conditions of ionic strength and temperature at which such hybrids are annealed and washed. Typically high, medium and low stringency encompass the following conditions or equivalent conditions thereto:
1) high stringency: 0.1 x SSPE or SSC, 0.1% SDS, 65°C 2) medium stringency: 0.2 x SSPE or SSC, 0.1% SDS, 50°C
3) low stringency: 1.0 x SSPE or SSC, 0.1 % SDS, 50°C. "Nucleic acid binding domain" (NABD) refers to a molecule, usually a protein, polypeptide, or peptide (but may also be a polycation) that binds nucleic acids, such as DNA or RNA. The NABD may bind to single or double strands of RNA or DNA or mixed RNA DNA hybrids. The nucleic acid binding domain may bind to a specific sequence or bind irrespective of the sequence.
As used herein, "nucleic acids" refer to RNA or DNA that are intended for internalization into a cell and includes, but are not limited to, DNA encoding a therapeutic protein, DNA encoding a cytotoxic protein, DNA encoding a prodrug, DNA encoding a cytocide, the complement of these DNAs, an antisense nucleic acid and other such molecules. Reference to nucleic acids includes duplex DNA, single-stranded DNA, RNA in any form, including triplex, duplex or single-stranded RNA, anti-sense RNA, polynucleotides, oligonucleotides, single nucleotides, chimeras, and derivatives thereof. Nucleic acids may be composed of the well-known deoxyribonucleotides and ribonucleotides composed of the bases adenosine, cytosine, guanine, thymidine, and uridine. As well, various other nucleotide derivatives and non-phosphate backbones or phosphate-derivative backbones may be used. For example, because normal phosphodiester oligonucleotides (referred to as PO oligonucleotides) are sensitive to DNA- and RNA-specific nucleases, several resistant types of oligonucleotides have been developed in which the phosphate group has been altered to a phosphotriester, methylphosphonate, or phosphorothioate (see U.S. Patent No. 5,218,088).
As used herein, "operative linkage" or operative association of heterologous DNA to regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, refers to the functional relationship between such DNA and such sequences of nucleotides. For example, operative linkage of heterologous DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA in reading frame.
As used herein, the term "polypeptide reactive with .an FGF receptor" refers to any polypeptide that specifically interacts with an FGF receptor, preferably the high-affinity FGF receptor and that is transported into the cell by virtue of its interaction with the FGF receptor. Polypeptides reactive with an FGF receptor are also called FGF proteins. Such polypeptides include, but are not limited to, FGF-1 to FGF- 9. For example, bFGF (FGF-2) should be generally understood to refer to polypeptides having substantially the same amino acid sequences and receptor-targeting activity as that of bovine bFGF or human bFGF. It is understood that differences in amino acid sequences can occur among FGFs of different species as well as among FGFs from individual organisms or species. In addition, chimeras or hybrids of any of FGF-1 through FGF-9, or FGFs that have deletions (see, e.g., PCT Application No. WO 90/02800), insertions or substitutions of amino acids are within the scope of FGF proteins, as long as the resulting peptide or protein specifically interacts with an FGF receptor and is internalized by virtue of this interaction. As used herein, a "prodrug" is a compound that metabolizes or otherwise converts an inactive, nontoxic compound to a biologically, pharmaceutically, therapeutically, of toxic active form of the compound. A prodrug may also be a pharmaceutically inactive compound that is modified upon administration to yield an active compound through metabolic or other processes. The prodrug may alter the metabolic stability or the transport characteristics of a drug, mask side effects or toxicity, improve or alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design inactive forms of the compound (see, e.g., Nogrady, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, 1985).
As used herein, "receptor-binding internalized ligand" or "ligand" refers to any peptide, polypeptide, protein or non-protein, such as a peptidomimetic, that is capable of binding to a cell-surface molecule and is internalized. Within the context of this invention, the receptor-binding internalized ligand is conjugated to a nucleic acid binding domain, either as a fusion protein or through chemical conjugation, and is used to deliver a cytocide-encoding or pro-drug encoding agent to a cell. In one aspect, the ligand is directly conjugated to a nucleic acid molecule, which may be further complexed with a nucleic acid binding domain. Such ligands include growth factors, cytokines, antibodies or fragments thereof, hormones, and the like. As used herein, "saporin" (abbreviated herein as SAP) refers to polypeptides that are isolated from the leaves or seeds of Saponaria officinalis, as well as modified forms that have amino acid substitutions, deletions, insertions or additions, which still express substantial ribosome inactivating activity. Purified preparations of saporin are frequently observed to include several molecular isoforms of the protein. It is understood that differences in amino acid sequences can occur in saporin from different species as well as between saporin molecules from individual organisms of the same species. Saporin for use herein may be purified from leaves, chemically synthesized, or synthesized by expression of DNA encoding a saporin polypeptide.
As used herein, a "targeted agent" is a nucleic acid molecule that is intended for internalization by complexing or linkage to a receptor-binding internalized ligand, and nucleic acid binding domain, and that upon internalization in some manner alters or affects cellular metabolism, growth, activity, viability or other property or characteristic of the cell.
As used herein, a "therapeutic nucleic acid" describes any nucleic acid molecule used in the context of the invention that modifies gene transcription or translation. This term also includes nucleic acids that bind to sites on proteins. It includes, but is not limited to, the following types of nucleic acids: nucleic acids encoding a protein, antisense RNA, DNA intended to form triplex molecules, extracellular protein binding oligonucleotides, and small nucleotide molecules. A therapeutic nucleic acid may be used to effect genetic therapy by serving as a replacement for a defective gene, by encoding a therapeutic product, such as TNF, or by encoding a cytotoxic molecule, especially an enzyme, such as saporin. The therapeutic nucleic acid may encode all or a portion of a gene, and may function by recombining with DNA already present in a cell, thereby replacing a defective portion of a gene. It may also encode a portion of a protein and exert its effect by virtue of co-suppression of a gene product.
PREPARATION OF RECEPTOR-BLNDING INTERNALIZED LIGAND, NUCLEIC ACID BINDING DOMAIN AND CYTOCIDE-ENCODING AGENT COMPLEXES As noted above, the present invention provides cytocide-encoding agents complexed with a conjugate of a receptor-binding internalized ligand and a nucleic acid binding domain. Upon binding to an appropriate receptor, the complex is internalized by the cell and is trafficked through the cell via the endosomal compartment, where at least a portion of the complex may be cleaved. A. Receptor-binding internalized ligands
As noted above, receptor-binding internalized ligands are used to deliver a cytocide-encoding agent to a cell expressing an appropriate receptor on its cell surface. Numerous molecules that bind specific receptors have been identified and are suitable for use in the present invention. Such molecules include growth factors, cytokines, and antibodies. Many growth factors and families of growth factors share structural and functional features and may be used in the present invention. One such family of growth factors specifically binds to heparin. The ability of heparin-binding growth factors to interact with heparin appears in general to be a reflection of a physiologically more relevant interaction occurring in vivo between these factors and heparin sulfate proteoglycan molecules, which are found on the surface of cells and in extracellular matrix. Heparin-binding growth factors include the fibroblast growth factors FGF-1 through FGF-9, vascular endothelial growth factor (VEGF), and heparin binding-epidermal growth factor (HBEGF). Antibodies that are specific to cell surface molecules expressed by a selected cell type are readily generated as monoclonals or polyclonal antisera. Many such antibodies are available (e.g., American Type Culture Collection, Rockville, MD). Other growth factors, such as PDGF (platelet-derived growth factor), EGF (epidermal growth factor), TGF-α (tumor growth factor), TGF-β, IGF-I (insulin-like growth factor), and IGF-II also bind to specific identified receptors on cell surfaces and may be used in the present invention. Cytokines, including interleukins, CSFs (colony stimulating factors), and interferons, have specific receptors, which are mostly found on hematopoeitic cells, and may be used as described herein. These ligands are discussed in more detail below.
Fragments of these ligands may be used within the present invention, so long as the fragment retains the ability to bind to the appropriate cell surface molecule. Likewise, ligands with substitutions or other alterations, but which retain binding ability, may also be used.
1. Fibroblast growth factors One family of growth factors that has a broad spectrum of activities is the fibroblast growth factor (FGF) family. These proteins share the ability to bind to heparin, induce intracellular receptor-mediated tyrosine phosphorylation and the expression of the c-fos mRNA transcript, and stimulate DNA synthesis and cell proliferation. This family of proteins includes FGFs designated FGF-1 (acidic FGF (aFGF)), FGF-2 (basic FGF (bFGF)), FGF-3 (int-2) (see, e.g., Moore et al., EMBO J. 5:919-924, 1986), FGF-4 (hst-1/K-FGF) (see, e.g., Sakamoto et al., Proc. Natl. Acad. Sci. USA 56:1836-1840, 1986; U.S. Patent No. 5,126,323), FGF-5 (see, e.g., U.S. Patent No. 5,155,217), FGF-6 (hst-2) (see, e.g., published European Application EP 0 488 196 A2; Uda et al., Oncogene 7:303-309, 1992), FGF-7 (keratinocyte growth factor) (KGF) (see, e.g., Finch et al, Science 245:752-755, 1985; Rubin et al., Proc. Natl. Acad. Sci. USA 56:802-806, 1989; and International Application WO 90/08771), FGF-8 (see, e.g., Tanaka et al., Proc Natl. Acad. Sci. USA 59:8528-8532, 1992); and FGF-9 (see, Miyamoto et al., Mol. Cell. Biol. 75:4251-4259, 1993).
DNA encoding FGF peptides and/or the amino acid sequences of FGFs are known to those of skill in the art. DNA encoding an FGF may be prepared synthetically based on a known amino acid or DNA sequence, isolated using methods known to those of skill in the art, or obtained from commercial or other sources. DNA encoding virtually all of the FGF family of peptides is known. For example, DNA encoding human FGF-1 (Jaye et al., Science 255:541-545, 1986; U.S. Patent No. 5,223,483), bovine FGF-2 (Abraham et al., Science 255:545-548, 1986; Esch et al., Proc. Natl. Acad. Sci. USA 52:6507-6511, 1985; and U.S. Patent No. 4,956,455), human FGF-2 (Abraham et al., EMBO J. 5:2523-2528, 1986; U.S. Patent No. 4,994,559; U.S. Patent No. 5,155,214; EP 470,183B; and Abraham et al., Quant. Biol. 57:657-668, 1986) and rat FGF-2 (see Shimasaki et al., Biochem. Biophys. Res. Comm., 1988, and Kurokawa et al., Nucleic Acids Res. 16:5201, 1988), FGF-3, FGF-6, FGF-7 and FGF-9 are known (see also U.S. Patent No. 5,155,214; U.S. Patent No. 4,956,455; U.S. Patent No. 5,026,839; U.S. Patent No. 4,994,559, EP 0,488,196 A2, DNASTAR, EMBL or GenBank databases, and references discussed above and below). DNA encoding an FGF may be produced from any of the preceding DNA fragments by substitution of degenerate codons. It is understood that once the complete amino acid sequence of a peptide, such as an FGF peptide, and the DNA fragment encoding such peptide are available to those of skill in this art, it is routine to substitute degenerate codons and produce any of the possible DNA fragments that encode such peptide. It is also generally possible to synthesize DNA encoding such peptide based on the amino acid sequence. Thus, as used herein, "FGF" refers to polypeptides having amino acid sequences of native FGF proteins, as well as modified sequences, having amino acid substitutions, deletions, insertions or additions in the native protein but retaining the ability to bind to FGF receptors and to be internalized. It is understood that differences in amino acid sequences can occur among FGFs of different species as well as among FGFs from individual organisms or species.
Reference to FGFs is intended to encompass proteins isolated from natural sources as well as those made synthetically, as by recombinant means or possibly by chemical synthesis. FGF also encompasses muteins that possess the ability to bind to FGF-receptor expressing cells. Such muteins include, but are not limited to, those produced by replacing one or more of the cysteines with serine as described herein or that have any other amino acids deleted or replaced as long as the resulting protein has the ability to bind to FGF-receptor bearing cells and internalize the linked targeted agent. Typically, such muteins will have conservative amino acid changes, such as those set forth below in Table 1. DNA encoding such muteins will, unless modified by replacement of degenerate codons, hybridize under conditions of at least low stringency to native DNA sequence encoding the starting FGF.
Acidic and basic FGF are about 55% identical at the amino acid level and are highly conserved among species. The other members of the FGF family have a high degree of amino acid sequence similarities and common physical and biological properties with FGF-1 and FGF-2, including the ability to bind to one or more FGF receptors. Basic FGF, int-2, hst-1 K-FGF, FGF-5, hst-2/FGF-6 and FGF-8 may have oncogenic potential; bFGF is expressed in melanomas, int-2 is expressed in mammary tumor virus and hst-1/K-FGF is expressed in angiogenic tumors. Acidic FGF, bFGF, KGF and FGF-9 are expressed in normal cells and tissues. FGFs exhibit a mitogenic effect on a wide variety of mesenchymal, endocrine and neural cells and are also important in differentiation and development. Of particular interest is their stimulatory effect on collateral vascularization and angiogenesis. In some instances, FGF-induced mitogenic stimulation may be detrimental. For example, cell proliferation and angiogenesis are an integral aspect of tumor growth. Members of the FGF family, including bFGF, are thought to play a pathophysiological role, for example, in tumor development, rheumatoid arthritis, proliferative diabetic retinopathies and other complications of diabetes.
The effects of FGFs are mediated by high affinity receptor tyrosine kinases present on the cell surface of FGF-responsive cells (see, e.g., PCT WO 91/00916, WO 90/05522, PCT WO 92/12948; Imamura et al., Biochem. Biophys. Res. Comm. 755:583-590, 1988; Huang et al., J. Biol. Chem. 267:9568-9571, 1986; Partanen et al., EMBO J. 10:1347, 1991; and Moscatelli, J Cell. Physiol. 757:123, 1987). Lower affinity receptors also appear to play a role in mediating FGF activities. The high affinity receptor proteins are single chain polypeptides with molecular weights ranging from 110 to 150 kD, depending on cell type that constitute a family of structurally related FGF receptors. Four FGF receptor genes have been identified, and three of these genes generate multiple mRNA transcripts via alternative splicing of the primary transcript.
2. Vascular endothelial growth factors
Vascular endothelial growth factors (VEGFs) were identified by their ability to directly stimulate endothelial cell growth, but do not appear to have mitogenic effects on other types of cells. VEGFs also cause a rapid and reversible increase in blood vessel permeability. The members of this family have been referred to variously as vascular endothelial growth factor (VEGF), vascular permeability factor (VPF) and vasculotropin (see, e.g., Plouet et al., EMBO J. 5:3801-3806, 1989). Herein, they are collectively referred to as VEGF.
VEGF was originally isolated from a guinea pig heptocarcinoma cell line, line 10 (see, e.g., U.S. Patent No. 4,456,550), and has subsequently been identified in humans and in normal cells. It is expressed during normal development and in certain normal adult organs. Purified VEGF is a basic, heparin-binding, homodimeric glycoprotein that is heat-stable, acid-stable and may be inactivated by reducing agents.
DNA sequences encoding VEGF and methods to isolate these sequences may be found primarily in U.S. Patent No. 5,240,848, U.S. Patent No. 5,332,671, U.S. Patent No. 5,219,739, U.S. Patent No. 5,194,596, and Houch et al., Mol. Endocrin. 5:180, 1991. As used herein, "DNA encoding a VEGF peptide or polypeptide" refers to any of the DNA fragments set forth herein as coding such peptides, to any such DNA fragments known to those of skill in the art, any DNA fragment that encodes a VEGF that binds to a VEGF receptor and is internalized thereby. VEGF DNA may be isolated from a human cell library, for example, using any of the preceding DNA fragments as a probe or any DNA fragment that encodes any of the VEGF peptides set forth in SEQ ID NOs. 1-4. It is understood that once the complete amino acid sequence of a peptide, such as a VEGF peptide, and the DNA fragment encoding such peptide are available to those of skill in this art, it is routine to substitute degenerate codons and produce any of the possible DNA fragments that encode such peptide. It is also generally possible to synthesize DNA encoding such peptide based on the amino acid sequence.
VEGF family members arise from a single gene organized as eight exons and spanning approximately 14 kb in the human genome. Four molecular species of VEGF result from alternative splicing of mRNA and contain 121, 165, 189 and 206 amino acids. The four species have similar biological activities, but differ markedly in their secretion patterns. The predominant isoform secreted by a variety of normal and transformed cells is VEGF165. Transcripts encoding VEGF121 and VEGFlg9 are detectable in most cells and tissues that express the VEGF gene. In contrast, VEGF206 is less abundant and has been identified only in a human fetal liver cDNA library. VEGF121 is a weakly acidic polypeptide that lacks the heparin binding domain and, consequently, does not bind to heparin. VEGFI89 and VEGF206 are more basic than VEGF165 and bind to heparin with greater affinity. Although not every identified VEGF isoform binds heparin, all isoforms are considered to be heparin-binding growth factors within the context of this invention. The secreted isoforms, VEGF,21 and VEGF165 are preferred VEGF proteins. The longer isoforms, VEGF,89 and VEGF206, are almost completely bound to the extracellular matrix and need to be released by an agent, such as suramin, heparin or heparinase, or plasmin. Other preferred VEGF proteins contain various combinations of VEGF exons, such that the protein still binds VEGF receptor and is internalized. It is not necessary that a VEGF protein used in the context of this invention either retain any of its in vivo biological activities, such as stimulating endothelial cell growth, or bind heparin. It is only necessary that the VEGF protein or fragment thereof bind the VEGF receptor and be internalized into the cell bearing the receptor. However, it may be desirable in certain contexts for VEGF to manifest certain of its biological activities. For example, if VEGF is used as a carrier for DNA encoding a molecule useful in wound healing, it would be desirable that VEGF exhibit vessel permeability activity and promotion of fibroblast migration and angiogenesis. It will be apparent from the teachings provided within the subject application which of the activities of VEGF are desirable to maintain.
VEGF promotes an array of responses in endothelium, including blood vessel hyperpermeability, endothelial cell growth, angiogenesis, and enhanced glucose transport. VEGF stimulates the growth of endothelial cells from a variety of sources (including brain capillaries, fetal and adult aortas, and umbilical veins) at low concentrations, but is reported to have no effect on the growth of vascular smooth muscle cells, adrenal cortex cells, keratinocytes, lens epithelial cells, or BHK-21 fibroblasts. VEGF also is a potent polypeptide regulator of blood vessel function; it causes a rapid but transient increase in microvascular permeability without causing endothelial cell damage or mast cell degranulation, and its action is not blocked by antihistamines. VEGF has also been reported to induce monocyte migration and activation and has been implicated as a tumor angiogenesis factor in some human gliomas. Also, VEGF is a chemoattractant for monocytes and VEGF has been shown to enhance the activity of the inflammatory mediator tumor necrosis factor (TNF).
Quiescent and proliferating endothelial cells display high-affinity binding to VEGF, and endothelial cell responses to VEGF appear to be mediated by high affinity cell surface receptors (see, e.g., International Application WO 92/14748, which is based on U.S. Applications Serial No. 08/657,236, de Vries et al., Science 255:989-91, 1992; Terman et al., Biochem. Biophys. Res. Commun. 757:1579-1586, 1992; Kendall et al., Proc. Natl. Acad. Sci. USA 90:10705-10709, 1993; and Peters et al., Proc. Natl. Acad. Sci. USA 90:8915-8919, 1993). Two tyrosine kinases have been identified as VEGF receptors. The first, known as fins-like tyrosine kinase or FLT is a receptor tyrosine kinase that is specific for VEGF. In adult and embryonic tissues, expression of FLT mRNA is localized to the endothelium and to populations of cells that give rise to endothelium. The second receptor, KDR (human kinase insert domain- containing receptor), and its mouse homologue FLK-1, are closely related to FLT. The KDR FLK-1 receptor is expressed in endothelium during the fetal growth stage, during earlier embryonic development, and in adult tissues. In addition, messenger RNA encoding FLT and KDR have been identified in tumor blood vessels and specifically by endothelial cells of blood vessels supplying glioblastomas. Similarly, FLT and KDR mRNAs are upregulated in tumor blood vessels in invasive human colon adenocarcinoma, but not in the blood vessels of adjacent normal tissues.
3. Heparin-binding epidermal growth factors
Several new mitogens in the epidermal growth factor protein family have recently been identified that display the ability to bind the glycosaminoglycan heparin. Among these is the mitogen known as heparin-binding EGF-like growth factor (HBEGF), which elutes from heparin-Sepharose™ columns at about 1.0 - 1.2 M NaCl and which was first identified as a secreted product of cultured human monocytes, macrophages, and the macrophage-like U-937 cell line (Higashiyama et al., Science 257:936-939, 1991; Besner et al., Cell Regul. 7:811-19, 1990). HBEGF has been shown to interact with the same high affinity receptors as EGF on bovine aortic smooth muscle cells and human A431 epidermoid carcinoma cells (Higashiyama, Science 257:936-939, 1991).
HBEGFs exhibit a mitogenic effect on a wide variety of cells including BALB/c 3T3 fibroblast cells and smooth muscle cells, but unlike VEGFs, are not mitogenic for endothelial cells (Higashiyama et al., Science 257:936-939, 1991). HBEGF also has a stimulatory effect on collateral vascularization and angiogenesis. Members of the HBEGF family are thought to play a pathophysiological role, for example, in a variety of tumors, such as bladder carcinomas, breast tumors and non- small cell lung tumors. Thus, these cell types are likely candidates for delivery of cytocide-encoded agents.
HBEGF isolated from U-937 cells is heterogeneous in structure and contains at least 86 amino acids and two sites of O-linked glycosyl groups (Higashiyama et al., J. Biol. Chem. 267:6205-6212, 1992). The carboxyl-terminal half of the secreted HBEGF shares approximately 35% sequence identity with human EGF, including six cysteines spaced in the pattern characteristic of members of the EGF protein family. In contrast, the amino-terminal portion of the mature factor is characterized by stretches of hydrophilic residues and has no structural equivalent in EGF. Site-directed mutagenesis of HBEGF and studies with peptide fragments have indicated that the heparin-binding sequences of HBEGF reside primarily in a 21 amino acid stretch upstream of and slightly overlapping the EGF-like domain.
The effects of HBEGFs are mediated by EGF receptor tyrosine kinases expressed on cell surfaces of HBEGF-responsive cells (see, e.g., U.S. Patent Nos. 5,183,884 and 5,218,090; and Ullrich et al., Nature 509:4113-425, 1984). The EGF receptor proteins, which are single chain polypeptides with molecular weights 170 kD, constitute a family of structurally related EGF receptors. Cells known to express the EGF receptors include smooth muscle cells, fibroblasts, keratinocytes, and numerous human cancer cell lines, such as the: A431 (epidermoid); KB3-1 (epidermoid); COLO 205 (colon); CRL 1739 (gastric); HEP G2 (hepatoma); LNCAP (prostate); MCF-7 (breast); MDA-MB-468 (breast); NCI 417D (lung); MG63 (osteosarcoma); U-251 (glioblastoma); D-54MB (glioma); and SW-13 (adrenal).
For the purposes of this invention, HBEGF need only bind a specific HBEGF receptor and be internalized. Any member of the HBEGF family, whether or not it binds heparin, is useful within the context of this invention as long as it meets the requirements set forth above. Members of the HBEGF family are those that have sufficient nucleotide identity to hybridize under normal stringency conditions (typically greater than 75% nucleotide identity). Subfragments or subportions of a full-length HBEGF may also be desirable. One skilled in the art may find from the teachings provided within that certain biological activities are more or less desirable, depending upon the application. DNA encoding an HBEGF peptide or polypeptide refers to any DNA fragment encoding an HBEGF, as defined above. Exemplary DNA fragments include: any such DNA fragments known to those of skill in the art; any DNA fragment that encodes an HBEGF or fragment that binds to an HBEGF receptor and is internalized thereby; and any DNA fragment that encodes any of the HBEGF polypeptides set forth in SEQ ID NOs. 5-8. Such DNA sequences encoding HBEGF fragments are available from publicly accessible databases, such as: EMBL, GenBank (Accession Nos. M93012 (monkey) and M60278 (human)); the plasmid pMTN-HBEGF (ATCC #40900) and pAX-HBEGF (ATCC #40899) (described in PCT Application WO/92/06705); and Abraham et al., Biochem. Biophys. Res. Comm. 790:125-133, 1993). Unless modified by replacement of degenerate codons, DNA encoding HBEGF polypeptides will hybridize under conditions of at least low stringency to DNA encoding a native human HBEGF (e.g., SEQ ID NO. 9). In addition, any DNA fragment that may be produced from any of the preceding DNA fragments by substitution of degenerate codons is also contemplated for use herein. It is understood that since the complete amino acid sequence of HBEGF polypeptides, and DNA fragments encoding such peptides, are available to those of skill in this art, it is routine to substitute degenerate codons and produce any of the possible DNA fragments that encode such HBEGF polypeptides. It is also generally possible to synthesize DNA encoding such peptides based on the amino acid sequence.
4. Other receptor-binding internalized ligands
Other receptor-binding ligands may be used in the present invention. Any protein, polypeptide, analogue, or fragment that binds to a cell-surface receptor and is internalized may be used. In general, in addition to the specific heparin-binding growth factors discussed above, other growth factors and cytokines are especially well suited for use. These ligands may be produced by recombinant or other means in preparation for conjugation to the nucleic acid binding domain. The DNA sequences and methods to obtain the sequences of these receptor-binding internalized ligands are well known. For example, these ligands include CSF-1 (GenBank Accession Nos. Ml 1038, M37435; Kawasaki et al., Science 250:291-296, 1985; Wong et al., Science 255:1504-1508, 1987); GM-CSF (GenBank Accession No. X03021; Miyatake et al., EMBO J. 4:2561-2568, 1985); IFN-α (interferon) (GenBank Accession No. A02076; Patent No. WO 8502862-A, July 4, 1985); IFN-γ (GenBank Accession No. A02137; Patent No. WO 8502624-A, June 20, 1985); hepatocyte growth factor (GenBank Accession No. X16323, S80567, X57574; Nakamura, et al, Nature 342:440-443, 1989; Nakamura et al., Prog. Growth Factor Res. 5:67-85, 1991; Miyazawa et al., Eur. J. Biochem. 797:15-22, 1991); IGF-Ia (Insulin-like growth factor la) (GenBank Accession No. X56773, S61841; Sandberg-Nordqvist et al., Brain Res. Mol. Brain Res. 12:275- 277, 1992; Sandberg, Sandberg-Nordqvist et al, Cancer Res. 55:2475-2478, 1993); IGF-Ib (GenBank Accession No. X56774 S61860; Sandberg-Nordqvist et al., Brain Res. Mol. Brain Res. 72:275-277, 1992; Sandberg-Nordqvist, A.C., Cancer Res. 55:2475-2478, 1993); IGF-I (GenBank Accession No. X03563, M29644; Dull et al., Nature 570:771-781, 1984; Rail et al., Meth. Enzymol. 746:239-248, 1987); IGF-II (GenBank Accession No. J03242; Shen et al., Proc. Natl. Acad. Sci. USA 55:1947- 1951, 1988); IL-l-α (interleukin 1 alpha) (GenBank Accession No. X02531, Ml 5329; March et al., Nature 575:641-647, 1985; Nishida et al., Biochem. Biophys. Res. Commun. 143:345-352, 1987); IL-l-β (interleukin 1 beta) (GenBank Accession No. X02532, M15330, M15840; March et al., Nature 575:641-647, 1985; Nishida et al., Biochem. Biophys. Res. Commun. 143:345-352, 1987; Bensi et al., Gene 52:95-101, 1987); IL-1 (GenBank Accession No. K02770, M54933, M38756; Auron et al, Proc. Natl. Acad. Sci. USA 57:7907-791 1, 1984; Webb et al., Adv. Gene Technol. 22:339-340, 1985); IL-2 (GenBank Accession No. A14844, A21785, X00695, X00200, X00201, X00202; Lupker et al., Patent No. EP 0307285-A, March 15, 1989; Perez et al., Patent No. EP 0416673-A, March 13, 1991; Holbrook et al., Nucleic Acids Res. 72:5005-5013, 1984; Degrave et al., EMBO J. 2:2349-2353, 1983; Taniguchi et al., Nature 502:305- 310, 1983); IL-3 (GenBank Accession No. M14743, M20137; Yang et al., Cell 47:3-10, 1986; Otsuka et al., J. Immunol. 740:2288-2295, 1988); IL-4 (GenBank Accession No. M13982; Yokota et al., Proc. Natl. Acad. Sci. USA 55:5894-5898, 1986); IL-5 (GenBank Accession No. X04688, J03478; Azuma et al., Nucleic Acids Res. 74:9149- 9158, 1986; Tanabe et al., J. Biol. Chem. 262:16580-16584, 1987); IL-6 (GenBank Accession No. Y00081, X04602, M54894, M38669, M14584; Yasukawa et al., EMBO J. 6:2939-2945, 1987; Hirano et al., Nature 524:73-76, 1986; Wong et al., Behring Inst. Mitt. 83:40-47, 1988; May et al., Proc. Natl. Acad. Sci. USA 55:8957-8961, 1986); IL-7 (GenBank Accession No. J04156; Goodwin et al., Proc. Natl. Acad. Sci. USA 56:302- 306, 1989); IL-8 (GenBank Accession No. Zl 1686; Kusner et al., Kidney Int. 59:1240- 1248, 1991); IL-10 (GenBank Accession No. X78437, M57627; Vieira et al, Proc. Natl. Acad. Sci. USA 55:1172-1176, 1991); IL-11 (GenBank Accession No. M57765 M37006; Paul et al., Proc. Natl. Acad. Sci. USA 57:7512-7516, 1990); IL-13 (GenBank Accession No. X69079, U10307; Minty et al., Nature 562:248-250, 1993; Smirnov, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, June 2, 1994); TNF-α (Tumor necrosis factor) (GenBank Accession No. A21522; Patent No. GB 2246569-A1 , February 5, 1992); TNF-β (GenBank Accession No. D12614; Matsuyama et al., FEBS LETTERS 502:141-144, 1992). DNA sequences of other suitable receptor-binding internalized ligands may be obtained from GenBank or EMBL DNA databases, reverse- synthesized from protein sequence obtained from PIR database or isolated by standard methods (Sambrook et al., supra) from cDNA or genomic libraries.
5. Modifications of receptor-binding internalized ligands
These ligands may be customized for a particular application. Means for modifying proteins is provided below. Briefly, additions, substitutions and deletions of amino acids may be produced by any commonly employed recombinant DNA method. An amino acid residue of FGF, VEGF, HBEGF or other receptor- binding internalized ligand is non-essential if the polypeptide that has been modified by deletion of the residue possesses substantially the same ability to bind to its receptor and internalize a linked agent as the unmodified polypeptide. As noted above, any polypeptide or peptide analogue, including peptidomimetics, that is reactive with an FGF receptor, a VEGF receptor, an HBEGF receptor, other growth factor receptor (e.g., PDGF receptor), cytokine receptor or other cell surface molecule including members of the families and fragments thereof, or constrained analogs of such peptides that bind to the receptor and internalize a linked targeted agent may be used in the context of this invention. Members of the FGF peptide family, including FGF-1 to FGF-9, are preferred. Modified peptides, especially those lacking proliferative function, and chimeric peptides, which retain the specific binding and internalizing activities are also contemplated for use herein.
A modification that is effected substantially near the N-terminus of a polypeptide is generally effected within the first about ten residues of the protein. Such modifications include the addition or deletion of residues, such as the addition of a cysteine to facilitate conjugation and form conjugates that contain a defined molar ratio, preferably a ratio of 1 : 1 of the polypeptides.
DNA encoding one of the receptor-binding internalized ligands discussed above may be mutagenized using standard methodologies to delete or replace any cysteine residues that are responsible for aggregate formation. If necessary, the identity of cysteine residues that contribute to aggregate formation may be determined empirically, by deleting and/or replacing a cysteine residue and ascertaining whether the resulting protein aggregates in solutions containing physiologically acceptable buffers and salts. In addition, fragments of these receptor-binding internalized ligands may be constructed and used. The binding region of many of these ligands have been delineated. Fragments may also be shown to bind and internalize by any one of the tests described herein.
Modification of the polypeptide may be effected by any means known to those of skill in this art. The preferred methods herein rely on modification of DNA encoding the polypeptide and expression of the modified DNA.
Merely by way of example, DNA encoding the FGF polypeptide may be isolated, synthesized or obtained from commercial sources (the amino acid sequences of FGF-1 - FGF-9 are set forth in SEQ ID NOs. 10-18; DNA sequences may be based on these amino acid sequences or may be obtained from public DNA databases and references (see, e.g., GenBank, see also U.S. Patent No. 4,956,455, U.S. Patent No. 5,126,323, U.S. Patent No. 5,155,217, U.S. Patent No. 4,868,113, PCT Application WO 90/08771, EP Application 0 488 196 A2, and Miyamoto et al., Mol. Cell. Biol. 75:4251-4259, 1993). Expression of a recombinant FGF-2 protein in yeast and E. coli is described in Barr et al., J. Biol. Chem. 265:16471-16478, 1988, in PCT Application Serial No. PCT/US93/05702 and United States Application Serial No. 07/901,718. Expression of recombinant FGF proteins may be performed as described herein or using methods known to those of skill in the art.
Similarly, DNA encoding any of the other receptor-binding internalized ligands, including VEGF, HBEGF, IL-1, IL-2, and other cytokines and growth factors may also be isolated, synthesized, or obtained from commercial sources. As noted above, DNA sequences are available in public databases, such as GenBank. Based on these sequences, oligonucleotide primers may be designed and used to amplify the gene from cDNA or mRNA by polymerase chain reaction technique as one means of obtaining DNA. Mutations may be made by any method known to those of skill in the art, including site-specific or site-directed mutagenesis of DNA encoding the protein and the use of DNA amplification methods using primers to introduce and amplify alterations in the DNA template, such as PCR splicing by overlap extension (SOE). Site-directed mutagenesis is typically effected using a phage vector that has single- and double-stranded forms, such as Ml 3 phage vectors, which are well-known and commercially available. Other suitable vectors that contain a single-stranded phage origin of replication may be used (see, e.g., Veira et al., Meth. Enzymol. 15:3, 1987). In general, site-directed mutagenesis is performed by preparing a single-stranded vector that encodes the protein of interest (i.e., a member of the FGF family or a cytotoxic molecule, such as a saporin). An oligonucleotide primer that contains the desired mutation within a region of homology to the DNA in the single-stranded vector is annealed to the vector followed by addition of a DNA polymerase, such as E. coli DNA polymerase I (Klenow fragment), which uses the double stranded region as a primer to produce a heteroduplex in which one strand encodes the altered sequence and the other the original sequence. The heteroduplex is introduced into appropriate bacterial cells and clones that include the desired mutation are selected. The resulting altered DNA molecules may be expressed recombinantly in appropriate host cells to produce the modified protein.
Suitable conservative substitutions of amino acids are well-known and may be made generally without altering the biological activity of the resulting molecule. For example, such substitutions may be made in accordance with those set forth in TABLE 1 as follows:
TABLE 1
Conservative
Original residue substitution
Ala (A) Gly; Ser
Arg (R) Lys
Asn (N) Gin; His
Cys (C) Ser
Gin (Q) Asn
Glu (E) Asp
Gly (G) Ala; Pro
His (H) Asn; Gin
He (I) Leu; Val
Leu (L) He; Val
Lys (K) Arg; Gin; Glu
Met (M) Leu; Tyr; He
Phe (F) Met; Leu; Tyr
Ser (S) Thr
Thr (T) Ser
Trp (W) Tyr
Tyr (Y) Trp; Phe
Val (V) He; Leu
Other similarly conservative substitutions may be made. If necessary, such substitutions may be determined empirically merely by testing the resulting modified protein for the ability to bind to and internalize upon binding to the appropriate receptors. Those that retain this ability are suitable for use in the conjugates and methods herein. In addition, muteins of the FGFs are known to those of skill in the art (see, e.g., U.S. Patent No. 5,175,147; PCT Application No. WO 89/00198, U.S. Serial No. 07/070,797; PCT Application No. WO 91/15229; and U.S. Serial No. 07/505,124).
B. Nucleic acid binding domains
As previously noted, nucleic acid binding domains (NABD) interact with the target nucleic acid either in a sequence-specific manner or a sequence-nonspecific manner. When the interaction is non-specific, the nucleic acid binding domain binds nucleic acid regardless of the sequence. For example, poly-L-lysine is a basic polypeptide that binds to oppositely charged DNA. Other highly basic proteins or polycationic compounds, such as histones, protamines, and spermidine, also bind to nucleic acids in a nonspecific manner.
Many proteins have been identified that bind specific sequences of DNA. These proteins are responsible for genome replication, transcription and repair of damaged DNA. The transcription factors regulate gene expression and are a diverse group of proteins. These factors are especially well suited for purposes of the subject invention because of their sequence-specific recognition. Host transcription factors have been grouped into seven well-established classes based upon the structural motif used for recognition. The major families include helix-turn-helix (HTH) proteins, homeodomains, zinc finger proteins, steroid receptors, leucine zipper proteins, the helix-loop-helix (HLH) proteins, and β-sheets. Other classes or subclasses may eventually be delineated as more factors are discovered and defined. Proteins from those classes or proteins that do not fit within one of these classes but bind nucleic acid in a sequence-specific manner, such as SV40 T antigen and p53 may also be used.
These families of transcription factors are generally well-known (see GenBank; Pabo and Sauer, Ann. Rev. Biochem. 67:1053-1095, 1992; and references below). Many of these factors are cloned and the precise DNA-binding region delineated in certain instances. When the sequence of the DNA-binding domain is known, a gene encoding it may be synthesized if the region is short. Alternatively, the genes may be cloned from the host genomic libraries or from cDNA libraries using oligonucleotides as probes or from genomic DNA or cDNA by polymerase chain reaction methods. Such methods may be found in Sambrook et al., supra.
Helix-turn-helix proteins include the well studied λ Cro protein, λcl, and E. coli CAP proteins (see Steitz et al., Proc. Natl. Acad. Sci. USA 79:3097-3100, 1982; Ohlendorf et al., J. Mol. Biol. 769:757-769, 1983). In addition, the lac repressor (Kaptein et al., J. Mol. Biol. 752:179-182, 1985) and Trp repressor (Scheritz et al., Nature 577:782-786, 1985) belong to this family. Members of the homeodomain family include the Drosophila protein Antennapaedia (Qian et al., Cell. 59:573-580, 1989) and yeast MATα2 (Wolberger et al., Cell. 67:517-528, 1991). Zinc finger proteins include TFIIIA (Miller et al., EMBO J. 4: 1609- 1614, 1985), Sp- 1 , zif 268, and many others (see generally Krizek et al., J. Am. Chem. Soc. 775:4518-4523, 1991). Steroid receptor proteins include receptors for steroid hormones, retinoids, vitamin D, thyroid hormones, as well as other compounds. Specific examples include retinoic acid, knirps, progesterone, androgen, glucocosteroid and estrogen receptor proteins. The leucine zipper family was defined by a heptad repeat of leucines over a region of 30 to 40 residues. Specific members of this family include C/ΕBP, c-fos, c-jun, GCN4, sis-A, and CRΕB (see generally O'Shea et al., Science 254:539-544, 1991). The helix-loop- helix (HLH) family of proteins appears to have some similarities to the leucine zipper family. Well-known members of this family include myoD (Weintraub et al., Science 257:761-766, 1991); c-myc; and AP-2 (Williams and Tijan, Science 257:1067-1071, 1991). The β-sheet family uses an antiparallel β-sheet for DNA binding, rather than the more common α-helix. The family contains the MeU (Phillips, Curr. Opin. Struc. Biol. 7:89-98, 1991), Arc (Breg et al., Nature 546:586-589, 1990) and Mnt repressors. In addition, other motifs are used for DNA binding, such as the cysteine-rich motif in yeast GAL4 repressor, and the GATA factor. Viruses also contain gene products that bind specific sequences. One of the most-studied such viral genes is the rev gene from HIV. The rev gene product binds a sequence called RRΕ (rev responsive element) found in the env gene. Other proteins or peptides that bind DNA may be discovered on the basis of sequence similarity to the known classes or functionally by selection. Several techniques may be used to select other nucleic acid binding domains (see U.S. Patent No. 5,270,170; PCT Application WO 93/14108; and U.S. Patent No. 5,223,409). One of these techniques is phage display. (See, for example, U.S. Patent No. 5,223,409.) In this method, DNA sequences are inserted into the gene III or gene VIII gene of a filamentous phage, such as Ml 3. Several vectors with multicloning sites have been developed for insertion (McLafferty et al., Gene 128:29- 36, 1993; Scott and Smith, Science 249:386-390, 1990; Smith and Scott, Methods Enzymol. 277:228-257, 1993). The inserted DNA sequences may be randomly generated or variants of a known DNA-binding domain. Generally, the inserts encode from 6 to 20 amino acids. The peptide encoded by the inserted sequence is displayed on the surface of the bacteriophage. Bacteriophage expressing a desired nucleic acid- binding domain are selected for by binding to the cytocide-encoding agent. This target molecule may be single stranded or double stranded DNA or RNA. When the cytocide- encoding agent to be delivered is single-stranded, such as RNA, the appropriate target is single-stranded. When the molecule to be delivered is double-stranded, the target molecule is preferably double-stranded. Preferably, the entire coding region of the cytocide-encoding agent is used as the target. In addition, elements necessary for transcription that are included for in vivo or in vitro delivery may be present in the target DNA molecule. Bacteriophage that bind the target are recovered and propagated. Subsequent rounds of selection may be performed. The final selected bacteriophage are propagated and the DNA sequence of the insert is determined. Once the predicted amino acid sequence of the binding peptide is known, sufficient peptide for use herein as an nucleic acid binding domain may be made either by recombinant means or synthetically. Recombinant means is used when the receptor-binding internalized ligand/nucleic acid binding domain is produced as a fusion protein. In addition, the peptide may be generated as a tandem array of two or more peptides, in order to maximize affinity or binding of multiple DNA molecules to a single polypeptide.
