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WO1996008274A2 - Conjugues d'un facteur de croissance semblable au facteur de croissance epidermique de type hbgf, et d'agents a cible definie - Google Patents

Conjugues d'un facteur de croissance semblable au facteur de croissance epidermique de type hbgf, et d'agents a cible definie Download PDF

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Publication number
WO1996008274A2
WO1996008274A2 PCT/US1995/012205 US9512205W WO9608274A2 WO 1996008274 A2 WO1996008274 A2 WO 1996008274A2 US 9512205 W US9512205 W US 9512205W WO 9608274 A2 WO9608274 A2 WO 9608274A2
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Prior art keywords
hbegf
conjugate
dna
cells
polypeptide
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PCT/US1995/012205
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English (en)
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WO1996008274A3 (fr
Inventor
Barbara A. Sosnowski
Lois Ann Chandler
L. L. Houston
Michael P. Nova
John R. Mcdonald
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Prizm Pharmaceuticals, Inc.
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Priority to AU37244/95A priority Critical patent/AU3724495A/en
Publication of WO1996008274A2 publication Critical patent/WO1996008274A2/fr
Publication of WO1996008274A3 publication Critical patent/WO1996008274A3/fr

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    • 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
    • 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
    • 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 factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
    • 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
    • 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
    • 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
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • This invention is related to the preparation and use of heparin-binding epidermal growth factor-like growth factor (HBEGF) conjugated to a targeted agent, such as a cytotoxic protein or a nucleic acid.
  • HEGF heparin-binding epidermal growth factor-like growth factor
  • the conjugates are for use as anti-tumor agents, for the treatment of disorders involving pathophysiological proliferation of smooth muscle cells, such as restenosis, and to effect genetic therapy of cells that bear receptors for heparin-binding epidermal growth factor.
  • 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 retroviral 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 conjugates of heparin-binding epidermal-like growth factor-like growth factor (HBEGF) polypeptide or a portion thereof and a targeted agent.
  • HBEGF and targeted agent are conjugated through a linker.
  • the linker is selected from the group consisting of protease substrates, linkers that increase the flexibility of the conjugate, linkers that increase the solubility of the conjugate, photocleavable linkers and acid cleavable linkers.
  • the HBEGF polypeptide may be mammalian HBEGF or HBEGF that is modified by addition of a cysteine residue or replacement of a nonessential amino acid residue within about 20 amino acids of the N-terminus or C-terminus.
  • the targeted agent is cytotoxic, preferably a ribosome inactivating protein, and most preferably saporin.
  • Other cytotoxic agents include a nucleic acid.
  • the conjugate has the formula: targeted agent n - (L)q-HBEGF m or HBEGF m -(L)q targeted agent n , wherein n and m. which may be the same or different, are at least 1.
  • methods of treating HBEGF-mediated pathophysiological conditions comprising administering to the animal a therapeutically effective amount of a conjugate between HBEGF and a cytotoxic agent.
  • the condition is a dermatological disorder involving epidermal cells, a neoplastic disorder of epidermal or mesodermal cells, an ophthalmic disorder involving proliferation of epithelial cells, or a disorder characterized by proliferation of smooth muscle cells.
  • Methods are also provided to inhibit proliferation of cells bearing HBEGF receptors, comprising contacting the cells with an effective amount of a HBEGF targeted agent conjugate.
  • methods of effecting gene therapy wherein cells are contacted with a conjugate having a targeted agent which is a nucleic acid, and the conjugate includes a nuclear translocation sequence linked to the targeted nucleic acid or HBEGF.
  • DNA fragments, encoding a conjugate between a targeted agent and HBEGF are provided.
  • the DNA conjugate may additionally comprise a linker.
  • Plasmids, vectors, and host cells are also provided.
  • methods of producing a fusion protein of HBEGF and a targeted agent comprising (a) cultiuing ceils transformed with a plasmid containing a DNA fragment according to claim 21, under conditions whereby the DNA fragment is transcribed and translated; (b) lysing the cells in a buffer containing urea; (c) eluting the protein from a cation-exchange chromatography resin; (d) passing the protein over an anion-exchange chromatography resin; (e) eluting the protein from a cation-exchange chromatography resin; (f) eluting the protein from a hydrophobic interaction chromatography resin; and (g) recovering the protein from a size exclusion chromatography resin.
  • a size exclusion chromatography resin comprising
  • compositions comprising the HBEGF targeted agent conjugate and a physiological acceptable excipient are also provided.
  • binding measures the capacity of the HBEGF conjugate or HBEGF polypeptide to specifically bind to a HBEGF receptor (known as EGF receptor) on smooth muscle or epidermoid cells, such as A431 cells, using a procedure substantially as described in Moscatelli (1987) J. Cell Physiol. 737. 123-130. Briefly, cells are grown to subconfluence and incubated in appropriate buffer with detectably labeled, such as radioiodinatcd HBEGF polypeptide in the presence of various concentrations ⁇ f an HBEGF polypeptide of interest. Binding affinity is measured by counting the membrane fraction that is solubilized in a suitable buffer containing a detergent, such as in 0.5% Triton X-100 in PBS (pH 8.1).
  • a detergent such as in 0.5% Triton X-100 in PBS (pH 8.1).
  • biologically active or reference to the "biological activity of a cytotoxic conjugate of HBEGF,” such as a conjugate containing HBEGF and saporin refers to the ability of such polypeptide to enzymatically inhibit protein synthesis by inactivation of ribosomes either in vivo or in vitro or to inhibit the growth of or kill cells upon internalization of the saporin-containing polypeptide by the cells.
  • biological or cytotoxic activity may be assayed by any method known to those of skill in the art including, but not limited to, the 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. Particularly preferred. however, are assays that assess cytotoxicity in targeted cells.
  • 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, however, may be defined with reference to particular in vitro activities, as measured in a defined assay. Thus, for example, reference herein to the biological activity of HBEGF or fragment thereof refers to the ability of the HBEGF to bind to cells bearing HBEGF receptors and internalize a linked agent. Such activity is typically assessed in vitro by linking the HBEGF (or fragment) to a cytotoxic agent, such as saporin.
  • a cytotoxic agent such as saporin.
  • in vitro activity should be extrapolatable to in vivo activity.
  • in vivo activity may be assessed using recognized animal models, such as the mouse xenograft model for anti-tumor activity (see, e.g., Beitz et al. (1992) Cancer Research 52:227-230; Houghton et al. (1982) Cancer Res. 42:535-539; Bogden et al. (1981) Cancer (Philadelphia) 48:10-20; Hoogenhout et al. (1983) Int. J. Radial. Oncol, Biol. Phys. 9:871-879; Stastny et al. (1993) Cancer Res. 53:5740-5744).
  • a "conjugate” refers to a molecule that contains at least one HBEGF moiety and at least one targeted agent 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.
  • cytotoxic agent refers to a molecule capable of inhibiting cell function.
  • the agent may inhibit cell growth, differentiation or proliferation or may be toxic to cells.
  • This term includes agents that, when internalized by a cell, interfere with or detrimentally alter cellular metabolism or in any manner inhibit cell growth or proliferation.
  • the term includes agents whose toxic effects are mediated when transported into the cell and also those whose toxic effects are mediated at the cell surface.
  • 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.
  • 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 that binds to an HBEGF receptor and is internalized thereby and may be isolated from a human cell library using any of the preceding DNA fragments as a probe; and any DNA fragment that encodes any of the HBEGF polypeptides set forth in SEQ ID NOs. 2-5.
  • Such DNA sequences encoding HBEGF fragments are available from publicly accessible databases, such as: DNA July, 1993 release from DNASTAR, Inc. Madison. WI, and Genbank Accession Nos.
  • HBEGF polypeptides will, unless modified by replacement of degenerate codons, hybridize under conditions of at least low stringency to DNA encoding a native HBEGF (e.g., SEQ ID NO. 1).
  • any DNA fragment that may be produced from any of the preceding DNA fragments by substitution of degenerate codons is also 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.
  • fusion protein refers to a polypeptide that contains at least two components, such as a HBEGF polypeptide and a targeted agent, a HBEGF polypeptide and linker, or a HBEGF polypeptide, linker, and targeted agent, and is produced by expression of DNA in a host cell.
  • HBEGF heparin-binding epidermal growth factor-like growth factor
  • HBEGF heparin-binding epidermal growth factor-like growth factor
  • Native HBEGF has a heparin-binding domain.
  • HBEGF refers to polypeptides having amino acid sequences of a native HBEGF polypeptide, as well as HBEGF polypeptides modified by amino acid substitutions, deletions, insertions or additions in the native protein, but retains the ability to bind to a HBEGF receptor and to be internalized in a cell bearing such receptor.
  • HBEGF polypeptides include, but are not limited to human HBEGF (SEQ ID NO. 2), monkey HBEGF (SEQ ID NO. 4) and rat HBEGF (SEQ ID NO. 5).
  • Reference to HBEGFs is intended to encompass HBEGF polypeptides isolated from natural sources as well as those made synthetically, as by recombinant means or by chemical synthesis.
  • HBEGF also encompasses muteins of HBEGF that possess the ability to target a targeted agent, such as a cytotoxic agent, including but not limited to ribosome- inactivating proteins, such as saporin, light activated porphyrin, and antisense nucleic acids, to HBEGF-receptor expressing cells.
  • a targeted agent such as a cytotoxic agent, including but not limited to ribosome- inactivating proteins, such as saporin, light activated porphyrin, and antisense nucleic acids, to HBEGF-receptor expressing cells.
  • Muteins of HBEGF include, but are not limited to, those produced by replacing one or more of the cysteines with serine as described herein or those that have any other amino acids deleted or replaced, as long as the resulting protein has the ability to bind to HBEGF-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 (1 X SSPE or SSC, 0-. l% SDS, 50°C, medium stringency; 0.2 X SSPE or SSC, 0.1% SDS.
  • mature HBEGF refers to processed HBEGFs.
  • Various isoforms of mature HBEGF have variable N-termini, and include, but are not limited to, those having N-termini corresponding to amino acid positions 63, 73, 74, 77 and 82 of the precursor protein (see, e.g., SEQ ID Nos. 1, 2, see also SEQ ID Nos. 4 and 5).
  • a "portion of a HBEGF” refers to a fragment or piece of HBEGF that is sufficient to bind to a receptor to which native HBEGF binds and internalize a linked targeted agent.
  • 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, but are not limited to, 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.
  • HBEGF receptor refers to a receptor that reacts with members of the HBEGF family of proteins and that is able to transport HBEGF into the cell.
  • HBEGF polypeptides interact with the high affinity EGF receptors (EGF-R) on bovine aortic smooth muscle cells and A431 epidermoid carcinoma cells (see Higashiyama et al. (1991) Science 251:936-939; Higashiyama et al. (1992; J. Biol. Chem. 267:6205-6212).
  • EGF-receptors which are also HBEGF- Rs, include the EGF receptors described in U.S. Patent Nos.
  • nucleic acids refer to RNA or DNA that are intended as targeted agents, which include, but are not limited to, DNA encoding therapeutic proteins, fragments of DNA for co-suppression, DNA encoding cytotoxic proteins, antisense nucleic acids 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 and derivatives thereof.
  • Nucleic acids may be composed of the well-known deoxyribonucleotides or 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 or type I normal phosphodiester oligonucleotides
  • X 0
  • PO oligonucleotides or type I normal phosphodiester oligonucleotides
  • B is a nucleotide base
  • X is OEt in phosphotriester (type II)
  • X is Me in methylphosphonate (type III; referred to as MP oligos)
  • X is S in phosphorothioate (referred to as PS oligos;
  • a "therapeutic nucleic acid” describes any nucleic acids used in the context of invention H0 that modify gene transcription or translation. This term also includes nucleic acids that bind to sites on proteins and to receptors. 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 oligo nucleotides and small nucleotide molecules.
  • a therapeutic nucleic acid may serve as a replacement for a defective gene or encode a therapeutic product, such as TNF or a cytotoxic molecule, 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.
  • restenosis refers to 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. 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. 96/08274 PC17US95/12205
  • substantially homologous with reference to an HBEGF polypeptide means that the protein is more homologous (i.e., shares more amino acid residues in common) to any of the mature HBEGF polypeptides included in SEQ ID Nos. 1-6 than is TGF- ⁇ .
  • DNA With reference to DNA it means that the DNA encodes a substantially homologous protein, and, but for substitution of degenerate codons, hybridizes under conditions of at least low stringency to DNA encoding any of the mature HBEGFs included in the sequences set forth in SEQ ID Nos. 1-6.
  • substantially pure DNA refers to DNA fragments purified according to standard techniques employed by those skilled in the art (see, e.g., Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.).
  • a "targeted agent” is any agent that is intended for internalization by linkage to a targeting moiety, as defined herein, and that upon internalization in some manner alters or affects cellular metabolism, growth, activity, viability or other property or characteristic of the cell.
  • the targeted agents include proteins, polypeptides, organic molecules, drugs, nucleic acids and other such molecules.
  • to target a targeted agent means to direct it to a cell that expresses a selected receptor by linking the agent to a polypeptide reactive with a HBEGF receptor. Upon binding to the receptor the targeted agent or targeted agent linked to the HBEGF is internalized by the cell.
  • 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.
  • HBEGFs Heparin-binding EGF-like growth factors
  • EGF epidermal growth factor
  • HBEGFs elute from heparin-Sepharose columns at about 1.0 - 1.2 M NaCl and were first identified as a secreted product of cultured human monocytes, macrophages, and the macrophage-like U-937 cell line (see, e.g., Higashiyama et al. (1991) Science 25 :936-939; Besner et al. (1990) Cell Regul. 7:811-819).
  • HBEGFs also called "heparin-binding EGF-homologous mitogen (HB-EHM)" (see WO 92/06705), are a family of growth factors that have a broad spectrum of activities, including a mitogenic effect on a variety of cells, such as BALB/c 3T3 fibroblast cells and smooth muscle cells. HBEGFs, however, are not mitogenic for endothelial cells (Higashiyama et al.
  • HBEGFs are heterogeneous in structure and contain up to 86 amino acids, including two sites of O-linked glycosylation (Higashiyama et al.
  • HBEGF The carboxyl-terminal half of the secreted human HBEGF shares approximately 35% sequence identity with human EGF, and includes six cysteines spaced in the pattern characteristic of members of the EGF protein family. HBEGF interacts with the same high affinity receptors as EGF on bovine aortic smooth muscle cells and human A431 epidermoid carcinoma cells (see, e.g., Higashiyama (1991) Science 251:936-939). The amino-terminal portion of the mature factor, which includes stretches of hydrophilic residues, has no structural equivalent in EGF.