As an example of the phage display selection technique, a DNA-binding domain/peptide that recognizes the coding region of saporin is isolated. Briefly, DNA fragments encoding saporin may be isolated from a plasmid containing these sequences. The plasmid FPFS1 contains the entire coding region of saporin. Digestion of the plasmid with Ncol and EcoRI restriction enzymes liberates the saporin specific sequence as a single fragment of approximately 780 bp. This fragment may be purified by any one of a number of methods, such as agarose gel electrophoresis and subsequent elution from the gel. The saporin fragment is fixed to a solid support, such as in the wells of a 96-well plate. If the double-stranded fragment does not bind well to the plate, a coating such as a positively charged molecule, may be used to promote DΝA adherence. The phage library is added to the wells and an incubation period allows for binding of the phage to the DΝA. Unbound phage are removed by a wash, typically containing 10 mM Tris, 1 mM ΕDTA, and without salt or with a low salt concentration. Bound phage are eluted starting at a 0.1 M ΝaCl containing buffer. The ΝaCl concentration is increased in a step-wise fashion until all the phage are eluted. Typically, phage binding with higher affinity will only be released by higher salt concentrations. Eluted phage are propagated in the bacteria host. Further rounds of selection may be performed to select for a few phage binding with high affinity. The DΝA sequence of the insert in the binding phage is then determined. In addition, peptides having a higher affinity may be isolated by making variants of the insert sequence and subjecting these variants to further rounds of selection.
C. Cytocide-encoding agents
A cytocide-encoding agent is a nucleic acid molecule (DΝA or RΝA) that, upon internalization by a cell, and subsequent transcription (if DΝA) and[/or] translation into a cytocidal agent, is cytotoxic to a cell or inhibits cell growth by inhibiting protein synthesis.
Cytocides include saporin, the ricins, abrin and other ribosome inactivating proteins, Pseudomonas exotoxin, diphtheria toxin, angiogenin, tritin, dianthins 32 and 30, momordin, pokeweed antiviral protein, mirabilis antiviral protein, bryodin, angiogenin, and shiga exotoxin, as well as other cytocides that are known to those of skill in the art. Alternatively, cytocide gene products may be noncytotoxic but activate a compound, which is endogenously produced or exogenously applied, from a nontoxic form to a toxic product that inhibits protein synthesis.
Especially of interest are DNA molecules that encode an enzyme that results in cell death or renders a cell susceptible to cell death upon the addition of another product. For example, saporin is an enzyme that cleaves rRNA and inhibits protein synthesis. Other enzymes that inhibit protein synthesis are especially well suited for use in the present invention. In addition, enzymes may be used where the enzyme activates a compound with little or no cytotoxicity into a toxic product that inhibits protein synthesis.
1. Ribosome inactivating proteins
Ribosome-inactivating proteins (RIPs), which include ricin, abrin, and saporin, are plant proteins that catalytically inactivate eukaryotic ribosomes. Ribosome- inactivating proteins inactivate ribosomes by interfering with the protein elongation step of protein synthesis. For example, the ribosome-inactivating protein saporin (hereinafter also referred to as SAP) has been shown to inactivate 60S ribosomes by cleavage of the N-glycosidic bond of the adenine at position 4324 in the rat 28S ribosomal RNA (rRNA). The particular region in which A4324 is located in the rRNA is highly conserved among prokaryotes and eukaryotes; A4324 in 28S rRNA corresponds to A2660 in E. coli 23 S rRNA. Several of the ribosome inactivating proteins also appear to interfere with protein synthesis in prokaryotes, such as E. coli.
Saporin is preferred as a cytocide, but other suitable ribosome inactivating proteins (RIPs) and toxins may be used. Other suitable RIPs include, but are not limited to, ricin, ricin A chain, maize ribosome inactivating protein, gelonin, diphtheria toxin, diphtheria toxin A chain, trichosanthin, tritin, pokeweed antiviral protein (PAP), mirabilis antiviral protein (MAP), Dianthins 32 and 30, abrin, monordin, bryodin, shiga (see, e.g., WO 93/24620) and others (see, e.g., Barbieri et al., Cancer Surveys 7:489-520, 1982, and European patent application No. 0466 222, incorporated herein by reference, which provide lists of numerous ribosome inactivating proteins and their sources; see also U.S. Patent No. 5,248,608 to Walsh et al.). Some ribosome inactivating proteins, such as abrin and ricin, contain two constituent chains: a cell- binding chain that mediates binding to cell surface receptors and internalization of the molecule and a chain responsible for toxicity. Single chain ribosome inactivating proteins (type I RIPS), such as the saporins, do not have a cell-binding chain. As a result, unless internalized, they are substantially less toxic to whole cells than the ribosome inactivating proteins that have two chains.
Several structurally related ribosome inactivating proteins have been isolated from seeds and leaves of the plant Saponaria officinalis (soapwort) (GB Patent 2,194,241 B; GP Patent 2,216,891; EP Patent 89306016). Saporin proteins for use in this invention have amino acid sequences found in the natural plant host Saponaria officinalis or modified sequences, having amino acid substitutions, deletions, insertions or additions, but which still express substantial ribosome inactivating activity. Purified preparations of saporin are frequently observed to include several molecular isoforms of the protein. It is understood that differences in amino acid sequences can occur in saporin from different species as well as between saporin molecules from individual organisms of the same species. Among these, SO-6 is the most active and abundant, representing 7% of total seed proteins. Saporin is very stable, has a high isoelectric point, does not contain carbohydrates, and is resistant to denaturing agents, such as sodium dodecyl sulfate (SDS), and a variety of proteases. The amino acid sequences of several saporin-6 isoforms from seeds are known, and there appear to be families of saporin ribosome inactivating proteins differing in few amino acid residues. Any of these saporin proteins or modified proteins that are cytotoxic may be used in the present invention.
a. Isolation of DNA encoding saporin Some of the DNA molecules provided herein encode saporin that has substantially the same amino acid sequence and ribosome inactivating activity as that of saporin-6 (SO-6), including any of four isoforms, which have heterogeneity at amino acid positions 48 and 91 (see, e.g., Maras et al., Biochem. Internat. 27:631-638, 1990, and Barra et al., Biotechnol. Appl. Biochem. 75:48-53, 1991; GB Patent 2,216,891 B and EP Patent 89306106; and SEQ ID NOs. 19-23). Other suitable saporin polypeptides include other members of the multi-gene family coding for isoforms of saporin-type ribosome inactivating proteins including SO-1 and SO-3 (Fordham- Skelton et al., Mol. Gen. Genet. 227:134-138, 1990), SO-2 (see, e.g., U.S. Application Serial No. 07/885,242; GB 2,216,891; see also Fordham-Skelton et al., Mol. Gen. Genet. 229:460-466, 1991), SO-4 (see, e.g., GB 2,194,241 B; see also Lappi et al., Biochem. Biophys. Res. Commun. 129:934-942, 1985) and SO-5 (see, e.g., GB 2,194,241 B; see also Montecucchi et al., 7«t. J. Peptide Protein Res. 55:263-267, 1989).
The saporin polypeptides for use in this invention include any of the isoforms of saporin that may be isolated from Saponaria officinalis or related species or modified forms that retain cytotoxic activity. In particular, such modified saporin may be produced by modifying the DNA encoding the protein (see, e.g., International PCT Application Serial No. PCT/US93/05702, and United States Application Serial No. 07/901,718; see also U.S. Patent Application No. 07/885,242, and Italian Patent No. 1,231,914) by altering one or more amino acids or deleting or inserting one or more amino acids. Any such protein, or portion thereof, that exhibits cytotoxicity in standard in vitro or in vivo assays within at least about an order of magnitude of the saporin conjugates described herein is contemplated for use herein.
Preferably, the saporin DNA sequence contains mammalian-preferred codons (SEQ. ID NO. 79). Preferred codon usage as exemplified in Current Protocols in Molecular Biology, infra, and Zhang et al. (Gene 705:61, 1991) for mammals, yeast, Drosophila, E. coli, and primates is established for saporin sequence.
The cytocide-encoding agent, such as saporin DNA sequence, is introduced into a plasmid in operative linkage to an appropriate promoter for expression of polypeptides in the organism. The presently preferred saporin proteins are SO-6 and SO-4. The DNA can optionally include sequences, such as origins of replication that allow for the extrachromosomal maintenance of the saporin-containing plasmid, or can be designed to integrate into the genome of the host (as an alternative means to ensure stable maintenance in the host). b. Nucleic acids encoding other ribosome inactivating proteins and cytocides
In addition to saporin discussed above, other cytocides that inhibit protein synthesis are useful in the present invention. The gene sequences for these cytocides may be isolated by standard methods, such as PCR, probe hybridization of genomic or cDNA libraries, antibody screenings of expression libraries, or clones may be obtained from commercial or other sources. The DNA sequences of many of these cytocides are well known, including ricin A chain (GenBank Accession No. X02388); maize ribosome inactivating protein (GenBank Accession No. L26305); gelonin (GenBank Accession No. LI 2243; PCT Application WO 92/03155; U.S. Patent No. 5,376,546; diphtheria toxin (GenBank Accession No. K01722); trichosanthin (GenBank Accession No. M34858); tritin (GenBank Accession No. D 13795); pokeweed antiviral protein (GenBank Accession No. X78628); mirabilis antiviral protein (GenBank Accession No. D90347); dianthin 30 (GenBank Accession No. X59260); abrin (GenBank Accession No. X55667); shiga (GenBank Accession No. M19437) and Pseudomonas exotoxin (GenBank Accession Nos. K01397, M23348). When DNA sequences or amino acid sequences are known, DNA molecules encoding these proteins may be synthesized, and preferably contain mammalian- preferred codons.
D. Prodrug-encoding agent
A nucleic acid molecule encoding a prodrug may alternatively be used within the context of the present invention. Prodrugs are inactive in the host cell until either a substrate is provided or an activating molecule is provided. Most typically, a prodrug activates a compound with little or no cytotoxicity into a toxic product. Two of the more often used prodrug molecules, both of which may be used in the present invention, are HSV thymidine kinase and E. coli cytosine deaminase.
Briefly, a wide variety of gene products which either directly or indirectly activate a compound with little or no cytotoxicity into a toxic product may be utilized within the context of the present invention. Representative examples of such gene products include HSVTK (herpes simplex virus thymidine kinase) and VZVTK (varicella zoster virus thymidine kinase), which selectively phosphorylate certain purine arabinosides and substituted pyrimidine compounds. Phosphoryation converts these compounds to metabolites that are cytotoxic or cytostatic. For example, exposure of the drugs ganciclovir, acyclovir, or any of their analogues (e.g., FIAU, FIAC, DHPG) to cells expressing HSVTK allows conversion of the drug into its corresponding active nucleotide triphosphate form.
Other gene products that may be utilized within the context of the present invention include E. coli guanine phosphoribosyl transferase, which converts thioxanthine into toxic thioxanthine monophosphate (Besnard et al., Mol. Cell. Biol. 7:4139-4141, 1987); alkaline phosphatase, which converts inactive phosphorylated compounds such as mitomycin phosphate and doxorubicin-phosphate to toxic dephosphorylated compounds; fungal (e.g., Fusarium oxysporum) or bacterial cytosine deaminase, which converts 5-fluorocytosine to the toxic compound 5-fluorouracil (Mullen, PNAS 59:33, 1992); carboxypeptidase G2, which cleaves glutamic acid from para-N-bis (2-chloroethyl) aminobenzoyl glutamic acid, thereby creating a toxic benzoic acid mustard; and Penicillin-V amidase, which converts phenoxyacetabide derivatives of doxorubicin and melphalan to toxic compounds (see generally, Vrudhula et al., J. of Med. Chem. 56(7) :919-923, 1993; Kern et al., Cane. Immun. Immunother. 57(4):202-206, 1990). Moreover, a wide variety of Herpesviridae thymidine kinases, including both primate and non-primate heφesviruses, are suitable. Such herpesviruses include Heφes Simplex Virus Type 1 (McKnight et al., Nuc. Acids Res 5:5949-5964, 1980), Heφes Simplex Virus Type 2 (Swain and Galloway, J Virol. 46:1045-1050, 1983), Varicella Zoster Virus (Davison and Scott, J. Gen. Virol. 67:1759-1816, 1986), marmoset heφesvirus (Otsuka and Kit, Virology 755:316-330, 1984), feline heφesvirus type 1 (Nunberg et al., J Virol. 65:3240-3249, 1989), pseudorabies virus (Kit and Kit, U.S. Patent No. 4,514,497, 1985), equine heφesvirus type 1 (Robertson and Whalley, Nuc. Acids Res. 76:11303-11317, 1988), bovine heφesvirus type 1 (Mittal and Field, J. Virol 70:2901-2918, 1989), turkey heφesvirus (Martin et al., J. Virol. 65:2847-2852, 1989), Marek's disease virus (Scott et al, J. Gen. Virol. 70:3055-3065, 1989), heφesvirus saimiri (Honess et al., J. Gen. Virol. 70:3003-3013, 1989) and Εpstein-Barr virus (Baer et al., Nature (London) 570:207-311, 1984). Such heφesviruses may be readily obtained from commercial sources such as the American Type Culture Collection ("ATCC", Rockville, Maryland).
Furthermore, as indicated above, a wide variety of inactive precursors may be converted into active inhibitors. For example, thymidine kinase can phosphorylate nucleosides (e.g., dT) and nucleoside analogues such as ganciclovir (9- {[2-hydroxy-l-(hydroxymethyl)ethoxyl methyl} guanosine), famciclovir, buciclovir, penciclovir, valciclovir, acyclovir (9-[2-hydroxy ethoxy)methyl] guanosine), trifluorothymidine, l-[2-deoxy, 2-fluoro, beta-D-arabino furanosyl]-5-iodouracil, ara-A (adenosine arabinoside, vivarabine), 1-beta-D-arabinofuranoxyl thymine, 5-ethyl-2'- deoxyuridine, 5-iodo-5'-amino-2,5'-dideoxyuridine, idoxuridine (5-iodo-2'- deoxyuridine), AZT (3' azido-3' thymidine), ddC (dideoxycytidine), AIU (5-iodo-5' amino 2', 5'-dideoxyuridine) and AraC (cytidine arabinoside).
E. Other nucleic acid molecules
The conjugates provided herein may also be used to deliver other types of nucleic acids to targeted cells. Such other nucleic acids include antisense RNA, antisense DNA, ribozymes, triplex-forming oligonucleotides, and oligonucleotides that bind proteins. The nucleic acids can also include RNA trafficking signals, such as viral packaging sequences (see, e.g., Sullenger et al. (1994) Science 262:1566-1569). The nucleic acids also include DNA molecules that encode proteins that replace defective genes, such as the gene associated with cystic fibrosis (see, e.g., PCT Application WO 93/03709, U.S. Application Serial No. 07/745,900; and Riordan et al. (1989) Science 245:1066-1073). Other DNA molecules may encode tumor-specific cytotoxic molecules, such as tumor necrosis factor, viral antigens and other proteins to render a cell susceptible to anti-cancer agents.
Nucleic acids and oligonucleotides for use as described herein can be synthesized by any method known to those of skill in this art (see, e.g., WO 93/01286, U.S. Application Serial No. 07/723,454; U.S.. Patent No. 5,218,088; U.S. Patent No. 5,175,269; U.S. Patent No. 5,109,124). Identification of oligonucleotides and ribozymes for use as antisense agents and DNA encoding genes for targeted delivery for genetic therapy involve methods well known in the art. For example, the desirable properties, lengths and other characteristics of such oligonucleotides are well known. Antisense oligonucleotides are typically designed to resist degradation by endogenous nucleolytic enzymes and include, but are not limited to: phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and other such linkages (see, e.g., Agrwal et al., Tetrehedron Lett. 25:3539-3542 (1987); Miller et al., J. Am. Chem. Soc. 95:6657-6665 (1971); Stec et al., Tetrehedron Lett. 26:2191-2194 (1985); Moody et al., Nucl. Acids Res. 72:4769-4782 (1989); Uznanski et al., Nucl. Acids Res. (1989); Letsinger et al., Tetrahedron 40:137- 143 (1984); Eckstein, Annu. Rev. Biochem. 54:367-402 (1985); Eckstein, Trends Biol. Sci. 74:97-100 (1989); Stein In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, Ed, Macmillan Press, London, pp. 97-117 (1989); Jager et al., Biochemistry 27:7237-7246 (1988)).
Antisense nucleotides are oligonucleotides that bind in a sequence- specific manner to nucleic acids, such as mRNA or DNA. When bound to mRNA that has complementary sequences, antisense prevents translation of the mRNA (see, e.g., U.S. Patent No. 5,168,053 to Altman et al.; U.S. Patent No. 5,190,931 to Inouye, U.S. Patent No. 5,135,917 to Burch; U.S. Patent No. 5,087,617 to Smith and Clusel et al. (1993) Nucl. Acids Res. 27:3405-3411, which describes dumbbell antisense oligonucleotides). Triplex molecules refer to single DNA strands that bind duplex DNA forming a colinear triplex molecule and thereby prevent transcription (see, e.g., U.S. Patent No. 5,176,996 to Hogan et al., which describes methods for making synthetic oligonucleotides that bind to target sites on duplex DNA).
Particularly useful antisense nucleotides and triplex molecules are molecules that are complementary or bind to the sense strand of DNA or mRNA that encodes an oncogene, such as bFGF, int-2, hst-1/K-FGF, FGF-5, hst-2/FGF-6, FGF-8. Other useful antisense oligonucleotides include those that are specific for IL-8 (see, e.g., U.S. Patent No. 5,241,049; and PCT Applications WO 89/004836; WO 90/06321; WO 89/10962; WO 90/00563; and WO 91/08483), which can be linked to bFGF for the treatment of psoriasis, anti-sense oligonucleotides that are specific for nonmuscle myosin heavy chain and/or c-myb (see, e.g., Simons et al. (1992) Circ. Res. 70:835-843; PCT Application WO 93/01286, U.S. application Serial No. 07/723,454: LeClerc et al. (1991) J. Am. Coll. Cardiol. 17 (2 Suppl. >:105A; Ebbecke et al. (1992) Basic Res. Cardiol 57:585-591), which can be targeted by an FGF to inhibit smooth muscle cell proliferation, such as that following angioplasty and thereby prevent restenosis or inhibit viral gene expression in transformed or infected cells.
A ribozyme is an RNA molecule that specifically cleaves RNA substrates, such mRNA, .and thus inhibits or interferes with cell growth or expression. There are at least five classes of ribozymes that are known that are involved in the cleavage and/or ligation of RNA chains. Ribozymes can be targeted to any RNA transcript and can catalytically cleave such transcript (see, e.g., U.S. Patent No. 5,272,262; U.S. Patent No. 5,144,019; and U.S. Patent Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246 to Cech et al., which described ribozymes and methods for production thereof). Any such ribosome may be linked to the growth factor for delivery to a cell bearing a receptor for a receptor-internalized binding ligand.
The ribozymes may be delivered to the targeted cells by DNA encoding the ribozyme linked to a eukaryotic promoter, such as a eukaryotic viral promoter, such that upon introduction into the nucleus, the ribozyme will be directly transcribed. In such instances, the construct will also include a nuclear translocation sequence, generally as part of the ligand or as part of a linker between the ligand and nucleic acid binding domain.
DNA that encodes a therapeutic product contemplated for use includes DNA encoding correct copies of defective genes, such as the defective gene (CFTR) associated with cystic fibrosis (see, e.g., International Application WO 93/03709, U.S. Application Serial No. 07/745,900; and Riordan et al. (1989) Science 245:1066-1073), and anticancer agents, such as tumor necrosis factors. The conjugate preferably includes an NTS. If the conjugate is designed such that the ligand and nucleic acid binding domain are cleaved in the cytoplasm, then the NTS should be included in a portion of the conjugate or linker that remains bound to the DNA. The nuclear translocation sequence (NTS) may be a heterologous sequence or a may be derived from the selected growth factor.
F. Construct containing cytocidal-encoding agent In the case of cytotocide molecules such as the ribosome inactivating proteins, very few molecules may need to be expressed to effect cell killing. Indeed, only a single molecule of diphtheria toxoid introduced into a cell was sufficient to kill the cell. With other cytocides or prodrugs, it may be that propagation or stable maintenance of the construct is necessary to attain a sufficient amount or concentration of the gene product for effective gene therapy. Examples of replicating and stable eukaryotic plasmids may be found in the scientific literature.
In general, constructs will also contain elements necessary for transcription and translation. If the cytocide-encoding agent is DNA, then it must contain a promoter. The choice of the promoter will depend upon the cell type to be transformed and the degree or type of control desired. Promoters can be constitutive or active in any cell type, tissue specific, cell specific, event specific temporal-specific or inducible. Cell-type specific promoters and event type specific promoters are preferred. Examples of constitutive or nonspecific promoters include the SV40 early promoter (U.S. Patent No. 5,118,627), the SV40 late promoter (U.S. Patent No. 5,118,627), CMV early gene promoter (U.S. Patent No. 5,168,062), and adenovirus promoter. In addition to viral promoters, cellular promoters are also amenable within the context of this invention. In particular, cellular promoters for the so-called housekeeping genes are useful. Viral promoters are preferred, because generally they are stronger promoters than cellular promoters. Tissue specific promoters are particularly useful when a certain tissue type is to be targeted for transformation. By using one of this class of promoters, an extra margin of specificity can be attained. For example, when the indication to be treated is ophthalmological (e.g., secondary lens clouding), either the alpha-crystalline promoter or gamma-crystalline promoter is preferred. When a tumor is the target of gene delivery, cellular promoters for specific tumor markers or promoters more active in tumor cells should be chosen. Thus, to treat prostate tumor, the prostate-specific antigen promoter is especially useful. Similarly, the tyrosinase promoter or tyrosinase- related protein promoter is a preferred promoter for melanoma treatment. For treatment of diseases that are angiogenic or exacerbated by angiogenesis, the VEGF receptor promoter is preferred. The VEGF receptor is expressed in developing capillaries. For treatment of breast cancer, the promoter from heat shock protein 27 is preferred; for treatment of colon or lung cancer, the promoter from carcinoembryonic antigen is preferred; for treatment of restenosis or other diseases involving smooth muscle cells, the promoter from α-actin or myosin heavy chain is preferred. For B lymphocytes, the immunoglobulin variable region gene promoter; for T lymphocytes, the TCR receptor variable region promoter; for helper T lymphocytes, the CD4 promoter; for liver, the albumin or α-fetoprotein promoter, are a few additional examples of tissue specific promoters. Many other examples of tissue specific promoters are readily available to one skilled in the art. Some of these promoters are temporally regulated, such as c-myc and cyclin D. Inducible promoters may also be used. These promoters include the
MMTV LTR (PCT WO 91/13160), which is inducible by dexamethasone, metallothionein, which is inducible by heavy metals, and promoters with cAMP response elements, which are inducible by cAMP. By using an inducible promoter, the nucleic acid may be delivered to a cell and will remain quiescent until the addition of the inducer. This allows further control on the timing of production of the therapeutic gene.
Event-type specific promoters are active or up-regulated only upon the occurrence of an event, such as tumorigenecity or viral infection. The HIV LTR is a well known example of an event-specific promoter. The promoter is inactive unless the tat gene product is present, which occurs upon viral infection. Another promoter is c-myc.
Additionally, promoters that are coordinately regulated with a particular cellular gene may be used. For example, promoters of genes that are coordinately expressed when a particular FGF receptor gene is expressed may be used. Then, the nucleic acid will be transcribed when the FGF receptor, such as FGFRl, is expressed, and not when FGFR2 is expressed. This type of promoter is especially useful when one knows the pattern of FGF receptor expression in a particular tissue, so that specific cells within that tissue may be killed upon transcription of a cytotoxic agent gene without affecting the surrounding tissues. If the domain binds in a sequence specific manner, the construct must contain the sequence that binds to the nucleic acid binding domain. As described below, the target nucleotide sequence may be contained within the coding region of the cytocide, in which case, no additional sequence need be incoφorated. Additionally, it may be desirable to have multiple copies of target sequence. If the target sequence is coding sequence, the additional copies must be located in non-coding regions of the cytocide-encoding agent. The target sequences of the nucleic acid binding domains are typically generally known. If unknown, the target sequence may be readily determined. Techniques are generally available for establishing the target sequence (e.g., see PCT Application WO 92/05285 and U.S. Serial No. 586,769).
G. Other Elements
1. Nuclear translocation signal
As used herein, a "nuclear translocation or targeting sequence" (NTS) is a sequence of amino acids in a protein that are required for translocation of the protein into a cell nucleus. Examples of NTSs are set forth in Table 2 below. Comparison with known NTSs, and if necessary testing of candidate sequences, should permit those of skill in the art to readily identify other amino acid sequences that function as NTSs. A heterologous NTS refers to an NTS that is different from the NTS that occurs in the wild-type peptide, polypeptide, or protein. For example, the NTS may be derived from another polypeptide, it may be synthesized, or it may be derived from another region in the same polypeptide. TABLE 2
Source Sequence SEQ ID NO.
SV40 large T Pro126LysLysArgLysValGlu 24
Polyoma large T Pro279 ProLysLysAlaArgGluVal 25
Human c-Myc 120
Pro AlaAlaLysArgValLysLeuAsp 26
Adenovirus El A 281
Lys ArgProArgPro 27
Yeast mat α2 Lys3IleProIleLys 28 c-Erb-A A. Gly22 LysArgLysArgLysSer 29
127
B. Ser LysArgValAlaLysArgLysLeu 30 31
C. Ser181HisT LysGlnLysArgLysPhe c-Myb Pro52 ' LeuLeuLysLysIleLysGln 32 p53 Pro316GlnProLysLysLysPro 33
Nucleolin Pro277GlyLysArgLysLysGluMetThrLysGlnLysGluVaIPro 34
HIV Tat Gly ArgLysLysArgArgGlnArgArgArgAlaPro 35
FGF-1 AsnTyrLysLysProLysLeu 36
FGF-2 HisPheLysAspProLysArg 37
FGF-3 AlaProArgArgArgLysLeu 38
FGF-4 IleLysArgLeuArgArg 39
FGF-5 GlyArgArg
FGF-6 IleLysArgGlnArgArg 40
FGF-7 IleArgValArgArg 41
Superscript indicates position in protein
In order to deliver the nucleic acid to the nucleus, the conjugate should include an NTS. If the conjugate is designed such that the receptor-binding internalized ligand and linked nucleic acid binding domain is cleaved or dissociated in the cytoplasm, then the NTS should be included in a portion of the complex that remains bound to the nucleic acid, so that, upon internalization, the conjugate will be trafficked to the nucleus. Thus, the NTS is preferably included in the nucleic acid binding domain, but may additionally be included in the ligand. An NTS is preferred if the cytocide-encoding agent is DNA. If the cytocide-encoding agent is mRNA, an NTS may be omitted. The nuclear translocation sequence (NTS) may be a heterologous sequence or a may be derived from the selected growth factor. All presently identified members of the FGF family of peptides contain an NTS (see, e.g., International Application WO 91/15229 and Table 2). A typical consensus NTS sequence contains an amino-terminal proline or glycine followed by at least three basic residues in a array of seven to nine amino acids (see, e.g., Dang et al., J. Biol. Chem. 264:18019-18023, 1989; Dang et al., Mol. Cell. Biol 5:4049-4058, 1988, and Table 2).
2. Cytoplasm-translocation signal Cytoplasm-translocation signal sequence is a sequence of amino acids in a protein that cause retention of proteins in the lumen of the endoplasmic reticulum and/or translocate proteins to the cytosol. The signal sequence in mammalian cells is KDEL (Lys-Asp-Glu-Leu) (SEQ ID NO. 42) (Munro and Pelham, Cell 45:899-907, 1987). Some modifications of this sequence have been made without loss of activity. For example, the sequences RDEL (Arg-Asp-Glu-Leu) (SEQ ID NO. 43) and KEEL (Lys-Glu-Glu-Leu) (SEQ ID NO. 44) confer efficient or partial retention, respectively, in plants (Denecke et al., Embo. J. 77:2345-2355, 1992).
A cytoplasm-translocation signal sequence may be included in either the receptor-internalized binding ligand or the nucleic acid binding domain part or both. If cleavable linkers are used to link the ligand with the nucleic acid binding domain, the cytoplasm-translocation signal is preferably included in the nucleic acid binding domain, which will stay bound to the cytocide-encoding agent. Additionally, a cytoplasmic-translocation signal sequence may be included in the receptor-internalized binding ligand, as long as it does not interfere with receptor binding. Similarly, the signal sequence placed in the nucleic acid binding domain should not interfere with binding to the cytocide-encoding agent.
3. Endosome-disruptive peptides
In addition, or alternatively, membrane-disruptive peptides may be incoφorated into the complexes. For example, adenoviruses are known to enhance disruption of endosomes. Virus-free viral proteins, such as influenza virus hemagglutinin HA-2, also disrupt endosomes and are useful in the present invention. Other proteins may be tested in the assays described herein to find specific endosome disrupting agents that enhance gene delivery. In general, these proteins and peptides are amphipathic (see Wagner et al., Adv. Drug. Del. Rev. 14: 113-135, 1994).
Endosome-disruptive peptides, sometimes called fusogenic peptides, may be incoφorated into the complex of receptor-internalized binding ligand, nucleic acid binding domain, and cytocide-encoding agent. Two such peptides derived from influenza virus are: GLFEAIEGFIENGWEGMIDGGGC (SEQ. ID NO. 45) and GLFEAIEGFIENGWEGMIDGWYGC (SEQ. ID NO. 46). Other peptides useful for disrupting endosomes may be identified by general characteristics: 25-30 residues in length, contain an alternating pattern of hydrophobic domains and acidic domains, and at low pH (e.g., pH 5) from amphipathic α-helices. A candidate endosome-disrupting peptide is tested by incoφorating it into the complex and determining whether it increases the total number of cells expressing the target gene. The peptides are added to a complex having excess negative charge. For example, a DNA construct is complexed with an FGF-poly-L-lysine chemical conjugate so that only a portion of the negative charge of the DNA is neutralized. Poly-L-lysine is added to further bind the DNA and a fusogenic peptide is then added. Optional ratios of DNA, poly-L-lysine and fusogenic peptide are determined using assays, such as gene expression and cell viability.
The fusogenic peptides may alternatively be incoφorated into the complex as a fusion protein with either the ligand or the nucleic acid binding domain or both. The endosome-disruptive peptide may be present as single or multiple copies at the N- or C- terminus of the ligand. A single fusion protein of the endosome-disruptive peptide, nucleic acid binding domain, and receptor-internalized binding ligand may be constructed and expressed. For insertion into a construct, DNA encoding the endosome-disruptive peptide may be synthesized by PCR using overlapping oligonucleotides and incoφorating a restriction site at the 5' and 3' end to facilitate cloning. The sequence may be verified by sequence analysis. 4. Linkers
As used herein, a "linker" is an extension that links the receptor-binding internalized ligand or fragment thereof and the nucleic acid binding domain. In certain instances, the linker is used to conjugate the ligand directly to the nucleic acid. The linkers provided herein confer specificity, enhance intracellular availability, serum stability and/or solubility on the conjugate and may serve to promote condensation of the nucleic acid.
The linkers provided herein confer specificity and serum stability on the cytotoxic conjugate, for example, by conferring specificity for certain proteases, particularly proteases that are present in only certain subcellular compartments or that are present at higher levels in tumor cells than normal cells. Specificity for proteases present in intracellular compartments and absent in blood is particularly preferred. The linkers may also include sorting signals that direct the conjugate to particular intracellular loci or compartments. Additionally, the linkers may reduce steric hindrance between the growth factor and other protein or linked nucleic acid by distancing the components of the conjugate. Linkers may also condense the nucleic acid. For this puφose, the linker comprises highly basic amino acids (e.g., Lys, Arg) and may even by poly-L-lysine.
In order to increase the serum stability, solubility and/or intracellular concentration or condense the targeted agent, one or more linkers (are) inserted between the receptor-binding internalized ligand and the nucleic acid binding domain. These linkers include peptide linkers, such as intracellular protease substrates, and chemical linkers, such as acid labile linkers, ribozyme substrate linkers and others. Peptides linkers may be inserted using heterobifunctional reagents, described below, or, preferably, are linked to FGF, other growth factors, including heparin-binding growth factors, or cytokines by linking DNA encoding the ligand to the DNA encoding the nucleic acid binding domain.
Chemical linkers may be inserted by covalently coupling the linker to the
FGF, other growth factor protein, or cytokine and the nucleic acid binding domain. The linker may be bound via the N- or C-terminus or an internal residue. The heterobifunctional agents, described below, may be used to effect such covalent coupling.
a. Protease substrates Peptides encoding protease-specific substrates may be introduced between the ligand and the nucleic acid binding domain. The peptides may be inserted using heterobifunctional reagents, as described below, or preferably inserted by recombinant means and expression of the resulting chimera.
Any protease specific substrate (see, e.g., O'Hare et al., FEBS 275:200- 204, 1990; Forsberg et al., J. Protein Chem. 70:517-526, 1991; Westby et al., Bioconjugate Chem. 5:375-381, 1992) may be introduced as a linker as long as the substrate is cleaved in an intracellular compartment. Preferred substrates include those that are specific for proteases that are expressed at higher levels in tumor cells, that are preferentially expressed in the endosome, or that are absent in blood. The following substrates are among those contemplated for use in accord with the methods herein: cathepsin B substrate, cathepsin D substrate, trypsin substrate, thrombin substrate, and recombinant subtilisin substrate.
b. Flexible linkers and linkers that increase the solubility of the conjugates
Flexible linkers, which reduce steric hindrance, and linkers that increase solubility of the conjugates are contemplated for use, either alone or with other linkers, such as the protease specific substrate linkers. Typically, these linkers are simple polymers of small amino acids (i.e., small side groups) with uncharged polar side groups. These amino acids (Gly, Ser, Thr, Cys, Tyr, Asn, Gin) are more soluble in water. Of these amino acids, Gly and Ser are preferred. Such linkers include, but are not limited to, (Gly4Ser)n, (Ser4Gly)n and (AlaAlaProAla)n in which n is 1 to 6, preferably 1-4, such as: a . Gly4Ser SEQ ID NO: 47 CCATGGGCGG CGGCGGCTCT GCCATGG b. (Gly4Ser)2 SEQ ID NO: 48 CCATGGGCGG CGGCGGCTCT GGCGGCGGCG GCTCTGCCAT GG c. (Ser4Gly)4 SEQ ID NO: 49
CCATGGCCTC GTCGTCGTCG GGCTCGTCGT CGTCGGGCTC GTCGTCGTCG GGCTCGTCGT CGTCGGGCGC CATGG d. (Ser4Gly)2 SEQ ID NO: 50 CCATGGCCTC GTCGTCGTCG GGCTCGTCGT CGTCGGGCGC CATGG e. (AlaAlaProAla)n. where n is 1 to 4, preferably 2 (see SEQ ID NO: 51)
c. Heterobifunctional cross-linking reagents Numerous heterobifunctional cross-linking reagents that are used to form covalent bonds between amino groups and thiol groups and to introduce thiol groups into proteins, are known to those of skill in this art (see, e.g., the PIERCE CATALOG, ImmunoTechnology Catalog & Handbook, 1992-1993, which describes the preparation of and use of such reagents and provides a commercial source for such reagents; see also, e.g., Cumber et al., Bioconjugate Chem. 5:397-401, 1992; Thoφe et al., Cancer Res. 47:5924-5931, 1987; Gordon et al., Proc. Natl. Acad Sci. 54:308-312, 1987; Walden et al., J. Mol. Cell Immunol. 2:191-197, 1986; Carlsson et al., Biochem. J. 775:723-737, 1978; Mahan et al., Anal. Biochem. 762:163-170, 1987; Wawryznaczak et al., Br. J. Cancer 66:361-366, 1992; Fattom et al., Infection & Immun. 60:584-589, 1992). These reagents may be used to form covalent bonds between the receptor- binding internalized ligands with protease substrate peptide linkers and nucleic acid binding domain. These reagents include, but are not limited to: N-succuιimidyl-3-(2- pyridyldithio)propionate (SPDP; disulfide linker); sulfosuccinimidyl 6-[3-(2- pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP); succinimidyloxycarbonyl-α- methyl benzyl thiosulfate (SMBT, hindered disulfate linker); succinimidyl 6-[3-(2- pyridyldithio) propionamido]hexanoate (LC-SPDP); sulfosuccmimidyl
4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (sulfo-SMCC); succinimidyl 3-(2-pyridyldithio)butyrate (SPDB; hindered disulfide bond linker); sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide) ethyl- l,3'-dithiopropionate (SAED); sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (SAMCA); sulfosuccinimidyl 6-[alpha-methyl-alpha-(2-pvridyldithio)toluamido]hexanoate (sulfo-LC-SMPT);
1 ,4-di-[3'-(2'-pyridyldithio)propionamido]butane (DPDPB);
4-succinimidyloxycarbonyl- -methyl- -(2-pyridylthio)toluene (SMPT, hindered disulfate linker); sulfosucciιώr-idyl6[ -methyl- -(2-pvridyldithio)tolι-ιamido]hexanoate (sulfo-LC- SMPT); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); m-maleimidobenzoyl- N-hydroxysulfosucciiiimide ester (sulfo-MBS); N-succinimidyl(4- iodoacetyl)aminobenzoate (SIAB; thioether linker); sulfosucciι-imidyl(4-iodoacetyl)amino benzoate (sulfo-SIAB); succi-t-i-τidyl4( 7-maleirnidophenyl)butyrate (SMPB); sulfosuccini- midy!4-( -7-maleimidophenyl)butyrate (sulfo-SMPB); azidobenzoyl hydrazide (ABH). These linkers should be particularly useful when used in combination with peptide linkers, such as those that increase flexibility.
d. Acid cleavable. photocleavable, and heat sensitive linkers
Acid cleavable linkers include, but are not limited to, bismaleimideothoxy propane, adipic acid dihydrazide linkers (see, e.g., Fattom et al., Infection & Immun. 60:584-589, 1992) and acid labile transferrin conjugates that contain a sufficient portion of transferrin to permit entry into the intracellular transferrin cycling pathway (see, e.g., Welhδner et al., J. Biol. Chem. 266:4309-4314, 1991). Conjugates linked via acid cleavable linkers should be preferentially cleaved in acidic intracellular compartments, such as the endosome.
Photocleavable linkers are linkers that are cleaved upon exposure to light (see, e.g., Goldmacher et al, Bioconj. Chem. 5:104-107, 1992), thereby releasing the targeted agent upon exposure to light. (Hazum et al., Proc. Eur. Pept. Symp., 16th, Brunfeldt, K (Ed), pp. 105-110, 1981; nitrobenzyl group as a photocleavable protective group for cysteine; Yen et al., Makromol. Chem 790:69-82, 1989; water soluble photocleavable copolymers, including hydroxypropylmethacrylamide copolymer, glycine copolymer, fluorescein copolymer and methylrhodamine copolymer; and Senter et al., Photochem. Photobiol 42:231-237, 1985; nitrobenzyloxycarbonyl chloride cross linking reagents that produce photocleavable linkages). Such linkers are particularly useful in treating dermatological or ophthalmic conditions and other tissues, such as blood vessels during angioplasty in the prevention or treatment of restenosis, that can be exposed to light using fiber optics. After administration of the conjugate, the eye or skin or other body part can be exposed to light, resulting in release of the targeted moiety from the conjugate. This should permit administration of higher dosages of such conjugates compared to conjugates that release a cytotoxic agent upon internalization. Heat sensitive linkers would also have similar applicability.
H. Expression vectors and host cells for expression of receptor-binding internalized ligands and nucleic acid binding domains Host organisms include those organisms in which recombinant production of heterologous proteins have been carried out, such as bacteria (for example, E. coli), yeast (for example, Saccharomyces cerevisiae and Pichia pastoris), mammalian cells, and insect cells. Presently preferred host organisms are E. coli bacterial strains. The DNA construct encoding the desired protein is introduced into a plasmid for expression in an appropriate host. In preferred embodiments, the host is a bacterial host. The sequence encoding the ligand or nucleic acid binding domain is preferably codon-optimized for expression in the particular host. Thus, for example, if human FGF-2 is expressed in bacteria, the codons would be optimized for bacterial usage. For small coding regions the gene can be synthesized as a single oligonucleotide. For larger proteins, splicing of multiple oligonucleotides, mutagenesis, or other techniques known to those in the art may be used. For example, the sequence of a bacterial-codon preferred FGF-SAP fusion is shown in SEQ. ID NO. 80. The sequences of nucleotides in the plasmids that are regulatory regions, such as promoters and operators, are operationally associated with one another for transcription. The sequence of nucleotides encoding the growth factor or growth factor-chimera may also include DNA encoding a secretion signal, whereby the resulting peptide is a precursor protein. The resulting processed protein may be recovered from the periplasmic space or the fermentation medium. In preferred embodiments, the DNA plasmids also include a transcription terminator sequence. As used herein, a "transcription terminator region" has either (a) a subsegment that encodes a polyadenylation signal and polyadenylation site in the transcript, and or (b) a subsegment that provides a transcription termination signal that terminates transcription by the polymerase that recognizes the selected promoter. The entire transcription terminator may be obtained from a protein-encoding gene, which may be the same or different from the inserted gene or the source of the promoter. Transcription terminators are optional components of the expression systems herein, but are employed in preferred embodiments.
The plasmids used herein include a promoter in operable association with the DNA encoding the protein or polypeptide of interest and are designed for expression of proteins in a bacterial host. It has been found that tightly regulatable promoters are preferred for expression of saporin. Suitable promoters for expression of proteins and polypeptides herein are widely available and are well known in the art. Inducible promoters or constitutive promoters that are linked to regulatory regions are preferred. Such promoters include, but are not limited to, the T7 phage promoter and other T7-like phage promoters, such as the T3, T5 and SP6 promoters, the tφ, lpp, and lac promoters, such as the lacUV5, from E. coli; the PI 0 or polyhedron gene promoter of baculovirus/insect cell expression systems (see, e.g., U.S. Patent Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784) and inducible promoters from other eukaryotic expression systems. For expression of the proteins such promoters are inserted in a plasmid in operative linkage with a control region such as the lac operon.
Preferred promoter regions are those that are inducible and functional in
E. coli. Examples of suitable inducible promoters and promoter regions include, but are not limited to: the E. coli lac operator responsive to isopropyl β
-D-thiogalactopyranoside (IPTG; see, et al. Nakamura et al., Cell 75:1109-1117, 1979); the metallothionein promoter metal-regulatory-elements responsive to heavy-metal (e.g., zinc) induction (see, e.g., U.S. Patent No. 4,870,009 to Evans et al.); the phage T71ac promoter responsive to IPTG (see, e.g., U.S. Patent No. 4,952,496; and Studier et al., Meth. Enzymol. 755:60-89, 1990) and the TAC promoter.
The plasmids also preferably include a selectable marker gene or genes that are functional in the host. A selectable marker gene includes any gene that confers a phenotype on bacteria that allows transformed bacterial cells to be identified and selectively grown from among a vast majority of untransformed cells. Suitable selectable marker genes for bacterial hosts, for example, include the ampicillin resistance gene (Ampr), tetracycline resistance gene (Tcr) and the kanamycin resistance gene (Kanr). The kanamycin resistance gene is presently preferred.
The plasmids may also include DNA encoding a signal for secretion of the operably linked protein. Secretion signals suitable for use are widely available and are well known in the art. Prokaryotic and eukaryotic secretion signals functional in E. coli may be employed. The presently preferred secretion signals include, but are not limited to, those encoded by the following E. coli genes: ompA, ompT, ompF, ompC, beta-lactamase, and alkaline phosphatase, and the like (von Heijne, J. Mol. Biol. 754:99-105, 1985). In addition, the bacterial pelB gene secretion signal (Lei et al., J. Bacteriol 169:4379, 1987), the phoA secretion signal, and the cek2 functional in insect cell may be employed. The most preferred secretion signal is the E. coli ompA secretion signal. Other prokaryotic and eukaryotic secretion signals known to those of skill in the art may also be employed (see, e.g., von Heijne, J. Mol. Biol. 754:99-105, 1985). Using the methods described herein, one of skill in the art can substitute secretion signals that are functional in either yeast, insect or mammalian cells to secrete proteins from those cells. Particularly preferred plasmids for transformation of E. coli cells include the pΕT expression vectors (see U.S patent 4,952,496; available from Novagen, Madison, WI; see also literature published by Novagen describing the system). Such plasmids include pΕT l la, which contains the T71ac promoter, T7 terminator, the inducible E. coli lac operator, and the lac repressor gene; pΕT 12a-c, which contains the T7 promoter, T7 terminator, and the E. coli ompT secretion signal; and pΕT 15b (Novagen, Madison, WI), which contains a His-Tag™ leader sequence for use in purification with a His column and a thrombin cleavage site that permits cleavage following purification over the column, the T7-lac promoter region and the T7 terminator. Other preferred plasmids include the pKK plasmids, particularly pKK 223-3, which contains the tac promoter, (available from Pharmacia; see also Brosius et al., Proc. Natl. Acad. Sci. 57:6929, 1984; Ausubel et al., Current Protocols in Molecular Biology; U.S. Patent Nos. 5,122,463, 5,173,403, 5,187,153, 5,204,254, 5,212,058, 5,212,286, 5,215,907, 5,220,013, 5,223,483, and 5,229,279). Plasmid pKK has been modified by replacement of the ampicillin resistance marker gene, by digestion with EcoRI, with a kanamycin resistance cassette with EcoRI sticky ends (purchased from Pharmacia; obtained from pUC4K, see, e.g., Vieira et al. (Gene 79:259-268, 1982; and U.S. Patent No. 4,719,179). Baculovirus vectors, such as pBlueBac (also called pJVΕTL and derivatives thereof), particularly pBlueBac III, (see, e.g., U.S. Patent Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784; available from Invitrogen, San Diego) may also be used for expression of the polypeptides in insect cells. The pBlueBacIII vector is a dual promoter vector and provides for the selection of recombinants by blue/white screening as this plasmid contains the β-galactosidase gene (lacZ) under the control of the insect recognizable ΕTL promoter and is inducible with IPTG. A DNA construct may be made in baculovirus vector pBluebac III and then co-transfected with wild type virus into insect cells Spodoptera frugiperda (sf9 cells; see, e.g., Luckow et al., Bio/technology 6:47-55, 1988, and U.S. Patent No. 4,745,051). Other plasmids include the pIN-IIIompA plasmids (see U.S. Patent
No. 4,575,013; see also Duffaud et al., Meth. Enz. 153:492-507, 1987), such as pIN- IIIompA2. The pIN-IIIompA plasmids include an insertion site for heterologous DNA linked in transcriptional reading frame with four functional fragments derived from the lipoprotein gene of E. coli. The plasmids also include a DNA fragment coding for the signal peptide of the ompA protein of E. coli, positioned such that the desired polypeptide is expressed with the ompA signal peptide at its amino terminus, thereby allowing efficient secretion across the cytoplasmic membrane. The plasmids further include DNA encoding a specific segment of the E. coli lac promoter-operator, which is positioned in the proper orientation for transcriptional expression of the desired polypeptide, as well as a separate functional E. coli lad gene encoding the associated repressor molecule that, in the absence of lac operon inducer, interacts with the lac promoter-operator to prevent transcription therefrom. Expression of the desired polypeptide is under the control of the lipoprotein (lpp) promoter and the lac promoter-operator, although transcription from either promoter is normally blocked by the repressor molecule. The repressor is selectively inactivated by means of an inducer molecule thereby inducing transcriptional expression of the desired polypeptide from both promoters.
Preferably, the DNA fragment is replicated in bacterial cells, preferably in E. coli. The preferred DNA fragment also includes a bacterial origin of replication, to ensure the maintenance of the DNA fragment from generation to generation of the bacteria. In this way, large quantities of the DNA fragment can be produced by replication in bacteria. Preferred bacterial origins of replication include, but are not limited to, the fl-ori and col El origins of replication. Preferred hosts contain chromosomal copies of DNA encoding T7 RNA polymerase operably linked to an inducible promoter, such as the lacUV promoter (see U.S. Patent No. 4,952,496). Such hosts include, but are not limited to, lysogens E. coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS, HMS174(DE3) and BL21(DE3). Strain BL21(DE3) is preferred. The pLys strains provide low levels of T7 lysozyme, a natural inhibitor of T7 RNA polymerase. The DNA fragments provided may also contain a gene coding for a repressor protein. The repressor protein is capable of repressing the transcription of a promoter that contains sequences of nucleotides to which the repressor protein binds. The promoter can be derepressed by altering the physiological conditions of the cell. For example, the alteration can be accomplished by adding to the growth medium a molecule that inhibits the ability to interact with the operator or with regulatory proteins or other regions of the DNA or by altering the temperature of the growth media. Preferred repressor proteins include, but are not limited to the E. coli lad repressor responsive to IPTG induction, the temperature sensitive λ cI857 repressor, and the like. The E. coli lad repressor is preferred. DNA encoding full-length FGF-2 or FGF-2 mutein is linked to DNA encoding an nucleic acid binding domain, such as protamine, and introduced into the pET vectors, including pET-l la and pET-12a expression vectors (Novagen, Madison,
WI), for intracellular and periplasmic expression, respectively, of FGF-protamine fusion proteins.
I. Preparation of complexes containing receptor-binding internalized ligands/nucleic acid binding domain conjugates and cytocide-encoding agents
Within the context of this invention, specificity of delivery is achieved through the ligand. Typically, a nucleic acid binding domain is coupled to a receptor- binding internalized ligand, either by chemical conjugation or as a fusion protein. As described below, the ligand may alternatively be coupled directly to the nucleic acid and then complexed with a nucleic acid binding protein, such as poly-lysine, which serves to condense the nucleic acid. Linkers as described above may optionally be used. The receptor-binding internalized ligand confers specificity of delivery in a cell-specific manner. The choice of the receptor-binding internalized ligand to use will depend upon the receptor expressed by the target cells. The receptor type of the target cell population may be determined by conventional techniques such as antibody staining, PCR of cDNA using receptor-specific primers, and biochemical or functional receptor binding assays. It is preferable that the receptor be cell type-specific or have increased expression or activity (i.e., higher rate of internalization) within the target cell population.
As described herein, the nucleic acid binding domain can be of two types, non-specific in its ability to bind nucleic acid, or highly specific so that the amino acid residues bind only the desired nucleic acid sequence. Nonspecific binding proteins, polypeptides, or compounds are generally polycationic or highly basic. Lys and Arg are the most basic of the 20 common amino acids; proteins enriched for these residues are candidates for nucleic acid binding domains. Examples of basic proteins include histones, protamines, and repeating units of lysine and arginine. Poly-L-lysine is an often-used nucleic acid binding domain (see U.S. Patent Nos. 5,166,320 and 5,354,844). Poly-L-lysine and protamine are preferred. Other polycations, such as spermine and spermidine, may also be used to bind nucleic acids. By way of example, the sequence-specific proteins, including gal4, Sp-1, AP-1, myoD and the rev gene product from HIV, may be used. Specific nucleic acid binding domains can be cloned in tandem, individually, or multiply to a desired region of the receptor-binding internalized ligand of interest. Alternatively, the ligand and binding domain can be chemically conjugated to each other.
The corresponding sequence that binds a sequence-specific domain is incoφorated into the construct to be delivered. Complexing the cytocidal-encoding agent to the receptor-binding internalized ligand/nucleic acid binding domain allows specific binding to the nucleic acid binding domain. Even greater specificity of binding may be achieved by identifying and using the minimal amino acid sequence that binds to the cytocidal-encoding agent of interest. For example, phage display methods can be used to identify amino acids residues of varying length that will bind to specific nucleic acid sequences with high affinity. (See U.S. Patent No. 5,223,409.) The peptide sequence can then be cloned into the receptor-binding internalized ligand as a single copy or multiple copies. Alternatively, the peptide may be chemically conjugated to the receptor-binding internalized ligand. Incubation of the cytocide-encoding agent with the conjugated proteins will result in a specific binding between the two.
These complexes may be used to deliver nucleic acids that encode saporin, other cytocidal proteins, or prodrugs into cells with appropriate receptors that are expressed, over-expressed or more active in internalization upon binding. The cytocide gene is cloned downstream of a mammalian promoter such as c-myc, SV40 early or late gene, CMV-IE, TK or adenovirus promoter. As described above, promoters of interest may be active in any cell type, active only in a tissue-specific manner, such as α-crystalline or tyrosinase, event specific, or inducible, such as the MMTV LTR. 1. Chemical conjugation a. Preparation of receptor-binding internalized ligands Receptor-binding internalized ligands are prepared as discussed by any suitable method, including recombinant DNA technology, isolation from a suitable source, purchase from a commercial source, or chemical synthesis. The selected linker or linkers is (are) linked to the receptor-binding internalized ligands by chemical reaction, generally relying on an available thiol or amine group on the receptor-binding internalized ligands. Heterobifunctional linkers are particularly suited for chemical conjugation. Alternatively, if the linker is a peptide linker, then the receptor-binding internalized ligands, linker and nucleic acid binding domain can be expressed recombinantly as a fusion protein.
Any protein that binds and internalizes through a receptor interaction may be used herein. In particular, any member of the FGF family of peptides or portion thereof that binds to an FGF receptor and internalizes a linked agent may be used herein. For the chemical conjugation methods the protein may be produced recombinantly, produced synthetically or obtained from commercial or other sources. For the preparation of fusion proteins, the DNA encoding the FGF may be obtained from any known source or synthesized according to its DNA or amino acid sequences (see discussion above). Although any of the growth factors may be conjugated in this manner,
FGF, VEGF, and HBEGF conjugation are discussed merely by way of example and not by way of limitation.
If necessary or desired, the heterogeneity of preparations of ligand (e.g., FGF) containing chemical conjugates and fusion proteins can be reduced by modifying the ligand by deleting or replacing a site(s) that causes the heterogeneity. Such sites in FGF are typically cysteine residues that upon folding of the protein remain available for interaction with other cysteines or for interaction with more than one cytotoxic molecule per molecule of FGF peptide. Thus, such cysteine residues do not include any cysteine residue that is required for proper folding of the FGF peptide or for binding to an FGF receptor and internalization. For chemical conjugation, one cysteine residue that in physiological conditions is available for interaction is not replaced but is used as the site for linking the cytotoxic moiety. The resulting modified FGF is thus conjugated with a single species of nucleic acid binding domain (or nucleic acid).
The polypeptide reactive with an FGF receptor may be modified by removing one or more reactive cysteines that are not required for receptor binding, but that are available for reaction with appropriately derivatized cytotoxic agent, so that the resulting FGF protein has only one cysteine residue available for conjugation with the cytotoxic agent. If necessary, the contribution of each cysteine to the ability to bind to FGF receptors may be determined empirically. Each cysteine residue may be systematically replaced with a conservative amino acid change (see Table 1, above) or deleted. The resulting mutein is tested for the requisite biological activity, the ability to bind to FGF receptors and internalize linked cytotoxic moieties. If the mutein retains at least 50% of wild-type activity, then the cysteine residue is not required. Additional cysteines are systematically deleted and replaced and the resulting muteins are tested for activity. In this manner the minimum number and identity of the cysteines needed to retain the ability to bind to an FGF receptor and internalize may be determined. The resulting mutant FGF is then tested for retention of the ability to target a cytotoxic agent to a cell that expresses an FGF receptor and to internalize the cytotoxic agent into such cells. Retention of proliferative activity is indicative, though not definitive, of the retention of such activities. Proliferative activity may be measured by any suitable proliferation assay, such as the assay, exemplified below, that measures the increase in cell number of bovine aortic endothelial cells.
It is noted, however, that modified or mutant FGFs may exhibit reduced or no proliferative activity, but may be suitable for use herein, if they retain the ability to target cytocide-encoding agent to cells bearing FGF receptors and result in internalization. Certain residues of FGF-2 have been associated with proliferative activity. Modification of these residues arg 116, lys 119, tyr 120, tφ 123 to ile 116, glu 119, ala 120, ala 123 may be made individually (see SEQ ID NOs. 81-84) to remove this function. The resulting protein is tested for proliferative activity by a standard assay. Any of FGF-1 - FGF-9 may be used. The complete amino acid sequence of each of FGF-1 - FGF- 9 is known (see, e.g., SEQ ID NO. 10 (FGF-1) and SEQ ID NOs. 12-18 (FGF-3 - FGF-9, respectively)). Comparison among the amino acid sequences of FGF-1 -FGF-9 reveals that one Cys is conserved among FGF family of peptides (see Table 3). These cysteine residues may be required for secondary structure and are not preferred residues to be altered. Each of the remaining cysteine residues may be systematically deleted and/or replaced by a serine residue or other residue that would not be expected to alter the structure of the protein. The resulting peptide is tested for biological activity. If the cysteine residue is necessary for retention of biological activity it is not deleted; if it not necessary, then it is preferably replaced with a serine or other residue that should not alter the secondary structure of the resulting protein.
The cysteine residues from each of FGF-1 - FGF-9 that appear to be essential for retention of biological activity and that are not preferred residues for deletion or replacement are as follows:
TABLE 3
FGF-1 cys98
FGF-2 cys101
FGF-3 cys"5
FGF-4 cys155
FGF-5 cys'60
FGF-6 cys147
FGF-7 cys137
FGF-8 cys127
FGF-9 cys134
For example, FGF-1 has cysteines at positions 31, 98 and 132; FGF-2 has cysteines at positions 34, 78, 96 and 101; FGF-3 has cysteines at positions 50 and 115; FGF-4 has cysteines at positions 88 and 155; FGF-5 has cysteines at positions 19, 93, 160 and 202; FGF-6 has cysteines at positions 80 and 147; FGF-7 has cysteines at positions 18, 23, 32, 46, 71, 133 and 137; FGF-8 has cysteines at positions 10, 19, 109 and 127; and FGF-9 has cysteines at positions 68 and 134.
Since FGF-3, FGF-4 and FGF-6 have only two cysteines, for puφoses of chemical conjugation, preferably neither cysteine is deleted or replaced, unless another residue, preferably one near either terminus, is replaced with a cysteine. With respect to the other FGF family members, at least one cysteine must remain available for conjugation with the cytotoxic conjugate and probably two cysteines, but at least the cysteine residues set forth in Table 3. A second cysteine may be required to form a disulfide bond. Thus, any FGF peptide that has more than three cysteines is be modified for chemical conjugation by deleting or replacing the other cysteine residues. FGF peptides that have three cysteine residues are modified by elimination of one cysteine, conjugated to a cytotoxic moiety and tested for the ability to bind to FGF receptors and internalize the cytotoxic moiety. In accord with the methods herein, several muteins of basic FGF for chemical conjugation have been produced (preparation of muteins for recombinant expression of the conjugate is described below). DNA, obtained from pFC80 (see PCT Application Serial No. PCT/US93/05702; United States Application Serial No. 07/901,718; see also SEQ ID NO. 52) encoding basic FGF has been mutagenized. Mutagenesis of cysteine 78 of basic FGF (FGF-2) to serine ([C78S]FGF) or cysteine 96 to serine ([C96SJFGF) produced two mutants that retain virtually complete proliferative activity of native basic FGF as judged by the ability to stimulate endothelial cell proliferation in culture. The activities of the two mutants and the native protein do not significantly differ as assessed by efficacy or maximal response. Sequence analysis of the modified DNA verified that each of the mutants has one codon for cysteine converted to that for serine. The construction and biological activity of FGF-1 with cysteine substitutions of one, two or all three cysteines has been disclosed (U.S. Patent No. 5,223,483). The mitogenic activity of the mutants was similar to or increased over the native protein. Thus, any of the cysteines may be mutated and FGF-1 will still bind and internalize. The resulting mutein FGF or unmodified FGF is reacted with a nucleic acid binding domain. The bFGF muteins may react with a single species of derivatized nucleic acid binding domain (mono-derivatized nucleic acid binding domain), thereby resulting in monogenous preparations of FGF-nucleic acid binding domain conjugates and homogeneous compositions of FGF-nucleic acid binding domain chemical conjugates. The resulting chemical conjugates do not aggregate and retain the requisite biological activities.
VEGF or HBEGF may be isolated from a suitable source or may be produced using recombinant DNA methodology, discussed below. To effect chemical conjugation herein, the growth factor protein is conjugated generally via a reactive amine group or thiol group to the nucleic acid binding domain directly or through a linker to the nucleic acid binding domain. The growth factor protein is conjugated either via its N-terminus, C-terminus, or elsewhere in the polypeptide. In preferred embodiments, the growth factor protein is conjugated via a reactive cysteine residue to the linker or to the nucleic acid binding domain. The growth factor can also be modified by addition of a cysteine residue, either by replacing a residue or by inserting the cysteine, at or near the amino or carboxyl terminus, within about 20, preferably 10 residues from either end, and preferably at or near the amino terminus.
In certain embodiments, the heterogeneity of preparations may be reduced by mutagenizing the growth factor protein to replace reactive cysteines, leaving, preferably, only one available cysteine for reaction. The growth factor protein is modified by deleting or replacing a site(s) on the growth factor that causes the heterogeneity. Such sites are typically cysteine residues that, upon folding of the protein, remain available for interaction with other cysteines or for interaction with more than one cytotoxic molecule per molecule of heparin-binding growth factor peptide. Thus, such cysteine residues do not include any cysteine residue that are required for proper folding of the growth factor or for retention of the ability to bind to a growth factor receptor and internalize. For chemical conjugation, one cysteine residue that, in physiological conditions, is available for interaction, is not replaced because it is used as the site for linking the cytotoxic moiety. The resulting modified heparin-binding growth factor is conjugated with a single species of cytotoxic conjugate.
Alternatively, the contribution of each cysteine to the ability to bind to VEGF, HBEGF or other heparin-binding growth factor receptors may be determined empirically as described herein. Each cysteine residue may be systematically replaced with a conservative amino acid change (see Table 1 , above) or deleted. The resulting mutein is tested for the requisite biological activity: the ability to bind to growth factor receptors and internalize linked nucleic acid binding domain and agents. If the mutein retains this activity, then the cysteine residue is not required. Additional cysteines are systematically deleted and replaced and the resulting muteins are tested for activity. Each of the remaining cysteine residues may be systematically deleted and/or replaced by a serine residue or other residue that would not be expected to alter the structure of the protein. The resulting peptide is tested for biological activity. If the cysteine residue is necessary for retention of biological activity it is not deleted; if it not necessary, then it is preferably replaced with a serine or other residue that should not alter the secondary structure of the resulting protein. In this manner the minimum number and identity of the cysteines needed to retain the ability to bind to a heparin-binding growth factor receptor and internalize may be determined. It is noted, however, that modified or mutant heparin-binding growth factors may exhibit reduced or no proliferative activity, but may be suitable for use herein, if they retain the ability to target a linked cytotoxic agent to cells bearing receptors to which the unmodified heparin-binding growth factor binds and result in internalization of the cytotoxic moiety. In the case of VEGF, VEGF121 contains 9 cysteines and each of VEGF]65, VEGF189 and VEGF206 contain 7 additional residues in the region not present in VEGF121. Any of the 7 are likely to be non-essential for targeting and internalization of linked cytotoxic agents. Recently, the role of Cys-25, Cys-56, Cys-67, Cys-101, and Cys-145 in dimerization and biological activity was assessed (Claffery et al., Biochem. Biophys. Acta 7246:1-9, 1995). Dimerization requires Cys-25, Cys-56, and Cys-67. Substitution of any one of these cysteine residues resulted in secretion of a monomeric VEGF, which was inactive in both vascular permeability and endothelial cell mitotic assays. In contrast, substitution of Cys 145 had no effect on dimerization, although biological activities were somewhat reduced. Substitution of Cys-101 did not result in the production of a secreted or cytoplasmic protein. Thus, substitution of Cys-145 is preferred.
The VEGF monomers are preferably linked via non-essential cysteine residues to the linkers or to the targeted agent. VEGF that has been modified by introduction of a Cys residue at or near one terminus, preferably the N-terminus is preferred for use in chemical conjugation. For use herein, preferably the VEGF is dimerized prior to linkage to the linker and/or targeted agent. Methods for coupling proteins to the linkers, such as the heterobifunctional agents, or to nucleic acids, or to proteins are known to those of skill in the art and are also described herein.
For recombinant expression using the methods described herein, up to all cysteines in the HBEGF polypeptide that are not required for biological activity can be deleted or replaced. Alternatively, for use in the chemical conjugation methods herein, all except one of these cysteines, which will be used for chemical conjugation to the cytotoxic agent, can be deleted or replaced. Each of the HBEGF polypeptides described herein have six cysteine residues. Each of the six cysteines may independently be replaced and the resulting mutein tested for the ability to bind to HBEGF receptors and to be internalized. Alternatively, the resulting mutein-encoding DNA is used as part of a construct containing DNA encoding the nucleic acid binding domain linked to the HBEGF-encoding DNA. The construct is expressed in a suitable host cell and the resulting protein tested for the ability to bind to HBEGF receptors and internalize. As long as this ability is retained the mutein is suitable for use herein.
Methods for chemical conjugation of proteins are known to those of skill in the art. The preferred methods for chemical conjugation depend on the selected components, but preferably rely on disulfide bond formation. For example, if the targeted agent is SPDP-derivatized saporin, then it is advantageous to dimerize the VEGF moiety prior coupling or conjugating to the derivatized saporin. If VEGF is modified to include a cysteine residue at or near the N-, preferably, or C- terminus, then dimerization should follow coupling to the nucleic acid binding domain. To effect chemical conjugation herein, the HBEGF polypeptide is linked via one or more selected linkers or directly to the nucleic acid binding domain.
b. Preparation of nucleic acid binding domains for chemical conjugation
A nucleic acid binding domain is prepared for chemical conjugation. For chemical conjugation, a nucleic acid binding domain may be derivatized with SPDP or other suitable chemicals. If the binding domain does not have a Cys residue available for reaction, one can be either inserted or substituted for another amino acid. If desired, mono-derivatized species may be isolated, essentially as described.
For chemical conjugation, the nucleic acid binding domain may be derivatized or modified such that it includes a cysteine residue for conjugation to the receptor-binding internalized ligand. Typically, derivatization proceeds by reaction with SPDP. This results in a heterogeneous population. For example, nucleic acid binding domain that is derivatized by SPDP to a level of 0.9 moles pyridine-disulfide per mole of nucleic acid binding domain includes a population of non-derivatized, mono-derivatized and di-derivatized SAP. nucleic acid binding domain proteins, which are overly derivatized with SPDP, may lose ability to bind nucleic acid because of reaction with sensitive lysines (Lambert et al., Cancer Treat. Res. 57:175-209, 1988). The quantity of non-derivatized nucleic acid binding domain in the preparation of the non-purified material can be difficult to judge and this may lead to errors in being able to estimate the correct proportion of derivatized nucleic acid binding domain to add to the reaction mixture.
Because of the removal of a negative charge by the reaction of SPDP with lysine, the three species, however, have a charge difference. The methods herein rely on this charge difference for purification of mono-derivatized nucleic acid binding domain by Mono-S cation exchange chromatography. The use of purified mono- derivatized nucleic acid binding domain has distinct advantages over the non-purified material. The amount of receptor-binding internalized ligand that can react with nucleic acid binding domain is limited to one molecule with the mono-derivatized material, and it is seen in the results presented herein that a more homogeneous conjugate is produced. There may still be sources of heterogeneity with the mono-derivatized nucleic acid binding domain used here but is acceptable as long as binding to the cytocide-encoding agent is not impacted.
Because more than one amino group on the nucleic acid binding domain may react with the succinimidyl moiety, it is possible that more than one amino group on the surface of the protein is reactive. This creates potential for heterogeneity in the mono-derivatized nucleic acid binding domain.
As an alternative to derivatizing to introduce a sulfhydryl, the nucleic acid binding domain can be modified by the introduction of a cysteine residue. Preferred loci for introduction of a cysteine residue include the N-terminus region, preferably within about one to twenty residues from the N-terminus of the nucleic acid binding domain.
Using either methodology (reacting mono-derivatized nucleic acid binding domain or introducing a Cys residue into nucleic acid binding domain), the resulting preparations of chemical conjugates are monogenous; compositions containing the conjugates also appear to be free of aggregates.
2. Fusion protein of receptor-binding internalized ligands and nucleic acid binding domain As a preferred alternative, heterogeneity can be avoided by producing a fusion protein of receptor-binding internalized ligand and nucleic acid binding domain, as described below. Expression of DNA encoding a fusion of a receptor-binding internalized ligand polypeptide linked to the nucleic acid binding domain results in a more homogeneous preparation of cytotoxic conjugates. Aggregate formation can be reduced in preparations containing the fusion proteins by modifying the receptor- binding internalized ligand, such as by removal of nonessential cysteines, and/or the nucleic acid binding domain to prevent interactions between conjugates via free cysteines. Optionally, one or more coding regions for endosome-disruptive peptide may be constructed as part of the fusion protein. DNA encoding the polypeptides may be isolated, synthesized or obtained from commercial sources or prepared as described herein. Expression of recombinant polypeptides may be performed as described herein; and DNA encoding these polypeptides may be used as the starting materials for the methods herein.
As described above, DNA encoding FGF, VEGF, HBEGF hepatocyte growth factor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-11, IL-13, TNF, GM- CSF, IFN and IGF polypeptides and/or the amino acid sequences of these factors are described above. DNA may be prepared synthetically based on the amino acid or DNA sequence or may be isolated using methods known to those of skill in the art, such as PCR, probe hybridization of libraries, and the like or obtained from commercial or other sources. For example, suitable methods are described in the Examples for amplifying FGF encoding cDNA from plasmids containing FGF encoding cDNA.
As described herein, such DNA may then be mutagenized using standard methodologies to delete or replace any cysteine residues that are responsible for aggregate formation. If necessary, the identity of cysteine residues that contribute to aggregate formation may be determined empirically, by deleting and/or replacing a cysteine residue and ascertaining whether the resulting growth factor with the deleted cysteine forms aggregates in solutions containing physiologically acceptable buffers and salts. Loci for insertion of cysteine residues may also be determined empirically. Generally, regions at or near (within 20, preferably 10 amino acids) the C- or, preferably, the N-terminus are preferred. The DNA construct encoding the fusion protein can be inserted into a plasmid and expressed in a selected host, as described above, to produce a recombinant receptor-binding internalized Uganda — nucleic acid binding domain conjugate. Multiple copies of the chimera can be inserted into a single plasmid in operative linkage with one promoter. When expressed, the resulting protein will then be a multimer. Typically, two to six copies of the chimera are inserted, preferably in a head to tail fashion, into one plasmid.
a. Preparation of muteins for recombinant production of the fusion protein Removal of cysteines not required for binding and internalization is preferred for both chemical conjugation and recombinant methods in the chemical conjugation methods, all except one cysteine, which is necessary for chemical conjugation are deleted or replaced. In practice, it appears that for FGF polypeptides only two cysteines (including each of the cysteine residues set forth in Table 3), and perhaps only the cysteines set forth in Table 3, are required for retention of the requisite biological activity of the FGF peptide. Thus, FGF peptides that have more than two cysteines are modified by replacing the remaining cysteines with serines. The resulting muteins may be tested for the requisite biological activity.
FGF peptides, such as FGF-3, FGF-4 and FGF-6, that have two cysteines can be modified by replacing the second cysteine, which is not listed in Table 3, and the resulting mutein used as part of a construct containing DNA encoding the cytotoxic agent linked to the FGF-encoding DNA. The construct is expressed in a suitable host cell and the resulting protein tested for the ability to bind to FGF receptors and internalize the cytotoxic agent. As exemplified herein, conjugates containing bFGF muteins in which Cys78 and Cys96 have been replaced with serine residues have been prepared.
b. DNA constructs and expression of the DNA constructs
To produce monogenous preparations of fusion protein, DNA encoding the FGF protein or other receptor-binding internalized ligand is modified so that, upon expression, the resulting FGF portion of the fusion protein does not include any cysteines available for reaction. In preferred embodiments, DNA encoding an FGF polypeptide is linked to DNA encoding a nucleic acid binding domain. The DNA encoding the FGF polypeptide or other receptor-binding internalized ligand is modified in order to remove the translation stop codon and other transcriptional or translational stop signals that may be present and to remove or replace DNA encoding the available cysteines. The DNA is then ligated to the DNA encoding the nucleic acid binding domain polypeptide directly or via a linker region of one or more codons between the first codon of the nucleic acid binding domain and the last codon of the FGF. The size of the linker region may be any length as long as the resulting conjugate binds and is internalized by a target cell. Presently, spacer regions of from about one to about seventy-five to ninety codons are preferred. The order of the receptor-binding internalized ligand and nucleic acid binding domain in the fusion protein may be reversed. If the nucleic acid binding domain is N-terminal, then it is modified to remove the stop codon and any stop signals. As discussed above, any heparin-binding protein, including FGF, VEGF,
HBEGF, cytokine, growth factor and the like may be modified and expressed in accord with the methods herein. Binding to an FGF receptor followed by internalization are the only activities required for an FGF protein to be suitable for use herein. All of the FGF proteins induce mitogenic activity in a wide variety of normal diploid mesoderm- derived and neural crest-derived cells and this activity is mediated by binding to an FGF cell surface receptor followed by internalization. A test of such "FGF mitogenic activity", which reflects the ability to bind to FGF receptors and to be internalized, is the ability to stimulate proliferation of cultured bovine aortic endothelial cells (see, e.g., Gospodarowicz et al., J. Biol. Chem. 257:12266-12278, 1982; Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 75:4120-4124, 1976).
If the FGF or other ligand has been modified so as to lack mitogenic activity or other biological activities, binding and internalization may still be readily assayed by any one of the following tests or other equivalent tests. Generally, these tests involve labeling the ligand, incubating it with target cells, and visualizing or measuring intracellular label. For example, briefly, FGF may be fluorescently labeled with FITC or radiolabeled with 125I. Fluorescein-conjugated FGF is incubated with cells and examined microscopically by fluorescence microscopy or confocal microscopy for internalization. When FGF is labeled with 125I, the labeled FGF is incubated with cells at 4°C. Cells are temperature shifted to 37°C and washed with 2 M NaCl at low pH to remove any cell-bound FGF. Label is then counted and thereby measuring internalization of FGF. Alternatively, the ligand can be conjugated with an nucleic acid binding domain by any of the methods described herein and complexed with a plasmid encoding saporin. As discussed below, the complex may be used to transfect cells and cytotoxicity measured. The DNA encoding the resulting receptor-binding internalized ligand — ucleic acid binding domain can be inserted into a plasmid and expressed in a selected host, as described above, to produce a monogenous preparation. Fusion proteins of FGF-2 and protamine are especially suitable for use in the present invention. Multiple copies of the modified receptor-binding internalized ligand/nucleic acid binding domain chimera can be inserted into a single plasmid in operative linkage with one promoter. When expressed, the resulting protein will be a multimer. Typically two to six copies of the chimera are inserted, preferably in a head to tail fashion, into one plasmid. Merely by way of example, DNA encoding human bFGF-SAP having
SEQ ID NO. 52 has been mutagenized as described in the Examples using splicing by overlap extension (SOE). Another preferred coding region is set forth in SEQ ID NO. 53. In both instances, in preferred embodiments, the DNA is modified by replacing the cysteines at positions 78 and 96 with serine. The codons encoding cysteine residues at positions 78 and 96 of FGF were converted to serine codons by SOE. Each application of the SOE method uses two amplified oligonucleotide products, which have complementary ends as primers and which include an altered codon at the locus at which the mutation is desired, to produce a hybrid product. A second amplification reaction that uses two primers that anneal at the non-overlapping ends amplify the hybrid to produce DNA that has the desired alteration.
3. Binding of the receptor-binding internalized ligand/nucleic acid binding domain conjugate to cytocide-encoding agents
The receptor-binding internalized ligand/nucleic acid binding domain is incubated with the cytocide-encoding agent, preferably a linear DNA molecule, to be delivered under conditions that allow binding of the nucleic acid binding domain to the agent. Conditions will vary somewhat depending on the nature of the nucleic acid binding domain, but will typically occur in 0.1M NaCl and 20 mM HEPES or other similar buffer. Alternatively, salt conditions can be varied to increase the packing or condensation of DNA. The extent of binding is preferably tested for each preparation. After complexing, additional nucleic acid binding domain, such as poly-L-lysine, may be added to further condense the nucleic acid.
Merely by way of example, test constructs have been made and tested. One construct is a chemical conjugate of bFGF and poly-L-lysine. The bFGF molecule is a variant in which the Cys residue at position 96 has been changed to a serine; thus, only the Cys at position 78 is available for conjugation. This bFGF is called FGF2-3. The poly-L-lysine was derivatized with SPDP and coupled to FGF2-3. This FGF2- 3/poly-L-lysine conjugate was used to deliver a plasmid able to express the β-galactosidase gene. The ability of a construct to bind nucleic acid molecules may be conveniently assessed by agarose gel electrophoresis. Briefly, a plasmid, such as pSVβ, is digested with restriction enzymes to yield a variety of fragment sizes. For ease of detection, the fragments may be labeled with 32P either by filling in of the ends with DNA polymerase I or by phosphorylation of the 5 '-end with polynucleotide kinase following dephosphorylation by alkaline phosphatase. The plasmid fragments are then incubated with the receptor-binding internalized ligand/nucleic acid binding domain in this case, FGF2-3/poly-L-lysine in a buffered saline solution, such as 20 mM HEPES, pH 7.3, 0.1M NaCl. The reaction mixture is electrophoresed on an agarose gel alongside similarly digested, but nonreacted fragments. If a radioactive label was incoφorated, the gel may be dried and autoradiographed. If no radioactive label is present, the gel may be stained with ethidium bromide and the DNA visualized through appropriate red filters after excitation with UV. Binding has occurred if the mobility of the fragments is retarded compared to the control. In the example case, the mobility of the fragments was retarded after binding with the FGF2-3/poly-L-lysine conjugate. If there is insufficient binding, poly-L-lysine may be additionally added until binding is observed.
Further testing of the conjugate is performed to show that it binds to the cell surface receptor and is internalized into the cell. It is not necessary that the receptor-binding internalized ligand part of the conjugate retain complete biological activity. For example, FGF is mitogenic on certain cell types. As discussed above, this activity may not always be desirable. If this activity is present, a proliferation assay is performed. Likewise, for each desirable activity, an appropriate assay may be performed. However, for application of the subject invention, the only criteria that need be met are receptor binding and internalization. Receptor binding and internalization may be measured by the following three assays. (1) A competitive inhibition assay of the complex to cells expressing the appropriate receptor demonstrates receptor binding. (2) Receptor binding and internalization may be assayed by measuring expression of a reporter gene, such as β-gal (e.g., enzymatic activity), in cells that have been transformed with a complex of a plasmid encoding a reporter gene and a conjugate of a receptor-binding internalized ligand and nucleic acid binding domain. This assay is particularly useful for optimizing conditions to give maximal transformation. Thus, the optimum ratio of receptor- binding internalized ligand/nucleic acid binding domain to nucleic acid and the amount of DNA per cell may readily be determined by assaying and comparing the enzymatic activity of β-gal. As such, these first two assays are useful for preliminary analysis and failure to show receptor binding or β-gal activity does not per se eliminate a candidate receptor-binding internalized ligand/nucleic acid binding domain conjugate or fusion protein from further analysis. (3) The preferred assay is a cytotoxicity assay performed on cells transformed with a cytocide-encoding agent bound by receptor-binding internalized ligand/nucleic acid binding domain. While, in general, any cytocidal molecule may be used, ribosome inactivating proteins are preferred and saporin, or another type I ribosome inactivating protein, is particularly preferred. A statistically significant reduction in cell number demonstrates the ability of the receptor-binding internalized ligand/nucleic acid binding domain conjugate or fusion to deliver nucleic acids into a cell.