  • HBEGF heparin-binding residues of HBEGF reside primarily in a twenty one-amino acid stretch upstream of and slightly overlapping the EGF-like domain. HBEGF appears to be a more potent mitogen for smooth muscle cells than either EGF or TGF- ⁇ , which also binds to EGF receptors.
  • Mammalian HBEGFs are derived from a 208 amino acid precursor protein.
  • the human and monkey precursor proteins share 97% sequence identity, the rat and mouse precursors are 92% identical; and there is 80% sequence identity between primate and rodent HBEGF precursor proteins (see Abraham et al. (1993) Biochem. and Biophys. Res. Comm. 790: 125-133).
  • the mature HBEGF polypeptides are heterogenous and range from about 75-86 amino acids in length.
  • HBEGFs have a molecular weight of approximately 19-23 kD, and have an isoelectric point between about 7.2-7.8.
  • HBEGFs are mediated at least in part by receptor tyrosine kinases on the cell surface membranes of HBEGF-responsive cells (see, e.g., U.S. Patent Nos. 5,183,884 and 5,218,090; and Ullrich et al. (1984) Nature 309:418-425, which are incorporated herein by reference).
  • the EGF receptor proteins which are single chain polypeptides with molecular weights of approximately 170 kD, depending on cell type, constitute a family of structurally related EGF receptors.
  • Cells that express the EGF receptors include, for example, smooth muscle cells, fibroblasts, keratinocytes, and numerous human cancer cell lines, such as the: A431 (epidermoid); KB3-1
  • MCF-7 (breast); MDA-MB-468 (breast); NCI 417D (lung); MG63 (osteosarcoma); U-251 (glioblastoma); D-54MB (glioma); and SW-13 (adrenal).
  • HBEGFs also bind to the heparan sulfate proteoglycans, which appear to internalize bound moieties via the endocytic pathway and contribute to internalization of HBEGFs.
  • polypeptides that are reactive with a HBEGF receptor include any molecule that (1) includes a receptor binding domain that is homologous to EGF and that is substantially homologous (more homologous than TGF- ⁇ ) to such domains in the mature HBEGFs having amino acid sequences set forth in
  • the polypeptides that are reactive with a HBEGF receptor include members of the HBEGF family of polypeptides, muteins of these polypeptides, chimeric or hybrid molecules that contain portions of any of these family members, and any portion thereof that binds to HBEGF receptors and internalizes a linked agent.
  • HBEGF for use herein also includes any fragment of a HBEGF polypeptide that retains the ability to bind to a HBEGF receptor and to be internalized by a cell bearing such receptors.
  • Mature human HBEGF as isolated has heterogenous amino acid lengths ranging from 75-86 (Higashiyama et al. (1992) Science 251:936-939).
  • various isoforms of mature HBEGF that have variable N-termini, and include, but are not limited to, those having N-termini corresponding to amino acid positions 63, 73. 74. 77 and 82 of the precursor protein (see, e.g., SEQ ID Nos. 1 and 2, see, also SEQ ID No. 3, for the presently preferred form).
  • a preferred HBEGF for use herein is the 77 amino acid form of human HBEGF beginning at amino acid 73 of the precursor protein (SEQ ID No. 3, which corresponds to amino acids 73-149 of SEQ ID NOs. 1 and 2; see 96/08274 PC17US95/12205
  • HBEGF polypeptides include SEQ ID NOs. 2-5, are particularly preferred. 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. All of the HBEGF polypeptides induce mitogenic activity in a wide variety of cells, and this activity is mediated by binding to an HBEGF cell surface receptor followed by internalization. Binding to a HBEGF receptor followed by internalization are the activities required for an HBEGF polypeptide to be suitable for use herein; mitogenic activity is not required.
  • a test for binding and internalization activity is the ability of the HBEGF-toxin conjugates to kill EGF-receptor containing cells.
  • An exemplary method for testing for such cytopathic activity is the Cell Proliferation Cytotoxicity Assay described in Example 4. Any HBEGF polypeptide that possesses such ability is intended for use herein. 2. Modifications of HBEGF If it is necessary or desired, the heterogeneity of preparations of HBEGF polypeptide-containing chemical conjugates can be reduced by modifying the HBEGF polypeptide by deleting or replacing a site(s) (that are non-essential for binding and internalization) on the HBEGF that cause the heterogeneity and/or by modifying the targeted agent.
  • 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 HBEGF polypeptide, but that are not required for binding to HBEGF receptors and internalization.
  • Such cysteine residues do not include any cysteine residue that are required for proper folding of the HBEGF polypeptide, or for retention of the ability to bind to a HBEGF receptor and internalize.
  • 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 HBEGF is conjugated with a single species of targeted agent, such as a RIP, antisense nucleic acid or therapeutic nucleic acid.
  • each cysteine residue may be systematically replaced with a conservative amino acid change or deleted.
  • the resulting mutein is tested for the requisite biological activity, the ability to bind to HBEGF receptors and internalize linked targeted moieties. 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. In this manner the minimum number and identity of the cysteines needed to retain the ability to bind to a HBEGF receptor and internalize may be determined.
  • the HBEGF polypeptide may also be modified by addition of one or more cysteine residues at or near the C- or N-terminus, preferably the N-terminus, in order to render it more amenable to chemical conjugation by providing a readily available non-essential cysteine residue.
  • HBEGF is modified herein by addition of Cys residues at or near the N-terminus in order to render them more amenable for chemical conjugation.
  • Any HBEGF may be modified for use herein by replacement of one or more cysteine residues that are not required for binding to a HBEGF receptor and internalization of the targeted agent. These modified forms of HBEGF are particularly suitable for chemical conjugation to linkers and or targeted agents.
  • Mutation may be effected 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 nucleic acid amplification splicing by overlap extension (SOE).
  • Site-specific 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. (1987) Meth. Enzymol. 15:3).
  • site-directed mutagenesis is performed by preparing a single-stranded vector that encodes the protein of interest (i.e.. a member of the HBEGF 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 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.
  • 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.
  • Suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule.
  • Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. co., p.224).
  • Such substitutions are preferably made in accordance with those set forth in TABLE 1 as follows:
  • HBEGF polypeptides may be isolated by methods known to those of skill in the art or may be prepared by expression of DNA encoding a HBEGF polypeptide (see, e.g., International Application WO/92/06705 (and the corresponding U.S. patent application serial No. 07/598,082), and Abraham et al. (1993) Biochem. Biophy. Res. Comm. 790:125-133 and SEQ ID NOs. 1-5 herein).
  • B. Targeted agents 1. Cytotoxic agents
  • Cytotoxic agent refers to a molecule capable of inhibiting cell function. Cytotoxic agents include any agent that, upon internalization, by a eukaryotic cell, inhibits growth or proliferation of the cell, either by killing the cell or inhibiting a metabolic pathway, transcription, or translation such that cell proliferation slows or stops. Any agent that, when internalized inhibits or destroys cell growth, cell proliferation or other essential cell functions is suitable for use herein. Cytotoxic agents include ribosome inactivating proteins, small metabolic inhibitors, antisense nucleic acids, toxic drugs, such as anticancer agents, and small molecules, such as light activated porphyrins. Ribosome inactivating proteins, such as saporin, are the preferred cytotoxic protein agents for use herein and nucleic acids are the preferred non- peptide agents.
  • Such cytotoxic agents include, but are not limited to, saporin, the ricins, abrin and other RIPs, Pseudomonas exotoxin, inhibitors of DNA, RNA or protein synthesis, including antisense nucleic acids and other metabolic inhibitors that are known to those of skill in this art.
  • Saporin is preferred, but other suitable RIPs include, but are not limited to, ricin, ricin A chain, maize RIP, gelonin, diphtheria toxin and diphtheria toxin A chain (see, e.g., U.S. Patent No.
  • the selected cytotoxic agent is, if necessary, derivatized to produce a group reactive with a cysteine on the selected HBEGF. If derivatization results in a mixture of reactive species, a mono-derivatized form of the cytotoxic agent can be isolated and then conjugated to the selected HBEGF.
  • Ribosome-inactivating-proteins which include ricin, abrin and saporin, are plant proteins that catalytically inactivate eukaryotic ribosomes. RIPs inactivate ribosomes by interfering with the protein elongation step of protein synthesis. 9 / PC17US95/12205
  • the RIP saporin (hereinafter also referred to as SAP) has been shown to enzymatically inactivate 60S ribosomes by cleavage of the n-glycosidic bond of the adenine at position 4324 in the rat 28S ribosomal RNA (rRNA).
  • Some RIPs such as the toxins abrin and ricin, contain two constituent chains: a cell-binding chain that mediates binding to cell surface receptors and internalization of the molecule; and an enzymatically active chain responsible for protein synthesis inhibitory activity.
  • Such RIPs are type II RIPs.
  • Other RIPs, such as the saporins are single chains and are designated type I RIPs.
  • saporin is a type I RIP, it does not possess a cell-binding chain. Consequently, its toxicity to whole cells is much lower than the other toxins, such as ricin and abrin. When internalized by eukaryotic cells, however, its cytotoxicity is 100- to 1000-fold more potent than ricin A chain.
  • Saporin is preferred herein.
  • the saporin polypeptides include any of the isoforms of saporin that may be isolated from Saponaria officinalis or related species or modified form that retain cytotoxic activity. Such modified forms have amino acid substitutions, deletions, insertions or additions but 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. In particular, such modified saporin may be produced by modifying the DNA encoding the protein (see, e.g..).
  • any such protein, or portion thereof, that, when conjugated to HBEGF as described herein, 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 SAP used herein includes any protein that is isolated from natural sources or that is produced by recombinant expression (see, e.g., copending published International PCT Application WO 93/25688 (Serial No. PCT US93/05702), which is a continuation-in-part of United States Application Serial No. 07/901,718, filed June 16, 1992; see, also Example 1, below).
  • DNA molecules provided herein encode saporin that has substantially the same amino acid sequence and ribosome-inactivating activity as that of preferred 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. Internal. 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. 8-12).
  • SO-6 preferred saporin-6
  • 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, which corresponds to 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.
  • SO-1 and SO-3 Forms of saporin-type ribosome-inactivating proteins
  • SO-2 see, e.g., U.S. Application Serial No. 07/885,242, which corresponds to GB 2,216,891 ; see, also,
  • the saporin polypeptides exemplified herein include those having substantially the same amino acid sequence as those listed in SEQ ID NOS. 8-12.
  • the isolation and expression of the DNA encoding these proteins is described in the Examples.
  • the saporin polypeptides 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, filed on June 14, 1993, and United States Application Serial No. 07/901,718; see, also, copending U.S. Patent Application No. 07/885,242 filed May 20, 1992, and Italian Patent No. 1 ,231,914) by altering one or more amino acids or deleting or inserting one or more amino acids.
  • 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 obtain clones 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. L12243; PCT Application WO 92/03155; U.S. Patent No. 5,376,546; diphtheria toxin (Genbank Accession No.
  • KOI 722 trichosanthin (Genbank Accession No. M34858); tritin (Genbank Accession No. D13795); 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. KOI 397, M23348).
  • DNA encoding SAP or any cytotoxic agent may be used in the recombinant methods provided herein.
  • the DNA may be modified to include a cysteine codon.
  • the codon may be inserted into any locus that does not reduce or reduces by less than about one order of magnitude the cytotoxicity of the resulting protein. Such locus may be determined empirically by modifying the protein and testing it for cytotoxicity in an assay, such as a cell-free protein synthesis assay.
  • the preferred loci in SAP for insertion of the cysteine residue is at or near the N-terminus (within about 20 residues, preferably 10 residues, of the N-terminus).
  • Host organisms include those organisms in which recombinant production of heterologous proteins have been carried out and in which the cytotoxic agent, such as saporin is not toxic or of sufficiently low toxicity to permit expression before cell death.
  • cytotoxic agent such as saporin
  • Presently preferred host organisms are strains of bacteria.
  • Most preferred host organisms are strains of E. coli, particularly, BL21(DE3) cells (Novagen, Madison, WI).
  • the DNA encoding the cytotoxic agent such as saporin protein
  • the DNA encoding the cytotoxic agent is introduced into a plasmid in operative linkage to an appropriate promoter for expression of polypeptides in a selected host organism.
  • the presently preferred saporin proteins are saporin proteins that have been modified by addition of a Cys residue or replacement of a non-essential residue at or near the amino- or carboxyl terminus of the saporin with Cys.
  • Saporin such as that of SEQ ID NO. 8 has been modified by insertion of Met-Cys residue at the N-terminus and has also been modified by replacement of the He or Asn residue at positions 4 and 10, respectively (see Example 4).
  • the DNA fragment encoding the saporin may also include a protein secretion signal that functions in the selected host to direct the mature polypeptide into the periplasm or culture medium.
  • the resulting saporin protein can be purified by methods routinely used in the art, including, methods described hereinafter in the Examples.
  • suitable host cells preferably bacterial cells, and more preferably E. coli cells, as well as methods applicable for culturing said cells containing a gene encoding a heterologous protein, are generally known in the art. See, for example, Sambrook et al. (1989) Molecular Clotting. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • the DNA construct encoding the saporin protein is introduced into the host cell by any suitable means, including, but not limited to transformation employing plasmids, bacterial phage vectors, transfection, electroporation, lipofection, and the like.
  • the heterologous 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).
  • Positive transformants can be characterized by Southern blot analysis (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) for the site of DNA integration; Northern blots for inducible-promoter-responsive saporin gene expression; and product analysis for the presence of saporin-containing proteins in either the cytoplasm, periplasm, or the growth media.
  • the desired saporin-containing protein is produced by subjecting the host cell to conditions under which the promoter is induced, whereby the operatively linked DNA is transcribed.
  • such conditions are those that induce expression from the E. coli lac operon.
  • the plasmid containing the DNA encoding the saporin-containing protein also includes the lac operator (O) region within the promoter and may also include the lac I gene encoding the lac repressor protein (see, e.g., Muller-Hill et al. (1968) Proc. Natl Acad. Sci. USA 59:1259-12649).
  • the lac repressor represses the expression from the lac promoter until induced by the addition of IPTG in an amount sufficient to induce transcription of the DNA encoding the saporin- containing protein.
  • the expression of saporin in E. coli is, thus accomplished in a two-stage process.
  • a culture of transformed E. coli cells is grown under conditions in which the expression of the saporin-containing protein within the transforming plasmid, preferably encoding a saporin, such as described in Example 4, is repressed by virtue of the lac repressor.
  • cell density increases.