4. Conjugation of ligand to nucleic acid and binding to nucleic acid binding domain
As an alternative, the receptor-internalized binding ligand may be conjugated to the nucleic acid, either directly or through a linker. Methods for conjugating nucleic acids, at the 5' ends, 3' ends and elsewhere, to the amino and carboxyl termini and other sites in proteins are known to those of skill in the art (for a review see, e.g., Goodchild, (1993) In: Perspectives in Bioconjugate Chemistry, Mears, Ed., American Chemical Society, Washington, D.C. pp. 77-99). For example, proteins have been linked to nucleic acids using ultraviolet irradiation (Sperling et al. (1978) Nucleic Acids Res. 5:2755-2773; Fiser et al. (1975) FEBS Lett. 52:281-283), bifunctional chemicals (Baumert et al. (1978 Eur. J. Biochem. 59:353-359; and Oste et al. (1979) Mol. Gen. Genet. 765:81-86) and photochemical cross-linking (Vanin et al. (1981) FEBS Lett. 724.89-92; Rinke et al. (1980) J. Mol. Biol. 757:301-314; Millon et al. (1980) Eur. J. Biochem. 770:485-454). In particular, the reagents (N-acetyl-N'-(p-glyoxylylbenzolyl)cystamine and 2-iminothiolane have been used to couple DNA to proteins, such as α-macroglobulin (C M) via mixed disulfide formation (see Cheng et al., Nucleic Acids Res. 77:659-669, 1983). N-acetyl-N'-(p-glyoxylylbenzolyl)cystamine reacts specifically with nonpaired guaninine residues and, upon reduction, generates a free sulfhydryl group. 2-iminothiolane reacts with proteins to generate sulfhydryl groups that are then conjugated to the derivatized DNA by an intermolecular disulfide interchange reaction. Any linkage may be used provided that the targeted nucleic acid is active upon internalization of the conjugate. Thus, it is expected that cleavage of the linkage may be necessary, although it is contemplated that for some reagents, such as DNA encoding ribozymes linked to promoters or DNA encoding therapeutic agents for delivery to the nucleus, such cleavage may not be necessary.
Thiol linkages, which are preferred, can be readily formed using heterbiofunctional reagents. Amines have also been attached to the terminal 5' phosphate of unprotected oligonucleotides or nucleic acids in aqueous solutions by reacting the nucleic acid with a water-soluble carbodiimide, such as l-ethyl-3'[3- dimethylaminopropyljcarbodiimide (EDC) or N-ethyl-N'(3-dimethylaminopropylcar- bodiimidehydrochloride (EDCI), in imidazole buffer at pH 6 to produce the 5'phosphorimidazolide. Contacting the 5'phosphorimidazolide with amine-containing molecules, such as an FGF, and ethylenediamine, results in stable phosphoramidates (see, e.g., Chu et al., Nucleic Acids Res. 77:6513-6529, 1983; and WO 88/05077). In particular, a solution of DNA is saturated with EDC, at pH 6 and incubated with agitation at 4°C overnight. The resulting solution is then buffered to pH 8.5 by adding, for example about 3 volutes of 100 mM citrate buffer, and adding about 5 μg - about 20 μg of an FGF, and agitating the resulting mixture at 4°C for about 48 hours. The unreacted protein may be removed from the mixture by column chromatography using, for example, Sephadex G75 (Pharmacia) using 0.1 M ammonium carbonate solution, pH 7.0 as an eluting buffer. The isolated conjugate may be lyophilized and stored until used.
U.S. Patent No. 5,237,016 provides methods for preparing nucleotides that are bromacetylated at their 5' termini and reacting the resulting oligonucleotides with thiol groups. Oligonucleotides derivatized at their 5'-termini bromoacetyl groups can be prepared by reacting 5'-aminohexyl-phosphoramidate oligonucleotides with bromoacetic acid-N-hydroxysuccinimide ester as described in U.S. Patent No. 5,237,016. This patent also describes methods for preparing thiol-derivatized nucleotides, which can then be reacted with thiol groups on the selected growth factor. Briefly, thiol-derivatized nucleotides are prepared using a 5 '-phosphorylated nucleotide in two steps: (1) reaction of the phosphate group with imidazole in the presence of a diimide and displacement of the imidazole leaving group with cystamine in one reaction step; and reduction of the disulfide bond of the cystamine linker with dithiothreitol (see, also, Orgel et al. ((1986) Nucl. Acids Res. 74:651, which describes a similar procedure). The 5'-phosphorylated starting oligonucleotides can be prepared by methods known to those of skill in the art (see, e.g., Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, p. 122).
The nucleic acid, such as a methylphosphonate oligonucleotide (MP- oligomer), may be derivatized by reaction with SPDP or SMPB. The resulting MP- oligomer may be purified by HPLC and then coupled to an FGF, such as an FGF or FGF mutein, modified by replacement of one or more cysteine residues, as described above. The MP-oligomer (about 0.1 μM) is dissolved in about 40-50 μl of 1 :1 acetonitrile/water to which phosphate buffer (pH 7.5, final concentration 0.1 M) and a 1 mg MP-oligomer in about 1 ml phosphate buffered saline is added. The reaction is allowed to proceed for about 5-10 hours at room temperature and is then quenched with about 15 μL 0.1 iodoacetamide. FGF-oligonucleotide conjugates can be purified on heparin sepharose Hi Trap columns (1 ml, Pharmacia) and eluted with a linear or step gradient. The conjugate should elute in 0.6 M NaCl. The ligand may be conjugated to the nucleic acid construct encoding the cytocide or cytotoxic agent or may be conjugated to a mixture of oligonucleotides complementary to one strand of the construct. The oligonucleotides are then added to single stranded construct produced by melting a double-stranded construct or grown and isolated as single-stranded. As a general guideline, the oligonucleotides should hybridize at a higher temperature than the construct alone, if a double-stranded construct is used as the starting material. The gaps are filled in by DNA polymerase I to generate a construct with one strand conjugated to ligand and one strand unconjugated. Oligonucleotides conjugated to ligand and complementary to the other strand may be used in addition to generate a mixture of constructs with different strands linked to ligand. Any remaining single stranded plasmid may be digested with a single strand specific endonuclease. The ligand-conjugated constructs are then mixed with a nucleic acid binding domain, such as protamine or polylysine, to effect condensation of the construct for delivery. Optimal ratios of ligand to DNA may be determined experimentally by receptor-mediated transfection of a construct containing a reporter gene.
J. Formulation and administration of pharmaceutical compositions
The conjugates and complexes provided herein are useful in the treatment and prevention of various diseases, syndromes, and hypeφroliferative disorders. As used herein, "treatment" means any manner in which the symptoms of a conditions, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein. As used herein, "amelioration" of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition. For example, these conjugates and complexes may be used to treat complications of the eye following laser surgery, glaucoma surgery, and removal of pterygii. Following these treatments, reoccurrence of the problem often ensues due to proliferation of cells in the cornea or eye. The conjugates and complexes inhibit the proliferation of these cells. The conjugates and complexes may be used in general to treat pathophysiological conditions, especially FGF-, VEGF-, or HBEGF-mediated pathophysiological conditions by specifically targeting to cells having corresponding receptors.
As used herein, "FGF-mediated pathophysiological condition" refers to a deleterious condition characterized by or caused by proliferation of cells that are sensitive to FGF mitogenic stimulation. Basic FGF-mediated pathophysiological conditions include, but are not limited to, melanoma, other tumors, rheumatoid arthritis, restenosis, Dupuytren's Contracture and certain complications of diabetes, such as proliferative retinopathy. As used herein, "HBEGF-mediated pathophysiological condition" refers to a deleterious condition characterized by or caused by proliferation of cells that are sensitive to HBEGF mitogenic stimulation. HBEGF-mediated pathophysiological conditions include conditions involving pathophysiological proliferation of smooth muscle cells, such as restenosis, certain tumors, such as solid tumors including breast and bladder tumors, tumors involving pathophysiological expression of EGF receptors, dermatological disorders, such as psoriasis, and ophthalmic disorders involving epithelial cells, such as recurrence of pterygii and secondary lens clouding.
Similarly, tumors and hypeφroliferating cells expressing cytokine receptors or growth factor receptors may be eliminated. Such diseases include restenosis, Dupuytren's Contracture, diabetic retinopathies, rheumatoid arthritis, Kaposi's sarcoma, lymphomas, leukemias, tumors such as renal cell carcinoma, colon carcinoma, breast cancer, bladder cancer, disorders with underlying vascular proliferation, such as diseases in the back of the eye (e.g., proliferative vitreoritinopathy, inacular degeneration and diabetic retinopathy). For treatment of the back of the eye especially, use of the VEGF-receptor promoter to control expression of the cytocide or cytotoxic agent is preferred. The conjugates may be used to prevent corneal haze or clouding that results from exposure of the cornea to laser radiation during eye surgery, particularly LRK. The haze or clouding appears to result from fibroblastic keratocyte proliferation in the subepithelial zone following photoablation of the cornea.
The conjugates may be used to treat a "hypeφroliferative skin disorder." As used herein, it is a disorder that is manifested by a proliferation of endothelial cells of the skin coupled with an underlying vascular proliferation, resulting in a localized patch of scaly or horny or thickened skin or a tumor of endothelial origin. Such disorders include actinic and atopic dermatitis, toxic eczema, allergic eczema, psoriasis, skin cancers and other tumors, such as Kaposi's sarcoma, angiosarcoma, hemangiomas, and other highly vascularized tumors, and vascular proliferative responses, such as varicose veins.
As well, the conjugates may be used to treat or prevent restenosis, a process and the resulting condition that occurs following angioplasty in which the arteries become reclogged. After treatment of arteries by balloon catheter or other such device, denudation of the interior wall of the vessel occurs, including removal of the endothelial cells that constitute the lining of the blood vessels. As a result of this removal and the concomitant vascular injury, smooth muscle cells (SMCs), which form the blood vessel structure, proliferate and fill the interior of the blood vessel. This process and the resulting condition is restenosis.
Pharmaceutical carriers or vehicles suitable for administration of the conjugates and complexes provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. In addition, the conjugates and complexes may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
The conjugates and complexes can be administered by any appropriate route, for example, orally, parenterally, including intravenously, intradermally, subcutaneously, or topically, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration. Preferred modes of administration depend upon the indication treated. Dermatological and ophthalmologic indications will typically be treated locally; whereas, tumors and restenosis, will typically be treated by systemic, intradermal, or intramuscular modes of administration.
The conjugates and complexes herein may be formulated into pharmaceutical compositions suitable for topical, local, intravenous and systemic application. For the ophthalmic uses herein, local administration, either by topical administration or by injection is preferred. Time release formulations are also desirable.
Effective concentrations of one or more of the conjugates and complexes are mixed with a suitable pharmaceutical carrier or vehicle. As used herein an "effective amount" of a compound for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Repeated administration may be required to achieve the desired amelioration of symptoms.
As used herein, "an ophthalmically effective amount" is that amount which, in the composition administered and by the technique administered, provides an amount of therapeutic agent to the involved eye tissues sufficient to prevent or reduce corneal haze following excimer laser surgery, prevent closure of a trabeculectomy, prevent or substantially slow the recurrence of pterygii, and other conditions.
The concentrations or amounts of the conjugates and complexes that are effective requires delivery of an amount, upon administration, that ameliorates the symptoms or treats the disease. Typically, the compositions are formulated for single dosage administration. Therapeutically effective concentrations and amounts may be determined empirically by testing the conjugates and complexes in known in vitro and in vivo systems, such as those described here; dosages for humans or other animals may then be extrapolated therefrom.
The conjugate is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The conjugates may be delivered as pharmaceutically acceptable salts, esters or other derivatives of the conjugates include any salts, esters or derivatives that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects. It is understood that number and degree of side effects depends upon the condition for which the conjugates and complexes are administered. For example, certain toxic and undesirable side effects are tolerated when treating life-threatening illnesses, such as tumors, that would not be tolerated when treating disorders of lesser consequence. The concentration of conjugate in the composition will depend on absoφtion, inactivation and excretion rates thereof, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
Preferably, the conjugate and complex are substantially pure. As used herein, "substantially pure" means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
Typically a therapeutically effective dosage should produce a serum concentration of active ingredient of from about 0.1 ng/ml to about 50-100 μg/ml. The pharmaceutical compositions typically should provide a dosage of from about 0.01 mg to about 100 - 2000 mg of conjugate, depending upon the conjugate selected, per kilogram of body weight per day. For example, for treatment of restenosis a daily dosage of about between 0.05 and 0.5 mg/kg (based on FGF-SAP chemical conjugate or an amount of conjugate provided herein equivalent on a molar basis thereto) should be sufficient. Local application for ophthalmic disorders and dermatological disorders should provide about 1 ng up to 100 μg, preferably about 1 ng to about 10 μg, per single dosage administration. It is understood that the amount to administer will be a function of the conjugate selected, the indication treated, and possibly the side effects that will be tolerated. Therapeutically effective concentrations and amounts may be determined for each application herein empirically by testing the conjugates and complexes in known in vitro and in vivo systems (e.g., murine, rat, rabbit, or baboon models), such as those described herein; dosages for humans or other animals may then be extrapolated therefrom. Demonstration that the conjugates and complexes prevent or inhibit proliferation of serum stimulated corneal keratocytes or fibroblasts explanted from eyes, as shown herein, and demonstration of any inhibition of proliferation of such tissues in rabbits should establish human efficacy. The rabbit eye model is a recognized model for studying the effects of topically and locally applied drugs (see, e.g., U.S. Patent Nos. 5,288,735, 5,263,992, 5,262,178, 5,256,408, 5,252,319, 5,238,925, 5,165,952; see also Mirate et al., Curr. Eye Res. 7:491-493, 1981).
The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
The conjugates and complexes may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracistemal or intraspinal application. Such solutions, particularly those intended for ophthalmic use, may be formulated as 0.01% -10% isotonic solutions, pH about 5-7, with appropriate salts. The ophthalmic compositions may also include additional components, such as hyaluronic acid. The conjugates and complexes may be formulated as aerosols for topical application (see, e.g., U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923).
Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of toxicity such as sodium chloride or dextrose. Parental preparations can be enclosed in ampules, disposable syringes or multiple dose vials made of glass, plastic or other suitable material. If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art.
Upon mixing or addition of the conjugate(s) with the vehicle, the resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the conjugate in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined based upon in vitro and/or in vivo data, such as the data from the mouse xenograft model for tumors or rabbit ophthalmic model. If necessary, pharmaceutically acceptable salts or other derivatives of the conjugates and complexes may be prepared. The active materials can also be mixed with other active materials, that do not impair the desired action, or with materials that supplement the desired action, including viscoelastic materials, such as hyaluronic acid, which is sold under the trademark HEALON (solution of a high molecular weight (MW of about 3 millions) fraction of sodium hyaluronate; manufactured by Pharmacia, Inc. see, e.g., U.S. Patent Nos. 5,292,362, 5,282,851, 5,273,056, 5,229,127, 4,517,295 and 4,328,803), VISCOAT (fluorine-containing (meth)acrylates, such as, lH,lH,2H,2H-hepta- decafluorodecylmethacrylate; see, e.g., U.S. Patent Nos. 5,278,126, 5,273,751 and 5,214,080; commercially available from Alcon Surgical, Inc.), ORCOLON (see, e.g., U.S. Patent Nos. 5,273,056; commercially available from Optical Radiation Coφoration), methylcellulose, methyl hyaluronate, polyacrylamide and polymethacrylamide (see, e.g., U.S. Patent No. 5,273,751). The viscoelastic materials are present generally in amounts ranging from about 0.5 to 5.0%, preferably 1 to 3% by weight of the conjugate material and serve to coat and protect the treated tissues. The compositions may also include a dye, such as methylene blue or other inert dye, so that the composition can be seen when injected into the eye or contacted with the surgical site during surgery.
The conjugates and complexes may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye. Such solutions, particularly those intended for ophthalmic use, may be formulated as 0.01%- 10% isotonic solutions, pH about 5-7, with appropriate salts. Suitable ophthalmic solutions are known (see, e.g., U.S. Patent No. 5,116,868, which describes typical compositions of ophthalmic irrigation solutions and solutions for topical application). Such solutions, which have a pH adjusted to about 7.4, contain, for example, 90-100 mM sodium chloride, 4-6 mM dibasic potassium phosphate, 4-6 mM dibasic sodium phosphate, 8-12 mM sodium citrate, 0.5-1.5 mM magnesium chloride, 1.5-2.5 mM calcium chloride, 15-25 mM sodium acetate, 10-20 mM D.L.-sodium β- hydroxybutyrate and 5-5.5 mM glucose. The conjugates and complexes may be prepared with carriers that protect them against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. For example, the composition may be applied during surgery using a sponge, such as a commercially available surgical sponges (see, e.g., U.S. Patent Nos. 3,956,044 and 4,045,238; available from Week, Alcon, and Mentor), that has been soaked in the composition and that releases the composition upon contact with the eye. These are particularly useful for application to the eye for ophthalmic indications following or during surgery in which only a single administration is possible. The compositions may also be applied in pellets (such as Elvax pellets(ethylene- vinyl acetate copolymer resin); about 1- 5 μg of conjugate per 1 mg resin) that can be implanted in the eye during surgery. Ophthalmologically effective concentrations or amounts of one or more of the conjugates and complexes are mixed with a suitable pharmaceutical carrier or vehicle. The concentrations or amounts of the conjugates and complexes that are effective requires delivery of an amount, upon administration, that prevents or substantially reduces corneal clouding, trabeculectomy closure, or pterygii recurrence. The conjugates and complexes herein are formulated into ophthalmologically acceptable compositions and are applied to the affected area of the eye during or immediately after surgery. In particular, following excimer laser surgery, the composition is applied to the cornea; following trabeculectomy the composition is applied to the fistula; and following removal of pterygii the composition is applied to the cornea. The compositions may also be used to treat pterygii. The conjugates and complexes are applied during and immediately following surgery and may, if possible be applied post-operatively, until healing is complete. The compositions are applied as drops for topical and subconjunctival application or are injected into the eye for intraocular application. The compositions may also be absorbed to a biocompatible support, such as a cellulosic sponge or other polymer delivery device, and contacted with the affected area.
The ophthalmologic indications herein are typically be treated locally either by the application of drops to the affected tissue(s), contacting with a biocompatible sponge that has absorbed a solution of the conjugates and complexes or by injection of a composition. For the indications herein, the composition will be applied during or immediately after surgery in order to prevent closure of the trabeculectomy, prevent a proliferation of keratocytes following excimer laser surgery, or to prevent a recurrence of pterygii. The composition may also be injected into the affected tissue following surgery and applied in drops following surgery until healing is completed. For example, to administer the formulations to the eye, it can be slowly injected into the bulbar conjunctiva of the eye.
Conjugates and complexes with photocleavable linkers are among those preferred for use in the methods herein. Upon administration of such composition to the affected area of the eye, the eye is exposed to light of a wavelength, typically visible or UV that cleaves the linker, thereby releasing the cytotoxic agent.
If oral administration is desired, the conjugate should be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.
Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the pi-upose of oral therapeutic administration, the active compound or compounds can be incoφorated with excipients and used in the form of tablets, capsules or troches.
Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.
The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth and gelatin; an excipient such as starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, and fruit flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The conjugates and complexes can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
The active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as cis-platin for treatment of tumors.
Finally, the compounds may be packaged as articles of manufacture containing packaging material, one or more conjugates and complexes or compositions as provided herein within the packaging material, and a label that indicates the indication for which the conjugate is provided. Many methods have been developed to deliver nucleic acid into cells including retroviral vectors, electroporation, CaPO4 precipitation and microinjection, but each of these methods has distinct disadvantages. Microinjecting nucleic acid into cells is very time consuming because each cell must be manipulated individually. Retroviral vectors can only hold a limited length of nucleic acid and can activate oncogenes depending upon the insertion site in the target chromosome. Conditions for electroporation and CaP04-mediated transfection are harsh and cause much cell death.
By comparison, receptor mediated gene delivery as described herein is a more desirable method of selectively targeting toxic genes into cells that have "more active" receptors or that overexpress the specific receptor on the cell surface. A receptor may be more active because it has a higher rate of internalization or higher cycling rate through the endosome to the cell surface. Advantages of this method over other gene delivery methods include increased specificity of delivery, the absence of nucleic acid length limitations, reduced toxicity, and reduced immunogenicity of the conjugate. These characteristics allow for repeated administration of the material with minimal harm to cells and may allow increased level of expression of the toxic protein. In addition, primary cultures can also be treated using this method.
The following examples are included for illustrative puφoses only and are not intended to limit the scope of the invention.
EXAMPLES
EXAMPLE 1 ISOLATION OF DNA ENCODING SAPORIN
A. Materials and methods
1. Bacterial Strains
E. coli strain JA221 (lpp- hdsM+ tφE5 leuB6 lacY recAl F'[lacl<e lac+ pro+]) is publicly available from the American Type Culture Collection (ATCC), Rockville, MD 20852, under the accession number ATCC 33875. (JA221 is also available from the Northern Regional Research Center (NRRL), Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604, under the accession number NRRL B-15211; see also U.S. Patent No. 4,757,013 to Inouye; and Nakamura et al., Cell 75:1109-1117, 1979). Strain INVlα is commercially available from Invitrogen, San Diego, CA.
2. DNA Manipulations
The restriction and modification enzymes employed herein are commercially available in the U.S. Native saporin and rabbit polyclonal antiserum to saporin were obtained as previously described in Lappi et al., Biochem. Biophys. Res.
Comm. 129:934-942. Ricin A chain is commercially available from Sigma, Milwaukee, WI. Antiserum was linked to Affi-gel 10 (Bio-Rad, Emeryville, CA) according to the manufacturer's instructions. Sequencing was performed using the Sequenase kit of United States Biochemical Coφoration (version 2.0) according to the manufacturer's instructions. Minipreparation and maxipreparation of plasmids, preparation of competent cells, transformation, Ml 3 manipulation, bacterial media, Western blotting, and ELISA assays were according to Sambrook et al., (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). The purification of DNA fragments was done using the Geneclean II kit (Bio 101) according to the manufacturer's instructions. SDS gel electrophoresis was performed on a Phastsystem (Pharmacia).
Western blotting was accomplished by transfer of the electrophoresed protein to nitrocellulose using the PhastTransfer system, as described by the manufacturer. The antiserum to SAP was used at a dilution of 1:1000. Horseradish peroxidase labeled anti-IgG was used as the second antibody (see Davis et al., Basic Methods In Molecular Biology, New York, Elsevier Science Publishing Co., pp 1-338, 1986).
B. Isolation of DNA encoding saporin
1. Isolation of genomic DNA and preparation of polymerase chain reaction fPCR) primers
Saponaria officinalis leaf genomic DNA was prepared as described in
Bianchi et al., Plant Mol. Biol. 77:203-214, 1988. Primers for genomic DNA amplifications were synthesized in a 380B automatic DNA synthesizer. The primer corresponding to the "sense" strand of saporin 5'- CTGCAGAATTCGCATGGATCCTGCTTCAAT-3' (SEQ ID NO. 54) includes an EcoR I restriction site adapter immediately upstream of the DNA codon for amino acid -15 of the native saporin N-terminal leader sequence. The primer 5'- CTGCAGAATTCGCCTCGTTTGACTACTTTG-3* (SΕQ ID NO. 55) corresponds to the "antisense" strand of saporin and complements the coding sequence of saporin starting from the last 5 nucleotides of the DNA encoding the carboxyl end of the mature peptide. Use of this primer introduced a translation stop codon and an EcoRI restriction site after the sequence encoding mature saporin.
2. Amplification of DNA encoding saporin Unfractionated Saponaria officinalis leaf genomic DNA (1 μl) was mixed in a final volume of 100 μl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.01% gelatin, 2 mM MgCl2, 0.2 mM dNTPs, 0.8 μg of each primer. Next, 2.5 U Taq DNA polymerase (Perkin Elmer Cetus) were added and the mixture was overlaid with 30 μl of mineral oil (Sigma). Incubations were done in a DNA Thermal Cycler (Ericomp). One cycle included a denaturation step (94°C for 1 min), an annealing step (60°C for 2 min), and an elongation step (72°C for 3 min). After 30 cycles, a 10 μl aliquot of each reaction was run on a 1.5% agarose gel to verify the structure of the amplified product.
The amplified DNA was digested with EcoRI and subcloned into EcoRI- restricted M13mpl8 (New England Biolabs, Beverly, MA; see also Yanisch-Perron et al. (1985), "Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mpl8 and pUC19 vectors", Gene 55:103). Single-stranded DNA from recombinant phages was sequenced using oligonucleotides based on internal points in the coding sequence of saporin (see Bennati et al., Eur. J. Biochem. 755:465- 470, 1989). Nine of the M13mpl8 derivatives were sequenced and compared. Of the nine sequenced clones, five had unique sequences, set forth as SEQ ID NOs. 19-23, respectively. The clones were designated M13mpl8-G4, -Gl, -G2, -G7, and -G9. Each of these clones contains all of the saporin coding sequence and 45 nucleotides of DNA encoding the native saporin N-terminal leader peptide. Saporin DNA sequence was also cloned in the pETl la vector. Briefly, the DNA encoding SAP-6 was amplified by polymerase chain reaction (PCR) from the parental plasmid pZlBl . The plasmid pZlBl contains the DNA sequence for human FGF-2 linked to SAP-6 by a two-amino-acid linker (Ala-Met). PZlBl also includes the T7 promoter, lac operator, ribosomal binding site, and T7 terminator present in the pET- l la vector. For SAP-6 DNA amplification, the 5' primer (5'
CATATGTGTGTCACATCAATCACATTAGAT 3') (SEQ ID NO. 105), corresponding to the sense strand of SAP-6, incoφorated a Ndeϊ restriction enzyme site used for cloning. It also contained a Cys codon at position -1 relative to the start site of the mature protein sequence. No leader sequence was included. The 3' primer (5' CAGGTTTGGATCCTTTACGTT 3') (SEQ ID NO. 106) corresponding to the antisense strand of SAP-6 had a BamHI site used for cloning. The amplified DNA was gel-purified and digested with Ndel and BamHI. The digested SAP-6 DNA fragment was subcloned into the Nc /ifømHI-digested pZlBl. This digestion removed FGF-2 and the 5' portion of SAP-6 (up to nucleotide position 650) from the parental rFGF2- SAP vector (pZlBl) and replaced this portion with a SAP-6 molecule containing a Cys at position -1 relative to the start site of the native mature SAP-6 protein. The resultant plasmid was designated as pZ50B. pZ50B was transformed into E. coli strain ΝovaBlue for restriction and sequencing analysis. The appropriate clone was then transformed into E. coli strain BL21(DE3) for expression and large-scale production.
C. Mammalian codon optimization of saporin cDΝA.
Mammalian expression plasmids encoding β-galactosidase (β-gal), pSV- β and pΝASS-β, were obtained from Clontech (Palo Alto, CA). Plasmid pSVβ expresses β-gal from the SV40 early promoter. Plasmid pΝASSb is a promoterless mammalian reporter vector containing the β-gal gene. The amino acid sequence for the plant protein saporin (SAP) was reverse translated using mammalian codons. The resulting mammalian optimized cDΝA was divided into 4 fragments (designated 5'-3' A-D) for synthesis by PCR using overlapping oligos. To facilitate subcloning of each fragment and piecing together of the entire cDΝA, restriction enzyme sites were added to the ends of each fragment, and added or removed within each fragment without changing the corresponding amino acid sequence. In addition, the 5' end of the cDΝA was modified to include a Kozak sequence for optimal expression in mammalian cells. Fragments A, B, and D were each synthesized by annealing 4 oligos (2 sense, 2 antisense) with 20 base overlaps and using PCR to fill-in and amplify the fragments. The PCR products were then purified using GeneClean (Biol 01), digested with restriction enzymes recognizing the sites in the primers, and subcloned into pBluescript (SK+) (Stratagene). The sequence of the inserts was verified using Sequenase Version 2.0 (United States Biochemical/Amersham). Fragment C was synthesized in two steps: The 5' and 3' halves of the fragment were independently synthesized by PCR using 2 overlapping oligos. The products of these using 2 reactions were then purified and combined and the full-length fragment C was generated by PCR using the outermost oligos as primers. Full-length fragment C was subcloned into pBluescript for sequencing. Fragments A and B were ligated together in pBluescript at an overlapping Kspl site. Fragments C and D were ligated together in pBluescript at an overlapping RvuII site. Fragments A-B and C-D were then joined in pBluescript at an overlapping Aval site to give the full-length mammalian optimized SAP cDNA. β-gal sequences were excised from the plasmids pNASS-β and pSV-β (Clontech) by digestion with NotI and replaced with the synthetic SAP gene, which has NotI ends. Orientation of the insert was confirmed by restriction enzyme digestion. Large scale plasmid preparations were performed using Qiagen Maxi 500 columns. The oligos used to synthesize each SAP fragment are (5 '-3'):
Al(sense):CGTATCAGGCGGCCGCCGCCATGGTGACCTCCATCACCCTGGACC TGGTGAACCCCACCGCCGGCC (SEQ ID NO.: 89)
A2(antisense):TTGGGGTCCTTCACGTTGTTGCGGATCTTGTCCACGAAGGAGG AGTACTGGCCGGCGGTGGGGTTCACC (SEQ ID NO.: 90)
A3(sense):AACAACGTGAAGGACCCCAACCTGAAGTACGGCGGCACCGACAT CGCCGTGATCGGCCCCCCCTC (SEQ ID NO.: 91)
A4(antisense):GTGCCGCGGGAGGACTGGAAGTTGATGCGCAGGAACTTCTCCT TGGAGGGGGGGCCGATCACGGC (SEQ ID NO.: 92)
B 1 (sense):CTCCCGCGGCACCGTGTCCCTGGGCCTGAAGCGCGACAACCTGTA CGTGGTGGCCTACCTGGCCATGGACAACAC (SEQ ID NO.: 93) B2(antisense):GCGGTCAGCTCGGCGGAGGTGATCTCGGACTTGAAGTAGTAGG CGCGGTTCACGTTGGTGTTGTCCATGGCCAGGTA (SEQ ID NO.: 94)
B3(sense):GCCGAGCTGACCGCCCTGTTCCCTGAGGCCACCACCGCCAACCAG AAGGCCCTGGAGTACACCGAGGACTACCAGTCC (SEQ ID NO.: 95)
B4(antisense):AGCCCGAGCTCCTTGCGGGACTTGTCGCCCTGGGTGATCTGGG CGTTCTTCTCGATGGACTGGTAGTCCTCGGTGT (SEQ ID NO.: 96)
C 1 (sense):TATAGAATTCCTCGGGCTGGGCATCGACCTGCTGCTGACCTTCATG GAGGCCGTGAACAAGAAGGCCCGCGTGG (SEQ ID NO.: 97)
C2(antisense):CGGCGGTCATCTGGATGGCGATCAGCAGGAAGCGGGCCTCGTT CTTC ACCACGCGGGCCTTCTTGTTC (SEQ ID NO. : 98)
C3(sense):CGCCATCCAGATGACCGCCGAGGTGGCCCGCTTCCGCTACATCCA GAACCTGGTGACCAAGAACTTCCCC (SEQ ID NO.: 99)
C4(antisense):GGCGGATCCCAGCTGACCTCGAACTGGATCACCTTGTTGTCGG AGTCGAACTTGTTGGGGAAGTTCTTGGTCACCA (SEQ ID NO.: 100)
Dl(sense):CCGGGATCCGTCAGCTGGCGCAAGATCTCCACCGCCATCTACGGC GACGCCAAGAACGGCG (SEQ ID NO.: 101)
D2(antisense):GCACCTTGCCGAAGCCGAAGTCGTAGTCCTTGTTGAACACGCC GTTCTTGGCGTCGCCGTAGAT (SEQ ID NO.: 102)
D3(sense):TTCGGCTTCGGCAAGGTGCGCCAGGTGAAGGACCTGCAGATGGGC CTGCTGATGTACC (SEQ ID NO.: 103) D4(antisense):TGAACGTGGCGGCCGCCTACTTGGGCTTGCCCAGGTACATCAG CAGGCCCAT (SEQ ID NO.: 104)
D. pQMPAG4 Plasmid Construction
Ml 3 mpl8-G4 was digested with EcoR I, and the resulting fragment was ligated into the EcoR I site of the vector pIN-IIIompA2 (see, e.g., see, U.S. Patent No. 4,575,013 to Inouye; and Duffaud et al., Meth. Enz. 153:492-507, 1987) using the methods described herein. The ligation was accomplished such that the DNA encoding saporin, including the N-terminal extension, was fused to the leader peptide segment of the bacterial ompA gene. The resulting plasmid pOMPAG4 contains the Ipp promoter (Nakamura et al., Cell 75:1109-1117, 1987), the E. coli lac promoter operator sequence (lac O) and the E. coli ompA gene secretion signal in operative association with each other and with the saporin and native N-terminal leader-encoding DNA listed in SΕQ ID NO. 19. The plasmid also includes the E. coli lac repressor gene (lac I).
The Ml 3 mpl8-Gl, -G2, -G7, and -G9 clones, containing SΕQ ID NOs. 20-23, respectively, are digested with EcoR I and ligated into EcoR I digested pIN- IIIompA2 as described for Ml 3 mpl8-G4 above in this example. The resulting plasmids, labeled pOMPAGl, ρOMPAG2, pOMPAG7, pOMPA9, are screened, expressed, purified, and characterized as described for the plasmid pOMPAG4.
INVlα competent cells were transformed with pOMPAG4 and cultures containing the desired plasmid structure were grown further in order to obtain a large preparation of isolated pOMPAG4 plasmid using methods described herein.
Ε. Saporin expression in E. coli
The pOMPAG4 transformed E. coli cells were grown under conditions in which the expression of the saporin-containing protein is repressed by the lac repressor until the end of the log phase of growth, at which time IPTG was added to induce expression of the saporin-encoding DNA. To generate a large-batch culture of pOMPAG4 transformed E. coli cells, an overnight culture (approximately 16 hours growth) of JA221 E. coli cells transformed with the plasmid pOMPAG4 in LB broth (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) containing 125 mg/ml ampicillin was diluted 1 :100 into a flask containing 750 ml LB broth with 125 mg/ml ampicillin. Cells were grown at logarithmic phase with shaking at 37°C until the optical density at 550 nm reached 0.9 measured in a spectrophotometer.
In the second step, saporin expression was induced by the addition of IPTG (Sigma) to a final concentration of 0.2 mM. Induced cultures were grown for 2 additional hours and then harvested by centrifugation (25 min., 6500 x g). The cell pellet was resuspended in ice cold 1.0 M TRIS, pH 9.0, 2 mM ΕDTA (10 ml were added to each gram of pellet). The resuspended material was kept on ice for 20-60 minutes and then centrifuged (20 min., 6500 x g) to separate the periplasmic fraction of E. coli, which corresponds to the supernatant, from the intracellular fraction corresponding to the pellet.
The E. coli cells containing C-SAP construct in pΕTl la were grown in a high-cell density fed-batch fermentation with the temperature and pH controlled at 30°C and 6.9, respectively. A glycerol stock (1 ml) was grown in 50 ml Luria broth until the A600 reached 0.6 Inoculum (10 ml) was injected into a 7-1-Applikon (Foster City CA) fermentor containing 21 complex batch medium consisting of 5 g/1 of glucose, 1.25 g/1 each of yeast extract and tryptone (Difco Laboratories), 7 g/1 of K2HPO4, 8 g/1 of KH2P04, 1.66 g/1 of (NH4)2SO4, 1 g/1 of MgS04 • 7H2O, 2 ml/1 of a trace metal solution (74 g/1 of trisodium citrate, 27 g/1 of FeCl3 • 6H2O, 2.0 g/1 of CoCl2 • 6H2O, 2.0 g/1 of Na2MoO4 • 2H2O, 1.9 g/1 of CuSO4 • 5H2O, 1.6 g/1 of MnCl2 » 4H2O, 1.4 g/1 of ZnCl2 • 4H20, 1.0 g/1 of CaCl2 • 2H20, 0.5 g/1 of H3B03). 2 ml/1 of a vitamin solution (6 g/1 of thiamin • HCl, 3.05 g/1 of niacin, 2.7 g/1 of pantothenic acid, 0.7 g/1 of pyridoxine • HCl, 0.21 g/1 of riboflavin, 0.03 g/1 of biotin, 0.02 g/1 of folic acid), and 100 mg/1 of carbenicillin. The culture was grown for 12 h before initiating the continuous addition of a 40x solution of complex batch media lacking the phosphates and containing only 25 ml/1, each, of trace metal and vitamin solutions. The feed addition continued until the Am of the culture reached 85, at which time (approximately 9 h) the culture was induced with 0.1 mM isopropyl β-D-thiogalactopyranoside. During 4 h of post-induction incubation, the culture was fed with a solution containing 100 g/1 of glucose, 100 g/1 of yeast extract, and 200 g/1 of tryptone. Finally, the cells were harvested by centrifugation (8000xg, 10 min) and frozen at -80°C until further processed.
The cell pellet («400 g wet mass) containing C-SAP was resuspended in
3 vol Buffer B (10 mM sodium phosphate pH 7.0, 5 mM EDTA, 5 mM EGTA, and 1 mM dithiothreitol). The suspension was passed through a microfluidizer three times at
124 Mpa on ice. The resultant lysate was diluted with NanoPure H2O until conductivity fell below 2.7 mS/cm. All subsequent procedures were performed at room temperature.
The diluted lysate was loaded onto an expanded bed of Streamline SP cation-exchange resin (300 ml) equilibrated with buffer C (20 mM sodium phosphate pH 7.0, 1 mM EDTA) at 100 ml/min upwards flow. The resin was washed with buffer C until it appeared clear. The plunger was then lowered at 2 cm/min while washing continued at 70 ml/min. Upwards flow was stopped when the plunger was approximately 8 cm away from the bed and the plunger was allowed to move to within 0.5 cm of the packed bed. The resin was further washed at 70 ml/min downwards flow until A28o reached baseline. Buffer C plus 0.25 M NaCl was then used to elute proteins containing C-SAP at the same flow rate.
The eluate was buffer exchanged into buffer D (50 mM sodium borate pH 8.5, 1 mM EDTA) using the Sartocon Mini crossflow filtration system with a 10000
NMolecular Massco module (Sartorius). The sample was then applied to a column of Source 15S (30 ml) equilibrated with buffer D. A 10-column- volume linear gradient of
0-0.3 M NaCl in buffer D was used to elute C-SAP at 30 ml/min.
F. Assay for cytotoxic activity
The ribosome inactivating protein activity of recombinant saporin was compared to the ribosome inactivating protein activity of native SAP in an in vitro assay measuring cell-free protein synthesis in a nuclease-treated rabbit reticulocyte lysate (Promega). Samples of immunoaffinity-purified saporin were diluted in PBS and 5 μl of sample was added on ice to 35 μl of rabbit reticulocyte lysate and 10 μl of a reaction mixture containing 0.5 μl of Brome Mosaic Virus RNA, 1 mM amino acid mixture minus leucine, 5 μCi of tritiated leucine and 3 μl of water. Assay tubes were incubated 1 hour in a 30°C water bath. The reaction was stopped by transferring the tubes to ice and adding 5 μl of the assay mixture, in triplicate, to 75 μl of 1 N sodium hydroxide, 2.5% hydrogen peroxide in the wells of a Millititer HA 96-well filtration plate (Millipore). When the red color had bleached from the samples, 300 μl of ice cold 25% trichloroacetic acid (TCA) were added to each well and the plate left on ice for another 30 min. Vacuum filtration was performed with a Millipore vacuum holder. The wells were washed three times with 300 μl of ice cold 8% TCA. After drying, the filter paper circles were punched out of the 96-well plate and counted by liquid scintillation techniques.
The IC50 for the recombinant and native saporin were approximately 20 pM. Therefore, recombinant saporin-containing protein has full protein synthesis inhibition activity when compared to native saporin.
EXAMPLE 2 PREPARATION OF FGF MUTEINS
A. Materials and Methods 1. Reagents
Restriction and modification enzymes were purchased from BRL (Gaithersburg, MD), Stratagene (La Jolla, CA) and New England Biolabs (Beverly, MA).
Plasmid pFC80, containing the basic FGF coding sequence, was a gift of
Drs. Paolo Sarmientos and Antonella Isacchi of Farmitalia Carlo Erba (Milan, Italy).
Plasmid pFC80, has been described in the PCT Application Serial No. WO 90/02800 and PCT Application Serial No. PCT/US93/05702, which are herein incoφorated in their entirety by reference. The sequence of DNA encoding bFGF in pFC80 is that set forth in PCT Application Serial No. PCT/US 93/05702 and in SEQ ID NO. 52.
Plasmid isolation, production of competent cells, transformation and Ml 3 manipulations were carried out according to published procedures (Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Purification of DNA fragments was achieved using the Geneclean II kit, purchased from Bio 101 (La Jolla, CA). Sequencing of the different constructions was performed using the Sequenase kit (version 2.0) of USB (Cleveland, OH).
2. Sodium dodecyl sulphate (SDS) gel electrophoresis and Western blotting SDS gel electrophoresis was performed on a PhastSystem utilizing 20% gels (Pharmacia). Western blotting was accomplished by transfer of electrophoresed protein to nitrocellulose using the PhastTransfer system (Pharmacia), as described by the manufacturer. The antisera to SAP and basic FGF were used at a dilution of 1 : 1000. Horseradish peroxidase labeled anti-IgG was used as the second antibody as described (Davis, L., Dibner et al. (1986) Basic Methods in Molecular Biology, p. 1, Elsevier Science Publishing Co., New York).