  • the second stage commences by addition of IPTG, which prevents binding of repressor to the operator thereby inducing the lac promoter and transcription of the saporin-encoding DNA.
  • the promoter is the T7 RNA polymerase promoter, which may be linked to the lac operator and the E. coli host strain includes DNA encoding T7 RNA polymerase operably linked to the lac operator and a promoter, preferably the lacUV5 promoter.
  • the presently preferred plasmid is pET 1 1a (Novagen, Madison, WI), which contains the T71ac promoter, T7 terminator, the inducible E. coli lac operator, and the lac repressor gene.
  • the plasmid pET 15b (Novagen, Madison, WI), which contains a His-Tag i M leader sequence (Seq. ID NO.
  • Transformed strains which are of the desired phenotype and genotype, are grown in fermentors by suitable methods well known in the art.
  • expression hosts are cultured in defined minimal medium lacking the inducing condition, preferably IPTG.
  • the inducer preferably IPTG
  • the inducer is added to the fermentation broth, thereby inducing expression of any DNA operatively linked to an IPTG-responsive promoter (a promoter region that contains lac operator). This last stage is the induction stage.
  • the resulting saporin-containing protein can be suitably isolated from the other fermentation products by methods routinely used in the art, e.g., using a suitable affinity column as described in the Examples; precipitation with ammonium sulfate; gel filtration; chromatography, preparative flat-bed iso-electric focusing; gel electrophoresis, high performance liquid chromatography (HPLC); and the like.
  • a method for isolating saporin is provided in Example 1 (see, also Lappi et al. ((1985) Biochem. Biophys. Res. Commun., 729:934-942).
  • the expressed saporin protein is isolated from either the cytoplasm, periplasm, or the cell culture medium (see, discussion below and see, e.g., Example 3 for preferred methods and saporin proteins).
  • Porphyrins are well known light activatable toxins that can be readily cross-linked to proteins (see, e.g., U.S. Patent No. 5,257,970; U.S. Patent No. 5,252,720; U.S. Patent No. 5,238,940; U.S. Patent No. 5,192,788; U.S. Patent No. 5,171,749; U.S. Patent No. 5,149,708; U.S. Patent No. 5,202,317; U.S. Patent No. 5,217,966; U.S. Patent No. 5,053,423; U.S. Patent No. 5,109,016; U.S. Patent No. 5,087,636; U.S. Patent No. 5,028,594; U.S. Patent No. 5,093,349; U.S. Patent No. 4,968,715; U.S. Patent No. 4,920,143 and International Application WO 93/02192).
  • Porphyrins are conjugated to proteins by direct, covalent bonds using. for example, a carbodiimide.
  • Linkage may be effected by treatment of HBEGF by l-ethyl-3-3-dimethylamino propyl) carbo diimide in the presence of a reaction medium such as DMSO.
  • a reaction medium such as DMSO.
  • the porphyrin HBEGF conjugates may be administered topically or systemically. Actuation of the porphyrin is by irradiating light chosen to match the maximum absorbance of the porphyrin-type photosensitizer.
  • the conjugates provided herein are also designed to deliver nucleic acids to targeted cells.
  • the nucleic acids include those intended to deliver a cytotoxic signal to a cell or to modify expression of genes and thereby effect genetic therapy.
  • Examples of nucleic acids include antisense RNA, DNA, ribozymes 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 intact genes or that encode proteins useful for gene therapy or for effecting cell cytotoxicity.
  • 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 the present invention.
  • Other enzymes may be used where the enzyme activates a compound with little or no cytotoxicity into a toxic product.
  • DNA (or RNA) that may be delivered to a cell to effect genetic therapy includes DNA that encodes tumor-specific cytotoxic molecules, such as tumor necrosis factor, viral antigens and other proteins to render a cell susceptible to anti-cancer agents, and DNA encoding genes, such as the such as the defective gene (CFTR) associated with cystic fibrosis (see, e.g., International Application WO 93/03709, which is based on U.S. Application Serial No. 07/745,900; and Riordan et al. (1989) Science 245 ⁇ 066- 1073), to replace defective genes.
  • CFTR defective gene
  • 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, which is based on 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 as well as selection of DNA encoding genes for targeted delivery for genetic therapy, is well within the skill in this art. For example, the desirable properties, lengths and other characteristics of such oligonucleotides are well known.
  • Antisense oligonucleotides are 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. (1987) Tetrehedron Lett. 25:3539-3542; Miller et al. (1971) J. Am. Chem. Soc. 95:6657- 6665; Stec et al. (1985) Tetrehedron Lett. 26:2191-2194; Moody et al. (1989) 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 Altaian 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.
  • antisense oligonucleotides include those that are specific for IL-8 (see, e.g., U.S. Patent No. 5,241,049; and International applications WO 89/004836; WO 90/06321; WO 89/10962; WO 90/00563; and WO 91/08483, and the corresponding U.S. applications for descriptions of DNA encoding IL-8 and amino acid sequences of IL-8), 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.
  • a ribozyme is an RNA molecule that specifically cleaves RNA substrates, such as messenger RNA, and thus inhibits or interferes with cell growth or expression.
  • RNA substrates such as messenger RNA
  • 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, 5,1 16,742 and 5,093,246 to Cech et al., which described ribozymes and methods for production thereof).
  • Any such ribozyme may be linked to the growth factor for delivery to HBEGF-receptor bearing cells.
  • the ribozymes may be delivered to the targeted cells, such as tumor cells that express a receptor to which HBEGF binds and upon binding is internalized, as DNA encoding the ribozyme linked to a eukaryotic promoter, such as a eukaryotic viral promoter, generally a later promoter, such that upon introduction into the nucleus, the ribozyme will be directly transcribed.
  • the construct will also include a nuclear translocation sequence (NTS; see Table 2, below), generally as part of the growth factor or as part of a linker between the growth factor and linked DNA.
  • NTS nuclear translocation sequence
  • DNA that encodes therapeutic products contemplated for use is 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. which is based on U.S. Application Serial No. 07/745,900; and Riordan et al. (1989) Science 24571066-1073), and anticancer agents, such as tumor necrosis factors, and cytotoxic agents, such as saporin.
  • the conjugate should include an NTS.
  • the nuclear translocation sequence may be a heterologous sequence or a may be derived from the selected growth factor.
  • NTS nuclear translocation sequence
  • Extracellular protein binding oligonucleotides refer to oligonucleotides that specifically bind to proteins.
  • Small nucleotide molecules refer to nucleic acids that target a receptor site.
  • the HBEGF protein is linked to the nucleic acid either directly or via one or more linkers.
  • 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. (1975) FEBS Lett.
  • the reagents N-acetyl-N'-(p-glyoxylylbenzolyl) cystamine and 2-iminothiolane have been used to couple DNA to proteins, such as ⁇ 2macroglobulin ( ⁇ 2M) via mixed disulfide formation (see, Cheng et al. (1983) Nucleic Acids Res. 77:659-669).
  • N-acetyl-N'-(p-glyoxylylbenzolyl)cystamine reacts specifically with nonpaired guanine 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, upon internalization of the conjugate the targeted nucleic acid is active. 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 can be readily formed using heterobifunctional 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-dimethylaminopropyl]carbodiimide (EDC) or N-ethyl-N'(3-dimethylaminopropylcarbodiimidehydrochloride (EDCI), in imidazole buffer at pH 6 to produce the 5'phosphorimidazolide.
  • a water-soluble carbodiimide such as l-ethyl-3'[3-dimethylaminopropyl]carbodiimide (EDC) or N-ethyl-N'(3-dimethylaminopropylcarbodiimidehydrochloride (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.
  • U.S. Patent No. 5,237,016 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'-phosphory- lated 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.
  • the antisense oligomer or nucleic acid such as a methylphosphonate oligonucleotide (MP-oligomer) may be derivatized by reaction with SPDP or SMPB.
  • MP-oligomer may be purified by HPLC and then coupled to HBEGF, which may be modified by replacement of one or more non-essential 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.
  • phosphate buffer pH 7.5, final concentration 0.1 M
  • the reaction is allowed to proceed for about 5-10 hours at room temperature and is then quenched with about 15 ⁇ L 0.1 iodoacetamide.
  • the HBEGF-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. f. Nucleic acids encoding cytocides
  • a cytocide-encoding agent is a nucleic acid molecule (DNA or RNA) that, upon internalization by a ceil, and subsequent transcription 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, diptheria 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.
  • 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 are examples.
  • saporin a preferred cytocide, 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.
  • 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 obtain clones 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. L12243; PCT Application WO 92/03155; U.S. Patent No. 5,376,546; diphtheria toxin (Genbank Accession No.
  • diphtheria toxoid introduced into a cell was sufficient to kill the cell.
  • propagation or stable maintenance of the construct is necessary to attain sufficient numbers or concentrations of the gene product for effective gene therapy. Examples of replicating and stable eukaryotic plasmids are 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 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. 5,1 18,627), the SV40 late promoter (U.S. Patent No. 5,1 18,627), 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.
  • Tissue specific promoters are particularly useful when a particular 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, 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 transform prostate tumor cells the prostate-specific antigen promoter is especially useful. Similarly, the tyrosinase promoter or tyrosinase-related protein promoter is a preferred promoter for melanoma treatment.
  • 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 promoter, are but a few examples of tissue specific promoters.
  • tissue specific promoters In certain applications, such as treatment of restenosis, a promoter for myosin light chain specific for smooth muscle cells is preferred. Many other examples of tissue specific promoters are readily available to one skilled in the art.
  • 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.
  • 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 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 unice the iut gene product is present, which occurs upon viral infection.
  • promoters that are coordinately regulated with a particular cellular gene may be used.
  • promoters of genes that are coordinately expressed when a particular HBEGF receptor gene is expressed may be used. Then, the nucleic acid will be transcribed when the HBEGF receptor is expressed. This type of promoter is especially useful when one knows the pattern of HBEGF 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.
  • 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.
  • the construct must contain the sequence that binds to the nucleic acid binding domain, if the domain binds in a sequence specific manner.
  • the target nucleotide sequence may be contained within the coding region of the cytocide, in which case, no additional sequence need be incorporated. 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. The target sequence may be readily determined, in any case. Techniques are generally available for establishing the target sequence (e.g.. see PCT Application WO 92/05285 and U.S. Serial No. 586,769).
  • Specificity of delivery is achieved by coupling a nucleic acid binding domain to a receptor-binding internalized ligand, either by chemical conjugation or by constructing a fusion protein. Linkers as described above may be used.
  • the receptor- binding internalized ligand part 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 polycations 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 a well-used nucleic acid binding domain (see U.S. Patent Nos. 5,166,320 and 5,354,844).
  • nucleic acids such as spermine and spermidine
  • sequence-specific proteins including 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 domains can be chemically conjugated to each other.
  • the corresponding response elements that bind sequence-specific domains are incorporated into the construct to be delivered.
  • Complexing the cytocidal- encoding agent to the receptor-binding internalized ligand/nucleic acid binding domain allows specific binding of response element 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.
  • 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 or other cytocidal proteins into cells that have 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 SV40, CMV, 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 tliiol 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.
  • 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.
  • HBEGF 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 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.
  • 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 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.
  • 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.
  • the HBEGF monomers are preferably linked via non-essential cysteine residues to the linkers or to the targeted agent. HBEGF 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. 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.
  • 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. 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.
  • 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.
  • the resulting preparations of chemical conjugates are monogenous; compositions containing the conjugates also appear to be free of aggregates.
  • heterogeneity can be avoided by producing a fusion protein of receptor-binding internalized ligand and nucleic acid binding domain, as described below.
  • 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.
  • 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 HBEGF 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.
  • 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.
  • Trie 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 ligand — 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 r Vtrl.ta f j-rlU ⁇ crlvllHU..
  • HBEGF DNA is modified so that, upon expression, the resulting HBEGF portion of the fusion protein does not include any cysteines available for reaction.
  • DNA encoding an HBEGF polypeptide is linked to DNA encoding a nucleic acid binding domain.
  • the DNA encoding the HBEGF 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 HBEGF.
  • 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.
  • HBEGF may be fluorescently labeled with FITC or radiolabeled with ⁇ 5j Fluorescein-conjugated HBEGF is incubated with cells and examined microscopically by fluorescence microscopy or confocal microscopy for internalization.
  • HBEGF is labeled with 125 ⁇ e labeled HBEGF 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 cytoxicity measured.
  • the DNA encoding the resulting receptor-binding internalized ligand — nucleic acid binding domain can be inserted into a plasmid and expressed in a selected host, as described above, to produce a monogenous preparation. 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.
  • DNA encoding human bFGF- has been mutagenized using splicing by overlap extension (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, typically a 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.
  • the desired application is the delivery of cytotocidal agents, such as saporin, in a non-toxic form.
  • cytotocidal agents such as saporin
  • the timing of cytotoxicity may be appropriately controlled. For example, if saporin is expressed under the control of a tissue-specific promoter, then uptake of the complex by cells having the tissue-specific factors necessary for promoter activation will result in the killing of those cells. On the other hand, if cells taking up the complex do not have those tissue-specific factors, the cells will be spared.
  • 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 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 incorporated, 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. Further testing of the conjugate is performed to show that it binds to the cell surface receptor and is internalized into the cell.
  • the receptor-binding internalized ligand part of the conjugate retain complete biological activity.
  • HBEGF 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.
  • 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 ⁇ -gal expression (e.g., enzymatic activity) in cells that have been transformed with a complex of a ⁇ -gal containing plasmid condensed with a receptor-binding internalized ligand/nucleic acid binding domain. 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.
  • these first two assays are useful for preliminary analysis and failure to show receptor binding or ⁇ -gal activity does not per se eliminate a candidate receptor-binding internalized ligand/nucleic acid binding domain conjugate or fusion protein from further analysis.
  • 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.
  • cytocidal molecule 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.
  • NTS nuclear translocation or targeting sequence
  • 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.
  • 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. (1989) J. Biol Chem. 264:18019-18023, Dang et al. (1988) Mol. Cell. Biol 5:4049-4058 and Table 2, which sets forth examples of NTSs and regions of proteins that share homology with known NTSs),
  • 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) (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) and KEEL (Lys-Glu-Glu-Leu) 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 saporin or, for conjugates of HBEGF with a nucleic acid binding domain, the sequence may reside in either part or both. If cleavable linkers are used in the conjugate, the cytoplasm- translocation signal is preferably included in saporin or the nucleic acid binding domain. Additionally, a cytoplasmic-translocation signal sequence may be included in HBEGF, as long as it is placed so as not to interfere with receptor binding.
  • membrane-disruptive peptides may be incorporated into complexes of HBEGF-nucleic acid binding domain and cytocide-encoding agent.