B. Preparation of the mutagenized FGF by site-directed mutagenesis
Cysteine to serine substitutions were made by oligonucleotide-directed mutagenesis using the Amersham (Arlington Heights, IL) in v tro-mutagenesis system 2.1. Oligonucleotides encoding the new amino acid were synthesized using a 380B automatic DNA synthesizer (Applied Biosystems, Foster City, CA).
1. Mutagenesis
The oligonucleotide used for in vitro mutagenesis of cysteine 78 was
AGGAGTGTCTGCTAACC (SEQ ID NO. 56), which spans nucleotides 225-241 of
SEQ ID NO. 52). The oligonucleotide for mutagenesis of cysteine 96 was TTCTAAATCGGTTACCGATGACTG (SEQ ID NO. 57), which spans nucleotides
279-302 of SEQ ID NO. 52). The mutated replicative form DNA was transformed into E. coli strain JM109 and single plaques were picked and sequenced for verification of the mutation. The FGF mutated gene was then cut out of Ml 3, ligated into the expression vector pFC80, which had the non-mutated form of the gene removed, and transformed into E. coli strain JM109. Single colonies were picked and the plasmids sequenced to verify the mutation was present. Plasmids with correct mutation were then transformed into the E. coli strain FICΕ 2 and single colonies from these transformations were used to obtain the mutant basic FGFs. Approximately 20 mg protein per liter of fermentation broth was obtained.
2. Purification of mutagenized FGF
Cells were grown overnight in 20 ml of LB broth containing 100 μg/ml ampicillin. The next morning the cells were pelleted and transferred to 500 ml of M9 medium with 100 μg/ml ampicillin and grown for 7 hours. The cells were pelleted and resuspended in lysis solution (10 mM TRIS, pH 7.4, 150 mM NaCl, lysozyme, 10 μ g/mL, aprotinin, 10 μg/mL, leupeptin, 10 μg/mL, pepstatin A, 10 μg/mL and 1 mM PMSF; 45-60 ml per 16 g of pellet) and incubated while stirring for 1 hour at room temperature. The solution was frozen and thawed three times and sonicated for 2.5 minutes. The suspension was centrifuged; the supernatant saved and the pellet resuspended in another volume of lysis solution without lysozyme, centrifuged again and the supernatants pooled. Extract volumes (40 ml) were diluted to 50 ml with lO mM TRIS, pH 7.4 (buffer A). Pools were loaded onto a 5 ml Hi-Trap heparin- Sepharose column (Pharmacia, Uppsala, Sweden) equilibrated in 150 mM sodium chloride in buffer A. The column was washed with 0.6 M sodium chloride and 1 M sodium chloride in buffer A and then eluted with 2 M sodium chloride in buffer A. Peak fractions of the 2 M elution, as determined by optical density at 280 nm, were pooled and purity determined by gel electrophoresis. Yields were 10.5 mg of purified protein for the Cys78 mutant and 10.9 mg for the Cys96 mutant.
The biological activity of [C78SJTGF and [C96S]FGF was measured on adrenal capillary endothelial cells in culture. Cells were plated at 3,000 per well in a 24 well plate in 1 ml of 10% calf serum-HDMEM. Cells were allowed to attach, and samples were added in triplicate at the indicated concentration and incubated for 48 h at 37°C. An equal quantity of samples was added and further incubated for 48 h. Medium was aspirated; cells were treated with trypsin (1 ml volume) to remove cells to 9 ml of Hematall diluent and counted in a Coulter Counter. The results show that the two mutants that retain virtually complete proliferative activity of native basic FGF as judged by the ability to stimulate endothelial cell proliferation in culture.
EXAMPLE 3 PREPARATION OF MONO-DERIVATIZED NUCLEIC ACID BINDING DOMAIN (M YOD)
MyoD at a concentration of 4.1 mg/ml is dialyzed against 0.1 M sodium phosphate, 0.1 M sodium chloride, pH 7.5. A 1.1 molar excess (563 μg in 156 μl of anhydrous ethanol) of SPDP (Pharmacia, Uppsala, Sweden) is added and the reaction mixture immediately agitated and put on a rocker platform for 30 minutes. The solution is then dialyzed against the same buffer. An aliquot of the dialyzed solution is examined for extent of derivatization according to the Pharmacia instruction sheet. The extent of derivatization is typically 0.79 to 0.86 moles of SPDP per mole of nucleic acid binding domain. Derivatized myoD (32.3 mg) is dialyzed in 0.1 M sodium borate, pH 9.0 and applied to a Mono S 16/10 column equilibrated with 25 mM sodium chloride in dialysis buffer. A gradient of 25 mM to 125 mM sodium chloride in dialysis buffer elutes free and derivatized nucleic acid binding domain. The flow rate is 4.0 ml/min, 4 ml fractions are collected. Aliquots of fractions were assayed for protein concentration (BCA Protein Assay, Pierce Chemical, Chicago, IL) and for pyridylthione released by reducing agent. Individual fractions (25 to 37) are analyzed for protein concentration and pyridyl-disulfide concentration. The data indicate a separation according to the level of derivatization by SPDP. The initial eluting peak is composed of myoD that is approximately di-derivatized; the second peak is mono-derivatized and the third peak shows no derivatization. The di-derivatized material accounts for approximately 20% of the three peaks; the second accounts for approximately 48% and the third peak contains approximately 32%. Material from the second peak is pooled and gives an average ratio of pyridyl-disulfide to myoD of 0.95. Fraction 33, which showed a divergent ratio of pyridine-2-thione to protein, was excluded from the pool. Fractions that showed a ratio of SPDP to myoD greater than 0.85 but less than 1.05 are pooled, dialyzed against 0.1 M sodium chloride, 0.1 M sodium phosphate, pH 7.5 and used for derivatization with basic FGF.
EXAMPLE 4
PREPARATION OF MODIFIED NUCLEIC ACID BINDING DOMAIN (MYOD)
As an alternative to derivatization, myoD is modified by addition of a cysteine residue at or near the N-terminus-encoding portion of the DNA. The resulting myoD can then react with an available cysteine on an FGF or react with a linker or a linker attached to an FGF to produce conjugates that are linked via the added Cys.
Modified myoD is prepared by modifying DNA encoding the myoD (GenBank Accession No. X56677). DNA encoding Cys is inserted at position -1 or at a codon within 10 or fewer residues of the N-terminus. The resulting DNA is inserted into pETl 1 a and pETl 5b and expressed in BL21 cells (NOVAGEN, Madison, WI).
A. Preparation of myoD with an added cysteine residue at the N-terminus
Primer #1 corresponding to the sense strand of myoD, nucleotides 121- 144, incoφorates a Ndel site and adds a Cys codon 5' to the start site for the mature protein
5'-CATATGTGTGAGCTACTGTCGCCACCGCTC-3' (SEQ ID NO. 58)
Primer #2 is an antisense primer complementing the coding sequence of nucleic acid binding domain spanning nucleotides 1054-1077 and contains a BamHI site. 5*-GGATCCGAGCACCTGGTATATCGGTGGGGG-3' (SEQ ID NO. 59)
MyoD DNA is amplified by PCR as follows using the above primers. A clone containing a full-length DNA (or cDNA) for myoD (1 μl) is mixed in a final volume of 100 μl containing lO mM Tris-HCl (pH 8.3), 50 mM KCl, 0.01% gelatin, 2 mM MgCl2, 0.2 mM dNTPs, 0.8 μg of each primer. Next, 2.5 U TaqI DNA polymerase (Boehringer Mannheim) is added and the mixture is overlaid with 30 μl of mineral oil (Sigma). Incubations are done in a DNA Thermal Cycler. Cycles include a denaturation step (94°C for 1 min), an annealing step (60°C for 2 min), and an elongation step (72°C for 3 min). After 35 cycles, a 10 μl aliquot of each reaction is run on a 1.5% agarose gel to verify the correct structure of the amplified product.
The amplified DNA is gel purified and digested with Ndel .and BamHI and subcloned into Ndel and TfømHI-digested plasmid containing FGF/myoD. This digestion and subcloning step removes the FGF-encoding DNA and 5' portion of SAP up to the BamHI site at nucleotides 555-560 (SEQ ID NO. 52) and replaces this portion with DNA encoding a myoD molecule that contains a cysteine residue at position -1 relative to the start site of the native mature SAP protein.
B. Preparation of nucleic acid binding domain with a cysteine residue at position 4 or 10 of the native protein
These constructs are designed to introduce a cysteine residue at position
4 or 10 of the native protein by replacing the Ser residue at position 4 or the Val residue at position 10 with cysteine. MyoD is amplified by polymerase chain reaction (PCR) from the parental plasmid encoding the FGF-nucleic acid binding domain fusion protein using primers that incoφorate a TGT or TGC codon at position 4 or 10.
The PCR conditions are performed as described above, using the following cycles: denaturation step 94°C for 1 minute, annealing for 2 minutes at 60°C, and extension for 2 minutes at 72°C for 35 cycles. The amplified DNA is gel purified, digested with Ndel and BamHI, and subcloned into Ndel and BamHI digested pETl la. This digestion removes the FGF and 5' portion of nucleic acid binding domain (up to the newly added BamHI) from the parental FGF- myoD vector and replaces this portion with a myoD molecule containing a Cys at position 4 or 10 relative to the start site of the native protein. The resulting plasmid is digested with NdellBamHl and inserted into pET15b (NOVAGEN, Madison, WI), which has a His-Tag™ leader sequence (SEQ ID NO. 60), that has also been digested NdellBamHl.
DNA encoding unmodified myoD can be similarly inserted into a pET5b or pETl 1 A and expressed as described below for the modified SAP-encoding DNA.
C. Expression of the modified nucleic acid binding domain-encoding DNA
BL21(DE3) cells are transformed with the resulting plasmids and cultured as described in Example 2, except that all incubations were conducted at 30°C instead of 37°C. Briefly, a single colony is grown in LB AMP100 to and OD600 of 1.0-1.5 and then induced with IPTG (final concentration 0.1 mM) for 2 h. The bacteria are spun down.
D. Purification of modified nucleic acid binding domain
Lysis buffer (20 mM NaPO4, pH 7.0, 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 0.5 μg/ml leupeptin, 1 μg/ml aprotinin, 0.7 μg/ml pepstatin) was added to the myoD cell paste (produced from pZ50Bl in BL21 cells, as described above) in a ratio of 1.5 ml buffer/g cells. This mixture is evenly suspended via a Polytron homogenizer and passed through a microfluidizer twice.
The resulting lysate is centrifuged at 50,000 rpm for 45 min. The supernatant is diluted with SP Buffer. A (20 mM NaPO4, 1 mM EDTA, pH 7.0) so that the conductivity is below 2.5 mS/cm. The diluted lysate supernatant is then loaded onto a SP-Sepharose column, and a linear gradient of 0 to 30% SP Buffer B (1 M NaCl, 20 mM NaP04, 1 mM EDTA, pH 7.0) in SP Buffer A with a total of 6 column volumes is applied. Fractions containing myoD are combined and the resulting rnucleic acid binding domain had a purity of greater than 90%. A buffer exchange step is used to get the SP eluate into a buffer containing 50 mM NaB03, 1 mM EDTA, pH 8.5 (S Buffer A). This sample is then applied to a Resource S column (Pharmacia, Sweden) pre- equilibrated with S Buffer A. Pure nucleic acid binding domain is eluted off the column by 10 column volumes of a linear gradient of 0 to 300 mM NaCl in SP Buffer A.
In this preparation, ultracentrifugation is used clarify the lysate; other methods, such as filtration and using floculents also can be used. In addition, Streamline S (PHARMACIA, Sweden) may also be used for large scale preparations.
EXAMPLE 5 PREPARATION OF CONJUGATES CONTAINING FGF MUTEINS
A. Coupling of FGF muteins to nucleic acid binding domain
1. Chemical Synthesis of FC78S")FGF-nucleic acid binding domain rCCFN2 and l"C96SlFGF-nucleic acid binding domain CCCFN3 [C78SJTGF or [C96SJFGF (1 mg; 56 nmol) that had been dialyzed against phosphate-buffered saline is added to 2.5 mg mono-derivatized nucleic acid binding domain (a 1.5 molar excess over the basic FGF mutants) and left on a rocker platform overnight. The next morning the ultraviolet-visible wavelength spectrum is taken to determine the extent of reaction by the release of pyridylthione, which adsorbs at 343 nm with a known extinction coefficient. The ratio of pyridylthione to basic FGF mutant for [C78S]FGF is 1.05 and for [C96S]FGF is 0.92. The reaction mixtures are treated identically for purification in the following manner: reaction mixture is passed over a HiTrap heparin- Sepharose column (1 ml) equilibrated with 0.15 M sodium chloride in buffer A at a flow rate of 0.5 ml/min. The column is washed with 0.6 M NaCl and 1.0 M NaCl in buffer A and the product eluted with 2.0 M NaCl in buffer A. Fractions (0.5 ml) are analyzed by gel electrophoresis and absorbance at 280 nm. Peak tubes are pooled and dialyzed versus 10 mM sodium phosphate, pH 7.5 and applied to a Mono-S 5/5 column equilibrated with the same buffer. A 10 ml gradient between 0 and 1.0 M sodium chloride in equilibration buffer is used to elute the product. Purity is determined by gel electrophoresis and peak fractions were pooled. Under these conditions, virtually 100%) of the mutant FGFs reacts with mono-derivatized myoD. Because the free surface cysteine of each mutant acts as a free sulfhydryl, it is unnecessary to reduce cysteines after purification from the bacteria.
The resulting product is purified by heparin-Sepharose (data not shown), thus establish- ing that heparin binding activity of the conjugate is retained.
2. Expression of the recombinant FGFC78/96S-nucleic acid binding domain fusion proteins (TPFN4)
A two-stage method is used to produce recombinant FGF[C78/96S]- myoD protein (hereinafter FPFN4). Two hundred and fifty ml of LB meditim containing ampicillin (100 μg/ml) are inoculated with a fresh glycerol stock of bacteria containing the plasmid. Cells are grown at 30°C in an incubator shaker to an OD600 of 0.7 and stored overnight at 4°C. The following day, cells are pelleted and resuspended in fresh LB medium (no ampicillin). The cells are divided into 5 1 -liter batches and grown at 30°C in an incubator shaker to an OD600 of 1.5. IPTG is added to a final concentration of 0.1 mM and growth is continued for about 2 to 2.5 hours at which time cells were harvested by centrifugation.
EXAMPLE 6
RECOMBINANT PRODUCTION OF FGF-NUCLEIC ACID BINDING DOMAIN FUSION PROTEIN
A. General Descriptions 1. Bacterial Strains and Plasmids
E. coli strains BL21(DE3), BL21(DE3)pLysS, HMS174(DE3) and HMS174(DE3)pLysS were purchased from NOVAGEN, Madison, WI. Plasmid pFC80, described below, has been described in the WIPO International Patent Application No. WO 90/02800, except that the bFGF coding sequence in the plasmid designated pFC80 herein has the sequence set forth as SEQ ID NO. 52, nucleotides 1-465. The plasmids described herein may be prepared using pFC80 as a starting material or, alternatively, by starting with a fragment containing the ell ribosome binding site (SEQ ID NO. 61) linked to the FGF-encoding DNA (SEQ ID NO. 52).
E. coli strain JA221 (lpp- hdsM+ tipE5 leuB6 lacY recAl F'[lacl<- lac+ pro+]) is publicly available from the American Type Culture Collection (ATCC), Rockville, MD 20852, under the accession number ATCC 33875. (JA221 is also available from the Northern Regional Research Center (NRRL), Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604, under the accession number NRRL B-15211; see also U.S. Patent No. 4,757,013 to Inouye; and Nakamura et al., Cell 75:1109-1117, 1979). Strain INVlα is commercially available from Invitrogen, San Diego, CA.
B. Construction of plasmids encoding FGF/nucleic acid binding domain fusion proteins 1. Construction of FGFM13 that contains DNA encoding the ci ribosome binding site linked to FGF
A Nco I restriction site is introduced into the nucleic acid binding domain-encoding DNA by site-directed mutagenesis using the Amersham in vitro- mutagenesis system 2.1. The oligonucleotide employed to create the Nco I restriction site is synthesized using a 380B automatic DNA synthesizer (Applied Biosystems). This oligonucleotide containing the Nco I site replaces the original nucleic acid binding domain-containing coding sequence.
In order to produce a bFGF coding sequence in which the stop codon was removed, the FGF-encoding DNA is subcloned into a Ml 3 phage and subjected to site-directed mutagenesis. Plasmid pFC80 is a derivative of pDS20 (see, e.g., Duester et al., Cell 50:855-864, 1982; see also U.S. Patent Nos. 4,914,027, 5,037,744, 5,100,784, and 5,187,261; see also PCT International Application No. WO 90/02800; and European Patent Application No. EP 267703 Al), which is almost the same as plasmid pKG1800 (see Bernardi et al., DNA Sequence 7:147-150, 1990; see also McKenney et al. (1981) pp. 383-415 in Gene Amplification and Analysis 2: Analysis of Nucleic Acids by Enzymatic Methods, Chirikjian et al. (eds.), North Holland Publishing Company, Amsterdam) except that it contains an extra 440 bp at the distal end of galK between nucleotides 2440 and 2880 in pDS20. Plasmid pKG1800 includes the 2880 bp EcoR l-Pvu II of pBR322 that contains the contains the ampicillin resistance gene and an origin of replication. Plasmid pFC80 is prepared from pDS20 by replacing the entire galK gene with the FGF-encoding DNA of SΕQ ID NO. 52, inserting the tφ promoter (SΕQ ID NO. 62) and the bacteriophage lambda ell ribosome binding site (SΕQ. ID No. 61; see, e.g., Schwarz et al., Nature 272:410, 1978) upstream of and operatively linked to the FGF-encoding DNA. The Tφ promoter can be obtained from plasmid pDR720 (Pharmacia PL Biochemicals) or synthesized according to SΕQ ID NO. 62. Plasmid pFC80, contains the 2880 bp EcoR 1-BamH I fragment of plasmid pSD20, a synthetic Sal l-Nde I fragment that encodes the Tφ promoter region:
£coR I AATTCCCCTGTTGACAATTAATCATCGAACTAGTTAACTAGTACGCAGCTTGGCTGCAG
and the ell ribosome binding site (SΕQ ID NO. 61)):
Sal I Nde I GTCGACCAAGCTTGGGCATACATTCAATCAATTGTTATCTAAGGAAATACTTACATATG
The FGF-encoding DNA is removed from pFC80 by treating it as follows. The pFC80 plasmid was digested by Hga l and Sai l, which produces a fragment containing the CII ribosome binding site linked to the FGF-encoding DNA. The resulting fragment is blunt ended with Klenow's reagent and inserted into M13mpl8 that has been opened by Sma l and treated with alkaline phosphatase for blunt-end ligation. In order to remove the stop codon, an insert in the ORI minus direction is mutagenized using the Amersham kit, as described above, using the following oligonucleotide (SΕQ ID NO. 63): GCTAAGAGCGCCATGGAGA, which contains one nucleotide between the FGF carboxy terminal serine codon and a Nco I restriction site; it replaces the following wild type FGF encoding DNA having SEQ ID NO. 64:
GCT AAG AGC TGA CCA TGG AGA Al a Lys Ser STOP Pro Trp Arg
The resulting mutant derivative of M13mpl8, lacking a native stop codon after the carboxy terminal serine codon of bFGF, was designated FGFM13. The mutagenized region of FGFM13 contained the correct sequence (SEQ ID NO. 65).
2. Preparation of a plasmid that encodes the FGF/MvoD fusion protein
Plasmid FGFM13 is cut with Nco I and S c I to yield a fragment containing the CII ribosome binding site linked to the bFGF coding sequence with the stop codon replaced. An M13mpl8 derivative containing the myoD coding sequence is also cut with restriction endonucleases Nco I and Sac I, and the bFGF coding fragment from FGFM13 was inserted by ligation to DΝA encoding the fusion protein bFGF- myoD into the M13mpl8 derivative to produce mpFGF- myoD, which contains the CII ribosome binding site linked to the FGF-nucleic acid binding domain fusion gene. Plasmid mpFGF- myoD is digested with Xba I and EcoR I and the resulting fragment containing the bFGF- myoD coding sequence is isolated and ligated into plasmid pΕT-l la (available from ΝOVAGΕΝ, Madison, WI; for a description of the plasmids see U.S. Patent No. 4,952,496; see also Studier et al., Meth. Enz. 755:60- 89, 1990; Studier et al., J. Mol. Biol. 759:113-130, 1986; Rosenberg et al., Gene 56: 125- 135, 1987) that has also been treated with EcoR I and Xba I.
E. coli strain BL21(DΕ3)pLysS (NOVAGEN, Madison WI) may be transformed with the plasmid containing the fusion gene.
Plasmid FGF/myoD may be digested with EcoR I, the ends repaired by adding nucleoside triphosphates and Klenow DNA polymerase, and then digested with Nde I to release the FGF-encoding DNA without the CII ribosome binding site. This fragment is ligated into pΕT 1 la, which is BamH I digested, treated to repair the ends, and digested with Nde I. The resulting plasmid includes the T7 transcription terminator and the pET-1 la ribosome binding site.
Plasmid FGF/myoD may be digested with EcoR I and Nde I to release the FGF-encoding DNA without the CII ribosome binding site and ends are repaired as described above. This fragment may be ligated into pΕT 12a, which had been BamH I digested and treated to repair the ends. The resulting plasmid includes DNA encoding the OMP T secretion signal operatively linked to DNA encoding the fusion protein.
3. Preparation of a plasmid that encodes FGF2-protamine fusion protein
Protamines are small basic DNA binding proteins, approximately 6.8 kD in molecular weight with a isoelectric point of 12.175. Twenty-four of the fifty one amino acids are strongly basic. Human protamine has been shown to condense genomic DNA for packaging into the sperm head. The positive charges of the protamine react with the negative charges of the phosphate backbone of the DNA.
A FGF-protamine fusion protein that has the ability to bind to the FGF receptor and bind DNA with high affinity is constructed for expression in E. coli. The sequence for the human protamine gene is obtained from GenBank (accession no. Y00443). Four overlapping oligonucleotides (60mers) are generated and used to amplify the protamine gene. The amplified product is purified and ligated into the bacterial expression vector pΕTl la (Novagen). To facilitate subcloning, a Ncol and BamHI site are incoφorated into the primers. The fragment is synthesized by annealing the 4 oligos (2 sense and 2 antisense) with 20 base overlaps and using PCR to fill-in and amplify the fragments. The PCR products are digested with Ncol and BamHI, and subcloned into pBluescript SK+. The insert sequence is verified. The sequenced product is then cloned downstream and in-frame with FGF2, which has been previously cloned into the pΕTl la expression plasmid. The oligos used to generate fragment A are (5'-3'):
PT1 :
TACATGCCATGGCCAGGTACAGATGCTGTCGCAGCCAGAGCCGGAGCAGAT ATTACCGCC(SΕQIDNO.:85) PT2:
GCAGCTCCGCCTCCTTCGTCTGCGACTTCTTTGTCTCTGGCGGTAATATCTGC TCCGGCT (SEQ ID NO.: 86)
PT3:
GACGAAGGAGGCGGAGCTGCCAGACACGGAGGAGAGCCATGAGGTGCTGC CGCCCCAGGT(SEQIDNO.:87)
PT4:
ATATATCCTAGGTTAGTGTCTTCTACATCTCGGTCTGTACCTGGGGCGGCAG CACCTCA (SEQ ID NO.: 88)
Competent bacterial cells, BL21 (DE3), are transformed with the pETl 1- FGF2-protamine construct. The cells are initially plated on LB agar plates containing 100 μg/ml ampicillin. A glycerol stock made from an individual colony added to 1 ml fresh LB broth and then to 250 ml of LB broth. The cells are grown to an OD600 of 0.7 and induced with IPTG. The culture is harvested 4 hours after induction. The suspension is centrifuged; the supernatant is saved and the pellet is resuspended in lysis buffer, centrifuged again and the supernatants pooled. A sample of the pellet and the supernatant are analyzed by Western analysis using antibodies to FGF2 to determine the percentage of fusion protein within each fraction. Soluble protein is purified. Briefly, the cells are pelleted and resuspended in buffer A (10 mM sodium phosphate, pH 6.0, containing 10 mM EDTA, 10 mM EGTA and 50 mM NaCl) and passed through a microfluidizer (Microfluidics Coφ., Newton, MA) to break open the bacteria and shear DNA. The resultant mixture is diluted and loaded onto an expanded bed Streamline SP cation-exchange resin. The column is washed with step gradients of increasing concentrations of NaCl. The eluted material is analyzed by Western analysis for fractions containing the fusion protein. These fractions are pooled, diluted, and loaded onto a Heparin-Sepharose affinity column. After washing, the bound proteins are eluted in a batch-wise manner in buffer containing 1 M NaCl and then in buffer containing 2 M NaCl. Peak fractions of the 2M elution, as determined by optical density at 280 nm, are pooled and the purity determined by gel electrophoresis and Western analysis. The final pool of material will be loaded onto a column of Sephacryl S-100 equilibrated with 20 mM HEPES pH 7.4, 150 mM NaCl.
Fusion protein located in the pellet is isolated, solubilized and refolded. Briefly, each culture pellet is thawed completely and resuspended in buffer A (10 mM Tris, 1 mM EDTA, pH 8.0 + 0.1 mg/ml lyzozyme). The mixture is sonicated on ice, centrifuged at 16,000 X g, and the supernatant discarded. Inclusion bodies are solubilized with solubilization buffer: (6 M guanidine-HCl, 100 mM Tris, 150 mM NaCl, 50 mM EDTA, 50 mM EGTA, pH 9.5,), vortexed, incubated for 30 minutes at room temperature, and centrifuged at 35,000 X g for 15 minutes. The supernatant is saved and diluted 1:10 in dilution buffer (100 mM Tris, 10 mM EDTA, 1% monothioglycerol, 0.25 M L-arginine, pH 9.5). The material is stirred, covered, at 4°C for 2 hours and then centrifuged at 35,000 X g for 20 minutes. The supernatant is dialyzed in against 5 liters PBS, pH 8.8, for 24 hours at 4°C with 3 changes of fresh PBS. The material is concentrated approximately 10-fold using size-exclusion spin columns. The soluble refolded material is then analyzed by gel electrophoresis.
Expression of the FGF-protamine fusion protein can be achieved in mammalian cells by excising the insert with restriction enzymes Ndel and BamHI and ligating into a mammalian expression vector.
C. Expression of the recombinant bFGF-nucleic acid binding domain fusion proteins A two-stage method is used to produce recombinant bFGF-myoD protein
(hereinafter bFGF-nucleic acid binding domain fusion protein).
Three liters of LB broth containing ampicillin (50 μg/ml) and chloramphenicol (25 μg/ml) are inoculated with pFS92 plasmid-containing bacterial cells (strain BL21(DE3)pLysS) from an overnight culture (1 :100 dilution). Cells are grown at 37°C in an incubator shaker to an OD600 of 0.7. IPTG (Sigma Chemical, St. Louis, MO) is added to a final concentration of 0.2 mM and growth was continued for 1.5 hours at which time cells were centrifuged.
Experiments have shown that growing BL21(DE3)pLysS cells at 30°C instead of 37°C improves yields. Thus, cells are grown at 30°C to an OD600 of 1.5 prior to induction. Following induction, growth is continued for about 2 to 2.5 hours at which time the cells are harvested by centrifugation.
The pellet is resuspended in lysis solution (45-60 ml per 16 g of pellet; 20 mM TRIS, pH 7.4, 5 mM EDTA, 10% sucrose, 150 mM NaCl, lysozyme, 100 μ g/ml, aprotinin, 10 μg/ml, leupeptin, 10 μg/ml, pepstatin A, 10 μg/ml and 1 mM PMSF) and incubated with stirring for 1 hour at room temperature. The solution is frozen and thawed three times and sonicated for 2.5 minutes. The suspension is centrifuged at 12,000 X g for 1 hour; the resulting first-supernatant saved and the pellet is resuspended in another volume of lysis solution without lysozyme. The resuspended material is centrifuged again to produce a second-supernatant, and the two supernatants are pooled and dialyzed against borate buffered saline, pH 8.3.
D. Affinity purification of bFGF-nucleic acid binding domain fusion protein
Thirty ml of the dialyzed solution containing the bFGF-nucleic acid binding domain fusion protein from Example 5.C. is applied to HiTrap heparin-Sepharose column (Pharmacia, Uppsala, Sweden) equilibrated with 0.15 M NaCl in 10 mM TRIS, pH 7.4 (buffer A). The column is washed first with equilibration buffer; second with 0.6 M NaCl in buffer A; third with 1.0 M NaCl in buffer A; and finally eluted with 2 M NaCl in buffer A into 1.0 ml fractions. Samples were assayed by the ELISA method. bFGF-nucleic acid binding domain fusion protein elutes from the heparin-Sepharose column at the same concentration (2 M NaCl) as native and recombinantly-produced bFGF, indicating that the heparin affinity is retained in the bFGF-SAP fusion protein. E. Characterization of the bFGF-nucleic acid binding domain fusion protein by Western blot
SDS gel electrophoresis is performed on a Phastsystem utilizing 20% acrylamide gels (Pharmacia). Western blotting is accomplished by transfer of the electrophoresed protein to nitrocellulose using the PhastTransfer system (Pharmacia), as described by the manufacturer. Antisera to bFGF is used at a dilution of 1 :1000.
Horseradish peroxidase labeled anti-IgG is used as the second antibody (Davis et al.,
Basic Methods in Molecular Biology, New York, Elsevier Science Publishing Co., pp 1-338, 1986). Anti-FGF antisera should bind to a protein with an approximate molecular weight of 53,000, which corresponds to the sum of the independent molecular weights of nucleic acid binding domain (35,000) and bFGF (18,000).
EXAMPLE 7
PREPARATION OF FGF-NUCLEIC ACID BINDING DOMAIN CONJUGATES THAT CONTAIN LINKERS ENCODING PROTEASE SUBSTRATES
A. Synthesis of oligos encoding protease substrates Complementary single-stranded oligos in which the sense strand encodes a protease substrate, have been synthesized either using a cyclone machine (Millipore, MA) according the instructions provided by the manufacturer, or were made by Midland Certified Reagent Co. (Midland, TX) or by National BioscienceSj, Inc. (MN). The following oligos have been synthesized.
1. Cathepsin B substrate linker
5 ' - CCATGGCCCTGGCCCTGGCCCTGGCCCTGGCCATGG SEQ ID NO: 66
2. Cathepsin D substrate linker
5 ' - CCATGGGCCGATCGGGCTTCCTGGGCTTCGGCTTCCTGG GCπCGCCAT GG -3 ' SEQ ID NO : 67
3. Trypsin substrate linker
5 ' - CCATGGGCCGATCGGGCGGTGGGTGCGCTGGTAATAGAGT CAGAAGATCAGTCGGAAGCAGCCTGTCTTGCGGTGGTCTC GACCTGCAGG CCATGG-3' SEQ ID NO: 68 4. Gly4Ser 5'- CCATGGGCGG CGGCGGCTCT GCCATGG -3' SEQ ID NO: 47 5.(Gly4Ser)2
5'- CCATGGGCGGCGGCGGCTCTGGCGGCGGCGGCTC TGCCATGG -3' SEQ ID NO: 48
6.(Ser4Gly)4
5"- CCATGGCCTCGTCGTCGTCGGGCTCGTCGTCGTC GGGCTCGTCGTCGTCGGGCTCGTCGTCGTCGGGC GCCATGG -3' SEQ ID NO: 49
7. (Ser4Gly)2
5 - CCATGGCCTCGTCGTCGTCGGGCTCGTCGTCGTC GGGCGCCATGG -3 ' SEQ ID NO: 50 8. Thrombin substrate linker
CTG GTG CCG CGC GGC AGC SEQ ID NO. 69 Leu Val Pro Arg Gly Ser
9. Enterokinase substrate linker GAC GAC GAC GAC CCA SEQ ID NO. 70 Asp Asp Asp Asp Lys
10. Factor Xa substrate ATC GAA GGT CGT SEQ ID NO. 71 H e Gl u Gly Arg
B. Preparation of DNA constructs encoding FGF-Linker-nucleic acid binding domain
The complementary oligos are annealed by heating at 95 °C for 15 min., cooled to room temperature, and then incubated at 4°C for a minute to about an hour.
Following incubation, the oligos are digested with Ncol and ligated overnight at a 3:1 (insert: vector) ratio at 15°C to Ncol-digested plasmid which has been treated with alkaline phosphatase (Boehringer Mannheim).
Bacteria (Νovablue (ΝOVAGEΝ, Madison, WI)) are transformed with the ligation mixture (1 μl) and plated on LB-amp or LB-Kan, depending upon the plasmid). Colonies are selected, clones isolated and sequenced to determine orientation of the insert. Clones with correct orientation are used to transform strain expression strain BL21(DE3) (NOVAGEN, Madison, WI). Glycerol stocks are generated from single transformed colonies. The transformed strains are cultured as described in Example 2 and fusion proteins with linkers were expressed.
The DNA and amino acid sequences of exemplary fusion proteins, containing cathepsin B substrate (FPFS9), cathepsin D substrate (FPFS5), Gly4Ser (FPFS7), (Gly4Ser)2 (FPFS8), trypsin substrate (FPFS6), (Ser4Gly)4 (FPFS12) and (Ser4Gly)2 (FPFS11) linkers, respectively, are set forth in SEQ ID NOs. 72-78.
EXAMPLE 8 FGF-POLY-L-L YSINE (FGF2-K) COMPLEXED WITH A
PLASMID ENCODING β-GALACτosiDASE
A. Derivatization of polv-L-lvsine
Polylysine polymer with average lengths of 13, 39, 89, 152, and 265 (K13, K39, K^, K152, K265) are purchased from a commercial vendor (Sigma, St. Louis, MO) and dissolved in 0.1 M NaPO4, 0.1 M NaCl, 1 mM EDTA, pH 7.5 (buffer A) at 3-5 mg/ml. Approximately 30 mg of poly-L-lysine solution is mixed with 0.187 ml of 3 mg/ml N-succinimidyl-3(pyridyldithio)proprionate (SPDP) in anhydrous ethanol resulting in a molar ratio of SPDP/poly-L-lysine of 1.5 and incubated at room temperature for 30 minutes. The reaction mixture is then dialyzed against 4 liters of buffer A for 4 hours at room temperature.
B. Conjugation of derivatized polylysine to FGF2-3
A solution containing 28.5 mg of poly-L-lysine-SPDP is added to 12.9 mg of FGF2-3 ([C96SJ-FGF2) in buffer A and incubated overnight at 4°C. The molar ratio of poly-L-lysine-SPDP/FGF2-3 is approximately 1.5. Following incubation, the conjugation reaction mixture is applied to a 6 ml Resource S (Pharmacia, Uppsala, Sweden) column. A gradient of 0.15 M to 2.1 M NaCl in 20 mM NaPO4, 1 mM EDTA, pH 8.0 (Buffer B) over 24 column volumes is used for elution. The FGF2- 3/poly-L-lysine conjugate, called FGF2-K, is eluted off the column at approximately 1.8-2 M NaCl concentration. Unreacted FGF2-3 is eluted off by 0.5-0.6 M NaCl. The fractions containing FGF2-K are concentrated and loaded onto a gel- filtration column (Sephacryl SI 00) for buffer exchange into 20 mM HEPES, 0.1 M NaCl, pH 7.3. The molecular weight of FGF-K152 as determined by size exclusion HPLC is approximately 42 kD. To determine if the conjugation procedure interferes with the ability of FGF2-3 to bind heparin, the chemical conjugate FGF2-K is loaded onto a heparin column and eluted off the column at 1.8- 2.0 M NaCl. In comparison, unconjugated FGF2-3 is eluted off heparin at 1.4 - 1.6 M NaCl. This suggests that poly-L-lysine contributes to FGF2-3 ability to bind heparin. The ability of poly-L- lysine 152 to bind heparin is not determined; poly-L-lysine 84 elutes at approximately 1.6 M NaCl. Histone Hl-polylysine was purchased and cytochrome C was conjugated to polylysine as described herein.
A sample of FGF2-K is electrophoresed on SDS-PAGE under non- reducing and reducing conditions. The protein migrates at the same molecular weight as FGF. Under non-reducing conditions the conjugate does not enter the gel because of its high charge density (Figure 1, lanes 1, 2, non-reducing; lanes 3, 4, reducing).
A standard proliferation assay using aortic bovine endothelial cells is performed to determine if the conjugation procedure reduced the ability of FGF2-3 ability to stimulate mitogenesis. The results reveal that FGF2-K is equivalent to FGF2- 3 in stimulating proliferation (Figure 2).
C. FGF2-3-polv-L-lysine-nucleic acid complex formation
Optimal conditions for complex formation are established. Varying quantities (0.2 to 200 μg) of β-galactosidase encoding plasmid nucleic acid pSVβ or pNASS-β (lacking a promoter) are slowly mixed with 100 μg of FGF2-K in 20 mM HEPES pH 7.3, 0.15 M NaCl. The reaction is incubated for 1 hour at room temperature. Nucleic acid binding to the FGF-lysine conjugate is confirmed by gel mobility shift assay using 32P-labeled SV40-β-gal nucleic acid cut with Hindi restriction endonuclease. In brief, SV40β-gal nucleic acid is digested with Hindi restriction endonucleases; ends are labeled by T4 PNK following dephosphorylation with calf intestinal alkaline phosphatase. To each sample of 35 ng of 32P-labeled nucleic acid increasing amounts of FGF-polylysine conjugate is added to the mixture. The protein/nucleic acid mixture is electrophoresed in an agarose gel with 1 X TAE buffer. Binding of the conjugate to the radiolabeled DNA is shown by a shift in the complex to the top of the well. (Figure 3.) As seen in Figure 3D, as little as 10 ng of K84 causes a complete shift of restriction fragments indicating binding. With K13, 100 ng of poly-L-lysine was required (Figure 3C). With K265, 10 ng was required (Figure 3E).
The optimal length of poly-L-lysine and weight ratios is determined by conjugation of FGF2-3 to poly-lysine of different lengths. DNA encoding β-galactosidase was complexed with the conjugates at 10:1, 5:1, 2:1, 1:1, and 0.5:1 (Figure 4, lanes 1-5, respectively) (w/w) ratios. The ability of these FGF2-K complexes to bind DNA was determined by measuring the ability of FGF to promote the uptake of plasmid DNA into cells. FGF2-K conjugates were evaluated at various protein to DNA ratios for their ability to deliver pSVβ-gal DNA into cells (Figure 4).
Briefly, the complexes were incubated for 1 hr at room temperature and then added to COS cells for 48 hrs. Cell extracts were prepared and assayed for β-gal enzyme activity. Briefly, cells are washed with 1 ml of PBS (Ca+2 and Mg+2 free) and lysed. The lysate was vortexed and cell debris removed by centrifugation. The lysate was assayed for β-gal activity as recommended by the manufacturer (Promega, Madison, WI). The β-gal activity was normalized to total protein. As seen in Figure 4, lane 3, a 2: 1 (w/w) ratio of FGF2-K:DNA gave maximal enzyme activity.
In addition, toroid formation, which correlates with increased gene expression, was assessed by electron microscopy. A representative toroid at a protein to DNA ratio of 2:1 is shown in Figure 5, upper panel. Toroidal structures are absent, or only partially formed, at low ratios (e.g., 0.5:1) (Figure 5, lower panel). A proliferation assay is performed to determine if the condensed nucleic acid had an effect on the ability of FGF2-K to bind to cognate receptor and stimulate mitogenesis. The proliferation assay shows that only the highest dose of nucleic acid (200 μg) has a slightly inhibitory effect on proliferation as compared to FGF2-3 plus poly-L-lysine + DNA (Figure 6). A FGF2-K84-DNA at a protein:DNA ratio of 2:1 is introduced into COS cells and an endothelial cell line, ABAE, both of which express FGF receptors. The cells are subsequently assayed for β-galactosidase enzyme activity. COS and ABAE cells are grown on coverslips and incubated with the different ratios of FGF2-K:DNA for 48 hours. The cells are then fixed and stained with X-gal. Maximal β-galactosidase enzyme activity is seen when 50 μg of pSVβ per 100 μg of FGF2-3 -polylysine conjugate is used.
FGF2-K84-ρSVβ-gal at a protein to DNA ratio of 2:1 was added to various cell lines and incubated for 48 hr. Cell extracts were prepared, assayed for β-gal activity and total protein. As shown in Figure 7A, COS, B16, NIH3T3, and BHK cell lines were all able to take up complex and express β-gal.
The expression of β-gal requires FGF2 for targeting into cells. pS Vβ or pNASSβ plasmid DNA was incubated with (Figure 7B, lanes 1, 2) or without (lanes 3, 4) FGF2-K84 for 1 hr at room temperature. Complexes were added to COS cells for 48 hr. Cell extracts were assayed for β-gal activity and normalized to total protein. Only background β-gal activity was seen unless the plasmid was complexed with FGF2/K84. Expression of β-gal is seen to be both time and dose-dependent (Figures 7C and 7D).
Sensitivity of the receptor mediated gene delivery system is determined using the optimized FGF2-K/DNA ratio for complex formation. Increasing amounts of the FGF2-K DNA complex is added to cells. 100 μg of FGF2-K was mixed with 50 ug of pS Vβ for 1 hour at room temperature. The COS and endothelial cells are incubated with increasing amounts of condensed material (0 ng, 1 ng, 10 ng, 100 ng, 1000 ng and 10,000 ng). The cells are incubated for 48 hours and then were assayed for β-galactosidase activity. In addition, cells grown on cover slips are treated with 1000 ng of FGF2-K-DNA for 48 hours, then fixed and stained using X-gal. The β-gal enzyme assay reveals that with increasing amounts of material there is an increase in enzyme activity. (Figure 7D) Cells incubated with X-gal show blue staining throughout the cytoplasm in approximately 3% of the cells on the coverslip.
Targeting of the complexes is specific for the FGF receptor. First, as seen in Figure 8A, FGF2-K84-pSVβ-gal resulted in enzyme activity (lane 1), while only background levels of activity were seen with FGF2+K84+DNA (lane 2), FGF2+DNA (lane 3), K84+DNA (lane 4), DNA (lane 5), FGF2-K84 (lane 6), FGF2 alone (lane 7) and K84 alone (lane 8). The expression of β-gal is specifically inhibited if free FGF2 is added during transfection (Figure 8B). Moreover, the addition of heparin attenuates the expression of β-gal (Figure 8C). Moreover, histone HI and cytochrome C were ineffective in delivering pSVβ-gal (Figure 8C).
Taken together, these findings support the hypothesis that the targeted DNA is introduced into receptor-bearing cells via the high affinity FGF receptor. Because histone can bind heparin sulfate yet fails to elicit a signal, the introduction of DNA appears independent of the low affinity FGF receptor or non-specific endocytosis.
D. Effect of endosome-disruptive peptides
Targeting is mediated by passage of the complex through endosomes. Chloroquine, which was added to complexes before transfection, resulted in an 8-fold increase in β-gal activity (Figure 9A).
Based on this, the effect of endosome disruptive peptides was evaluated. The peptide INF7, GLF EAIEGFIEN GWEGMIDGWYGC, derived from influenza virus, was synthesized. A complex between FGF2-K84 (5 μg) .and pSVβ-gal plasmid DNA (5 μg) was formed. At this ratio, approximately half of the negative charge of the DNA was neutralized by the conjugate. K84, poly-L-lysine, was further added to saturate binding to the remaining DNA. The INF7 peptide was added 30 minutes later. The complex is added to COS cells and β-gal activity is assayed 48 or 72 hr later.
The amount of free polylysine necessary to neutralize the DNA and allow INF7 to complex was determined. Polylysine was added at 4, 10, or 25 μg to the FGF2-K84/pSVβ-gal complex. To each of these complexes four different concentrations of INF7 were added. Maximal β-gal expression was seen with 4 μg of K84 and 12 μg of INF7 (Figure 13 A). When higher amounts of poly-lysine were used, more cell death resulted. The optimal amount of INF7 was determined using 4 μg of polylysine. As seen in Figure 13B, 24 μg of INF7 gave maximal β-gal activity. At 72 hr, 48 μg of INF7 gave maximal β-gal activity (approximately 20-32 fold enhancement) (Figure 13C).
When an endosome disruptive peptide was included in the complex, expression of β-gal was increased 26-fold (Figure 9B). Concomitant with this increased level of expression was an increase in the number of cells expressing β-gal. As seen in
Figure 9C, when endosome disruptive peptide (EDP) was present (right panel), 1 %-5% of cells express β-gal in comparison to 0.1%-0.3% without EDP added (left panel).
EXAMPLE 9
CYTOTOXIC ACTIVITY OF FGF/POLY-L-LYSINE BOUND TO SAP DNA PLASMID
The cytotoxicity assay measures viable cells after transfection with a cytocide-encoding agent. When FGF-2 is the receptor-binding internalized ligand,
COS7 cells, which express FGFR, may be used as targets, and T47D, which does not express a receptor for FGF-2 at detectable levels, may be used as negative control cells.
Cells are plated at 38,000 cells/well and 48,000 cells/well in a 12-well tissue culture plate in RPMI 1640 supplemented with 5% FBS. The complex FGF2- K pZ200M (a plasmid which expresses saporin) is incubated with COS7 or T47D cells for 48 hrs. Controls include FGF2-K alone, pZ200M alone, and FGF-2 plus poly-L-lysine plus pZ200M. Following incubation, cells are rinsed in PBS lacking Mg4-1" and Ca"1-1". Trypsin at 0.1% is added for 10 min and cells are harvested and washed. Cell number from each well is determined by a Coulter particle counter (or equivalent method). A statistically significant decrease in cell number for cells incubated with FGF2-K/pZ200M compared to FGF2-K or pZ200M alone indicates sufficient cytotoxicity.
FGF2-polylysine-DNASAP complexes show selective cytotoxicity. To optimize the expression of the plant RIP, saporin, in mammalian cells, a synthetic saporin gene using preferred mammalian codons and introduced a "Kozak" sequence for translation initiation. The synthetic gene was then cloned into SV40 promoter and promoterless expression vectors. Because the expression of SAP from SAP-encoding DNA would only be feasible if the mammalian ribosome can synthesize the protein (SAP) prior to its inactivation by the SAP synthesized, the enzymatic activity of saporin encoded by the synthetic gene was tested. SAP was cloned into a T7/SP6 promoter plasmid and sense RNA was generated using T7 RNA polymerase. The RNA was then added to a mammalian in vitro translation assay. The results from this cell-free in vitro translation assay clearly show that the saporin expressed in a mammalian system can inhibit the expression of protein mutagenesis (Figure 10). When added above to the lysate, SAP mRNA is translated into a protein that has the anticipated molecular weight of the saporin protein (lane 2). Similarly, when luciferase mRNA is added to the lysate, a molecule consistent with the luciferase protein is detected (lane 3). In contrast, if SAP mRNA is added to the lysate along with or 30 minutes prior to luciferase mRNA, saporin activity is detected (lanes 4 and 5). Transfection of cells with SAP DNA demonstrates cytotoxicity. When a mammalian expression vector encoding saporin is transiently expressed in NIH 3T3 cells using CaPO4, there is a >65% decrease in cell survival (lane 3) compared to cells mock transfected (lane 1) or transfected with DNA encoding β-gal (lane 2) (Figure 11).
To determine whether the FGF2-K can transfer plasmid DNA encoding SAP into FGF receptor bearing cells, FGF2-K was condensed with the pSV40-SAP plasmid DNA at a ratio of 2:1 (w:w). BHK 21 .and NIH 3T3 cells were used as the target cells. The cells (24,000 cells/well) were incubated with either FGF2-K-DNASAP or an FGF2-K-DNAβ-gal complex. After 72 hours of incubation, cell number was determined. As shown in Figure 12, there is a significant decrease in cell number when cells are incubated with the FGF2-K-DNASAP complex compared to cells incubated with the FGF2-K-DNAβ-gal complex.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for puφoses of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Prizm Pharmaceuticals. Inc.
(ii) TITLE OF INVENTION: COMPOSITIONS CONTAINING NUCLEIC ACIDS AND LIGANDS FOR THERAPEUTIC TREATMENT
(iii) NUMBER OF SEQUENCES: 106
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SEED and BERRY
(B) STREET: 6300 Columbia Center. 701 Fifth Avenue
(C) CITY: Seattle
(D) STATE: Washington
(E) COUNTRY: USA
(F) ZIP: 98104-7092
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0. Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 16-MAY-1996
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Nottenburg Ph.D.. Carol
(B) REGISTRATION NUMBER: 39.317
(C) REFERENCE/DOCKET NUMBER: 760100.415PC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (206) 622-4900
(B) TELEFAX: (206) 682-6031
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 473 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 13..456
( D) OTHER INFORMATION : /product= "VEGF121-encodi ng DNA" (i x) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 13..90
(D) OTHER INFORMATION: /product= leader-encoding sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GGATCCGAAA CC ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTT 48 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 1 5 10
GCC TTG CTG CTC TAC CTC CAC CAT GCC AAG TGG TCC CAG GCT GCA CCC 96 Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gin Ala Ala Pro 15 20 25
ATG GCA GAA GGA GGA GGG CAG AAT CAT CAC GAA GTG GTG AAG TTC ATG 144 Met Ala Glu Gly Gly Gly Gin Asn His His Glu Val Val Lys Phe Met 30 35 40
GAT GTC TAT CAG CGC AGC TAC TGC CAT CCA ATC GAG ACC CTG GTG GAC 192 Asp Val Tyr Gin Arg Ser Tyr Cys His Pro lie Glu Thr Leu Val Asp 45 50 55 60
ATC TTC CAG GAG TAC CCT GAT GAG ATC GAG TAC ATC TTC AAG CCA TCC 240 He Phe Gin Glu Tyr Pro Asp Glu He Glu Tyr He Phe Lys Pro Ser 65 70 75
TGT GTG CCC CTG ATG CGA TGC GGG GGC TGC TGC AAT GAC GAG GGC CTG 288 Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu 80 85 90
GAG TGT GTG CCC ACT GAG GAG TCC AAC ATC ACC ATG CAG ATT ATG CGG 336 Glu Cys Val Pro Thr Glu Glu Ser Asn He Thr Met Gin He Met Arg 95 100 105
ATC AAA CCT CAC CAA GGC CAG CAC ATA GGA GAG ATG AGC TTC CTA CAG 384 He Lys Pro His Gin Gly Gin His He Gly Glu Met Ser Phe Leu Gin 110 115 120
CAC AAC AAA TGT GAA TGC AGA CCA AAG AAA GAT AGA GCA AGA CAA GAA 432 His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gin Glu 125 130 135 140
AAA TGT GAC AAG CCG AGG CGG TGATGAATGA ATGAGGATCC 473
Lys Cys Asp Lys Pro Arg Arg 145
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 605 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 13..588
(D) OTHER INFORMATION : /product^ "VEGF165-encodi ng DNA"
(i x) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 13..90
(D) OTHER INFORMATION: /product= "leader sequence-encoding DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GGATCCGAAA CC ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTT 48 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 1 5 10
GCC TTG CTG CTC TAC CTC CAC CAT GCC AAG TGG TCC CAG GCT GCA CCC 96 Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gin Ala Ala Pro 15 20 25
ATG GCA GAA GGA GGA GGG CAG AAT CAT CAC GAA GTG GTG AAG TTC ATG 144 Met Ala Glu Gly Gly Gly Gin Asn His His Glu Val Val Lys Phe Met 30 35 40
GAT GTC TAT CAG CGC AGC TAC TGC CAT CCA ATC GAG ACC CTG GTG GAC 192 Asp Val Tyr Gin Arg Ser Tyr Cys His Pro lie Glu Thr Leu Val Asp 45 50 55 60
ATC TTC CAG GAG TAC CCT GAT GAG ATC GAG TAC ATC TTC AAG CCA TCC 240 He Phe Gin Glu Tyr Pro Asp Glu He Glu Tyr He Phe Lys Pro Ser 65 70 75
TGT GTG CCC CTG ATG CGA TGC GGG GGC TGC TGC AAT GAC GAG GGC CTG 288 Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu 80 85 90
GAG TGT GTG CCC ACT GAG GAG TCC AAC ATC ACC ATG CAG ATT ATG CGG 336 Glu Cys Val Pro Thr Glu Glu Ser Asn He Thr Met Gin He Met Arg 95 100 105
ATC AAA CCT CAC CAA GGC CAG CAC ATA GGA GAG ATG AGC TTC CTA CAG 384 He Lys Pro His Gin Gly Gin His He Gly Glu Met Ser Phe Leu Gin 110 115 120
CAC AAC AAA TGT GAA TGC AGA CCA AAG AAA GAT AGA GCA AGA CAA GAA 432 His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gin Glu 125 130 135 140
AAT CCC TGT GGG CCT TGC TCA GAG CGG AGA AAG CAT TTG TTT GTA CAA 480 Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gin 145 150 155 GAT CCG CAG ACG TGT AAA TGT TCC TGC AAA AAC ACA GAC TCG CGT TGC 528 Asp Pro Gin Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys 160 165 170
AAG GCG AGG CAG CTT GAG TTA AAC GAA CGT ACT TGC AGA TGT GAC AAG 576 Lys Ala Arg Gin Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys 175 180 185
CCG AGG CGG TGATGAATGA ATGAGGATCC 605
Pro Arg Arg 190
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 677 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 13..657
(D) OTHER INFORMATION : /product= "VEGF189-encodi ng DNA"
(i x) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 13..90
(D) OTHER INFORMATION: /product= "leader sequence-encoding DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3
GGATCCGAAA CC ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTT 48 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 1 5 10
GCC TTG CTG CTC TAC CTC CAC CAT GCC AAG TGG TCC CAG GCT GCA CCC 96 Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gin Ala Ala Pro 15 20 25
ATG GCA GAA GGA GGA GGG CAG AAT CAT CAC GAA GTG GTG AAG TTC ATG 144 Met Ala Glu Gly Gly Gly Gin Asn His His Glu Val Val Lys Phe Met 30 35 40
GAT GTC TAT CAG CGC AGC TAC TGC CAT CCA ATC GAG ACC CTG GTG GAC 192 Asp Val Tyr Gin Arg Ser Tyr Cys His Pro He Glu Thr Leu Val Asp 45 50 55 60
ATC TTC CAG GAG TAC CCT GAT GAG ATC GAG TAC ATC TTC AAG CCA TCC 240 He Phe Gin Glu Tyr Pro Asp Glu He Glu Tyr He Phe Lys Pro Ser 65 70 75
TGT GTG CCC CTG ATG CGA TGC GGG GGC TGC TGC AAT GAC GAG GGC CTG 288 Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu 80 85 90 GAG TGT GTG CCC ACT GAG GAG TCC AAC ATC ACC ATG CAG ATT ATG CGG 336 Glu Cys Val Pro Thr Glu Glu Ser Asn He Thr Met Gin He Met Arg 95 100 105
ATC AAA CCT CAC CAA GGC CAG CAC ATA GGA GAG ATG AGC TTC CTA CAG 384 He Lys Pro His Gin Gly Gin His He Gly Glu Met Ser Phe Leu Gin 110 115 120
CAC AAC AAA TGT GAA TGC AGA CCA AAG AAG GAT AGA GCA AGA CAA GAA 432 His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gin Glu 125 130 135 140
AAA AAA TCA GTT CGA GGA AAG GGA AAG GGG CAA AAA CGA AAG CGC AAG 480 Lys Lys Ser Val Arg Gly Lys Gly Lys Gly Gin Lys Arg Lys Arg Lys 145 150 155
AAA TCC CGG TAT AAG TCC TGG AGC GTT CCC TGT GGG CCT TGC TCA GAG 528 Lys Ser Arg Tyr Lys Ser Trp Ser Val Pro Cys Gly Pro Cys Ser Glu 160 165 170
CGG AGA AAG CAT TTG TTT GTA CAA GAT CCG CAG ACG TGT AAA TGT TCC 576 Arg Arg Lys His Leu Phe Val Gin Asp Pro Gin Thr Cys Lys Cys Ser 175 180 185
TGC AAA AAC ACA GAC TCG CGT TGC AAG GCG AGG CAG CTT GAG TTA AAC 624 Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gin Leu Glu Leu Asn 190 195 200
GAA CGT ACT TGC AGA TGT GAC AAG CCG AGG CGG TGATGAATGA ATGAGGATCC 677 Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg 205 210 215
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 728 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 13..711
(D) OTHER INFORMATION : /product= "VEGF206-encodi ng DNA" (ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 13..90
(D) OTHER INFORMATION: /product= leader sequence encoding DNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GGATCCGAAA CC ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTT 48 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 1 5 10
GCC TTG CTG CTC TAC CTC CAC CAT GCC AAG TGG TCC CAG GCT GCA CCC 96 Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gin Ala Ala Pro 15 20 25
ATG GCA GAA GGA GGA GGG CAG AAT CAT CAC GAA GTG GTG AAG TTC ATG 144 Met Ala Glu Gly Gly Gly Gin Asn His His Glu Val Val Lys Phe Met 30 35 40
GAT GTC TAT CAG CGC AGC TAC TGC CAT CCA ATC GAG ACC CTG GTG GAC 192 Asp Val Tyr Gin Arg Ser Tyr Cys His Pro He Glu Thr Leu Val Asp 45 50 55 60
ATC πC CAG GAG TAC CCT GAT GAG ATC GAG TAC ATC TTC AAG CCA TCC 240 He Phe Gin Glu Tyr Pro Asp Glu He Glu Tyr He Phe Lys Pro Ser 65 70 75
TGT GTG CCC CTG ATG CGA TGC GGG GGC TGC TGC AAT GAC GAG GGC CTG 288 Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu 80 85 90
GAG TGT GTG CCC ACT GAG GAG TCC AAC ATC ACC ATG CAG ATT ATG CGG 336 Glu Cys Val Pro Thr Glu Glu Ser Asn He Thr Met Gin He Met Arg 95 100 105
ATC AAA CCT CAC CAA GGC CAG CAC ATA GGA GAG ATG AGC TTC CTA CAG 384 He Lys Pro His Gin Gly Gin His He Gly Glu Met Ser Phe Leu Gin 110 115 120
CAC AAC AAA TGT GAA TGC AGA CCA AAG AAG GAT AGA GCA AGA CAA GAA 432 His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gin Glu 125 130 135 140
AAA AAA TCA GTT CGA GGA AAG GGA AAG GGG CAA AAA CGA AAG CGC AAG 480 Lys Lys Ser Val Arg Gly Lys Gly Lys Gly Gin Lys Arg Lys Arg Lys 145 150 155
AAA TCC CGG TAT AAG TCC TGG AGC GTT TAC GTT GGT GCC CGC TGC TGT 528 Lys Ser Arg Tyr Lys Ser Trp Ser Val Tyr Val Gly Ala Arg Cys Cys 160 165 170
CTA ATG CCC TGG AGC CTC CCT GGC CCC CAT CCC TGT GGG CCT TGC TCA . 576 Leu Met Pro Trp Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser 175 180 185
GAG CGG AGA AAG CAT TTG TTT GTA CAA GAT CCG CAG ACG TGT AAA TGT 624 Glu Arg Arg Lys His Leu Phe Val Gin Asp Pro Gin Thr Cys Lys Cys 190 195 200
TCC TGC AM AAC ACA GAC TCG CGT TGC AAG GCG AGG CAG CTT GAG TTA 672 Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gin Leu Glu Leu 205 210 215 220 AAC GAA CGT ACT TGC AGA TGT GAC AAG CCG AGG CGG TGATGAATGA 718 Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg 225 230 235
ATGAGGATCC 728
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..627
(D) OTHER INFORMATION: /note "human HBEGF precursor"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Met Lys Leu Leu Pro Ser Val Val Leu Lys Leu Phe Leu Ala Ala Val 1 5 10 15
Leu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Arg Leu Arg Arg Gly 20 25 30
Leu Ala Ala Gly Thr Ser Asn Pro Asp Pro Pro Thr Val Ser Thr Asp 35 40 45
Gin Leu Leu Pro Leu Gly Gly Gly Arg Asp Arg Lys Val Arg Asp Leu 50 55 60
Gin Glu Ala Asp Leu Asp Leu Leu Arg Val Thr Leu Ser Ser Lys Pro 65 70 75 80
Gin Ala Leu Ala Thr Pro Asn Lys Glu Glu His Gly Lys Arg Lys Lys 85 90 95
Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys Leu Arg Lys Tyr 100 105 110
Lys Asp Phe Cys He His Gly Glu Cys Lys Tyr Val Lys Glu Leu Arg 115 120 125
Ala Pro Ser Cys He Cys His Pro Gly Tyr His Gly Glu Arg Cys His 130 135 140
Gly Leu Ser Leu Pro Val Glu Asn Arg Leu Tyr Thr Tyr Asp His Thr 145 150 155 160
Thr He Leu Ala Val Val Ala Val Val Leu Ser Ser Val Cys Leu Leu 165 170 175 Val He Val Gly Leu Leu Met Phe Arg Tyr His Arg Arg Gly Gly Tyr 180 185 190
Asp Val Glu Asn Glu Glu Lys Val Lys Leu Gly Met Thr Asn Ser His 195 200 205
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 77 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(D) OTHER INFORMATION: /note "human mature HBEGF"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Arg Val Thr Leu Ser Ser Lys Pro Gin Ala Leu Ala Thr Pro Asn Lys 1 5 10 15
Glu Glu His Gly Lys Arg Lys Lys Lys Gly Lys Gly Leu Gly Lys Lys 20 25 30
Arg Asp Pro Cys Leu Arg Lys Tyr Lys Asp Phe Cys He His Gly Glu 35 40 45
Cys Lys Tyr Val Lys Glu Leu Arg Ala Pro Ser Cys He Cys His Pro 50 55 60
Gly Tyr His Gly Glu Arg Cys His Gly Leu Ser Leu Pro 65 70 75
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(D) OTHER INFORMATION: /note "monkey HBEGF precursor"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met Lys Leu Leu Pro Ser Val Val Leu Lys Leu Leu Leu Ala Ala Val 1 5 10 15
Leu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Gin Leu Arg Arg Gly 20 25 30 Leu Ala Ala Gly Thr Ser Asn Pro Asp Pro Ser Thr Gly Ser Thr Asp 35 40 45
Gin Leu Leu Arg Leu Gly Gly Gly Arg Asp Arg Lys Val Arg Asp Leu 50 55 60
Gin Glu Ala Asp Leu Asp Leu Leu Arg Val Thr Leu Ser Ser Lys Pro 65 70 75 80
Gin Ala Leu Ala Thr Pro Ser Lys Glu Glu His Gly Lys Arg Lys Lys 85 90 95
Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys Leu Arg Lys Tyr 100 105 110
Lys Asp Phe Cys He His Gly Glu Cys Lys Tyr Val Lys Glu Leu Arg 115 120 125
Ala Pro Ser Cys He Cys His Pro Gly Tyr His Gly Glu Arg Cys His 130 135 140
Gly Leu Ser Leu Pro Val Glu Asn Arg Leu Tyr Thr Tyr Asp His Thr 145 150 155 160
Thr He Leu Ala Val Val Ala Val Val Leu Ser Ser Val Cys Leu Leu 165 170 175
Val He Val Gly Leu Leu Met Phe Arg Tyr His Arg Arg Gly Gly Tyr 180 185 190
Asp Val Glu Asn Glu Glu Lys Val Lys Leu Gly Met Thr Asn Ser His 195 200 205
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(D) OTHER INFORMATION: /note "rat HBEGF precursor"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Lys Leu Leu Pro Ser Val Val Leu Lys Leu Phe Leu Ala Ala Val 1 5 10 15
Leu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Arg Leu Arg Arg Gly 20 25 30 Leu Ala Ala Ala Thr Ser Asn Pro Asp Pro Pro Thr Gly Thr Thr Asn 35 40 45
Gin Leu Leu Pro Thr Gly Ala Asp Arg Ala Gin Glu Val Gin Asp Leu 50 55 60
Glu Gly Thr Asp Leu Asp Leu Phe Lys Val Ala Phe Ser Ser Lys Pro 65 70 75 80
Gin Ala Leu Ala Thr Pro Gly Lys Glu Lys Asn Gly Lys Lys Lys Arg 85 90 95
Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys Leu Lys Lys Tyr 100 105 110
Lys Asp Tyr Cys He His Gly Glu Cys Arg Tyr Leu Lys Glu Leu Arg 115 120 125
He Pro Ser Cys His Cys Leu Pro Gly Tyr His Gly Gin Arg Cys His 130 135 140
Gly Leu Thr Leu Pro Val Glu Asn Pro Leu Tyr Thr Tyr Asp His Thr 145 150 155 160
Thr Val Leu Ala Val Val Ala Val Val Leu Ser Ser Val Cys Leu Leu 165 170 175
Val He Val Gly Leu Leu Met Phe Arg Tyr His Arg Arg Gly Gly Tyr 180 185 190
Asp Leu Glu Ser Glu Glu Lys Val Lys Leu Gly Met Ala Ser Ser His 195 200 205
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 627 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: both
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..627
(D) OTHER INFORMATION: /note "human HBEGF precursor"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
ATG AAG CTG CTG CCG TCG GTG GTG CTG AAG CTC TTT CTG GCT GCA GTT 48 Met Lys Leu Leu Pro Ser Val Val Leu Lys Leu Pne Leu Ala Ala Val 1 5 10 15
CTC TCG GCA CTG GTG ACT GGC GAG AGC CTG GAG CGG CTT CGG AGA GGG 96 Leu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Arg Leu Arg Arg Gly 20 25 30 CTA GCT GCT GGA ACC AGC AAC CCG GAC CCT CCC ACT GTA TCC ACG GAC 144 Leu Ala Ala Gly Thr Ser Asn Pro Asp Pro Pro Thr Val Ser Thr Asp 35 40 45
CAG CTG CTA CCC CTA GGA GGC GGC CGG GAC CGG AAA GTC CGT GAC TTG 192 Gin Leu Leu Pro Leu Gly Gly Gly Arg Asp Arg Lys Val Arg Asp Leu 50 55 60
CAA GAG GCA GAT CTG GAC CTT TTG AGA GTC ACT TTA TCC TCC AAG CCA 240 Gin Glu Ala Asp Leu Asp Leu Leu Arg Val Thr Leu Ser Ser Lys Pro 65 70 75 80
CAA GCA CTG GCC ACA CCA AAC AAG GAG GAG CAC GGG AAA AGA AAG AAG 288 Gin Ala Leu Ala Thr Pro Asn Lys Glu Glu His Gly Lys Arg Lys Lys 85 90 95
AAA GGC AAG GGG CTA GGG AAG AAG AGG GAC CCA TGT CTT CGG AAA TAC 336 Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys Leu Arg Lys Tyr 100 105 110
AAG GAC TTC TGC ATC CAT GGA GAA TGC AAA TAT GTG AAG GAG CTC CGG 384 Lys Asp Phe Cys He His Gly Glu Cys Lys Tyr Val Lys Glu Leu Arg 115 120 125
GCT CCC TCC TGC ATC TGC CAC CCG GGT TAC CAT GGA GAG AGG TGT CAT 432 Ala Pro Ser Cys He Cys His Pro Gly Tyr His Gly Glu Arg Cys His 130 135 140
GGG CTG AGC CTC CCA GTG GAA AAT CGC TTA TAT ACC TAT GAC CAC ACA 480 Gly Leu Ser Leu Pro Val Glu Asn Arg Leu Tyr Thr Tyr Asp His Thr 145 150 155 160
ACC ATC CTG GCC GTG GTG GCT GTG GTG CTG TCA TCT GTC TGT CTG CTG 528 Thr He Leu Ala Val Val Ala Val Val Leu Ser Ser Val Cys Leu Leu 165 170 175
GTC ATC GTG GGG CTT CTC ATG TTT AGG TAC CAT AGG AGA GGA GGT TAT 576 Val He Val Gly Leu Leu Met Phe Arg Tyr His Arg Arg Gly Gly Tyr 180 185 190
GAT GTG GAA AAT GAA GAG AAA GTG AAG TTG GGC ATG ACT AAT TCC CAC 624 Asp Val Glu Asn Glu Glu Lys Val Lys Leu Gly Met Thr Asn Ser His 195 200 205
TGA 627
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (ix) FEATURE:
(D) OTHER INFORMATION: /note- "FGF-1"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Ala Glu Gly Glu He Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5 10 15
Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30
Asn Gly Gly His Phe Leu Arg He Leu Pro Asp Gly Thr Val Asp Gly 35 40 45
Thr Arg Asp Arg Ser Asp Gin His He Gin Leu Gin Leu Ser Ala Glu 50 55 60
Ser Val Gly Glu Val Tyr He Lys Ser Thr Glu Thr Gly Gin Tyr Leu 65 70 75 80
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gin Thr Pro Asn Glu 85 90 95
Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110
He Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125
Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gin Lys Ala 130 135 140
He Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 155 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
( i x) FEATURE :
(D) OTHER INFORMATION : /note= "FGF-2"
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 11 :
Met Al a Al a Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15
Gly Ser Gly Al a Phe Pro Pro Gly Hi s Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45
Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu 50 55 60
Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn 65 70 75 80
Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95
Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110
Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 ' 125
Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 130 135 140
Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 239 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(i i ) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "FGF-3"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Gly Leu He Trp Leu Leu Leu Leu Ser Leu Leu Glu Pro Gly Trp 1 5 10 15
Pro Ala Ala Gly Pro Gly Ala Arg Leu Arg Arg Asp Ala Gly Gly Arg 20 25 30
Gly Gly Val Tyr Glu His Leu Gly Gly Ala Pro Arg Arg Arg Lys Leu 35 40 45
Tyr Cys Ala Thr Lys Tyr His Leu Gin Leu His Pro Ser Gly Arg Val 50 55 60
Asn Gly Ser Leu Glu Asn Ser Ala Tyr Ser He Leu Glu He Thr Ala 65 70 75 80
Val Glu Val Gly He Val Ala He Arg Gly Leu Phe Ser Gly Arg Tyr 85 90 95 Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr Ser 100 105 110
Ala Glu Cys Glu Phe Val Glu Arg He His Glu Leu Gly Tyr Asn Thr 115 120 125
Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser Ser Thr Pro Gly Ala Arg 130 135 140
Arg Gin Pro Ser Ala Glu Arg Leu Trp Tyr Val Ser Val Asn Gly Lys 145 150 155 160
Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gin Lys Ser Ser 165 170 175
Leu Phe Leu Pro Arg Val Leu Asp His Arg Asp His Glu Met Val Arg 180 185 190
Gin Leu Gin Ser Gly Leu Pro Arg Pro Pro Gly Lys Gly Val Gin Pro 195 200 205
Arg Arg Arg Arg Gin Lys Gin Ser Pro Asp Asn Leu Glu Pro Ser His 210 215 220
Val Gin Ala Ser Arg Leu Gly Ser Gin Leu Glu Ala Ser Ala His 225 230 235
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 206 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "FGF-4"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Ser Gly Pro Gly Thr Ala Ala Val Ala Leu Leu Pro Ala Val Leu 1 5 10 15
Leu Ala Leu Leu Ala Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala Pro 20 25 30
Thr Ala Pro Asn Gly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu 35 40 45
Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gin Pro 50 55 60
Lys Glu Ala Ala Val Gin Ser Gly Ala Gly Asp Tyr Leu Leu Gly He 65 70 75 80 Lys Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly He Gly Phe His Leu 85 90 95
Gin Ala Leu Pro Asp Gly Arg He Gly Gly Ala His Ala Asp Thr Arg 100 105 110
Asp Ser Leu Leu Glu Leu Ser Pro Val Glu Arg Gly Val Val Ser He 115 120 125
Phe Gly Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys 130 135 140
Leu Tyr Gly Ser Pro Phe Phe Thr Asp Glu Cys Thr Phe Lys Glu He 145 150 155 160
Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly 165 170 175
Met Phe He Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg 180 185 190
Val Ser Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu 195 200 205
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "FGF-5"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu He Leu 1 5 10 15
Ser Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro Lys Gly Gin Pro 20 25 30
Gly Pro Ala Ala Thr Asp Arg Asn Pro He Gly Ser Ser Ser Arg Gin 35 40 45
Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala 50 55 60
Ala Ser Leu Gly Ser Gin Gly Ser Gly Leu Glu Gin Ser Ser Phe Gin 65 70 75 80
Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly 85 90 95 He Gly Phe His Leu Gin He Tyr Pro Asp Gly Lys Val Asn Gly Ser 100 105 110
His Glu Ala Asn Met Leu Ser Val Leu Glu He Phe Ala Val Ser Gin 115 120 125
Gly He Val Gly He Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met 130 135 140
Ser Lys Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys 145 150 155 160
Lys Phe Arg Glu Arg Phe Gin Glu Asn Ser Tyr Asn Thr Tyr Ala Ser 165 170 175
Ala He His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu 180 185 190
Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro 195 200 205
Gin His He Ser Thr His Phe Leu Pro Arg Phe Lys Gin Ser Glu Gin 210 215 220
Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro 225 230 235 240
Ser Pro He Lys Ser Lys He Pro Leu Ser Ala Pro Arg Lys Asn Thr 245 250 255
Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 198 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
( i x) FEATURE :
(D) OTHER INFORMATION : /note= "FGF-6"
( xi ) SEQUENCE DESCRI PTION : SEQ ID NO : 15 :
Met Ser Arg Gly Al a Gly Arg Leu Gi n Gly Thr Leu Trp Al a Leu Val 1 5 10 15
Phe Leu Gly H e Leu Va l Gly Met Val Val Pro Ser Pro Al a Gly Thr 20 25 30
Arg Al a Asn Asn Thr Leu Leu Asp Ser Arg Gly Trp Gly Thr Leu Leu 35 40 45 Ser Arg Ser Arg Ala Gly Leu Ala Gly Glu He Ala Gly Val Asn Trp 50 55 60
Glu Ser Gly Tyr Leu Val Gly He Lys Arg Gin Arg Arg Leu Tyr Cys 65 70 75 80
Asn Val Gly He Gly Phe His Leu Gin Val Leu Pro Asp Gly Arg He 85 90 95
Ser Gly Thr His Glu Glu Asn Pro Tyr Ser Leu Leu Glu He Ser Thr 100 105 110
Val Glu Arg Gly Val Val Ser Leu Phe Gly Val Arg Ser Ala Leu Phe 115 120 125
Val Ala Met Asn Ser Lys Gly Arg Leu Tyr Ala Thr Pro Ser Phe Gin 130 135 140
Glu Glu Cys Lys Phe Arg Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala 145 150 155 160
Tyr Glu Ser Asp Leu Tyr Gin Gly Thr Tyr He Ala Leu Ser Lys Tyr 165 170 175
Gly Arg Val Lys Arg Gly Ser Lys Val Ser Pro He Met Thr Val Thr 180 185 190
Figure imgf000137_0001
(2) INFORMATION FOR SEQ ID NO 16
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 194 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY unknown
(n) MOLECULE TYPE peptide
(ix) FEATURE
(D) OTHER INFORMATION /note= "FGF-7"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 16
Met His Lys Trp He Leu Thr Trp He Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15
Ser Cys Phe His He He Cys Leu Val Gly Thr He Ser Leu Ala Cys 20 25 30
Asn Asp Met Thr Pro Glu Gin Met Ala Thr Asn Val Asn Cys Ser Ser 35 40 45
Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp He 50 55 60 Arg Val Arg Arg Leu Phe Cys Arg Thr Gin Trp Tyr Leu Arg He Asp 65 70 75 80
Lys Arg Gly Lys Val Lys Gly Thr Gin Glu Met Lys Asn Asn Tyr Asn 85 90 95
He Met Glu He Arg Thr Val Ala Val Gly He Val Ala He Lys Gly 100 105 110
Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115 120 125
Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu He Leu 130 135 140
Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly 145 150 155 160
Gly Glu Met Phe Val Ala Leu Asn Gin Lys Gly He Pro Val Arg Gly 165 170 175
Lys Lys Thr Lys Lys Glu Gin Lys Thr Ala His Phe Leu Pro Met Ala 180 185 190
He Thr
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "FGF-8"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu 1 5 . 10 15
Val Leu Cys Leu Gin Ala Gin Val Thr Val Gin Ser Ser Pro Asn Phe 20 25 30
Thr Gin His Val Arg Glu Gin Ser Leu Val Thr Asp Gin Leu Ser Arg 35 40 45
Arg Leu He Arg Thr Tyr Gin Leu Tyr Ser Arg Thr Ser Gly Lys His 50 55 60
Val Gin Val Leu Ala Asn Lys Arg He Asn Ala Met Ala Glu Asp Gly 65 70 75 80 Asp Pro Phe Ala Lys Leu He Val Glu Thr Asp Thr Phe Gly Ser Arg 85 90 95
Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr He Cys Met Asn Lys 100 105 110
Lys Gly Lys Leu He Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys Val 115 120 125
Phe Thr Glu He Val Leu Glu Asn Asn Tyr Thr Ala Leu Gin Asn Ala 130 135 140
Lys Tyr Glu Gly Trp Tyr Met Ala Phe Thr Arg Lys Gly Arg Pro Arg 145 150 155 160
Lys Gly Ser Lys Thr Arg Gin His Gin Arg Glu Val His Phe Met Lys 165 170 175
Arg Leu Pro Arg Gly His His Thr Thr Glu Gin Ser Leu Arg Phe Glu 180 185 190
Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser Gin Arg 195 200 205
Thr Trp Ala Pro Glu Pro Arg 210 215
(2) INFORMATION FOR SEQ ID NO 18
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 208 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY unknown
(n) MOLECULE TYPE peptide
(ix) FEATURE
(D) OTHER INFORMATION /note= "FGF-9"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 18
Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gin Asp Ala 1 5 10 15
Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu 20 25 30
Leu Ser Asp His Leu Gly Gin Ser Glu Ala Gly Gly Leu Pro Arg Gly 35 40 45
Pro Ala Val Thr Asp Leu Asp His Leu Lys Gly He Leu Arg Arg Arg 50 55 60
Gin Leu Tyr Cys Arg Thr Gly Phe His Leu Glu He Phe Pro Asn Gly 65 70 75 80 Thr He Gin Gly Thr Arg Lys Asp His Ser Arg Phe Gly He Leu Glu 85 90 95
Phe He Ser He Ala Val Gly Leu Val Ser He Arg Gly Val Asp Ser 100 105 110
Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 115 120 125
Lys Leu Thr Gin Glu Cys Val Phe Arg Glu Gin Phe Glu Glu Asn Trp 130 135 140
Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg 145 150 155 160
Arg Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175
Arg Thr Lys Arg His Gin Lys Phe Thr His Phe Leu Pro Arg Pro Val 180 185 190
Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp He Leu Ser Gin Ser 195 200 205
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..804
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..804
(D) OTHER INFORMATION: /note= "Nucleotide sequence corresponding to the clone M13 mpl8-G4 in Example I.B.2."
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 46..804
(D) OTHER INFORMATION: /product= ""Saporin""
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48 Ala Trp He Leu Leu Gin Phe Ser Ala Trp Thr Thr Thr Asp Ala Val -15 -10 -5 1 ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA 96 Thr Ser He Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser 5 10 15
TCT TTT GTG GAT AAA ATC CGA AAC AAT GTA AAG GAT CCA AAC CTG AAA 144 Ser Phe Val Asp Lys He Arg Asn Asn Val Lys Asp Pro Asn Leu Lys 20 25 30
TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA 192 Tyr Gly Gly Thr Asp He Ala Val He Gly Pro Pro Ser Lys Glu Lys 35 40 45 πC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC 240
Phe Leu Arg He Asn Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly
50 55 60 65
CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC 288
Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn 70 75 80
ACG AAT GTT AAT CGG GCA TAT TAC TTC AAA TCA GAA ATT ACT TCC GCC 336
Thr Asn Val Asn Arg Ala Tyr Tyr Phe Lys Ser Glu He Thr Ser Ala 85 90 95
GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT 384 Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala 100 105 110
TTA GAA TAC ACA GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA 432 Leu Glu Tyr Thr Glu Asp Tyr Gin Ser He Glu Lys Asn Ala Gin He 115 120 125
ACA CAG GGA GAT AAA AGT AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA 480 Thr Gin Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly He Asp Leu 130 135 140 145
CTT TTG ACG TTC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA 528 Leu Leu Thr Phe Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys 150 155 160
AAC GAA GCT AGG TTT CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA 576 Asn Glu Ala Arg Phe Leu Leu He Ala He Gin Met Thr Ala Glu Val 165 170 175
GCA CGA TTT AGG TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC 624 Ala Arg Phe Arg Tyr He Gin Asn Leu Val Thr Lys Asn Phe Pro Asn 180 185 190
AAG TTC GAC TCG GAT AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT 672 Lys Phe Asp Ser Asp Asn Lys Val He Gin Phe Glu Val Ser Trp Arg 195 200 205
AAG ATT TCT ACG GCA ATA TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT 720 Lys He Ser Thr Ala He Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn 210 215 220 225 AAA GAT TAT GAT TTC GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG 768 Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg Gin Val Lys Asp Leu 230 235 240
CAA ATG GGA CTC CTT ATG TAT TTG GGC AAA CCA AAG 804
Gin Met Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys 245 250
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..804
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..804
(D) OTHER INFORMATION: /note= "Nucleotide sequence corresponding to the clone M13 mpl8-Gl in Example I.B.2."