  • Adenoviruses are known to enhance disruption of endosomes.
  • Virus-free viral proteins such as influenza virus hemagglutinin HA-2, may be 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. 74: 1 13- 135, 1994).
  • a linker is a peptide or other molecule that couples a HBEGF polypeptide to the targeted agent.
  • the linker may be bound via the N- or C-terminus or an internal reside, but, typically within about 20 amino acids of either terminus of a HBEGF and/or targeted agent.
  • the linkers provided herein increase intracellular availability, serum stability, specificity and solubility of the conjugate or provide increased flexibility or relieve steric hindrance in the conjugate.
  • specificity or intracellular availability of the targeted agent may be conferred by including a linker that is a substrate for certain proteases, such as a protease that is present in only certain subcellular compartments or that is present at higher levels in tumor cells than normal cells.
  • one or more linkers is(are) inserted between the HBEGF protein and the targeted moiety.
  • linkers include peptide linkers, such as intracellular protease substrates and peptides that increase flexibility or solubility of the linked moieties, and chemical linkers, such as acid labile linkers, ribozyme substrate linkers and others.
  • Peptide linkers may be inserted using heterobiofunctional reagents, described below, or, preferably, are linked to HBEGF by linking DNA encoding the substrate to the DNA encoding the HBEGF protein and expressing the resulting chimera.
  • the targeted agent is a protein, such as a RIP
  • the DNA encoding the linker can be inserted between the DNA encoding the HBEGF protein and the DNA encoding the targeted protein agent.
  • Chemical linkers may be inserted by covalently coupling the linker to the HBEGF protein and the targeted agent.
  • the heterobifunctional agents described below, may be used to effect such covalent coupling.
  • Peptides encoding protease-specific substrates are introduced between the HBEGF protein and the targeted moiety.
  • the peptides may be inserted using heterobiofunctional reagents, described below, or, preferably, are linked to HBEGF by linking DNA encoding the substrate to the DNA encoding the HBEGF protein and expressing the resulting chimera.
  • the targeted agent is a protein, such as a RIP
  • the DNA encoding the linker can be inserted between the DNA encoding the HBEGF protein and the DNA encoding the targeted protein agent.
  • DNA encoding substrates specific for intracellular proteases has been inserted between the DNA encoding the HBEGF protein and a targeted agent, such as saporin. Any protease specific substrate (see, e.g., O ⁇ are et al. (1990) FEBS
  • Bioconjuugate Chem. 5:375-381 may be introduced as a linker between the HBEGF polypeptide and linked targeting agent 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 or that are preferentially expressed in the endosome.
  • cathepsin B substrate cathepsin D substrate
  • trypsin substrate trypsin substrate
  • thrombin substrate recombinant subtilisin substrate
  • XaaAspGluLeu SEQ ID NO. 50 particularly, PheAlaHisTyr, SEQ ID NO. 49.
  • linkers 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.
  • linkers include, but are not limited to, (Gly4Ser) n , (Ser4Gly) n and (AlaAlaProAla) n (see, SEQ ID NO. 48) in which n is 1 to 6, preferably 1 -4, such as:
  • Gly4Ser SEQ ID NO. 40 CCATGGGCGG CGGCGGCTCT GCCATGG
  • reagents include, but are not limited to: N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP; disulfide linker); sulfosuccinimidyl 6-[3-(2-pyridyldithio)propion- amidojhexanoate (sulfo-LC-SPDP); succinimidyloxycarbonyl- ⁇ -methyl benzyl thiosulfate (SMBT, hindered disulfate linker); succinimidyl 6-[3-(2-pyridyldithio) propionamido]hexanoate (LC-SPDP); sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxy late (sulfo-SMCC); succinimidyl 3-(2- pyridyldithio)butyrate (SPDB; hindered disulfide bond
  • Acid cleavable linkers include, but are not limited to, bismaleimideothoxy propane; and adipic acid dihydrazide linkers (see, e.g., Fattom et al. (1992) Infection & lmmun. 60:584-589) and acid labile transferrin conjugates that contain a sufficient portion of transferrin to permit entry into the intracellular transferrin cycling pathway (see, e.g., Welhoner et al. (1991) J. Biol Chem. 266:4309-4314). 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. (1992) Bioconj. Chem. 3:104-107, which linkers are herein incorporated by reference), thereby releasing the targeted agent upon exposure to light.
  • Photocleavable linkers that are cleaved upon exposure to light are known (see, e.g., Hazum et al. (1981) in Pept., Proc. Eur. Pept. Symp, 16th, Brunfeldt, K (Ed), pp. 105- 110, which describes the use of a nitrobenzyl group as a photocleavable protective group for cysteine; Yen et al.
  • UV light 350 nm
  • Senter et al. (1985) Photochem. Photobiol 42:231-237 which describes nitrobenzyloxycarbonyl chloride cross linking reagents that produce photocleavable linkages), thereby releasing the targeted agent upon exposure to light.
  • linkers would have particular use in treating dermatological or ophthalmic conditions that can be exposed to light using fiber optics.
  • the eye or skin or other body part can be exposed to light, resulting in release of the targeted moiety from the conjugate. If the toxic moiety is a light activated porphyrin, light-exposure will also activate the porphyrin, thereby causing cell death.
  • Use of photocleavable linkers 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.
  • vector or plasmid refers to discrete elements that are used to introduce heterologous DNA into cells for either expression of the heterologous DNA or for replication of the cloned heterologous DNA. Selection and use of such vectors and plasmids are well within the level of skill of the art. Expression refers to the process by which nucleic acid is transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the nucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA.
  • expression vector includes vectors capable of expressing DNA fragments that are in operative linkage with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments.
  • an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA.
  • Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or may integrate into the host cell genome.
  • operative linkage or operative association of heterologous DNA to regulatory and effector sequences of nucleotides 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.
  • the NTS may be derived from another polypeptide, it may be synthesized, or it may be derived from another region in the same polypeptide.
  • transfection refers to the taking up of DNA or RNA by a host cell. Transformation refers to this process performed in a manner such that the DNA is replicable, either as an extrachromosomal element or as part of the chromosomal DNA of the host.
  • Methods and means for effecting transfection and transformation are well known to those of skill in this art (see, e.g., Wigler et al. (1979) Proc. Natl Acad. Sci. USA 76: 1373-1376; Cohen et al. (1972) Proc. Natl. Acad. Sci. USA 69:2 ⁇ ⁇ 0).
  • DNA encoding the selected HBEGF or a portion thereof, HBEGF conjugate or polypeptide targeted agent is inserted into a suitable vector and expressed in a suitable prokaryotic or eukaryotic host.
  • suitable hosts and vectors are known and available to those of skill in this art and may be purchased commercially or constructed according to published protocols using well known and available starting materials.
  • Suitable eukaryotic host cells include insect cells, yeast cells, and animal cells. Insect cells and bacterial host cells are presently preferred.
  • Suitable prokaryotic host cells include E. coli, strains oi Bacillus and Streptomyces.
  • the plasmids used herein must 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.
  • a promoter region refers to the portion of DNA of a gene that controls transcription of DNA to which it is operatively linked. A portion of the promoter region includes specific sequences of DNA that are sufficient for RNA polymerase recognition, binding and transcription initiation. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. For use herein, inducible promoters are preferred. The promoters are recognized by an RNA polymerase that is expressed by the host.
  • the RNA polymerase may be endogenous to the host or may be introduced by genetic engineering into the host, either as part of the host chromosome or on an episomal element, including a plasmid containing the DNA encoding the saporin-containing polypeptide.
  • Most preferred promoters for use herein are tightly regulated such that, absent induction, the DNA encoding the saporin-containing protein is not expressed. 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. 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 or early genes in vectors of viral origin.
  • suitable inducible promoters and promoter regions include, but are not limited to: the E. coli lac operator responsive to isopropyl ⁇ -D-thiogalactopyranoside (IPTG; see, et al. Nakamura et al. (1979) Cell 75: 1 109-1 1 17); the metallothionein promoter metal-regulatory-elements responsive to heavy-metal (e.g., zinc) induction (see, e.g., U.S. Patent No.
  • the phage T71ac promoter responsive to IPTG see, e.g., U.S. Patent No. 4,952,496; and Studier et al. (1990) Meth. Enzymol. 755:60-89
  • Other 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 trp, l ⁇ , and lac promoters, such as the lacUV5, from E. coli; the PI 0 or polyhedrin 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.
  • the DNA construct is introduced into a plasmid suitable for expression in the selected host.
  • 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 HBEGF, HBEGF chimera or cytotoxic agent may also include DNA encoding a secretion signal, whereby the resulting peptide is a precursor protein.
  • Secretion signals suitable for use are widely available and are well known in the art. Secretion signal refers to a peptide region within the precursor protein that directs secretion of the precursor protein from the cytoplasm of the host into the periplasmic space or into the extracellular growth medium.
  • Such signals may be either at the amino terminus or carboxyl terminus of the precursor protein.
  • the preferred secretion signal is linked to the amino terminus and may be heterologous to the protein to which it is linked.
  • 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, o pT, ompF, ompC, beta-lactamase, pelB and bacterial alkaline phosphatase. and the like (von Heijne (1985) J. Mol. Biol. 754:99-105).
  • the bacterial pelB gene secretion signal (Lei et al.
  • the phoA secretion signal, and the cek2 secretion signal, functional in insect cells may be employed.
  • the most preferred secretion signal for bacterial expression is the E. coli ompA secretion signal.
  • the signals from secreted proteins, such as insulin, growth hormone, mellitin, and mammalian alkaline phosphatase are of interest herein.
  • Other prokaryotic and eukaryotic secretion signals known to those of skill in the art may also be employed (see, e.g., von Heijne (1985) J. Mol. Biol. 754:99-105).
  • the methods described herein can substitute secretion signals that are functional in either yeast, insect or mammalian cells to secrete the heterologous protein from those cells.
  • the resulting processed protein may be recovered from the periplasmic space or the fermentation medium or growth medium.
  • the plasmids may also 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 ampiciilin resistance gene (Amp r ), tetracycline resistance gene (Tc r ) and the kanamycin resistance gene (Kan r ). The kanamycin resistance gene is presently preferred.
  • coli cells include the pET expression vectors (see, U.S patent 4,952,496; available from Novagen, Madison, WI; see, also literature published by Novagen describing the system).
  • plasmids include pET 11a, which contains the T71ac promoter, T7 terminator, the inducible E. coli lac operator, and the lac repressor gene; pET 12a-c, which contains the T7 promoter, T7 terminator, and the E. coli ompT secretion signal; and pET 15b (Novagen, Madison, WI), which contains a His-Tag M leader sequence (Seq. ID NO.
  • plasmid 23 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
  • Plasmid pKK has been modified by insertion of a kanamycin resistance cassette with EcoRI sticky ends (purchased from Pharmacia; obtained from pUC4K, see, e.g., Vieira et al. (1982) Gene 79:259-268; and U.S. Patent No. 4,719,179) into the ampiciilin resistance marker gene.
  • pP -lambda inducible expression vector pTrc99A
  • tac promoter vector pDR450 see, e.g., U.S. Patent Nos. 5,281.525, 5,262,309, 5,240,831, 5,231,008, 5,227,469, 5,227,293, ; available from Pharmacia P.L. Biochemicals, see; also Mott, et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 52:88; and De Boer et al. (1983) Proc. Natl Acad. Sci. U.S.A.
  • baculovirus vectors such as a pBlueBac vector (also called pJVETL and derivatives thereof; 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), including pBlueBac III.
  • pBlueBac vector also called pJVETL and derivatives thereof; 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
  • plasmids include the pIN-IIIompA plasmids (see, U.S. Patent No. 4,575,013 to Inouye; see, also, Duffaud et al. (1987) Meth. Enz. 153:492-507), such as pIN-IIIompA2 .
  • the pIN-IIIompA plasmids include an insertion site for heterologous DNA linked in transcriptional reading frame with 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.
  • 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 (Ipp) 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 repressor protein may be encoded by the plasmid containing the construct or a second plasmid that contains a gene encoding 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 the addition to the growth medium of a molecule that inhibits, for example, 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.
  • the constructs 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 gene, which is the source of the promoter.
  • Preferred transcription terminator regions are those that are functional in E.
  • the DNA fragment is replicated in bacterial cells, preferably in E. coli.
  • the DNA fragment also typically 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 bacterial 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, lysogenic 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.
  • Preferred eukaryotic hosts are the insect cells Spodoptera frugiperda (sf9 cells; see, e.g., Luckow et al. (1988) Bio/technology 6:47-55 and U.S. Patent No. 4,745,051).
  • baculovirus vectors such as a pBlueBac vector (also called pJVETL 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.
  • pBlueBac vector also called pJVETL 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 ETL promoter and is inducible with IPTG.
  • a DNA construct is introduced into a baculovirus vector pBluebac III (INVITROGEN, San Diego, CA) and then co-transfected with wild type virus into insect cells Spodoptera frugiperda (sf9 cells; see, e.g., Luckow et al. (1988) Bio/technology 6:47-55 and U.S. Patent No. 4,745,051).
  • Other baculovirus vectors such as pPbac and pMbac (available from
  • DNA encoding full-length HBEGF, HBEGF-SAP, SAP-HBEGF with and without linkers, and other such constructs has been introduced into the pET vectors pET 1 1a (Novagen, Madison, WI).
  • DNA encoding SAP has also been introduced in pET 15b (Novagen, Madison, WI).
  • 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 ETL promoter and is inducible with IPTG.
  • lacZ ⁇ - galactosidase gene
  • the HBEGF construct or other construct is inserted into this vector under control of the polyhedrin promoter.
  • the DNA is then cotransfected, such as by CaP ⁇ 4 transfection or liposomes, into Spodoptera frugiperda cells (sf9 cells) with wild type baculovirus and grown in tissue culture flasks or in suspension cultures.
  • Blue occlusion minus viral plaques are selected and plaque purified and screened for the presence of HBEGF-encoding DNA by any standard methodology, such as western blots using HBEGF anti-sera or Southern blots using an appropriate HBEGF probe.
  • DNA encoding an HBEGF with and without linkers is introduced into a Bluebac vector for expression in baculovirus. Details are set forth in the Examples. E. Methods of preparation of HBEGF-targeted agent conjugates
  • Cytotoxic conjugates with linked targeted agents can be prepared either by chemical conjugation, recombinant DNA technology, or combinations of recombinant expression and chemical conjugation.
  • the methods herein are exemplified with particular reference to HBEGF and saporin. It is understood, however, that the same methods may be used to prepare and use conjugates of any HBEGF polypeptide with any cytotoxic agent, such as a RIP, a nucleic acid or any other targeted agent either directly or via linkers as described herein.
  • the growth factor and targeted agent may be linked in any orientation and more than one growth factor and/or targeted agent may be present in a conjugate.
  • HBEGF polypeptides linked, either directly or via a linker, to one or more targeted agents are provided.
  • the presently preferred HBEGF polypeptides are those having sequences set forth in SEQ ID NOs. 1 - 5. Human HBEGF is particularly preferred.
  • the conjugates provided herein contain the following components:
  • HBEGF HBEGF n , (L)q, and (targeted agent) m in which: at least one HBEGF moiety is linked with or without a linker (L) to at least one targeted agent, n is 1 or more, typically is between 2 and 6, generally 1 or 2; q is 0 or more as long as the resulting conjugate binds to the targeted receptor, is internalized and delivers the targeted agent, q is generally 1 to 4; m is 1 or more, generally 1 or 2; L refers to a linker, and the targeted agent is any agent, such as a cytotoxic agent or a nucleic acid, or a drug, such as methotrexate, intended for internalization by a cell that expresses a receptor to which HBEGF binds and upon binding is internalized.
  • n 1 or more, typically is between 2 and 6, generally 1 or 2
  • q is 0 or more as long as the resulting conjugate binds to the targeted receptor, is internalized and delivers the targeted agent, q
  • HBEGF and targeted agent may be linked in any order and through any appropriate linkage, as long as the resulting conjugate binds to a receptor to which HBEGF binds and internalizes the targeted agent(s) in cells bearing the receptor.
  • the HBEGF polypeptide may be linked to the targeted agent or linker at or near its N-terminus or at or near its C-terminus.
  • the HBEGF may be linked to a second HBEGF polypeptide, which may be the same or a different HBEGF polypeptide; and one or more targeted agents may be linked to the HBEGF or may be linked to each other.
  • the linkage may be at any locus, although the C- or N- terminus region of HBEGF (within about 20. preferably 10. amino acids from the terminus) is preferred. If more than one targeted agent is included, the second agent may be the same or different from the first agent.
  • the conjugates provided herein may be represented by the formulae (I):
  • HBEGF refers to a polypeptide that is reactive with a HBEGF receptor (also referred to herein as a HBEGF polypeptide), such as HBEGF
  • L refers to a linker, which may be present or absent
  • q is 0 or more as long as the resulting conjugate binds to a targeted receptor and the targeted agent is internalized
  • m, n and p are, independently, 1 or more, and generally less than or equal to 4, and preferably 1 or 2
  • the targeted agent is any agent, such as a cytotoxic agent or a nucleic acid, or a drug, such as methotrexate, intended for internalization by a cell that expresses a HBEGF receptor; and the HBEGF may be linked to the linker or targeted agent via its N-terminus or C-terminus or any other locus in polypeptide, such as derivatized cys residues.
  • conjugates are preferably linked via cysteine residues present or introduced into HBEGF.
  • conjugates in which non-essential cysteines in the HBEGF polypeptides and/or targeted agent, if the agent is a polypeptide, are deleted or replaced with Ser or other conservative substitution are provided.
  • Compositions of such conjugates should exhibit reduced aggregation compared to conjugates that contain non- essential cysteines.
  • Non-essential cysteines may be identified empirically.
  • the linker is selected to increase the specificity, toxicity, solubility, serum stability, and/or intracellular availability of the targeted moiety. More preferred linkers are those that can be incorporated in fusion proteins and expressed in a host cell, such as E. coli. Such linkers include: enzyme substrates, such as cathepsin B substrate, cathepsin D substrate, trypsin substrate, thrombin substrate, subtilisin substrate, factor Xa substrate, and enterokinase substrate; linkers that increase solubility, flexibility. and/or intracellular cleavability, such as (glymser) n and (sermgly)n * i n which n is 1 to 6.
  • linkers are those, such as cathepsin D substrate, that are preferentially cleaved in the endosome or cytoplasm following internalization of the conjugate linker; other such linkers, such as (glymser) n and (ser m gly)n> also increase the flexibility, serum stability and/or solubility of the conjugate or the availability of the region joining the HBEGF and targeted agent for cleavage.
  • linkers that are the same or different may be included in order to take advantage of desired properties of each linker.
  • Linkers are suitable for incorporation into chemically produced conjugates.
  • Linkers that are suitable for chemically linking conjugates include disulfide bonds, thioether bonds, hindered disulfide bonds, and covalent bonds between free reactive groups, such as amine and thiol groups. These bonds are produced using heterobifunctional reagents to produce reactive thiol groups on one or both of the polypeptides and then reacting the thiol groups on one polypeptide with reactive thiol groups or amine groups to which reactive maleimido groups or thiol groups can be attached on the other.
  • linkers include acid cleavable linkers, such as bismaleimideothoxy propane, acid labile-transferrin conjugates and adipic acid diihydrazide, that would be cleaved in more acidic intracellular compartments and cross linkers that are cleaved upon exposure to UV or visible light and linkers.
  • acid cleavable linkers such as bismaleimideothoxy propane, acid labile-transferrin conjugates and adipic acid diihydrazide
  • the targeted agents or moieties include any molecule that, when internalized, alter the metabolism or gene expression in the cell .
  • agents include cytotoxic agents, such as ribosome inactivating proteins DNA encoding cytotoxic agents, and antisense nucleic acids, that result in inhibition of growth or cell death.
  • cytotoxic agents such as ribosome inactivating proteins DNA encoding cytotoxic agents
  • antisense nucleic acids that result in inhibition of growth or cell death.
  • Other such agents also include antisense RNA, DNA, and truncated proteins that alter gene expression via interactions with the DNA, or co-suppression or other mechanism.
  • the conjugates herein may also be used to deliver DNA and thereby serve as agents for gene therapy or to deliver agents that, upon, transcription and/or translation thereof, result in cell death.
  • Cytotoxic agents include, but are not limited to, ribosome inactivating proteins, inhibitors of DNA, RNA and/or protein synthesis, including antisense nucleic acids, and other metabolic inhibitors.
  • the cytotoxic agent is a ribosome-inactivating protein (RIP), such as, for example, saporin. although other cytotoxic agents can also be advantageously used.
  • RIP ribosome-inactivating protein
  • the targeted agents may also be modified to render them more suitable for conjugation with the linker and/or a HBEGF protein or to increase their intracellular activity.
  • modifications include, but are not limited to, the introduction of a Cys residue at or near the N-terminus or C-terminus, derivatization to introduce reactive groups, such as thiol groups, and addition of sorting signals, such as (XaaAspGluLeu)n (SEQ ID NO. 50), where Xaa is Lys or Arg, preferably Lys, and n is 1 to 6, preferably 1-3, at, preferably, the carboxy-terminus (see, e.g., Seetharam et al. (1991) J. Biol. Chem.
  • Conjugates that contain a plurality of HBEGF polypeptides linked to the cytotoxic agent are also provided. These conjugates that contain several, typically two to about six, monomers can be produced by linking multiple copies of DNA encoding the HBEGF fusion protein under the transcriptional control of a single promoter region. In addition conjugates that contain, more than one targeted agent per HBEGF, such as
  • SAP-HBEGF-SAP linked with or without linkers are contemplated herein.
  • HBEGF polypeptides for chemical conjugation HBEGF may be isolated from a suitable source or may be produced using recombinant DNA methodology, discussed below.
  • 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
  • the HBEGF polypeptide is conjugated generally via a reactive amine group or thiol group to the targeted agent or to a linker, which has been or is subsequently linked to the targeted agent.
  • the HBEGF polypeptide is conjugated either via its N-terminus, C-terminus, or elsewhere in the polypeptide.
  • the HBEGF polypeptide is conjugated via a reactive cysteine residue to the linker or to the targeted agent.
  • the HBEGF 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 HBEGF polypeptide can be modified by mutagenesis to replace reactive cysteines, leaving, preferably, only one available cysteine for reaction.
  • the HBEGF polypeptide is modified by deleting or replacing a site(s) on the HBEGF 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 HBEGF polypeptide.
  • cysteine residues do not include any cysteine residues that are required for proper folding of the HBEGF polypeptide or for retention of the ability to bind to a HBEGF receptor and internalize.
  • cytotoxic moiety 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 HBEGF is conjugated with a single species of cytotoxic conjugate.
  • the contribution of each cysteine to the ability to bind to HBEGF receptors may be determined empirically as described herein. b. Preparation of targeted proteins for chemical conjugation If the targeted agent is a polypeptide it may be directly linked to the
  • HBEGF or HBEGF with linker or to a linker by reaction of a reactive group in the polypeptide may react at only a single locus, so that the resulting preparation of conjugates is homogeneous.
  • the targeted agent can be derivatized and then a single species isolated, or can be modified so that it only has one reactive group, such as a cysteine, for a particular set of conditions and reagents.
  • saporin has been derivatized and a single species isolated. Saporin has also been modified by introduction of a single cysteine residue.
  • the SAP may be derivatized or modified such that it includes a cysteine residue for conjugation to the HBEGF protein.
  • Saporin for chemical conjugation may be produced by isolating the protein from the leaves or seeds of Saponaria officinalis or using recombinant methods and the DNA provided herein or known to those of skill in the art or obtained by screening appropriate libraries (see, e.g., International PCT Application WO 93/25688, which describes the isolation of saporin, plasmids containing DNA encoding saporin, expression of saporin and isolation of purified saporin).
  • DNA encoding saporin may also include an N-terminal extension sequence linked to the amino terminus of the saporin that encodes a linker so that, if desired, the SAP and linker can be expressed as a fusion protein.
  • the sequence of DNA encoding saporin is set forth in SEQ ID Nos. 8-12.
  • 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. (1990) Biochem. Internal. 27:631-638 and Barra et al. (1991) Biotechnol Appl.
  • saporin polypeptides include other members of the multi-gene family coding for isoforms of saporin-type RIPs including SO-1 and SO-3 (Fordham-Skelton et al. (1990) Mol Gen. Genet. 227:134-138), SO-2 (see, e.g., U.S. Application Serial No. 07/885,242, which corresponds to GB 2.216,891; see, also, Fordham-Skelton et al. (1991) Mol Gen. Genet. 229:460-466), SO-4 (see, e.g., GB 2,194,241 B; see, also, Lappi et al.
  • SO-1 and SO-3 Forms of saporin-type RIPs
  • SO-2 see, e.g., U.S. Application Serial No. 07/885,242, which corresponds to GB 2.216,891; see, also, Fordham-Skelton et al. (1991) Mol Gen. Genet. 229:460-466
  • SO-4 which includes the N-terminal 40 amino acids set forth in SEQ ID NO.
  • the saporin can be modified by the introduction of a cysteine residue into the SAP such that the resulting modified saporin protein reacts with a HBEGF monomer or a linker (and then to a HBEGF monomer) to produce a conjugate.
  • Preferred loci for introduction of a cysteine residue include the N- terminus region, preferably within about one to twenty residues, more preferably one to about ten residues, from the N-terminus of the cytotoxic agent, such as SAP.
  • DNA encoding SAP has been modified by inserting a DNA encoding Met-Cys (ATG TGT or ATG TGC) at the N-terminus immediately adjacent to the codon for first residue of the mature protein.
  • Muteins in which a cysteine residue has been added at the N-terminus and muteins in which the amino acid at position 4 or 10 has been replaced with cysteine have been prepared by modifying the DNA encoding saporin (see, Example 3).
  • the modified DNA may be expressed and the resulting saporin protein purified, as described herein for expression and purification of the resulting SAP.
  • the modified saporin can then be reacted with an HBEGF, to form disulfide linkages between the HBEGF and the cysteine residue on the modified SAP.
  • SAP is derivatized by reaction with SPDP.
  • SPDP is derivatized by reaction with SPDP.
  • SAP that is derivatized by SPDP to a level of 0.9 moles pyridine-disulfide per mole of SAP includes a population of non-derivatized, mono-derivatized and di-derivatized SAP.
  • Methods for isolation of mono-derivatized saporin are described, for example, in Lappi et al. (1993) Anal. Biochem. 272:446-451, copending U.S. Application Serial No. 08/099,924).
  • the methods rely on the charge differences among the three species of SAP that are produced upon reaction of one or more lysines in saporin with SPDP.
  • the mono-derivatized saporin species is purified by Mono-S cation exchange chromatogiaphy and pooling of the fractions that contain the monoderivatized species.
  • the initial eluting peak is composed of SAP that is approximately di-derivatized; the second peak is mono-derivatized and the third peak shows no derivatization.
  • the di-derivatized material accounts for 20% of the three peaks; the second accounts for 48% and the third peak contains 32%.
  • Fractions that have a ratio of SPDP to SAP greater than 0.85 but less than 1.05 are pooled, dialyzed against an appropriate buffer, such as 0.1 M sodium chloride, 0.1 M sodium phosphate, pH 7.5, used for coupling to a linker, to a HBEGF or to HBEGF with linker.
  • an appropriate buffer such as 0.1 M sodium chloride, 0.1 M sodium phosphate, pH 7.5
  • the resulting preparation although more uniform, is heterogeneous because native saporin as purified from the seed is a mixture of four isoforms, as judged by protein sequencing (see, e.g., copending published International PCT Application WO 93/25688 (Serial No. PCT/US93/05702), which is a continuation-in-part of copending United States Application Serial No. 07/901,718; see also, Montecucchi et al. (1989) Int. J. Pept. Prot. Res. 53:263-267; Maras et al. (1990) Biochem. lnternat. 27:631-638; and Barra et al. (1991) Biotechnol Appl. Biochem. 73:48-53).
  • the HBEGF polypeptides are preferably linked via non-essential cysteine residues to the linkers or to the targeted agent.
  • HBEGF that has been modified by introduction of a cys residue at or near one terminus; the N-terminus is preferred; is used in chemical conjugation (see Examples for preparation of such modified HBEGF).
  • 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.
  • 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.
  • DNA encoding the HBEGF polypeptide may be isolated, synthesized or obtained from commercial sources or prepared as described herein in Example 4 and in International Application WO/92/06705 (and the corresponding U.S. patent application serial No. 07/598,082), and Abraham et al. (1993) Biochem. Biophy. Res. Comm. 790:125-133. Expression of recombinant HBEGF polypeptides may be performed as described herein; and DNA encoding HBEGF polypeptides may be used as the starting materials for the methods herein.
  • DNA encoding HBEGF polypeptides and/or the amino acid sequences of HBEGFs are known to those of skill in this art (see, e.g., SEQ ID NOs. 1-5).