(ix) FEATURE:
(A) NAME/KEY: mat peptide
(B) LOCATION: 46.7804
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48 Ala Trp He Leu Leu Gin Phe Ser Ala Trp Thr Thr Thr Asp Ala Val -15 -10 -5 1
ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA 96 Thr Ser He Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser 5 10 15
TCT TTT GTG GAT AAA ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG AAA 144 Ser Phe Val Asp Lys He Arg Asn Asn Val Lys Asp Pro Asn Leu Lys 20 25 30
TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA 192 Tyr Gly Gly Thr Asp He Ala Val He Gly Pro Pro Ser Lys Glu Lys 35 40 45
TTC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC 240 Phe Leu Arg He Asn Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly 50 55 60 65 CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC 288 Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn 70 75 80
ACG AAT GTT AAT CGG GCA TAT TAC TTC AGA TCA GAA ATT ACT TCC GCC 336 Thr Asn Val Asn Arg Ala Tyr Tyr Phe Arg Ser Glu He Thr Ser Ala 85 90 95
GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT 384 Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala 100 105 110
TTA GAA TAC ACA GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA 432 Leu Glu Tyr Thr Glu Asp Tyr Gin Ser He Glu Lys Asn Ala Gin He 115 120 125
ACA CAG GGA GAT AAA TCA AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA 480 Thr Gin Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly He Asp Leu 130 135 140 145
CH TTG ACG TCC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA 528 Leu Leu Thr Ser Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys 150 155 160
AAC GAA GCT AGG TTT CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA 576 Asn Glu Ala Arg Phe Leu Leu He Ala He Gin Met Thr Ala Glu Val 165 170 175
GCA CGA TTT CGG TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC 624 Ala Arg Phe Arg Tyr He Gin Asn Leu Val Thr Lys Asn Phe Pro Asn 180 185 190
AAG TTC GAC TCG GAT AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT 672 Lys Phe Asp Ser Asp Asn Lys Val He Gin Phe Glu Val Ser Trp Arg 195 200 205
AAG ATT TCT ACG GCA ATA TAC GGA GAT GCC AAA AAC GGC GTG TTT AAT 720 Lys He Ser Thr Ala He Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn 210 215 220 225
AAA GAT TAT GAT TTC GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG 768 Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg Gin Val Lys Asp Leu 230 235 240
CAA ATG GGA CTC CTT ATG TAT TTG GGC AAA CCA AAG 804
Gin Met Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys 245 250
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA (i x) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..804
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..804
(D) OTHER INFORMATION: /note= "Nucleotide sequence corresponding to the clone M13 mpl8-G2 in Example I.B.2."
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 46..804
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48 Ala Trp He Leu Leu Gin Phe Ser Ala Trp Thr Thr Thr Asp Ala Val -15 -10 -5 1
ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACT GCG GGT CAA TAC TCA 96 Thr Ser He Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser 5 10 15
TCT Tπ GTG GAT AAA ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG AAA 144 Ser Phe Val Asp Lys He Arg Asn Asn Val Lys Asp Pro Asn Leu Lys 20 25 30
TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAT AAA 192 Tyr Gly Gly Thr Asp He Ala Val He Gly Pro Pro Ser Lys Asp Lys 35 40 45
TTC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC 240 Phe Leu Arg He Asn Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly 50 55 60 65
CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC 288 Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn 70 75 80
ACG AAT GTT AAT CGG GCA TAT TAC TTC AAA TCA GAA ATT ACT TCC GCC 336 Thr Asn Val Asn Arg Ala Tyr Tyr Phe Lys Ser Glu He Thr Ser Ala 85 90 95
GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT 384 Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala 100 105 110
TTA GAA TAC ACA GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA 432 Leu Glu Tyr Thr Glu Asp Tyr Gin Ser He Glu Lys Asn Ala Gin He 115 120 125
ACA CAG GGA GAT AAA AGT AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA 480 Thr Gin Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly He Asp Leu 130 135 140 145 CTT πG ACG TTC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA 528 Leu Leu Thr Phe Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys 150 155 160
AAC GAA GCT AGG Tπ CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA 576 Asn Glu Ala Arg Phe Leu Leu He Ala He Gin Met Thr Ala Glu Val 165 170 175
GCA CGA πT AGG TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC 624 Ala Arg Phe Arg Tyr He Gin Asn Leu Val Thr Lys Asn Phe Pro Asn 180 185 190
AAG TTC GAC TCG GAT AAC AAG GTG ATT CAA πT GAA GTC AGC TGG CGT 672 Lys Phe Asp Ser Asp Asn Lys Val He Gin Phe Glu Val Ser Trp Arg 195 200 205
AAG ATT TCT ACG GCA ATA TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT 720 Lys He Ser Thr Ala He Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn 210 215 220 225
AAA GAT TAT GAT πC GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG 768 Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg Gin Val Lys Asp Leu 230 235 240
CAA ATG GGA CTC Cπ ATG TAT πG GGC AAA CCA AAG 804
Gin Met Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys 245 250
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..804
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 1..804
(D) OTHER INFORMATION: /note= "Nucleotide sequence corresponding to the clone M13 mpl8-G7 in Example I.B.2."
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 46..804
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22: GCA TGG ATC CTG Cπ CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48 Ala Trp He Leu Leu Gin Phe Ser Ala Trp Thr Thr Thr Asp Ala Val -15 -10 -5 1
ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA 96 Thr Ser He Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser 5 10 15
TCT Tπ GTG GAT AAA ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG AAA 144 Ser Phe Val Asp Lys He Arg Asn Asn Val Lys Asp Pro Asn Leu Lys 20 25 30
TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA 192 Tyr Gly Gly Thr Asp He Ala Val He Gly Pro Pro Ser Lys Glu Lys 35 40 45 πC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC 240
Phe Leu Arg He Asn Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly
50 55 60 65
CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT Cπ GCA ATG GAT AAC 288 Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn 70 75 80
ACG AAT GTT AAT CGG GCA TAT TAC πC AGA TCA GAA ATT ACT TCC GCC 336 Thr Asn Val Asn Arg Ala Tyr Tyr Phe Arg Ser Glu He Thr Ser Ala 85 90 95
GAG πA ACC GCC Cπ πC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT 384 Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala 100 105 110
TTA GAA TAC ACA GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA 432 Leu Glu Tyr Thr Glu Asp Tyr Gin Ser He Glu Lys Asn Ala Gin He 115 120 125
ACA CAG GGA GAT AAA TCA AGA AAA GAA CTC GGG TTG GGG ATC GAC πA 480 Thr Gin Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly He Asp Leu 130 135 140 145
CTT πG ACG TCC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG Gπ AAA 528 Leu Leu Thr Ser Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys 150 155 160
AAC GAA GCT AGA TTC CTT CTT ATC GCT ATT CAG ATG ACG GCT GAG GCA 576 Asn Glu Ala Arg Phe Leu Leu He Ala He Gin Met Thr Ala Glu Ala 165 170 175
GCA CGA TTT AGG TAC ATA CAA AAC TTG GTA ATC AAG AAC TTT CCC AAC 624 Ala Arg Phe Arg Tyr He Gin Asn Leu Val He Lys Asn Phe Pro Asn 180 185 190
AAG TTC AAC TCG GAA AAC AAA GTG ATT CAG TTT GAG GTT AAC TGG AAA 672 Lys Phe Asn Ser Glu Asn Lys Val He Gin Phe Glu Val Asn Trp Lys 195 200 205 AAA ATT TCT ACG GCA ATA TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT 720 Lys He Ser Thr Ala He Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn 210 215 220 225
AAA GAT TAT GAT πC GGG πT GGA AAA GTG AGG CAG GTG AAG GAC TTG 768 Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg Gin Val Lys Asp Leu 230 235 240
CAA ATG GGA CTC Cπ ATG TAT TTG GGC AAA CCA AAG 804
Gin Met Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys 245 250
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 804 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..804
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1..804
(D) OTHER INFORMATION: /note= "Nucleotide sequence corresponding to the clone M13 mpl8-G9 in Example I.B.2."
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 46..804
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48 Ala Trp He Leu Leu Gin Phe Ser Ala Trp Thr Thr Thr Asp Ala Val -15 -10 -5 1
ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA 96 Thr Ser He Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser 5 10 15
TCT TTT GTG GAT AAA ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG AAA 144 Ser Phe Val Asp Lys He Arg Asn Asn Val Lys Asp Pro Asn Leu Lys 20 25 30
TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA 192 Tyr Gly Gly Thr Asp He Ala Val He Gly Pro Pro Ser Lys Glu Lys 35 40 45 πC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC 240 Phe Leu Arg He Asn Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly 50 55 60 65
CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC 288 Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn 70 75 80
ACG AAT GTT AAT CGG GCA TAT TAC TTC AGA TCA GAA ATT ACT TCC GCC 336 Thr Asn Val Asn Arg Ala Tyr Tyr Phe Arg Ser Glu He Thr Ser Ala 85 90 95
GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT 384 Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala 100 105 110 πA GAA TAC ACA GAA GAT TAT CAG TCG Aπ GAA AAG AAT GCC CAG ATA 432 Leu Glu Tyr Thr Glu Asp Tyr Gin Ser He Glu Lys Asn Ala Gin He 115 120 125
ACA CAA GGA GAT CAA AGT AGA AAA GAA CTC GGG πG GGG ATT GAC πA 480 Thr Gin Gly Asp Gin Ser Arg Lys Glu Leu Gly Leu Gly He Asp Leu 130 135 140 145
CTT TCA ACG TCC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA 528 Leu Ser Thr Ser Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys 150 155 160
GAC GAA GCT AGA TTC Cπ CTT ATC GCT ATT CAG ATG ACG GCT GAG GCA 576 Asp Glu Ala Arg Phe Leu Leu He Ala He Gin Met Thr Ala Glu Ala 165 170 175
GCG CGA TTT AGG TAC ATA CAA AAC TTG GTA ATC AAG AAC TTT CCC AAC 624 Ala Arg Phe Arg Tyr He Gin Asn Leu Val He Lys Asn Phe Pro Asn 180 185 190
AAG TTC AAC TCG GAA AAC AAA GTG ATT CAG TTT GAG GTT AAC TGG AAA 672 Lys Phe Asn Ser Glu Asn Lys Val He Gin Phe Glu Val Asn Trp Lys 195 200 205
AAA ATT TCT ACG GCA ATA TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT 720 Lys He Ser Thr Ala He Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn 210 215 220 225
AAA GAT TAT GAT TTC GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG 768 Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg Gin Val Lys Asp Leu 230 235 240
CAA ATG GGA CTC CTT ATG TAT TTG GGC AAA CCA AAG 804
Gin Met Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys 245 250
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..7
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Pro Lys Lys Arg Lys Val Glu 1 5
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..8
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Pro Pro Lys Lys Ala Arg Glu Val
1 5
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..9
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5
(2) INFORMATION FOR SEQ ID NO:27: 148
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..5
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Lys Arg Pro Arg Pro 1 5
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..5
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Lys He Pro He Lys 1 5
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..9
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
Gly Lys Arg Lys Arg Lys Ser 1 5 (2) INFORMATION FOR SEQ ID NO 30
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 9 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY unknown
(n) MOLECULE TYPE peptide
(ix) FEATURE
(A) NAME/KEY CDS
(B) LOCATION 1 9
(D) OTHER INFORMATION /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION SEQ ID NO 30
Ser Lys Arg Val Ala Lys Arg Lys Leu 1 5
(2) INFORMATION FOR SEQ ID NO 31
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 9 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY unknown
(n) MOLECULE TYPE peptide
(ix) FEATURE
(A) NAME/KEY CDS
(B) LOCATION 1 9
(D) OTHER INFORMATION /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION SEQ ID NO 31
Ser His Trp Lys Gin Lys Arg Lys Phe 1 5
(2) INFORMATION FOR SEQ ID NO 32
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 8 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY unknown
(n) MOLECULE TYPE peptide
(ix) FEATURE
(A) NAME/KEY CDS
(B) LOCATION 1 8
(D) OTHER INFORMATION /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Pro Leu Leu Lys Lys He Lys Gin 1 5
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..7
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Pro Gin Pro Lys Lys Lys Pro
1 5
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Pro Gly Lys Arg Lys Lys Glu Met Thr Lys Gin Lys Glu Val Pro 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 1..12
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Ala Pro 1 5 10
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..7
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
Asn Tyr Lys Lys Pro Lys Leu 1 5
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..7
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
His Phe Lys Asp Pro Lys Arg 1 5
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (i x) FEATURE :
(A) NAME/ KEY : CDS
(B) LOCATION: 1. .7
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Ala Pro Arg Arg Arg Lys Leu 1 5
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..6
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
He Lys Arg Leu Arg Arg 1 5
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..6
(D) OTHER INFORMATION: /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
He Lys Arg Gin Arg Arg 1 5
(2) INFORMATION FOR SEQ ID N0:41: (l) SEQUENCE CHARACTERISTICS
(A) LENGTH 5 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY unknown
(n) MOLECULE TYPE peptide
(ix) FEATURE
(A) NAME/KEY CDS
(B) LOCATION 1 5
(D) OTHER INFORMATION /product= nuclear translocation sequence
(xi) SEQUENCE DESCRIPTION SEQ ID NO 41
He Arg Val Arg Arg 1 5
(2) INFORMATION FOR SEQ ID NO 42
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 4 amino acids
(B) TYPE amino acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note= "Cytoplasmic Translocation Signal"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 42
Lys Asp Glu Leu
1
(2) INFORMATION FOR SEQ ID NO 43
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 4 ammo acids
Figure imgf000155_0001
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note= "Cytoplasmic Translocation Signal"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 43
Arg Asp Glu Leu
1 (2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Cytoplasmic Translocation Signal"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Lys Glu Glu Leu 1
(2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: /note= "Endosome-disruptive peptide INF"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
Gly Leu Phe Glu Ala He Glu Gly Phe He Glu Asn Gly Trp Glu Gly 1 5 10 15
Met He Asp Gly Gly Gly Cys 20
(2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i x) FEATURE :
(D) OTHER INFORMATION: /note= "Endosome-di srupti ve peptide INF"
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 46 :
Gly Leu Phe Gl u Al a H e Gl u Gly Phe H e Glu Asn Gly Trp Gl u Gly 1 5 10 15
Met He Asp Gly Trp Tyr Gly Cys 20 (2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY CDS
(B) LOCATION 3..26 (A) NAME/KEY Gly4Ser with Ncol ends
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47: CCATGGGCGG CGGCGGCTCT GCCATGG 27
(2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY CDS
(B) LOCATION 3..41 (A) NAME/KEY (Gly4Ser)2 with Ncol ends
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: CCATGGGCGG CGGCGGCTCT GGCGGCGGCG GCTCTGCCAT GG 42
(2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY CDS
(B) LOCATION 3..74 (A) NAME/KEY (Ser4Gly)4 with Ncol ends
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49: CCATGGCCTC GTCGTCGTCG GGCTCGTCGT CGTCGGGCTC GTCGTCGTCG GGCTCGTCGT 60 CGTCGGGCGC CATGG 75
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..45
(A) NAME/KEY: (Ser4Gly)2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: CCATGGCCTC GTCGTCGTCG GGCTCGTCGT CGTCGGGCGC CATGG 45 (2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..8
(D) OTHER INFORMATION: /product= Flexible linker
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
Ala Ala Pro Ala Ala Ala Pro Ala 1 5
(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..465
(ix) FEATURE: (A) NAME/KEY: mat_peptide
(B) LOCATION: 1..465
(D) OTHER INFORMATION: /product= "bFGF"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC πG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15
GGC AGC GGC GCC πC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30
TAC TGC AAA AAC GGG GGC πC πC CTG CGC ATC CAC CCC GAC GGC CGA 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45
Gπ GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu 50 55 60
CAA GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC 240 Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn 65 70 75 80
CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT 288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95
GTT ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110
AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA TTG AAA 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125
CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA 432 Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 130 135 140
GCT ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC 465
Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1230 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA ( i x) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..1230
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 1..465
(D) OTHER INFORMATION: /product= "bFGF"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 472..1230
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
ATG GCT GCT GGT TCT ATC ACT ACT CTG CCG GCT CTG CCG GAA GAC GGT 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15
GGT TCT GGT GCT πC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30
TAC TGC AAA AAC GGG GGC TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45
Gπ GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu 50 55 60
CAA GCA GAA GAG AGA GGA Gπ GTG TCT ATC AAA GGA GTG TGT GCT AAC 240 Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn 65 70 75 80
CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT 288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95
GTT ACG GAT GAG TGT TTC TTT TTT GAA CGA πG GAA TCT AAT AAC TAC 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110
AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA TTG AAA 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125
CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA 432 Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 130 135 140
GCT ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC GCC ATG GTC ACA TCA 480 Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Val Thr Ser 145 150 155 160 ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA TCT Tπ 528 He Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser Ser Phe 165 170 175
GTG GAT AAA ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG AAA TAC GGT 576 Val Asp Lys He Arg Asn Asn Val Lys Asp Pro Asn Leu Lys Tyr Gly 180 185 190
GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA TTC CTT 624 Gly Thr Asp He Ala Val He Gly Pro Pro Ser Lys Glu Lys Phe Leu 195 200 205
AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC CTA AAA 672 Arg He Asn Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly Leu Lys 210 215 220
CGC GAT AAC πG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC ACG AAT 720 Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn Thr Asn 225 230 235 240
GTT AAT CGG GCA TAT TAC πC AAA TCA GAA Aπ ACT TCC GCC GAG πA 768 Val Asn Arg Ala Tyr Tyr Phe Lys Ser Glu He Thr Ser Ala Glu Leu 245 250 255
ACC GCC Cπ TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT TTA GAA 816 Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala Leu Glu 260 265 270
TAC ACA GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA ACA CAG 864 Tyr Thr Glu Asp Tyr Gin Ser He Glu Lys Asn Ala Gin He Thr Gin 275 280 285
GGA GAT AAA AGT AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA CTT πG 912 Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly He Asp Leu Leu Leu 290 295 300
ACG TTC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA AAC GAA 960 Thr Phe Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys Asn Glu 305 310 315 320
GCT AGG TTT CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA GCA CGA 1008 Ala Arg Phe Leu Leu He Ala He Gin Met Thr Ala Glu Val Ala Arg 325 330 335
TTT AGG TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC AAG TTC 1056 Phe Arg Tyr He Gin Asn Leu Val Thr Lys Asn Phe Pro Asn Lys Phe 340 345 350
GAC TCG GAT AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT AAG ATT 1104 Asp Ser Asp Asn Lys Val He Gin Phe Glu Val Ser Trp Arg Lys He 355 360 365
TCT ACG GCA ATA TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT AAA GAT 1152 Ser Thr Ala He Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn Lys Asp 370 375 380 TAT GAT TTC GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG CAA ATG 1200 Tyr Asp Phe Gly Phe Gly Lys Val Arg Gin Val Lys Asp Leu Gin Met 385 390 395 400
GGA CTC CTT ATG TAT TTG GGC AAA CCA AAG 1230
Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys 405 410
(2) INFORMATION FOR SEQ ID NO 54
(ι) SEQUENCE CHARACTERISTICS
(A) LENGTH 30 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(n) MOLECULE TYPE DNA (genomic)
(ix) FEATURE
(A) NAME/KEY ππ sc_recomb
(B) LOCATION 6 11
(D) OTHER INFORMATION /standard_name= "EcoRI Restriction Site"
(ix) FEATURE
(A) NAME/KEY sιg_peptιde
(B) LOCATION 12 30
(D) OTHER INFORMATION /functιon= "N-terminal extension" /product= "Native saporin signal peptide"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 54
CTGCAGAATT CGCATGGATC CTGCTTCAAT 30
(2) INFORMATION FOR SEQ ID NO 55
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 30 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(n) MOLECULE TYPE DNA (genomic) (iv) ANTI-SENSE YES
(ix) FEATURE
(A) NAME/KEY mιsc_recomb
(B) LOCATION 6 11
(D) OTHER INFORMATION /standard_name= "EcoRI Restriction Site"
(ix) FEATURE
(A) NAME/KEY terminator
(B) LOCATION 23 25
(D) OTHER INFORMATION /note= "Anti-sense stop codon' (ix) FEATURE :
(A) NAME/KEY: mat_peptide
(B) LOCATION: 26..30
(D) OTHER INFORMATION: /note= "Anti-sense to carboxyl terminus of mature peptide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55
CTGCAGAAπ CGCCTCGTTT GACTACπTG 30
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: AGGAGTGTCT GCTAACC 17
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57: TTCTAAATCG GTTACCGATG ACTG 24
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58: CATATGTGTG AGCTACTGTC GCCACCGCTC 30 (2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59: GGATCCGAGC ACCTGGTATA TCGGTGGGGG 30
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: GGATCCGCCT CGπTGACTA CTT 23
(2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(D) OTHER INFORMATION/product= bacteriophage lambda CII ribosome binding site
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
GTCGACCAAG CTTGGGCATA CATTCAATCA ATTGTTATCT AAGGAAATAC TTACATATG 59
(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 59 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(D) OTHER INFORMATION: /product= trp promoter (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: AATTCCCCTG TTGACAAπA ATCATCGAAC TAGTTAACTA GTACGCAGCT TGGCTGCAG 59 (2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_recomb
(B) LOCATION: 11..16
(D) OTHER INFORMATION: /standard_name= "Nco I restriction enzyme recognition site.
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 1..10
(D) OTHER INFORMATION: /product= "Carboxy terminus of mature FGF protein"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
GCTAAGAGCG CCATGGAGA 19
(2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..12
(D) OTHER INFORMATION: /product= "Carboxy terminus of wild type FGF"
(ix) FEATURE:
(A) NAME/KEY: misc_recomb
(B) LOCATION: 13..18
(D) OTHER INFORMATION: /standard_name= "Nco I restriction enzyme recognition site"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
GCT AAG AGC TGACCATGGA GA 21
Ala Lys Ser
1 (2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..96
(D) OTHER INFORMATION: /product= "pFGFNcoI"
/note= "Equals the plasmid pFC80 wih native FGF stop codon removed."
(ix) FEATURE:
(A) NAME/KEY: misc_recomb
(B) LOCATION: 29..34
(D) OTHER INFORMATION: /standard_name= "Nco I restriction enzyme recognition site"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
CTT TTT CTT CCA ATG TCT GCT AAG AGC GCC ATG GAG ATC CGG CTG AAT 48 Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Glu He Arg Leu Asn 1 5 10 15
GGT GCA GTT CTG TAC CGG TTT TCC TGT GCC GTC πT CAG GAC TCC TGAAATCTT 102 Gly Ala Val Leu Tyr Arg Phe Ser Cys Ala Val Phe Gin Asp Ser 20 25 30
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY CDS
(B) LOCATION 3..35 (A) NAME/KEY Cathepsin B linker
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: CCATGGCCCT GGCCCTGGCC CTGGCCCTGG CCATGG 36 (2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY CDS
(B) LOCATION 3..50 (A) NAME/KEY Cathepsin D linker
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: CCATGGGCCG ATCGGGCπC CTGGGCTTCG GCTTCCTGGG CTTCGCCATGG 51
(2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 96 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..95
(A) NAME/KEY: "Trypsin linker"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:68: CCATGGGCCG ATCGGGCGGT GGGTGCGCTG GTAATAGAGT CAGAAGATCA GTCGGAAGCA 60 GCCTGTCTTG CGGTGGTCTC GACCTGCAGG CCATGG 96
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..18
(D) OTHER INFORMATION: /product= Thrombin substrate linker (xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
CTG GTG CCG CGC GGC AGC 18
Leu Val Pro Arg Gly Ser 1 5
(2) INFORMATION FOR SEQ ID N0:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..15
(D) OTHER INFORMATION: /product= Enterokinase substrate linker
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:70:
GAC GAC GAC GAC CCA 15
Asp Asp Asp Asp Lys 1 5
(2) INFORMATION FOR SEQ ID NO-71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1.-12
(D) OTHER INFORMATION: /product= Factor Xa substrate
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
ATC GAA GGT CGT 12
He Glu Gly Arg 1
(2) INFORMATION FOR SEQ ID N0:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1260 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA (i x) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1- .1260
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 1..465
(D) OTHER INFORMATION: /product= "bFGF"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 466...501
(D) OTHER INFORMATION: /product= "Cathepsin B linker"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 502..1260
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:
ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15
GGC AGC GGC GCC πC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30
TAC TGC AAA AAC GGG GGC TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45
GTT GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu 50 55 60
CAA GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC 240 Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn 65 70 75 80
CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT 288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95
GTT ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110
AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA TTG AAA 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125
CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA 432 Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 130 135 140 GCT ATA CTT TTT Cπ CCA ATG TCT GCT AAG AGC GCC ATG GCC CTG GCC 480 Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Ala Leu Ala 145 150 155 160
CTG GCC CTG GCC CTG GCC ATG GTC ACA TCA ATC ACA TTA GAT CTA GTA 528 Leu Ala Leu Ala Leu Ala Met Val Thr Ser He Thr Leu Asp Leu Val
165 170 175
AAT CCG ACC GCG GGT CAA TAC TCA TCT πT GTG GAT AAA ATC CGA AAC 576 Asn Pro Thr Ala Gly Gin Tyr Ser Ser Phe Val Asp Lys He Arg Asn 180 185 190
AAC GTA AAG GAT CCA AAC CTG AAA TAC GGT GGT ACC GAC ATA GCC GTG 624 Asn Val Lys Asp Pro Asn Leu Lys Tyr Gly Gly Thr Asp He Ala Val 195 200 205
ATA GGC CCA CCT TCT AAA GAA AAA TTC CTT AGA ATT AAT TTC CAA AGT 672 He Gly Pro Pro Ser Lys Glu Lys Phe Leu Arg He Asn Phe Gin Ser 210 215 220
TCC CGA GGA ACG GTC TCA CTT GGC CTA AAA CGC GAT AAC TTG TAT GTG 720 Ser Arg Gly Thr Val Ser Leu Gly Leu Lys Arg Asp Asn Leu Tyr Val 225 230 235 240
GTC GCG TAT CTT GCA ATG GAT AAC ACG AAT GTT AAT CGG GCA TAT TAC 768 Val Ala Tyr Leu Ala Met Asp Asn Thr Asn Val Asn Arg Ala Tyr Tyr 245 250 255 πC AAA TCA GAA Aπ ACT TCC GCC GAG TTA ACC GCC CTT TTC CCA GAG 816 Phe Lys Ser Glu He Thr Ser Ala Glu Leu Thr Ala Leu Phe Pro Glu 260 265 270
GCC ACA ACT GCA AAT CAG AAA GCT TTA GAA TAC ACA GAA GAT TAT CAG 864 Ala Thr Thr Ala Asn Gin Lys Ala Leu Glu Tyr Thr Glu Asp Tyr Gin 275 280 285
TCG ATC GAA AAG AAT GCC CAG ATA ACA CAG GGA GAT AAA AGT AGA AAA 912 Ser He Glu Lys Asn Ala Gin He Thr Gin Gly Asp Lys Ser Arg Lys 290 295 300
GAA CTC GGG TTG GGG ATC GAC TTA CTT TTG ACG TTC ATG GAA GCA GTG 960 Glu Leu Gly Leu Gly He Asp Leu Leu Leu Thr Phe Met Glu Ala Val 305 310 315 320
AAC AAG AAG GCA CGT GTG GTT AAA AAC GAA GCT AGG TTT CTG CTT ATC 1008 Asn Lys Lys Ala Arg Val Val Lys Asn Glu Ala Arg Phe Leu Leu He 325 330 335
GCT ATT CAA ATG ACA GCT GAG GTA GCA CGA TTT AGG TAC ATT CAA AAC 1056 Ala He Gin Met Thr Ala Glu Val Ala Arg Phe Arg Tyr He Gin Asn 340 345 350
TTG GTA ACT AAG AAC TTC CCC AAC AAG TTC GAC TCG GAT AAC AAG GTG 1104 Leu Val Thr Lys Asn Phe Pro Asn Lys Phe Asp Ser Asp Asn Lys Val 355 360 365 ATT CAA TTT GAA GTC AGC TGG CGT AAG ATT TCT ACG GCA ATA TAC GGG 1152 He Gin Phe Glu Val Ser Trp Arg Lys He Ser Thr Ala He Tyr Gly 370 375 380
GAT GCC AAA AAC GGC GTG TTT AAT AAA GAT TAT GAT TTC GGG TTT GGA 1200 Asp Ala Lys Asn Gly Val Phe Asn Lys Asp Tyr Asp Phe Gly Phe Gly 385 390 395 400
AAA GTG AGG CAG GTG AAG GAC TTG CAA ATG GGA CTC CTT ATG TAT TTG 1248 Lys Val Arg Gin Val Lys Asp Leu Gin Met Gly Leu Leu Met Tyr Leu 405 410 415
GGC AAA CCA AAG 1260
Gly Lys Pro Lys 420
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1275 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
' (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1275
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 1..465
(D) OTHER INFORMATION: /product= "bFGF"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 466...516
(D) OTHER INFORMATION: /product= "Cathepsin D linker"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 517..1275
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15
GGC AGC GGC GCC TTC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 TAC TGC AAA AAC GGG GGC πC πc CTG CGC ATC CAC CCC GAC GGC CGA 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45
GTT GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA Cπ 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu 50 55 60
CAA GCA GAA GAG AGA GGA Gπ GTG TCT ATC AAA GGA GTG TGT GCT AAC 240
Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn
65 70 75 80
CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT 288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95
Gπ ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110
AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA TTG AAA 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125
CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA 432 Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 130 135 140
GCT ATA CTT πT Cπ CCA ATG TCT GCT AAG AGC GCC ATG GGC CGA TCG 480 Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Gly Arg Ser 145 150 155 160
GGC πC CTG GGC πC GGC TTC CTG GGC πC GCC ATG GTC ACA TCA ATC 528 Gly Phe Leu Gly Phe GLy Phe Leu GLy Phe Ala Met Val Thr Ser He
165 170 175
ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA TCT TTT GTG 576 Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser Ser Phe Val 180 185 190
GAT AAA ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG AAA TAC GGT GGT 624 Asp Lys He Arg Asn Asn Val Lys Asp Pro Asn Leu Lys Tyr Gly Gly 195 200 205
ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA TTC CTT AGA 672 Thr Asp He Ala Val He Gly Pro Pro Ser Lys Glu Lys Phe Leu Arg 210 215 220
ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC CTA AAA CGC 720 He Asn Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly Leu Lys Arg 225 230 235 240
GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC ACG AAT GTT 768 Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn Thr Asn Val 245 250 255 AAT CGG GCA TAT TAC πc AAA TCA GAA ATT ACT TCC GCC GAG TTA ACC 816 Asn Arg Ala Tyr Tyr Phe Lys Ser Glu He Thr Ser Ala Glu Leu Thr 260 265 270
GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT TTA GAA TAC 864 Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala Leu Glu Tyr 275 280 285
ACA GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA ACA CAG GGA 912 Thr Glu Asp Tyr Gin Ser He Glu Lys Asn Ala Gin He Thr Gin Gly 290 295 300
GAT AAA AGT AGA AAA GAA CTC GGG πG GGG ATC GAC TTA CTT TTG ACG 960 Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly He Asp Leu Leu Leu Thr 305 310 315 320
TTC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA AAC GAA GCT 1008 Phe Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys Asn Glu Ala 325 330 335
AGG πT CTG CTT ATC GCT Aπ CAA ATG ACA GCT GAG GTA GCA CGA πT 1056 Arg Phe Leu Leu He Ala He Gin Met Thr Ala Glu Val Ala Arg Phe 340 345 350
AGG TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC AAG TTC GAC 1104 Arg Tyr He Gin Asn Leu Val Thr Lys Asn Phe Pro Asn Lys Phe Asp 355 360 365
TCG GAT AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT AAG ATT TCT 1152 Ser Asp Asn Lys Val He Gin Phe Glu Val Ser Trp Arg Lys He Ser 370 375 380
ACG GCA ATA TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT AAA GAT TAT 1200 Thr Ala He Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn Lys Asp Tyr 385 390 395 400
GAT TTC GGG πT GGA AAA GTG AGG CAG GTG AAG GAC TTG CAA ATG GGA 1248 Asp Phe Gly Phe Gly Lys Val Arg Gin Val Lys Asp Leu Gin Met Gly 405 410 415
CTC CTT ATG TAT TTG GGC AAA CCA AAG 1275
Leu Leu Met Tyr Leu Gly Lys Pro Lys 420 425
(2) INFORMATION FOR SEQ ID N0:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1251 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1251 (ix) FEATURE :
(A) NAME/KEY: mat_peptide
(B) LOCATION: 1..465
(D) OTHER INFORMATION: /product= "bFGF"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 466..492
(D) OTHER INFORMATION: /product= " Gly4Ser linker"
(ix) FEATURE:
(A) NAME/KEY: mat peptide
(B) LOCATION: 4937.1251
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:74:
ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15
GGC AGC GGC GCC TTC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30
TAC TGC AAA AAC GGG GGC πC πC CTG CGC ATC CAC CCC GAC GGC CGA 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45
GTT GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu 50 55 60
CAA GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC 240 Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn 65 70 75 80
CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT 288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95
GTT ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110
AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA TTG AAA 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125
CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA 432 Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 130 135 140
GCT ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC GCC ATG GGC GGC GGC 480 Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Gly Gly Gly 145 150 155 160 GGC TCT GCC ATG GTC ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC 528 Gly Ser Ala Met Val Thr Ser He Thr Leu Asp Leu Val Asn Pro Thr
165 170 175
GCG GGT CAA TAC TCA TCT πT GTG GAT AAA ATC CGA AAC AAC GTA AAG 576 Ala Gly Gin Tyr Ser Ser Phe Val Asp Lys He Arg Asn Asn Val Lys 180 185 190
GAT CCA AAC CTG AAA TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA 624 Asp Pro Asn Leu Lys Tyr Gly Gly Thr Asp He Ala Val He Gly Pro 195 200 205
CCT TCT AAA GAA AAA TTC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA 672 Pro Ser Lys Glu Lys Phe Leu Arg He Asn Phe Gin Ser Ser Arg Gly 210 215 220
ACG GTC TCA CTT GGC CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT 720 Thr Val Ser Leu Gly Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr 225 230 235 240
CTT GCA ATG GAT AAC ACG AAT GTT AAT CGG GCA TAT TAC TTC AAA TCA 768 Leu Ala Met Asp Asn Thr Asn Val Asn Arg Ala Tyr Tyr Phe Lys Ser 245 250 255
GAA ATT ACT TCC GCC GAG πA ACC GCC Cπ πC CCA GAG GCC ACA ACT 816 Glu He Thr Ser Ala Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr 260 265 270
GCA AAT CAG AAA GCT TTA GAA TAC ACA GAA GAT TAT CAG TCG ATC GAA 864 Ala Asn Gin Lys Ala Leu Glu Tyr Thr Glu Asp Tyr Gin Ser He Glu 275 280 285
AAG AAT GCC CAG ATA ACA CAG GGA GAT AAA AGT AGA AAA GAA CTC GGG 912 Lys Asn Ala Gin He Thr Gin Gly Asp Lys Ser Arg Lys Glu Leu Gly 290 295 300
TTG GGG ATC GAC TTA CTT TTG ACG TTC ATG GAA GCA GTG AAC AAG AAG 960 Leu Gly He Asp Leu Leu Leu Thr Phe Met Glu Ala Val Asn Lys Lys 305 310 315 320
GCA CGT GTG GTT AAA AAC GAA GCT AGG TTT CTG CTT ATC GCT ATT CAA 1008 Ala Arg Val Val Lys Asn Glu Ala Arg Phe Leu Leu He Ala He Gin 325 330 335
ATG ACA GCT GAG GTA GCA CGA TTT AGG TAC ATT CAA AAC TTG GTA ACT 1056 Met Thr Ala Glu Val Ala Arg Phe Arg Tyr He Gin Asn Leu Val Thr 340 345 350
AAG AAC TTC CCC AAC AAG TTC GAC TCG GAT AAC AAG GTG ATT CAA πT 1104 Lys Asn Phe Pro Asn Lys Phe Asp Ser Asp Asn Lys Val He Gin Phe 355 360 365
GAA GTC AGC TGG CGT AAG ATT TCT ACG GCA ATA TAC GGG GAT GCC AAA 1152 Glu Val Ser Trp Arg Lys He Ser Thr Ala He Tyr Gly Asp Ala Lys 370 375 380 AAC GGC GTG TTT AAT AAA GAT TAT GAT TTC GGG TTT GGA AAA GTG AGG 1200 Asn Gly Val Phe Asn Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg 385 390 395 400
CAG GTG AAG GAC πG CAA ATG GGA CTC CTT ATG TAT πG GGC AAA CCA 1248 Gin Val Lys Asp Leu Gin Met Gly Leu Leu Met Tyr Leu Gly Lys Pro
405 410 415
AAG 1251
Lys
(2) INFORMATION FOR SEQ ID N0:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1266 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1266
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 1..465
(D) OTHER INFORMATION: /product- "bFGF"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 466..507
(D) OTHER INFORMATION: /product= " (Gly4Ser)2 linker"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 508..1266
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:75:
ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15
GGC AGC GGC GCC TTC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30
TAC TGC AAA AAC GGG GGC TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45
GTT GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu 50 55 60 CAA GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC 240
Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn
65 70 75 80
CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT 288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95
Gπ ACG GAT GAG TGT TTC TTT πT GAA CGA πG GAA TCT AAT AAC TAC 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110
AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA TTG AAA 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125
CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA 432 Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 130 135 140
GCT ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC GCC ATG GGC GGC GGC 480 Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Gly Gly Gly 145 150 155 160
GGC TCT GGC GGC GGC GGC TCT GCC ATG GTC ACA TCA ATC ACA πA GAT 528 Gly Ser Gly Gly Gly Gly Ser Ala Met Val Thr Ser He Thr Leu Asp 165 170 175
CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA TCT Tπ GTG GAT AAA ATC 576 Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser Ser Phe Val Asp Lys He 180 185 190
CGA AAC AAC GTA AAG GAT CCA AAC CTG AAA TAC GGT GGT ACC GAC ATA 624 Arg Asn Asn Val Lys Asp Pro Asn Leu Lys Tyr Gly Gly Thr Asp He 195 200 205
GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA TTC CTT AGA ATT AAT TTC 672 Ala Val He Gly Pro Pro Ser Lys Glu Lys Phe Leu Arg He Asn Phe 210 215 220
CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC CTA AAA CGC GAT AAC TTG 720 Gin Ser Ser Arg Gly Thr Val Ser Leu Gly Leu Lys Arg Asp Asn Leu 225 230 235 240
TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC ACG AAT GTT AAT CGG GCA 768 Tyr Val Val Ala Tyr Leu Ala Met Asp Asn Thr Asn Val Asn Arg Ala 245 250 255
TAT TAC TTC AAA TCA GAA ATT ACT TCC GCC GAG TTA ACC GCC Cπ TTC 816 Tyr Tyr Phe Lys Ser Glu He Thr Ser Ala Glu Leu Thr Ala Leu Phe 260 265 270
CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT TTA GAA TAC ACA GAA GAT 864 Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala Leu Glu Tyr Thr Glu Asp 275 280 285 TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA ACA CAG GGA GAT AAA AGT 912 Tyr Gin Ser He Glu Lys Asn Ala Gin He Thr Gin Gly Asp Lys Ser 290 295 300
AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA Cπ TTG ACG TTC ATG GAA 960 Arg Lys Glu Leu Gly Leu Gly He Asp Leu Leu Leu Thr Phe Met Glu 305 310 315 320
GCA GTG AAC AAG AAG GCA CGT GTG Gπ AAA AAC GAA GCT AGG TTT CTG 1008 Ala Val Asn Lys Lys Ala Arg Val Val Lys Asn Glu Ala Arg Phe Leu 325 330 335
CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA GCA CGA TTT AGG TAC Aπ 1056 Leu He Ala He Gin Met Thr Ala Glu Val Ala Arg Phe Arg Tyr He 340 345 350
CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC AAG TTC GAC TCG GAT AAC 1104 Gin Asn Leu Val Thr Lys Asn Phe Pro Asn Lys Phe Asp Ser Asp Asn 355 360 365
AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT AAG ATT TCT ACG GCA ATA 1152 Lys Val He Gin Phe Glu Val Ser Trp Arg Lys He Ser Thr Ala He 370 375 380
TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT AAA GAT TAT GAT TTC GGG 1200 Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn Lys Asp Tyr Asp Phe Gly 385 390 395 400
TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG CAA ATG GGA CTC CTT ATG 1248 Phe Gly Lys Val Arg Gin Val Lys Asp Leu Gin Met Gly Leu Leu Met 405 410 415
TAT TTG GGC AAA CCA AAG 1266
Tyr Leu Gly Lys Pro Lys 420
(2) INFORMATION FOR SEQ ID N0:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1320 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1- .1320
(ix) FEATURE:
(A) NAME/KEY: mat_peptιde
(B) LOCATION: 1..465
(D) OTHER INFORMATION: /product= "bFGF" ( i x) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 466..561
(D) OTHER INFORMATION: /product= "Trypsin linker"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 562..1320
(D) OTHER INFORMATION: /product- "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
ATG GCA GCA GGA TCA ATA ACA ACA πA CCC GCC πG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15
GGC AGC GGC GCC TTC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30
TAC TGC AAA AAC GGG GGC TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45
Gπ GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu 50 55 60
CAA GCA GAA GAG AGA GGA Gπ GTG TCT ATC AAA GGA GTG TGT GCT AAC 240 Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn 65 70 75 80
CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT 288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95
GTT ACG GAT GAG TGT TTC TTT TTT GAA CGA πG GAA TCT AAT AAC TAC 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110
AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA TTG AAA 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125
CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA 432 Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 130 135 140
GCT ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC GCC ATG GGC CGA TCG 480 Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Gly Arg Ser 145 150 155 160
GGC GGT GGG TGC GCT GGT AAT AGA GTC AGA AGA TCA GTC GGA AGC AGC 528 Gly Gly Gly Cys Ala Gly Asn Arg Val Arg Arg Ser Val Gly Ser Ser 165 170 175 CTG TCT TGC GGT GGT CTC GAC CTG CAG GCC ATG GTC ACA TCA ATC ACA 576 Leu Ser Cys Gly Gly Leu Asp Leu Gin Ala Met Val Thr Ser He Thr 180 185 190
TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA TCT TTT GTG GAT 624 Leu Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser Ser Phe Val Asp 195 200 205
AAA ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG AAA TAC GGT GGT ACC 672 Lys He Arg Asn Asn Val Lys Asp Pro Asn Leu Lys Tyr Gly Gly Thr 210 215 220
GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA πC CTT AGA Aπ 720 Asp He Ala Val He Gly Pro Pro Ser Lys Glu Lys Phe Leu Arg He 225 230 235 240
AAT πC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC CTA AAA CGC GAT 768 Asn Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly Leu Lys Arg Asp 245 250 255
AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC ACG AAT GTT AAT 816 Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn Thr Asn Val Asn 260 265 270
CGG GCA TAT TAC πC AAA TCA GAA Aπ ACT TCC GCC GAG TTA ACC GCC 864 Arg Ala Tyr Tyr Phe Lys Ser Glu He Thr Ser Ala Glu Leu Thr Ala 275 280 285
CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT TTA GAA TAC ACA 912 Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala Leu Glu Tyr Thr 290 295 300
GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA ACA CAG GGA GAT 960 Glu Asp Tyr Gin Ser He Glu Lys Asn Ala Gin He Thr Gin Gly Asp 305 310 315 320
AAA AGT AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA CTT TTG ACG TTC 1008 Lys Ser Arg Lys Glu Leu Gly Leu Gly He Asp Leu Leu Leu Thr Phe 325 330 335
ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA AAC GAA GCT AGG 1056
Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys Asn Glu Ala Arg 340 345 350
TTT CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA GCA CGA TTT AGG 1104
Phe Leu Leu He Ala He Gin Met Thr Ala Glu Val Ala Arg Phe Arg 355 360 365
TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC AAG TTC GAC TCG 1152
Tyr He Gin Asn Leu Val Thr Lys Asn Phe Pro Asn Lys Phe Asp Ser 370 375 380
GAT AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT AAG ATT TCT ACG 1200
Asp Asn Lys Val He Gin Phe Glu Val Ser Trp Arg Lys He Ser Thr 385 390 395 400 GCA ATA TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT AAA GAT TAT GAT 1248 Ala He Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn Lys Asp Tyr Asp 405 410 415
TTC GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG CAA ATG GGA CTC 1296 Phe Gly Phe Gly Lys Val Arg Gin Val Lys Asp Leu Gin Met Gly Leu 420 425 430
CTT ATG TAT TTG GGC AAA CCA AAG 1320
Leu Met Tyr Leu Gly Lys Pro Lys 435 440
(2) INFORMATION FOR SEQ ID N0:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1299 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1299
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 1..465
(D) OTHER INFORMATION: /product- "bFGF"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 466..540
(D) OTHER INFORMATION: /product- "(Ser4Gly)4linker"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 541..1299
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15
GGC AGC GGC GCC TTC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30
TAC TGC AAA AAC GGG GGC TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45 GTT GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu 50 55 60
CAA GCA GAA GAG AGA GGA Gπ GTG TCT ATC AAA GGA GTG TGT GCT AAC 240 Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn 65 70 75 80
CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA πA CTG GCT TCT AAA TGT 288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95
Gπ ACG GAT GAG TGT TTC TTT Tπ GAA CGA πG GAA TCT AAT AAC TAC 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110
AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA TTG AAA 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125
CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA 432 Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 130 135 140
GCT ATA cπ πT cπ CCA ATG TCT GCT AAG AGC GCC ATG GCC TCG TCG 480 Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Ala Ser Ser 145 150 155 160
TCG TCG GGC TCG TCG TCG TCG GGC TCG TCG TCG TCG GGC TCG TCG TCG 528 Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser 165 170 175
TCG GGC GCC ATG GTC ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC 576
Ser Gly Ala Met Val Thr Ser He Thr Leu Asp Leu Val Asn Pro Thr 180 185 190
GCG GGT CAA TAC TCA TCT TTT GTG GAT AAA ATC CGA AAC AAC GTA AAG 624
Ala Gly Gin Tyr Ser Ser Phe Val Asp Lys He Arg Asn Asn Val Lys 195 200 205
GAT CCA AAC CTG AAA TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA 672 Asp Pro Asn Leu Lys Tyr Gly Gly Thr Asp He Ala Val He Gly Pro 210 215 220
CCT TCT AAA GAA AAA TTC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA 720 Pro Ser Lys Glu Lys Phe Leu Arg He Asn Phe Gin Ser Ser Arg Gly 225 230 235 240
ACG GTC TCA CTT GGC CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT 768 Thr Val Ser Leu Gly Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr 245 250 255
CTT GCA ATG GAT AAC ACG AAT GTT AAT CGG GCA TAT TAC TTC AAA TCA 816 Leu Ala Met Asp Asn Thr Asn Val Asn Arg Ala Tyr Tyr Phe Lys Ser 260 265 270 GAA ATT ACT TCC GCC GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT 864 Glu He Thr Ser Ala Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr 275 280 285
GCA AAT CAG AAA GCT TTA GAA TAC ACA GAA GAT TAT CAG TCG ATC GAA 912 Ala Asn Gin Lys Ala Leu Glu Tyr Thr Glu Asp Tyr Gin Ser He Glu 290 295 300
AAG AAT GCC CAG ATA ACA CAG GGA GAT AAA AGT AGA AAA GAA CTC GGG 960 Lys Asn Ala Gin He Thr Gin Gly Asp Lys Ser Arg Lys Glu Leu Gly 305 310 315 320 πG GGG ATC GAC πA Cπ πG ACG πC ATG GAA GCA GTG AAC AAG AAG 1008 Leu Gly He Asp Leu Leu Leu Thr Phe Met Glu Ala Val Asn Lys Lys 325 330 335
GCA CGT GTG Gπ AAA AAC GAA GCT AGG TTT CTG CTT ATC GCT ATT CAA 1056 Ala Arg Val Val Lys Asn Glu Ala Arg Phe Leu Leu He Ala He Gin 340 345 350
ATG ACA GCT GAG GTA GCA CGA TTT AGG TAC ATT CAA AAC TTG GTA ACT 1104 Met Thr Ala Glu Val Ala Arg Phe Arg Tyr He Gin Asn Leu Val Thr 355 360 365
AAG AAC TTC CCC AAC AAG πC GAC TCG GAT AAC AAG GTG ATT CAA TTT 1152 Lys Asn Phe Pro Asn Lys Phe Asp Ser Asp Asn Lys Val He Gin Phe 370 375 380
GAA GTC AGC TGG CGT AAG ATT TCT ACG GCA ATA TAC GGG GAT GCC AAA 1200 Glu Val Ser Trp Arg Lys He Ser Thr Ala He Tyr Gly Asp Ala Lys 385 390 395 400
AAC GGC GTG TTT AAT AAA GAT TAT GAT TTC GGG πT GGA AAA GTG AGG 1248 Asn Gly Val Phe Asn Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg 405 410 415
CAG GTG AAG GAC TTG CAA ATG GGA CTC CTT ATG TAT πG GGC AAA CCA 1296 Gin Val Lys Asp Leu Gin Met Gly Leu Leu Met Tyr Leu Gly Lys Pro
420 425 430
AAG 1299
Lys
(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1269 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1269 (ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 1..465
(D) OTHER INFORMATION: /product- "bFGF"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 466..510
(D) OTHER INFORMATION: /product- "(Ser4Gly)2 linker"
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 511..1269
(D) OTHER INFORMATION: /product= "Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15
GGC AGC GGC GCC πC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30
TAC TGC AAA AAC GGG GGC TTC πC CTG CGC ATC CAC CCC GAC GGC CGA 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 35 40 45
GTT GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu 50 55 60
CAA GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC 240 Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn 65 70 75 80
CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT 288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95
GTT ACG GAT GAG TGT TTC πT TTT GAA CGA πG GAA TCT AAT AAC TAC 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110
AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA TTG AAA 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125
CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA 432 Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 130 135 140
GCT ATA CTT TTT Cπ CCA ATG TCT GCT AAG AGC GCC ATG GCC TCG TCG 480 Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Ala Ser Ser 145 150 155 160 TCG TCG GGC TCG TCG TCG TCG GGC GCC ATG GTC ACA TCA ATC ACA πA 528 Ser Ser Gly Ser Ser Ser Ser Gly Ala Met Val Thr Ser He Thr Leu 165 170 175
GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA TCT TTT GTG GAT AAA 576 Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser Ser Phe Val Asp Lys 180 185 190
ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG AAA TAC GGT GGT ACC GAC 624 He Arg Asn Asn Val Lys Asp Pro Asn Leu Lys Tyr Gly Gly Thr Asp 195 200 205
ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA TTC CTT AGA ATT AAT 672
He Ala Val He Gly Pro Pro Ser Lys Glu Lys Phe Leu Arg He Asn 210 215 220
TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC CTA AAA CGC GAT AAC 720
Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly Leu Lys Arg Asp Asn
225 230 235 240
TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC ACG AAT GTT AAT CGG 768
Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn Thr Asn Val Asn Arg 245 250 255
GCA TAT TAC TTC AAA TCA GAA ATT ACT TCC GCC GAG TTA ACC GCC CTT 816 Ala Tyr Tyr Phe Lys Ser Glu He Thr Ser Ala Glu Leu Thr Ala Leu 260 265 270
TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT TTA GAA TAC ACA GAA 864
Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala Leu Glu Tyr Thr Glu 275 280 285
GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA ACA CAG GGA GAT AAA 912
Asp Tyr Gin Ser He Glu Lys Asn Ala Gin He Thr Gin Gly Asp Lys 290 295 300
AGT AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA CTT TTG ACG TTC ATG 960
Ser Arg Lys Glu Leu Gly Leu Gly He Asp Leu Leu Leu Thr Phe Met
305 310 315 320
GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA AAC GAA GCT AGG TTT 1008
Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys Asn Glu Ala Arg Phe 325 330 335
CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA GCA CGA TTT AGG TAC 1056 Leu Leu He Ala He Gin Met Thr Ala Glu Val Ala Arg Phe Arg Tyr 340 345 350
ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC AAG TTC GAC TCG GAT 1104 He Gin Asn Leu Val Thr Lys Asn Phe Pro Asn Lys Phe Asp Ser Asp 355 360 365
AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT AAG ATT TCT ACG GCA 1152 Asn Lys Val He Gin Phe Glu Val Ser Trp Arg Lys He Ser Thr Ala 370 375 380 ATA TAC GGG GAT GCC AAA AAC GGC GTG Tπ AAT AAA GAT TAT GAT TTC 1200 He Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn Lys Asp Tyr Asp Phe 385 390 395 400
GGG TTT GGA AAA GTG AGG CAG GTG AAG GAC TTG CAA ATG GGA CTC CTT 1248 Gly Phe Gly Lys Val Arg Gin Val Lys Asp Leu Gin Met Gly Leu Leu 405 410 415
ATG TAT πG GGC AAA CCA AAG 1269
Met Tyr Leu Gly Lys Pro Lys 420
(2) INFORMATION FOR SEQ ID N0:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 765 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..762
(D) OTHER INFORMATION: /product- "Mammalian codon optimized saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
ATG GTG ACC TCC ATC ACC CTG GAC CTG GTG AAC CCC ACC GCC GGC CAG 48 Met Val Thr Ser He Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gin 1 5 10 15
TAC TCC TCC TTC GTG GAC AAG ATC CGC AAC AAC GTG AAG GAC CCC AAC 96 Tyr Ser Ser Phe Val Asp Lys He Arg Asn Asn Val Lys Asp Pro Asn 20 25 30
CTG AAG TAC GGC GGC ACC GAC ATC GCC GTG ATC GGC CCC CCC TCC AAG 144 Leu Lys Tyr Gly Gly Thr Asp He Ala Val He Gly Pro Pro Ser Lys 35 40 45
GAG AAG TTC CTG CGC ATC AAC TTC CAG TCC TCC CGC GGC ACC GTG TCC 192 Glu Lys Phe Leu Arg He Asn Phe Gin Ser Ser Arg Gly Thr Val Ser 50 55 60
CTG GGC CTG AAG CGC GAC AAC CTG TAC GTG GTG GCC TAC CTG GCC ATG 240 Leu Gly Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met 65 70 75 80
GAC AAC ACC AAC GTG AAC CGC GCC TAC TAC TTC AAG TCC GAG ATC ACC 288 Asp Asn Thr Asn Val Asn Arg Ala Tyr Tyr Phe Lys Ser Glu He Thr 85 90 95 TCC GCC GAG CTG ACC GCC CTG TTC CCT GAG GCC ACC ACC GCC AAC CAG 336
Ser Ala Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin 100 105 110
AAG GCC CTG GAG TAC ACC GAG GAC TAC CAG TCC ATC GAG AAG AAC GCC 384
Lys Ala Leu Glu Tyr Thr Glu Asp Tyr Gin Ser He Glu Lys Asn Ala 115 120 125
CAG ATC ACC CAG GGC GAC AAG TCC CGC AAG GAG CTC GGG CTG GGC ATC 432 Gin He Thr Gin Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly He 130 135 140
GAC CTG CTG CTG ACC TTC ATG GAG GCC GTG AAC AAG AAG GCC CGC GTG 480 Asp Leu Leu Leu Thr Phe Met Glu Ala Val Asn Lys Lys Ala Arg Val 145 150 155 160
GTG AAG AAC GAG GCC CGC TTC CTG CTG ATC GCC ATC CAG ATG ACC GCC 528 Val Lys Asn Glu Ala Arg Phe Leu Leu He Ala He Gin Met Thr Ala 165 170 175
GAG GTG GCC CGC πC CGC TAC ATC CAG AAC CTG GTG ACC AAG AAC TTC 576 Glu Val Ala Arg Phe Arg Tyr He Gin Asn Leu Val Thr Lys Asn Phe 180 185 190
CCC AAC AAG TTC GAC TCC GAC AAC AAG GTG ATC CAG TTC GAG GTC AGC 624 Pro Asn Lys Phe Asp Ser Asp Asn Lys Val He Gin Phe Glu Val Ser 195 200 205
TGG CGC AAG ATC TCC ACC GCC ATC TAC GGC GAC GCC AAG AAC GGC GTG 672 Trp Arg Lys He Ser Thr Ala He Tyr Gly Asp Ala Lys Asn Gly Val 210 215 220 πC AAC AAG GAC TAC GAC TTC GGC TTC GGC AAG GTG CGC CAG GTG AAG 720 Phe Asn Lys Asp Tyr Asp Phe Gly Phe Gly Lys Val Arg Gin Val Lys 225 230 235 240
GAC CTG CAG ATG GGC CTG CTG ATG TAC CTG GGC AAG CCC AAG 762
Asp Leu Gin Met Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys
245 250
TAG 765
(2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1233 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (i x) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..1230
(D) OTHER INFORMATION: /product- "E. coli codon optimized FGF-SAP"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
ATG GCA GCG GGT TCT ATT ACT ACC CTG CCG GCG CTG CCG GAG GAC GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 255 260 265 270
GGT TCT GGC GCT πC CCA CCG GGC CAC Tπ AAG GAC CCG AAA CGC CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 275 280 285
TAT TGC AAA AAC GGT GGT TTT TTC CTG CGT ATC CAC CCG GAT GGC CGC 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg He His Pro Asp Gly Arg 290 295 300
GTC GAT GGC GTC CGC GAA AAG TCT GAT CCG CAC ATC AAA CTG CAA TTG 192 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu 305 310 315
CAA GCA GAG GAA CGC GGT GTT GTA AGC ATC AAG GGC GTT TGC GCG AAT 240 Gin Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn 320 325 330
CGT TAC CTG GCG ATG AAA GAG GAT GGC CGC CTG CTG GCC TCC AAG TGT 288 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 335 340 345 350
GTA ACC GAT GAA TGC πC TTC TTT GAA CGT CTG GAG TCG AAC AAT TAT 336 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 355 360 365
AAC ACC TAT CGT AGC CGT AAG TAC ACC TCG TGG TAC GTA GCA TTG AAA 384 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 370 375 380
CGC ACC GGT CAG TAC AAA CTG GGT TCG AAG ACG GGT CCA GGT CAG AAA 432 Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys 385 390 395
GCA ATT CTG TTC CTG CCA ATG TCG GCC AAA TCG GCC ATG GTC ACT TCT 480 Ala He Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Val Thr Ser 400 405 410
ATC ACG CTG GAT CTG GTC AAC CCG ACC GCT GGT CAG TAC AGC TCG TTT 528 He Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser Ser Phe 415 420 425 430
GTC GAT AAG ATT CGT AAT AAT GTG AAA GAT CCG AAT TTA AAA TAC GGT 576 Val Asp Lys He Arg Asn Asn Val Lys Asp Pro Asn Leu Lys Tyr Gly 435 440 445 GGC ACG GAT ATT GCA GTG ATT GGC CCG CCG TCT AAG GAA AAG TTC πG 624 Gly Thr Asp He Ala Val He Gly Pro Pro Ser Lys Glu Lys Phe Leu 450 455 460
CGT Aπ AAC TTT CAA AGC TCT CGC GGC ACT GTG TCT CTG GGC TTA AAA 672 Arg He Asn Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly Leu Lys 465 470 475
CGC GAT AAT TTG TAC GTT GTA GCG TAC CTG GCG ATG GAT AAT ACC AAT 720 Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn Thr Asn 480 485 490
GTA AAC CGT GCT TAC TAT TTC AAA AGC GAA Aπ ACC TCT GCT GAA CTG 768 Val Asn Arg Ala Tyr Tyr Phe Lys Ser Glu He Thr Ser Ala Glu Leu 495 500 505 510
ACT GCA TTA TTC CCG GAA GCG ACT ACT GCC AAT CAG AAA GCC CTG GAA 816 Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala Leu Glu 515 520 525
TAT ACC GAA GAT TAT CAG TCG ATT GAA AAA AAC GCG CAA ATT ACC CAG 864 Tyr Thr Glu Asp Tyr Gin Ser He Glu Lys Asn Ala Gin He Thr Gin 530 535 540
GGC GAC AAA TCG CGC AAA GAG TTG GGT CTG GGT ATT GAC CTG CTG CTG 912 Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly He Asp Leu Leu Leu 545 550 555
ACG TTT ATG GAG GCG GTC AAC AAA AAA GCT CGT GTA GTG AAA AAC GAA 960 Thr Phe Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys Asn Glu 560 565 570
GCT CGC TTT CTG CTG ATC GCT Aπ CAA ATG ACT GCT GAA GTT GCT CGT 1008 Ala Arg Phe Leu Leu He Ala He Gin Met Thr Ala Glu Val Ala Arg 575 580 585 590
TTC CGT TAC ATT CAG AAC TTG GTT ACT AAG AAC TTT CCG AAC AAA TTC 1056 Phe Arg Tyr He Gin Asn Leu Val Thr Lys Asn Phe Pro Asn Lys Phe 595 600 605
GAC TCC GAT AAT AAG GTT ATT CAG TTC GAA GTG AGC TGG CGC AAG ATT 1104 Asp Ser Asp Asn Lys Val He Gin Phe Glu Val Ser Trp Arg Lys He 610 615 620
TCG ACG GCT ATT TAT GGC GAT GCC AAA AAC GGC GTA TTT AAC AAA GAT 1152 Ser Thr Ala He Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn Lys Asp 625 630 635
TAT GAC TTC GGT TTT GGC AAG GTT CGT CAG GTG AAA GAT TTG CAG ATG 1200 Tyr Asp Phe Gly Phe Gly Lys Val Arg Gin Val Lys Asp Leu Gin Met 640 645 650
GGT CTG CTG ATG TAC TTG GGC AAG CCG AAA TAA 1233 Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys 655 660 (2) INFORMATION FOR SEQ ID N0:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..462
(D) OTHER INFORMATION: /product- "FGF 2 - He Mutation at Residue 116"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
ATG GCA GCA GGA TCA ATA ACA ACA πA CCC GCC TTG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 415 420 425
GGC AGC GGC GCC πC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 430 435 440
TAC TGC AAA AAC GGG GGC TTC TTC CTG CGC CAC CCC GAC GGC CGA GTT 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg His Pro Asp Gly Arg Val 445 450 455
GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT CAA 192 Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu Gin 460 465 470
GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC CGT 240 Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn Arg 475 480 485 490
TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT GTT 288 Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val 495 500 505
ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC AAT 336 Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn 510 515 520
ACT TAC ATA TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA TTG AAA CGA 384 Thr Tyr He Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys Arg 525 530 535
ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA GCT 432 Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys Ala 540 545 550 ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC TAA 465 He Leu Phe Leu Pro Met Ser Ala Lys Ser 555 560
(2) INFORMATION FOR SEQ ID N0:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..462
(D) OTHER INFORMATION: /product- "FGF 2 - Glu Mutation at Residue 119"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 155 160 165 170
GGC AGC GGC GCC πC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 175 180 185
TAC TGC AAA AAC GGG GGC TTC πC CTG CGC CAC CCC GAC GGC CGA GTT 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg His Pro Asp Gly Arg Val 190 195 200
GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA Cπ CAA 192 Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu Gin 205 210 215
GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC CGT 240 Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn Arg 220 225 230
TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT GTT 288 Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val 235 240 245 250
ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC AAT 336 Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn 255 260 265
ACT TAC CGG TCA AGG GAA TAC ACC AGT TGG TAT GTG GCA TTG AAA CGA 384 Thr Tyr Arg Ser Arg Glu Tyr Thr Ser Trp Tyr Val Ala Leu Lys Arg 270 275 280 ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA GCT 432 Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys Ala 285 290 295
ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC TAA 465 He Leu Phe Leu Pro Met Ser Ala Lys Ser 300 305
(2) INFORMATION FOR SEQ ID N0:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..462
(D) OTHER INFORMATION: /product- "FGF 2 - Ala Mutation at Residue 120"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:83:
ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC 48 Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 155 160 165 170
GGC AGC GGC GCC TTC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 175 180 185
TAC TGC AAA AAC GGG GGC TTC TTC CTG CGC CAC CCC GAC GGC CGA GTT 144 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg His Pro Asp Gly Arg Val 190 195 200
GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT CAA 192 Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu Gin 205 210 215
GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC CGT 240 Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn Arg 220 225 230
TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT GTT 288 Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val 235 240 245 250
ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC AAT 336 Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn 255 260 265 ACT TAC CGG TCA AGG AAA GCA ACC AGT TGG TAT GTG GCA TTG AAA CGA 384 Thr Tyr Arg Ser Arg Lys Ala Thr Ser Trp Tyr Val Ala Leu Lys Arg 270 275 280
ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA GCT 432 Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys Ala 285 290 295
ATA CTT πT CTT CCA ATG TCT GCT AAG AGC TAA 465 He Leu Phe Leu Pro Met Ser Ala Lys Ser 300 305
(2) INFORMATION FOR SEQ ID N0:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 465 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..462
(D) OTHER INFORMATION: /product- "FGF 2 - Trp Mutation at Residue 123"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:84:
ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC 48
Met Ala Ala Gly Ser He Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 155 160 165 170
GGC AGC GGC GCC πC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG 96
Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 175 180 185
TAC TGC AAA AAC GGG GGC TTC TTC CTG CGC CAC CCC GAC GGC CGA GTT 144
Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg His Pro Asp Gly Arg Val 190 195 200
GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTT CAA CTT CAA 192
Asp Gly Val Arg Glu Lys Ser Asp Pro His He Lys Leu Gin Leu Gin
205 210 215
GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC CGT 240
Ala Glu Glu Arg Gly Val Val Ser He Lys Gly Val Cys Ala Asn Arg 220 225 230
TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT GTT 288
Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val 235 240 245 250 ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC AAT 336 Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn 255 260 265
ACT TAC CGG TCA AGG AAA TAC ACC AGT GCA TAT GTG GCA TTG AAA CGA 384 Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Ala Tyr Val Ala Leu Lys Arg 270 275 280
ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA GCT 432 Thr Gly Gin Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gin Lys Ala 285 290 295
ATA Cπ TTT CTT CCA ATG TCT GCT AAG AGC TAA 465 He Leu Phe Leu Pro Met Ser Ala Lys Ser 300 305
(2) INFORMATION FOR SEQ ID NO 85
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 60 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for Protamine"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 85 TACATGCCAT GGCCAGGTAC AGATGCTGTC GCAGCCAGAG CCGGAGCAGA TATTACCGCC 60
(2) INFORMATION FOR SEQ ID NO 86
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 60 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- 'Primer for Protamine"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 86 GCAGCTCCGC CTCCTTCGTC TGCGACTTCT TTGTCTCTGG CGGTAATATC TGCTCCGGCT 60 (2) INFORMATION FOR SEQ ID NO 87
(ι) SEQUENCE CHARACTERISTICS
(A) LENGTH 60 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for Protamine"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 87 GACGAAGGAG GCGGAGCTGC CAGACACGGA GGAGAGCCAT GAGGTGCTGC CGCCCCAGGT 60
(2) INFORMATION FOR SEQ ID NO 88
(ι) SEQUENCE CHARACTERISTICS
(A) LENGTH 59 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for Protamine"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 88 ATATATCCTA GGTTAGTGTC TTCTACATCT CGGTCTGTAC CTGGGGCGGC AGCACCTCA 59 (2) INFORMATION FOR SEQ ID NO 89
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 66 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 89 CGTATCAGGC GGCCGCCGCC ATGGTGACCT CCATCACCCT GGACCTGGTG AACCCCACCG 60 CCGGCC 66 (2) INFORMATION FOR SEQ ID N0:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:90: πGGGGTCCT TCACGπGTT GCGGATCTTG TCCACGAAGG AGGAGTACTG GCCGGCGGTG 60 GGGTTCACC 69
(2) INFORMATION FOR SEQ ID N0:91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: /note- "Primer for Mammalian Codon Preferred Saporin"
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: AACAACGTGA AGGACCCCAA CCTGAAGTAC GGCGGCACCG ACATCGCCGT GATCGGCCCC 60 CCCTC 65
(2) INFORMATION FOR SEQ ID N0:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: /note- "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID N0:92: GTGCCGCGGG AGGACTGGAA GπGATGCGC AGGAACTTCT CCTTGGAGGG GGGGCCGATC 60 ACGGC 65
(2) INFORMATION FOR SEQ ID N0:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: CTCCCGCGGC ACCGTGTCCC TGGGCCTGAA GCGCGACAAC CTGTACGTGG TGGCCTACCT 60 GGCCATGGAC AACAC 75
(2) INFORMATION FOR SEQ ID N0:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 77 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:94: GCGGTCAGCT CGGCGGAGGT GATCTCGGAC TTGAAGTAGT AGGCGCGGTT CACGTTGGTG 60 TTGTCCATGG CCAGGTA 77
(2) INFORMATION FOR SEQ ID N0:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 78 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ix) FEATURE:
(D) OTHER INFORMATION: /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:95: GCCGAGCTGA CCGCCCTGTT CCCTGAGGCC ACCACCGCCA ACCAGAAGGC CCTGGAGTAC 60 ACCGAGGACT ACCAGTCC 78
(2) INFORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 76 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: AGCCCGAGCT CCTTGCGGGA CTTGTCGCCC TGGGTGATCT GGGCGTTCTT CTCGATGGAC 60 TGGTAGTCCT CGGTGT 76
(2) INFORMATION FOR SEQ ID NO:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 74 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(D) OTHER INFORMATION: /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: TATAGAATTC CTCGGGCTGG GCATCGACCT GCTGCTGACC TTCATGGAGG CCGTGAACAA 60 GAAGGCCCGC GTGG 74 (2) INFORMATION FOR SEQ ID NO 98
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 68 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 98 CGGCGGTCAT CTGGATGGCG ATCAGCAGGA AGCGGGCCTC GTTCTTCACC ACGCGGGCCT 60 TCTTGTTC 68
(2) INFORMATION FOR SEQ ID NO 99
(ι) SEQUENCE CHARACTERISTICS
(A) LENGTH 70 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 99 CGCCATCCAG ATGACCGCCG AGGTGGCCCG CTTCCGCTAC ATCCAGAACC TGGTGACCAA 60 GAACTTCCCC 70
(2) INFORMATION FOR SEQ ID NO 100
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 76 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for Mammalian Codon Preferred Saporin' (xi) SEQUENCE DESCRIPTION SEQ ID NO 100 GGCGGATCCC AGCTGACCTC GAACTGGATC ACCTTGTTGT CGGAGTCGAA CTTGπGGGG 60 AAGTTCTTGG TCACCA 76
(2) INFORMATION FOR SEQ ID NO 101
(ι) SEQUENCE CHARACTERISTICS
(A) LENGTH 61 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 101 CCGGGATCCG TCAGCTGGCG CAAGATCTCC ACCGCCATCT ACGGCGACGC CAAGAACGGC 60 G 61
(2) INFORMATION FOR SEQ ID NO 102
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 64 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 102 GCACCTTGCC GAAGCCGAAG TCGTAGTCCT TGTTGAACAC GCCGTTCTTG GCGTCGCCGT 60 AGAT 64
(2) INFORMATION FOR SEQ ID NO 103
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 58 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear (ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 103 πCGGCTTCG GCAAGGTGCG CCAGGTGAAG GACCTGCAGA TGGGCCTGCT GATGTACC 58 (2) INFORMATION FOR SEQ ID NO 104
(ι) SEQUENCE CHARACTERISTICS
(A) LENGTH 52 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for Mammalian Codon Preferred Saporin"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 104 TGAACGTGGC GGCCGCCTAC TTGGGCTTGC CCAGGTACAT CAGCAGGCCC AT 52
(2) INFORMATION FOR SEQ ID NO 105
(ι) SEQUENCE CHARACTERISTICS
(A) LENGTH 30 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ix) FEATURE
(D) OTHER INFORMATION /note- "Primer for SAP-6"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 105 CATATGTGTG TCACATCAAT CACATTAGAT 30
(2) INFORMATION FOR SEQ ID NO 106
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 21 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear (ix) FEATURE:
(D) OTHER INFORMATION: /note- "Primer for SAP-6"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:106: CAGGTπGGA TCCTTTACGT T 21

Claims

Claims
1. A pharmaceutical composition having the formula: receptor-binding internalized Uganda — nucleic acid binding domain — cytocide- encoding agent, wherein: receptor-binding internalized ligand is a polypeptide reactive with a cell surface receptor; nucleic acid binding domain binds to a nucleic acid, the domain being conjugated or fused to the receptor-binding internalized ligand; cytocide-encoding agent is a nucleic acid molecule encoding a cytocide, the agent being bound to the nucleic acid binding domain; and wherein the receptor-binding internalized ligands — nucleic acid binding domain — cytocide-encoding agent binds to the cell surface receptor and internalizes the cytocide-encoding agent in cells bearing the receptor.
2. A pharmaceutical composition having the formula: receptor-binding internalized ligand — nucleic acid binding domain — prodrug- encoding agent, wherein: receptor-binding internalized ligand is a polypeptide reactive with a cell surface receptor; nucleic acid binding domain binds to a nucleic acid, the domain being conjugated or fused to the receptor-binding internalized ligand; prodrug-encoding agent is a nucleic acid molecule encoding a prodrug, the agent being bound to the nucleic acid binding domain; and wherein the receptor-binding internalized ligand — nucleic acid binding domain — prodrug-encoding agent binds to the cell surface receptor and internalizes the cytocide- encoding agent in cells bearing the receptor.
3. The composition of either of claims 1 or 2 wherein the receptor- binding internalized ligand is a polypeptide reactive with an FGF receptor.
4. The composition of either of claims 1 or 2 wherein the receptor- binding internalized ligand is selected from the group consisting of a polypeptide reactive with a VEGF receptor and a polypeptide reactive with an HBEGF receptor.
5. The composition of either of claims 1 or 2 wherein the receptor- binding internalized ligand is a cytokine.
6. The composition of claim 1 wherein the cytocide-encoding agent encodes a protein that inhibits protein synthesis.
7. The composition of claim 6 wherein the protein is a ribosome inactivating protein.
8. The composition of claim 7 wherein the ribosome inactivating protein is saporin.
The composition of claim 7 wherein the ribosome inactivating protein is gelonin.
10. The composition of claim 6 wherein the protein inhibits elongation factor 2.
11. The composition of claim 10 wherein the protein is diphtheria toxin.
12. The composition of claim 2 wherein the prodrug-encoding agent encodes HSV-thymidine kinase.
13. The composition of either of claims 1 or 2 wherein the growth factor is a polypeptide reactive with the FGF receptor and the nucleic acid binding domain is poly-L- lysine.
14. The composition of either of claims 1 or 2 wherein the nucleic acid binding domain is selected from the group consisting of helix-turn-helix motif proteins, homeodomain proteins, zinc finger motif proteins, steroid receptor proteins, leucine zipper motif proteins, helix-loop-helix motif proteins, and β-sheet motif proteins.
15. The composition of either of claims 1 or 2 wherein the nucleic acid binding domain is selected from the group consisting of AP-1, Sp-1, rev, GCN4, λcro, λcl, TFIIA, myoD, retinoic acid receptor, glucocosteroid receptor, SV40 large T antigen, and GAL4.
16. The composition of either of claims 1 or 2 wherein the nucleic acid binding domain is selected from the group consisting of poly-L-lysine, protamine, histone and spermine.
17. The composition of claim 1 wherein the nucleic acid binding domain binds a DNA molecule that encodes a ribosome inactivating protein.
18. The composition of claim 1 wherein the nucleic acid binding domain binds the coding region of saporin DNA.
19. The composition of claim 1 wherein the cytocide-encoding agent further comprises a tissue-specific promoter.
20. The composition of claim 2 wherein the prodrug-encoding agent further comprises a tissue-specific promoter.
21. The composition of either of claims 19 or 20 wherein the tissue- specific promoter is selected from the group consisting of alpha-crystalline, tyrosinase, α-fetoprotein, prostate specific antigen, CEA, α-actin, VEGF receptor, erbB-2, C-myc, cyclin D, FGF receptor and gamma-crystalline promoter.
22. The composition of any one of claims 1-21, further comprising at least one linker that increases the serum stability or intracellular availability of the nucleic acid binding domain, the addition of said linker(s) resulting in the formula: receptor-binding internalized ligand — (L)q — ucleic acid binding domain- cytocide encoding agent or the formula: receptor-binding internalized ligand — (L)q — nucleic acid binding domain- prodrug encoding agent wherein:
L is at least one linker; and q is 1 or more, such that the conjugate retains the ability to bind to a cell surface receptor and internalize the cytocide-encoding agent, and wherein the cytocide- encoding agent is bound to the nucleic acid binding domain.
23. The composition of claim 22 wherein the linker increases the flexibility of the conjugate.
24. The composition of claim 23 wherein the linker is selected from the group consisting of (GlymSerp)n, (SerrnGlyp)n and (AlaAlaProAla)π in which n is 1 to 6, m is 1 to 6 and p is 1 to 4.
25. The composition of claim 24 wherein m is 4, p is 1 and n is 2 to 4.
26. The composition of claim 22 wherein the linker is a disulfide bond.
27. A therapeutically effective amount of a pharmaceutical composition according to any one of claims 1 , 2 or 22, for use in the manufacture of a medicament for preventing excessive cell proliferation in the eye, comprising contacting the eye with a cell proliferation-inhibiting amount, wherein: the inhibited cells are epithelial cells, endothelial cells, fibroblast cells or keratocytes; and the excessive amount is an amount greater than that required to heal the surgical wound.
28. A therapeutically effective amount of a pharmaceutical composition according to any one of claims 1, 2 or 22, for use in the manufacture of a medicament for treating cancer, comprising contacting the cancer cells with an amount of the composition sufficient for inhibiting proliferation of the cancer cells.
29. A therapeutically effective amount of a pharmaceutical composition according to any one of claims 1 , 2 or 22, for use in the manufacture of a medicament for treating smooth muscle cell hyperplasia, comprising contacting the smooth muscle cells with an amount of the composition sufficient for inhibiting hyperplasia of smooth muscle cells.
30. A pharmaceutical composition having the formula: receptor-binding internalized ligand-cytocide-encoding agent nucleic acid binding domain, wherein: receptor-binding internalized ligand is a polypeptide reactive with a cell surface receptor; cytocide-encoding agent is a nucleic acid molecule encoding a cytocide, the agent being conjugated to the receptor-binding internalized ligand; and wherein the cytocide- encoding agent is bound to the nucleic acid binding domain; and wherein the receptor- binding internalized ligand-cytocide-encoding agent nucleic acid binding domain binds to the cell surface receptor and is internalized in cells bearing the receptor.
31. The composition of claim 30 wherein the nucleic acid binding domain is poly-L-lysine.
32. The composition of claim 30 wherein the receptor binding internalized ligand is a polypeptide reactive with an FGF receptor.
PCT/US1996/007164 1995-05-16 1996-05-16 Compositions containing nucleic acids and ligands for therapeutic treatment WO1996036362A1 (en)

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EP1188448A3 (en) * 1994-03-15 2002-04-17 Prizm Pharmaceuticals, Inc. Heparin-binding growth factors for gene therapy and anterior eye disorders
JP2002534119A (en) * 1999-01-14 2002-10-15 ボルダー バイオテクノロジー， インコーポレイテッド Method for producing a protein having a free cysteine residue
US6537813B1 (en) 1998-02-13 2003-03-25 Selective Genetics, Inc. Concurrent flow mixing methods and apparatuses for the preparation of gene therapy vectors and compositions prepared thereby
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US9925243B2 (en) 2013-10-21 2018-03-27 Salk Institute For Biological Studies Chimeric fibroblast growth factor (FGF) 2/FGF1 peptides and methods of use
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US7959909B2 (en) 1997-07-14 2011-06-14 Bolder Biotechnology, Inc. Cysteine variants of interferon gamma
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US7795396B2 (en) 1997-07-14 2010-09-14 Bolder Biotechnology, Inc. COOH-terminally added cysteine variant of the beta interferon
US8748392B2 (en) 1997-07-14 2014-06-10 Bolder Biotechnology Inc. Methods of treatment using cysteine variants of interleukin-11
US8618256B2 (en) 1997-07-14 2013-12-31 Bolder Biotechnology Cysteine variants of interferon gamma
US7964184B2 (en) 1997-07-14 2011-06-21 Bolder Biotechnology, Inc. Cysteine variants of interferon-gamma
US10329337B2 (en) 1997-07-14 2019-06-25 Bolder Biotechnology, Inc. Method to increase the number of circulating platelets by administering PEGylated cysteine variants of IL-11
US7824669B2 (en) 1997-07-14 2010-11-02 Bolder Biotechnology, Inc. In vivo stimulation of peripheral blood progenitor cells by granulocyte-macrophage colony stimulating factor (GM-CSF) cysteine muteins and their PEGylated variants
US8859497B2 (en) 1997-07-14 2014-10-14 Bolder Biotechnology, Inc. Method of treatment using cysteine mutants of beta interferon
US6537813B1 (en) 1998-02-13 2003-03-25 Selective Genetics, Inc. Concurrent flow mixing methods and apparatuses for the preparation of gene therapy vectors and compositions prepared thereby
US6903077B1 (en) 1999-01-04 2005-06-07 University Of Vermont And State Agricultural College Methods and products for delivering nucleic acids
WO2000040723A2 (en) * 1999-01-04 2000-07-13 University Of Vermont And State Agricultural College Methods and products for delivering nucleic acids
WO2000040723A3 (en) * 1999-01-04 2000-11-02 Univ Vermont Methods and products for delivering nucleic acids
US8957023B2 (en) 1999-01-14 2015-02-17 Bolder Biotechnology Inc. Methods for making proteins containing free cysteine residues
JP2014064594A (en) * 1999-01-14 2014-04-17 Bolder Biotechnology Inc Methods for making proteins containing free cysteine residues
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