  • DNA may be prepared synthetically based on the amino acid sequence or known DNA sequence of an HBEGF or may be isolated using methods known to those of skill in the art or obtained from commercial or other sources known to those of skill in this art. For example, suitable methods are described in Example 4 for amplifying HBEGF encoding cDNA from well known plasmids (e.g., pMTN-HBEGF, ATCC #40900 and pAX- HBEGF, ATCC #40899) containing HBEGF encoding cDNA.
  • plasmids e.g., pMTN-HBEGF, ATCC #40900 and pAX- HBEGF, ATCC #40899
  • Such DNA may then be mutagenized using standard methodologies to delete or delete and replace any cysteine residues, as described herein, that are responsible for aggregate formation.
  • identity of cysteine residues that contribute to aggregate formation may be determined empirically, by deleting and/or deleting and replacing a cysteine residue and ascertaining whether the resulting HBEGF 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 conjugate can be inserted into a plasmid and expressed in a selected host, as described above, to produce a recombinant HBEGF-toxin conjugate.
  • Multiple copies of the modified HBEGF-cytotoxic agent chimera or modified HBEGF-cytotoxic agent chimera can be inserted into a single plasmid in operative linkage with one promoter. When expressed, the resulting protein will be an HBEGF-cytotoxic agent multimer.
  • two to six copies of the chimera are inserted, preferably in a head to tail fashion, into one plasmid.
  • cysteines in the HBEGF polypeptide that are not required for biological activity can be deleted or replaced.
  • all except for 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 cytotoxic agent 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 the cytotoxic agent. As long as this ability is retained the mutein is suitable for use herein. c. DNA constructs and expression of the constructs
  • DNA encoding HBEGF conjugates is expressed in any suitable host, particularly bacterial and insect hosts. Methods and plasmids for such expression are set forth in the Examples (see, also TABLE 3). Using the methods and materials described above and in the Examples numerous chemical conjugates and fusion proteins have been synthesized. These include those set forth in TABLE 3 below.
  • constructs have been prepared and have been or can be inserted into plasmids including pET 1 1 (with and without the T7 transcription terminator), pET 12 and pET 15 (INVITROGEN, San Diego), ⁇ pPL and pKK223-3 (PHARMACIA, P.L.) and derivatives of pKK223-3.
  • the resulting plasmids have been and can be transformed into bacterial hosts including BL21(DE3), BL21(DE3)+pLYS S, HMS174(DE3), HMS174(DE3)+pLYS S (Novagen, Madison, WI) and N4830(cI857) (see, Gottesman et al. (1980) J. Mol Biol. 740:57-75, commercially available from PL Biochemicals, Inc, also, see, e.g., U.S. Patent Nos.
  • N4830 harbors a heavily deleted phage lambda prophage carrying the mutant cl857 temperature sensitive repressor and an active N gene.
  • the constructs have also been introduced into a baculovirus vector sold commercially under the name pBLUEBACIII (INVITROGEN, San Diego CA; see the INVITROGEN CATALOG; see, also, Vialard et al. (1990) J. Virol.
  • 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 ETL promoter and is inducible with IPTG.
  • lacZ ⁇ -galactosidase gene
  • the baculovirus vector is then co- transfected with wild type virus into insect host cells Spodoptera frugiperda (sf9; see, e.g., Luckow et al. (1988) Bio/technology 6:47-55 and U.S. Patent No. 4,745,051).
  • the plasmids such as PZ1A1 are designated with (i) a PZnumber (PZ1), followed by (ii) a letter (A), and optionally (iii) followed by a number (1).
  • PZnumber - refers to the construct number
  • the next letter refers to the plasmid into which the construct was cloned
  • A pET 11 wthout the T7 transcription terminator
  • B pET 11 with the T7 transcription terminator.
  • c pET 13
  • D pET 12
  • G pKK 223-3
  • H PRZ 1 (pKK223-3+Kan R ).
  • I pBlueBac III
  • cell paste is suspended in 3-4 volumes of cell lysis buffer containing 10 mM sodium citrate, pH 6.0, 1 M urea, 5 mM EDTA, 5 mM EGTA and 50 mM NaCl.
  • the lysate is passaged 3 times through a micro fluidizer and diluted to 10 volumes with lysis buffer.
  • concentration of urea should be less than 8 M to reduce viscosity, and it is not necessary to include a cocktail of protease inhibitors.
  • Urea is necessary for isolation of active protein.
  • the extract is loaded onto an expanded bed of Streamline SP cation- exchange resin equilibrated with lysis buffer.
  • Proteins are eluted with 2 buffers containing increasing NaCl concentrations: the first buffer contains 0.25 M NaCl and the second buffer contains 0.8 M NaCl.
  • the second eluate is diluted in buffer without NaCl and subjected to anion-exchange chromatography on Q-Sepharose to remove DNA, endotoxins and contaminating proteins, and cation-exchange chromatography on SP-Sepharose to remove other contaminants. Proteins bound to the S-Sepharose column are eluted with a gradient of 0.25 to 1 M NaCl in buffer. Ammonium sulfate is added to the fusion proteins.
  • the protein is loaded onto a phenyl-Sepharose HP column and eluted with buffer containing 2 M ammonium sulfate. Monothioglycerol is added to the fusion protein.
  • the protein is dialyzed and subjected to size -exclusion chromatography on S-100 resin. No heparin affinity chromatography is performed and a refolding protocol is not necessary to attain active material in the case of conjugates.
  • equivalent resins and buffers may be readily substituted at each step in accordance with the purpose of each purification step. That is. for example, other equivalent cation exchange resins may be used in place of SP-Sepharose.
  • FPHS2 and FPHS1 fusion proteins are purified as above except that a heparin sepharose FF affinity column was additionally used prior to the S-100 column.
  • the conjugates provided herein can be used in vitro to identify cells. particularly tumor cells that express receptors to which the conjugate selectively binds and which internalizes the conjugates.
  • the cells are contacted with the conjugates and assayed for proliferation.
  • Cells in which proliferation is inhibited express receptors to which HBEGF binds. If such cells are derived from a tumor, such tumor will be a candidate for treatment with the HBEGF conjugate. If such cells are a cell line, such cell line will be useful in drug screening assays for identification of compounds that modulate the activity of HBEGF receptors (see, e.g., U.S. Patent Nos. 5.208,145, 5,071,773, 4,981,784, 4,603,106, which describe such assays for other receptors).
  • HBEGF-containing conjugates produced by the methods described herein can be tested, using a variety of well known in vitro and in vivo assays, for their ability to exert a cytopathic effect.
  • the Promega (Madison, WI) CellTiter 96 Cell Proliferation/Cytotoxicity Assay described above and in Example 4 may be employed.
  • in vitro cytotoxicity assays described in, for example, Kreitman et al. (1991) Bioconjugate Chem. 3:63-68; Epstein et al. (1991) Circulation 54:778-787, and the like may be employed to test the conjugates produced herein.
  • EGF-receptor expressing cells are plated in 96-well tissue culture plates at 1000-3000 cells/ ell in iheir respective medium. One day later, the medium is removed, and medium containing 1 pM to 1 ⁇ M of the conjugate HBEGF-SAP, free SAP, free HBEGF, and HBEGF+SAP are added. Cells are treated in triplicate and maintained at 37° C and 5% CO2. Forty-eight hours after the treatment is initiated, the MTT colorometric assay is utilized to measure cell sensitivity to HBEGF-SAP conjugates (Mossman, T. (1983) J. Immunological Meth. 65:55-63).
  • Results are expressed as the mean optical density from treated wells, normalized to media controls, as a function of the HBEGF-SAP, free SAP, free HBEGF, and HBEGF+SAP concentration.
  • the 50% inhibition values are calculated from dose-response curves and represent the concentration which resulted in a 50% reduction in cell number.
  • treatment means any manner in which the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered, reduced or relieved. 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.
  • the conjugates herein may be formulated into pharmaceutical compositions suitable for topical, local, intravenous and systemic application. Effective concentrations of one or more of the conjugates are mixed with a suitable pharmaceutical carrier or vehicle. The concentrations or amounts of the conjugates 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 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 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 xeiiograft model for tumors or rabbit ophthalmic model. If necessary, pharmaceutically acceptable salts or other derivatives of the conjugates may be prepared.
  • compositions suitable for administration of the conjugates include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the conjugates may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • 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 and that either are pharmaceutically active or are prodrugs.
  • a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound.
  • the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes.
  • the prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug.
  • the conjugates can be administered by any appropriate route, for example, orally, parenterally, 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 vascular proliferative disorders, will typically be treated by systemic, intradermal or intramuscular, modes of administration.
  • 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. It is understood that number and degree of side effects depends upon the condition for which the conjugates are administered. For example, certain toxic and undesirable side effects are tolerated when treating iife- 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 absorption, inactivation and excretion rates thereof, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
  • 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.
  • 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.
  • a daily dosage of about between 0.05 and 0.5 mg/kg should be sufficient and can be administered as a bolus or continuous infusion.
  • Local application for ophthalmic disorders should provide about 1 ng up to 100 ⁇ g, 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. Dosages can be empirically determined using recognized models for each disorder.
  • 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.
  • 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.
  • Solutions or suspensions used for parerelial, iiitradermal, 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 tonicity 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.
  • 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
  • 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.
  • the conjugates may be prepared with carriers that protect them against rapid elimination from the body, such as time release formulations or coatings.
  • 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, polylacetic acid and others. These are particularly useful for application to the eye for ophthalmic indications following or during surgery in which only a single administration is possible. Methods for preparation of such formulations are known to those skilled in the art.
  • the conjugates 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 intracisternal 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 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).
  • 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 release., 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 incorporated 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.
  • 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 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 or compositions as provided herein within the packaging material, and a label that indicates the indication for which the conjugate is provided. H. Therapeutic uses of the HBEGF conjugates
  • the conjugates provided herein can be used in pharmaceutical compositions to treat HBEGF-mediated pathophysiological conditions by targeting to cells that bear HBEGF receptors and inhibiting proliferation of or causing death of the cells.
  • pathophysiological conditions include, for example, certain tumors, such as renal cell carcinomas and breast and bladder tumors, psoriasis, ophthalmic disorders involving epithelial cells, such as recurrence of pterygii and secondary lens clouding.
  • the treatment is effected by administering a therapeutically effective amount of the HBEGF conjugate, for example, in a physiological vehicle suitable for local or systemic application.
  • the conjugate is formulated for topical, local or intralesional application to the skin and is applied topically, locally or intralesional.
  • Atherosclerosis which results from the development of an intimal lesion and the subsequent narrowing of the vessel lumen, commonly results from the buildup of plaque which lines the interior of blood vessels, particularly the arteries.
  • a number of surgical procedures have been developed that interarterially remove such plaque, often by balloon catheterization or other such treatments, either by compressing it against or scraping it away from the interior surface of the artery.
  • the patient so treated experiences a recurrence of narrowing of the vessel lumen in a relatively short period thereafter. This narrowing following treatment to remove plaque is referred to as restenosis.
  • Methods are provided herein for treating restenosis by administering an effective amount of an HBEGF cytotoxic conjugate, such that the HBEGF conjugate inhibits smooth muscle cell proliferation in the lining of vessels that have been injured without inhibiting proliferation of endothelial cells that is necessary for preventing or treating restenosis following vascular injury. It can be administered locally or intravenously.
  • a medicament containing an HBEGF-toxin, preferably saporin, conjugate will be targeted to proliferating smooth muscle cells in the treated arteries and relatively few infusions (or a few, i.e., up to about 3-5) should prevent restenosis.
  • the medicament containing the conjugate is administered intravenously (IV), although treatment by localized administration of the conjugate may be tolerated in some instances.
  • IV intravenously
  • the medicament containing the conjugate is injected into the circulatory system of a patient in order to deliver a dose of cytotoxin to the targeted cells by first binding the conjugate to high HBEGF receptors expressed by such cells.
  • a cytotoxin such as saporin or a Ricin A chain or a similar RIP, can inhibit protein synthesis and consequently interfere with DNA synthesis is fairly widely known.
  • the dosage of the conjugate that is administered will, to some extent, depend upon the particular cytotoxin chosen; however, doses of the conjugate in the general range of about 0.01 mg to about 100 mg of the conjugate per kilogram of body weight are expected to be employed as a daily dosage. There may be particular advantages in administering a daily dosage of about 0.1 mg/kg (i.e. between 0.05 and 0.3 mg/kg). 2. Treatment of tumors
  • Tumors particularly solid tumors, including bladder, breast, ovarian, pancreatic and some colon carcinomas, have receptors to which HBEGFs bind.
  • the susceptibility of particular tumors can be ascertained by isolating the cell, and contacting them with an HBEGF cytotoxic conjugate and determining sensitivity to the conjugate by a standard proliferation assay. This should identify those tumors that would be amenable to treatment and also identifies tumor cells that express receptors to which HBEGF binds.
  • Cytotoxic conjugates such as HBEGF conjugated with the saporin molecule (HBEGF-SAP), are inhibitors of cell growth in vitro for cell lines that express HBEGF receptors. Such in vitro activity should be extrapolatable to in vivo activity.
  • JJQ vivo activity may be assessed using recognized animal models, such as the mouse xenograft model for anti-tumor activity (see, e.g., Beitz et al. (1992) Cancer Research 52:227-230; Houghton et al. (1982) Cancer Res. 42:535-539; Bogden et al. (1981) Cancer (Philadelphia) 45:10-20; Hoogenhout et al. (1983) Int. J. Radiat. Oncol, Biol. Phys. 9:871-879; Stastny et al. (1993) Cancer Res. 53:5740-5744).
  • HBEGF-SAP conjugates Cell lines that are sensitive to the cytotoxic HBEGF conjugates can be grown subcutaneously as solid tumor xenografts in nude mice, and administration of HBEGF-SAP conjugates to such mice should show rapid reduction in tumor volume in those cell lines which responded to treatment of the conjugate.
  • the conjugates are used to target cytotoxic agents to human solid tumors, including bladder or breast tumors, to inhibit the proliferation of such cells.
  • the conjugates are also used to target HBEGF receptor-expressing cells in similar tumorigenic pathophysiological conditions.
  • E. coli strain JA221 (lpp " hdsM+ trpE5 leuB6 lacY recAl F'flacl Q lac + pro “1” ]) 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-1521 1 ; see, also, U.S. Patent No. 4,757,013 to Inouye; and Nakamura et al. (1979) Cell 75: 1109-1117). Strain INVl ⁇ is commercially available from Invitrogen, San Diego, CA; and strains Novablue and BL21(DE3) are commercially available (Novagen, Madison WI). 3. DNA Manipulations
  • 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. (1985) Biochem. Biophys. Res. Comm. 729:934-942. 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 Corporation (version 2.0) according to the manufacturer's instructions.
  • 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 PhastTransfcr system (Pharmacia), 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 as described (Davis, L., Dibner et al. (1986) Basic Methods in Molecular Biology, pp. 1-338, Elsevier Science Publishing Co., New York).
  • the RIP activity of saporin can be and is determined in an in vitro assay measuring cell-free protein synthesis in a nuclease-treated rabbit reticulocyte lysate (Promega). Samples of saporin are 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 are incubated 1 hour in a 30 C water bath.
  • the reaction is 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).
  • a Millititer HA 96-well filtration plate Millititer HA 96-well filtration plate (Millipore).
  • 300 ⁇ l of ice cold 25% trichloroacetic acid (TCA) are added to each well and the plate left on ice for another 30 min. Vacuum filtration is performed with a Millipore vacuum holder.
  • the wells are washed three times with 300 ⁇ l of ice cold 8% TCA.
  • CTGCAGAATTCGCCTCGTTTGACTACTTTG-3' 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 EcoRl restriction site after the sequence encoding mature saporin.
  • Unfractionated Saponaria officinalis leaf genomic DNA (1 ⁇ l) was mixed in a final volume of 100 ⁇ l containing 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 0.01% gelatin, 2 mM MgCl2, 0.2 mM dNTPs, 0.8 ⁇ g of each primer.
  • 2.5 U TaqI DNA polymerase (Perkin Elmer Cetus) was added and the mixture was overlaid with 30 ⁇ l 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 ⁇ l aliquot of each reaction was run on a 1.5% agarose gel to verify the correct size of the amplified product.
  • the amplified DNA was digested with EcoRl and subcloned into EcoRI I-restricted M13mpl 8 (NEW ENGLAND BIOLABS, Beverly, MA; see, also, Yanisch- Perron et al. (1985), "Improved Ml 3 phage cloning vectors and host strains: Nucleotide sequences of the M13mpl8 and pUC19 vectors", Gene 33:103).
  • Single- stranded DNA from recombinant phages was sequenced using oligonucleotides based on internal points in the coding sequence of saporin (see, Bennati et al. (1989) Eur. J. Biochem. 753:465-470).
  • Ml 3 mpl8-G4 containing the clone containing saporin of SEQ ID NO. 8 from Example LB.
  • 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 lpp promoter (Nakamura et al. (1979) Cell 75: 1109-1 1 17), 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 SEQ ID NO 8.
  • the plasmid also includes the E. coli lac repressor gene (lac I).
  • the Ml 3 mpl8-Gl, -G2, -G7, and -G9 clones obtained from Example 1.B.2, containing SEQ ID NOs. 9-12, respectively, are digested with EcoR I and ligated into EcoR I digested prN-IIIompA2 as described for Ml 3 mpl 8-G4 above in this example.
  • the resulting plasmids, labeled pOMPAGl , pOMPAG2, 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 in Example l .A. D. 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 to an O.D. in or at the end of the log phase of growth after which IPTG was added to induce expression of the saporin-encoding DNA.
  • 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 EDTA (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 RIP activity of recombinant saporin was compared to the RIP 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 (see, e.g., Lappi et al. (1985) Biochem. Biophys. Res. Comm.
  • 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).
  • a Millititer HA 96-well filtration plate Millititer HA 96-well filtration plate (Millipore).
  • TCA trichloroacetic acid
  • 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.
  • 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.
  • 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.
  • bFGF coding sequence in the plasmid designated pFC80 herein has the sequence set forth as SEQ ID NO. 34, 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 CII ribosome binding site (SEQ ID NO. 19) linked to the FGF-encoding DNA (SEQ ID NO. 34).
  • the restriction and modification enzymes employed here are commercially available in the U.S. Native SAP, chemically conjugated bFGF-SAP and rabbit polyclonal antiserum to SAP were obtained as described, for example, in Lappi et al. (1985) Biochem. Biophys. Res. Comm. 129:934-942, Lappi et al. (1989) Biochem. Biophys., Res. Comm. 760:917-923 and U.S. Patent No. 5,191 ,067.
  • the pET System Induction Control was purchased from Novagen, Madison, WI.
  • the sequencing of the different constructions was done using the Sequenase kit of United States Biochemical Corporation (version 2.0).
  • MEL28 cells 2500 cells are plated per well.
  • a Nco I restriction site was introduced into the SAP-encoding DNA of the M13mpl8-G4 clone, described in Example 1, by site-directed mutagenesis method using the Amersham In v/fro-mutagenesis system 2.1.
  • the oligonucleotide employed to create the Nco I restriction site was synthesized using a 380B automatic DNA synthesizer (Applied Biosystems) and is has the sequence (SEQ ID NO. 17): CAACAACTGCCATGGTCACATC. This oligonucleotide containing the Nco. I site replaced the original SAP-containing coding sequence at SEQ ID NO. 8, nts 32-53.
  • the resulting M13mpl8-G4 derivative was designated mpNG4.
  • Plasmid pFC80 is a derivative of pDS20 (see, e.g., Duester et al. (1982) Cell 50:855-864; 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.
  • Plasmid pKG1800 includes the 2880 bp EcoR I-Pvu II of pBR322 that contains the ampiciilin resistance gene and an origin of replication.
  • Plasmid ⁇ FC80 was prepared from pDS20 by replacing the entire galK gene with the FGF-encoding DNA of SEQ ID NO. 34, inserting the tip promoter (SEQ ID NO. 18) and the bacteriophage lambda CII ribosome binding site (SEQ. ID NO. 19; see, e.g., Schwarz et al. (1978) Nature 272:410) upstream of and operatively linked to the FGF-encoding DNA.
  • the Tip promoter can be obtained from plasmid pDR720 (Pharmacia PL Biochemicals) or synthesized according to SEQ ID NO. 18.
  • Plasmid pFC80 contains the 2880 bp EcoR 1-BamH I fragment of plasmid pSD20, a synthetic Sal l-Nde I fragment that encodes the Tip promoter region (SEQ ID NO. 18):
  • the FGF-encoding DNA was removed from pFC80 by treating it as follows.
  • the pFC80 plasmid was digested by Hga I and Sal I, which produces a fragment containing the CII ribosome binding site linked to the FGF-encoding DNA.
  • the resulting fragment was blunt ended with DNA pol I (Klenow fragment) and inserted into M13mpl 8 that had been opened by Sma l and treated with alkaline phosphatase for blunt-end ligation.
  • an insert in the ORI minus direction was mutagenized using the Amersham kit, as described above, using the following oligonucleotide (SEQ ID NO.
  • GCTAAGAGCGCCATGGAGA which contains 1 nucleotide between the FGF carboxy terminal serine codon and a Nco I restriction site, and it replaced the following wild type FGF encoding DNA having SEQ ID NO. 21 :
  • 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. 22).
  • Plasmid FGFM13 was cut with Nco I and Sac I to yield a fragment containing the CII ribosome binding site linked to the bFGF coding sequence with the stop codon replaced.
  • the M13mpl 8 derivative mp ⁇ G4 containing the saporin coding sequence was also cut with restriction endonucleases Nco I and Sac I, and the bFGF coding fragment from FGFM13 was inserted by ligation to DNA encoding the fusion protein bFGF-SAP into the M13mpl 8 derivative to produce mpFGF-SAP, which contains the CII ribosome binding site linked to the FGF-SAP fusion gene.
  • the sequence of the fusion gene is set forth in SEQ ID NO. 34 and indicates that the FGF protein carboxy terminus and the saporin protein amino terminus are separated by 6 nucleotides (SEQ ID NOs. 34 and 35, nts 466-471) that encode two amino acids Ala Met.
  • Plasmid FGF-SAP was digested with Xba I and EcoR I and the resulting fragment containing the bFGF-SAP coding sequence was isolated and ligated into plasmid p ⁇ T 11a (available from Novagen, Madison, WI; for a description of the plasmids see U.S. Patent No. 4,952.496; see, also Studier et al. (1990) Meth. Enz. 755:60-89; Studier et al. (1986) J. Mol Biol 759:113-130; Rosenberg et al. (1987) Gene 56:125-135) that had also been treated with EcoR I and Xba I. The resulting plasmid was designated pFS92. It was renamed PZIA.
  • Plasmid pFS92 (or PZIA) contains DNA encoding the entire basic FGF protein (S ⁇ Q ID NO. 34), a 2-amino acid long connecting peptide, and amino acids 1 to 253 of the mature SAP protein. Plasmid pFS92 also includes the CII ribosome binding site linked to the FGF-SAP fusion protein and the T7 promoter region from p ⁇ T 1 la. E. coli strain BL21(D ⁇ 3)pLysS (Novagen, Madison WI) was transformed with pFS92 according to manufacturer's instructions and the methods described in Example 2.A.2. b.
  • Plasmid PZ1B Plasmid pFS92 was 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 ell ribosome binding site. This fragment was ligated into p ⁇ T 1 la. which had been BamH I digested, treated to repair the ends, and digested with Nde I. The resulting plasmid was designated PZ1B. PZ1B includes the T7 transcription terminator and the p ⁇ T 1 la ribosome binding site.
  • E. coli strain BL21(D ⁇ 3) (Novagen, Madison WI) was transformed with PZ1B according to manufacturer's instructions and the methods described in Example 2.A.2.
  • Plasmid PZIC Plasmid PZIC was prepared similarly to PZ1B but contains a kanamycin resistance gene and is based on the pET 13a vector.
  • Plasmid PZID Plasmid PZID
  • Plasmid pFS92 was digested with EcoR I and Nde I to release the FGF- encoding DNA without the CII ribosome binding site and the ends were repaired. This fragment was ligated into p ⁇ T 12a, which had been BamH I digested and treated to repair the ends. The resulting plasmid was designated PZID.
  • PZID includes DNA encoding the OMP T secretion signal operatively linked to DNA encoding the fusion protein.
  • E. coli strains BL21(D ⁇ 3), BL21(DE3)pLysS, HMS174(DE3) and HMS174(DE3)pLysS were transformed with PZID according to manufacturer's instructions and the methods described in Example 2.
  • Saporin was modified by addition of a cysteine residue at the N- terminus-encoding portion of the DNA or by the addition of a cysteine at position 4 or 10.
  • the resulting saporin is then reacted with an available cysteine or sulfhydryl- reacting moiety on a targeting agent to produce conjugates that are linked via the added Cys or Met-Cys on saporin.
  • Modified SAP has been prepared by altering the DNA encoding the SAP by inserting DNA encoding Met-Cys at position -1 or by replacing the He or the Asn codon within 10 or fewer residues of the N-terminus with Cys.
  • the resulting DNA has been inserted into pET 1 1a and pET 15b and expressed in BL21(DE3) cells.
  • the resulting saporin proteins are designated FPS1 (saporin with Cys at -1), FPS2 (saporin with Cys at position 4) and FPS3 (saporin with Cys at position 10).
  • a plasmid that encodes FPS1 and that has been used for expression of FPS1 has been designated PZ50B.
  • Plasmids that encode FPS2 and that have been used for expression of FPS2 have been designated PZ51B (pETl la-based plasmid) and PZ51E (pET15b-based plasmid). Plasmids that encode FPS3 and that have been used for expression of FPS3 have been designated PZ52B (pETl la-based plasmid) and PZ52E (pET 15b-based plasmid).
  • DNA manipulations were performed as described in Examples 1 and 2.
  • Plasmid PZ1B (designated PZ1B1 (the "1 " at the end refers to the bacterial host strain.
  • Example 2 BL21(DE3) described in Example 2 was used as the DNA template.
  • Primer #2 - Antisense primer complements the coding sequence of saporin spanning nucleotides 547-567 of SEQ ID NO. 34 and contains a BantHl site:
  • PZIB DNA was amplified as follows using the above primers.
  • PZ1B DNA (1 ⁇ l) was mixed in a final volume of 100 ⁇ l containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.01% gelatin, 2 mM MgCl2, 0.2 mM dNTPs, 0.8 ⁇ g of each primer.
  • 2.5 U TaqI DNA polymerase (Boehringer Mannheim) was added and the mixture was overlaid with 30 ⁇ l 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 35 cycles, a 10 ⁇ l aliquot of each reaction was run on a 1.5% agarose gel to verify the correct size of the amplified product.
  • the amplified DNA was gel purified and digested with Ndel and BamHl and subcloned into Ndel and BamHl -digested pZlB.
  • This digestion and subclor.ing step removed the FGF-encoding DNA and 5' portion of SAP up to the BamHl site at nucleotides 555-560 (SEQ ID NO. 34) and replaced this portion with DNA encoding a saporin molecule that contains a cysteine residue at position -1 relative to the start site of the native mature SAP protein (see, SEQ ID NO. 58).
  • the resulting plasmid is designated pZ50B.
  • Primer #2 - Antisense primer complements the coding sequence of saporin spanning nucleotides 547-567 of SEQ ID NO. 34 and contains a BamHl site (SEQ ID NO. 16): CAGGTTTGGATCCTTTACGTT .
  • the nucleic acid amplification reactions were performed as described above, using the following cycles: denaturation step 94°C for 1 min, annealing for 2 min at 60°C, and extension for 2 min at 72°C for 35 cycles.
  • the amplified DNA was gel purified, digested with Ndel and BamHl. and subcloned into Ndel and BamHl digested pZlB. This digestion removed the FGF and 5' portion of SAP (up to the BamHl site) from the parental FGF-SAP vector (pZlB) and replaced this portion with a SAP molecule containing a CYS at position +4 or +10 relative to the start site of the native mature SAP protein (see SEQ ID NOs. 36 and 37. respectively).
  • the resulting plasmids are designated pZ51 B and pZ52B. respectively.
  • the initial step in this construction was the mutagenesis of the internal BamHl site at nucleotides 555-560 (SEQ ID NO. 34) in pZlB using a sense primer corresponding to nucleotides 543-570 (SEQ ID NO. 34) but changing the G at nucleotide 555 (the third position in the Lys codon) to an A.
  • the complement of the sense primer was used as the antisense primer: 5'
  • TTTCAGGTTTGGATCTTTTACGTTGTTT 3' (SEQ ID NO. 61).
  • the first round of amplification used amplification reactions, conducted as in B above, with primers having SEQ ID NOs. 15 and 61 (set forth above) and primers having SEQ ID NOs. 62 and 24 as follows: 5 ' AAACAACGTAAAAGATCCAAACCTGAAA 3 ' (SEQ ID NO . 62) 5 ' GGATCCGCCTCGTTTGACTACTT 3 ' ( SEQ ID NO . 24 ) .
  • the resulting fragment was digested with NdellBamHl and inserted into pET15b (Novagen, Madison, WI), which has a His-TagT leader sequence (SEQ ID NO. 23), that had also been digested with NdellBamHl.
  • the sequence of SAP-Cys-1 is set forth in SEQ ID NO. 58).
  • This construction was performed similarly to the SAP-Cys-1 using pZlB as the starting material, and splice overlap extension (SOE) using PZlB as the starting plasmid, including mutagenesis of the internal BamHl site at nucleotides 555-560 (SEQ ID NO. 34) in pZlB using a sense primer corresponding to nucleotides 543-570 (SEQ ID NO. 34) but changing the G at nucleotide 555 (the third position in the Lys codon) to an A and introduction of the cys at position 4 or 10 in place of the native amino acid.
  • the first round of amplification used primers of SEQ ID NOs.
  • Amplification conditions were as follows: denaturation for 1 min at 94° C, annealing for 2 min at 70° C and extension for 2 min at 72° C for 35 cycles. Individual fragments were gel purified and subjected to a second round of amplification, following the same protocol, using only the external oligonucleotides of SEQ ID NO. 24 and SEQ ID NO. 59 for the cys+4 mutant or SEQ ID NOs. 60 and 24 for the cys+10 mutant. The resulting fragments had a Ndel site on the 5' end of the saporin-encoding DNA and a BamHl site for cloning and a stop codon on the 3' end.
  • DNA encoding unmodified SAP (EXAMPLE 1 ) can be similarly inserted into a pET15b or pETl IA and expressed as described below for the modified SAP-encoding DNA.
  • Lysis buffer (20 mM NaPO pH 7.0, 5 mM EDTA, 5 M EGTA. 1 mM D ' lT, 0.5 ⁇ g/ml leupeptin, 1 ⁇ g/ml aprotinin, 0.7 ⁇ g/ml pepstatin) was added to the rSAP cell paste (produced from pZ50B in BL21(DE3) cells, as described above) in a ratio of 1.5 ml buffer/g cells. This mixture was evenly suspended via a Polytron homogenizer and passed through a microfluidizer twice.
  • the resulting lysate was centrifuged at 50.000 rpm for 45 min.
  • the supernatant was diluted with SP Buffer A (20 mM NaP ⁇ 4, 1 mM EDTA. pH 7.0) so that the conductivity was below 2.5 mS/cm.
  • the diluted lysate supernatant was then loaded onto a SP-Sepharose column, and a linear gradient of 0 to 30% SP Buffer B (1 M NaCl, 20 mM NaP ⁇ 4, 1 mM EDTA, pH 7.0) in SP Buffer A with a total of 6 column volumes was applied. Fractions containing rSAP were combined and the resulting rSAP had a purity of greater than 90%.
  • a buffer exchange step was used to get the eluate into a buffer containing 50 mM NaBO ⁇ , 1 mM EDTA, pH 8.5 (S Buffer A). This sample was then applied to a Resource S column (Pharmacia, Sweden) pre-equilibrated with S Buffer A. Pure rSAP was eluted off the column by 10 column volumes of a linear gradient of 0 to 300 mM NaCl in SP Buffer A. The final rSAP was approximately 98% pure and the overall yield of rSAP was about 50% (the amount of rSAP in crude lysate was determined by ELISA).
  • E. coli strains BL21(DE3), BL21(DE3)pLysS. HMS174(DE3) and HMS174(DE3)pLysS were purchased from Novagen. Madison. WI.
  • restriction and modification enzymes employed here are commercially available in the U.S. Minipreparation and maxipreparations of plasmids. preparation of competent cells, transformation. Ml 3 manipulation, bacterial media and Western blotting were performed using routine methods (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. NY). The purification of DNA fragments was done using the Geneclean II kit, purchased from Bio 101. SDS gel electrophoresis was performed on a Phastsystem (Pharmacia).
  • the plasmid PZ1B1 contains DNA encoding FGF linked to DNA encoding saporin via a spacer region encoding two amino acids (Ala-Met).
  • the fusion gene is cloned into the Ndel and BamHl sites of the plasmid vector pETl la (Novagen).
  • the vector provides a T71ac promoter, a lac operator, and a ribosome binding site upstream of the fusion gene, and a T7 terminator downstream of the fusion gene.
  • PZ1B1 was digested with the restriction enzymes Ndel and Ncol.
  • Ndel cuts once within the plasmid at a position encompassing the translation initiation codon (ATG) for FGF.
  • Ncol also cuts once within the plasmid at a site within the two amino acid linker region of the plasmid PZ1B1.
  • Digestion of PZ1B 1 with Ndel and Ncol thus generates two fragments: a FGF-fragment and a fragment containing vector (pETl la) and saporin-encoding sequence.
  • the digestion products were resolved in an agarose gel and the vector/saporin fragment was purified using the Geneclean II kit.
  • No. 08/598.082 contains a 2.36 kb human HBEGF cDNA fragment (with added EcoRl linkers) cloned into the EcoRl site of pUC9 (see, e.g.. Viera et al. (1982) Gene 19:259-
  • HBEGF HBEGF
  • Any plasmid that contains the cDNA encoding full length precursor protein e.g., pMTN-HBEGF, ATCC #40900 and pAX- HBEGF, ATCC #40899 may also be used for HBEGF amplification as described herein.
  • the region of the DNA that encodes a 77 amino acid form of mature HBEGF (corresponding to nucleotides 217-447 or amino acids 73-149 in the precursor) was amplified.
  • the primer (SEQ ID NO. 25) corresponding to the "sense" strand of mature HBEGF includes an Ndel restriction site adaptor (and a Met codon) upstream of the codon for amino acid 73 of precursor HBEGF (SEQ ID No. 1 ) and spans the first 14 nucleotides of the DNA sequence encoding mature HBEGF (SEQ ID NO. 3):
  • HBEGF complements 23 nucleotides encoding amino acids 144-149 of the precursor peptide and introduces an Real restriction site downstream of the HBEGF-encoding DNA (SEQ NO. 26):
  • amplification reaction was performed in which plasmid pJMU2-l DNA (200 ng) was mixed in a final volume of 100 ⁇ l containing 10 mM Tris-HCl (pH 8.3). 50 mM KCl, 1.5 mM MgCl2, 200 ⁇ M dNTPs, 100 pmole of each primer, and 2.5 U TaqI polymerase (Boehringer Mannheim). Amplifications were done in a TwinBlock DNA thermal cycler (Ericomp). The first cycle was a denaturation step (94°C for 5 min).
  • the second cycle was repeated 30 times and included a denaturation step, an annealing step, and an elongation step (94°C for 1 min; 62°C for 2 min; 72 °C for 2 min).
  • the third cycle was an elongation step (72 °C for 7 min).
  • An aliquot of the reaction was run on an agarose gel and the amplified product was purified using the Geneclean II kit (Bio 101 ).
  • the purified amplified product encoding HBEGF was then digested with Ndel and Real (which generates an end compatible with Ncol) and ligated into the NdellNcol sites of PZlB 1.
  • Ndel and Real which generates an end compatible with Ncol
  • PZlB 1 The purified amplified product encoding HBEGF was then digested with Ndel and Real (which generates an end compatible with Ncol) and ligated into the NdellNcol sites of PZlB 1.
  • NovaBlue Novagen
  • a positive clone designated pZ31Bl was sequenced starting within the vector sequence (using the T7 promoter primer) and extending through the HBEGF-coding sequence, the Val-Met two amino acid linker (generated by ligation at the 7?c ⁇ l and Ncol sites), and into the saporin sequence.
  • the positive pZ31Bl plasmid gave the proper nucleotide sequence (i.e., SEQ ID NO. 6) for the HBEGF-SAP fusion gene.
  • the fusion protein encoded by the plasmid pZ31Bl contains 78 amino acids of HB-EGF (including a methionine introduced by the Ndel restriction site), a two amino acid (Val-Met) linker and 253 amino acids of saporin (SEQ ID NO. 6).
  • the pZ31Bl encoded fusion protein is therefore 333 amino acids long with a predicted molecular weight of about 37.6 kD and an isoelectric point of 9.6.
  • HBEGF-SAP fusion protein recombinant HBEGF-SAP protein encoded by pZ31 B l .
  • Plasmid pZ31Bl was transformed into E. coli strain BL21(DE3). which contains chromosomal copies of the T7 RNA polymerase gene linked to an IPTG-inducible lacUV promoter.
  • the transformed E. coli cells were grown in 50 ml cultures of LB broth + ampiciilin (100 ⁇ g/ml) at 30°C to an OD600 o 0.7. The second stage was commenced by the addition of IPTG (0.1 mM) to induce expression of the T7 RNA polymerase gene. Cultures were continued at 30° C. One ml aliquots of the culture were removed just prior to IPTG addition and then hourly thereafter. Aliquots were centrifuged. resuspended in 1 ml lysis buffer (10 mM Tris pH 8.0. 2 mM EDTA. 0.01 mg/ml lysozyme. 10 mM DTT) and incubated for 1 hour at room temperature.
  • the Western blot analysis demonstrated induction of a soluble protein with the predicted molecular weight. Aliquots of the bacterial lysates were also analyzed by an ELISA assay (using an anti-SAP antibody). The results of this assay confirmed the induced expression of HBEGF-SAP.
  • the column was washed with buffer B (10 mM Na-Phosphate, 5 mM EDTA, 5 mM EGTA at pH 8.0) containing 0.2 M NaCl until the A280 of the eluate reached baseline.
  • the HBEGF fusion protein was eluted with buffer B containing 0.8 M NaCl.
  • HBEGF-containing fractions were pooled, diluted 4x with buffer B and applied to a Q-Sepharose (Pharmacia) column equilibrated with buffer B. The flow through was applied directly to a SP-Sepharose Fast Flow cation exchange column (Pharmacia) equilibrated with buffer B containing 0.2 M NaCl. The HBEGF fusion protein was eluted with a 0.2-1.0 M NaCl gradient.
  • Fractions containing fusion protein were pooled and loaded onto a heparin-Sepharose CL6B (Pharmacia) column equilibrated with buffer C (10 mM NaCitrate-pH 6.0, 1 mM EDTA, 1 mM EGTA, 0.2 M NaCl). The fusion protein was eluted with a 0.2-1.2 M NaCl gradient. Fractions containing fusion protein were pooled and NH4SO4 was added to 2.0 M. Following filtration, the material was applied to a Phenyl-Sepharose HP column equilibrated with buffer C containing 2 M NH4SO4.
  • the fusion protein was eluted with a 2.0-0.0 M NH4SO4 gradient. Fractions containing fusion protein were pooled and applied to a SI 00 size exclusion column equilibrated with 10 mM NaCitrate (pH 6.0). 0.1 M EDTA, 0.14 M NaCl. Fractions containing purified HBEGF fusion protein were then selected. G. Characterization of the HBEGF-SAP fusion protein
  • HBEGF fusion protein encoded by pZ31 B l was assayed as described in Example 1 for saporin. The results indicated that the IC50 of the HBEGF-SAP fusion protein exhibits activity in this assay.
  • Cytotoxicity experiments are performed with the Promega (Madison. WI) CellTiter 96 Cell Proliferation/Cytotoxicity Assay. About 1,500 A431 cells (ATCC Accession No. CRL 1555). an epidermoid carcinoma cell line, are plated per well in a 96 well plate in 90 ⁇ l HDMEM plus 10% FCS and incubated overnight at 37° C. 5% C ⁇ 2- The following morning 10 ⁇ l of media alone or 10 ⁇ l of media containing various concentrations of the fusion protein, HBEGF polypeptide or saporin are added to the wells. The plate is incubated for 72 hours at 37 C.
  • the number of living cells is determined by measuring the incorporation and conversion of the commonly available dye MTT supplied as a part of the Promega kit. Fifteen ⁇ l of the MTT solution is added to each well, and incubation is continued for approximately 4 hours. Next, 100 ⁇ l of the standard solubilization solution supplied as a part of the Promega kit is added to each well. The plate is allowed to stand overnight at room temperature and the absorbance at 560 nm is read on an ELISA plate reader (e.g.. Titertek Multiskan PLUS, ICN, Flow. Costa Mesa. CA).
  • an ELISA plate reader e.g. Titertek Multiskan PLUS, ICN, Flow. Costa Mesa. CA.
  • reaction mixtures are treated 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 HBEGF-SAP conjugate should be cytotoxic to each cell type that expresses an EGF receptor.
  • Plasmid plasmid pJMU2-l as described in Example 4, was used as the amplification template for preparatin of the linker-amenable HBEGF-SAP plasmid pZ32Bl .
  • HBEGF was mutated in separate amplification reactions.
  • a "sense" primer was constructed that corresponds to the nucleotides encoding amino acids 13-19 (SEQ ID NO:
  • nucleotides 37-57 in the HBEGF precursor and includes a P ⁇ tl site SEQ ID NO. 51:
  • an "antisense" primer spanning the nucleotides that encode amino acids 1 14 to 129 in the HBEGF precursor was designed that introduces a single base mutation (T ⁇ C in the sense strand) that destroys an Ncol site while maintaining a codon for the amino acid histidine at position 1 18 (SEQ ID NO. 29):
  • the antisense primer also spans a S ⁇ cl site. Amplification of the DNA encoding HBEGF using these two primers generates a fragment with a Pstl site at its 5' end and a S ⁇ cl site at the 3' end. The amplification was carried out under the same conditions previously described in Example 4.C., except that the second cycle was repeated only 20 times.
  • the second Ncol site within the mature HBEGF coding sequence was mutated using a "sense" primer that spans the nucleotides encoding amino acids 124-142 in the HBEGF precursor (SEQ ID NO. 30):
  • This sense primer includes a S ⁇ cl site, and introduces a single base mutation (C ⁇ T) that destroys the Ncol site while maintaining a codon for the amino acid tyrosine at position 138 of SEQ ID NO. 1.
  • An antisense primer that spans a region of the HBEGF-encoding DNA downstream of the precursor HBEGF coding region was designed to introduce an EcoRl site adaptor at the 3' end of the amplified DNA fragment (S ⁇ Q ID NO. 31 ):
  • Amplification of HB ⁇ GF-encoding DNA using these two primers generates a fragment with a S ⁇ cl site at its 5' end and an EcoRl site at the 3' end.
  • the amplified HB ⁇ GF fragments generated by the two above amplification reactions overlap at a S ⁇ cl site.
  • the first product was digested with Pstl and S ⁇ cl and the second product was digested with S ⁇ cl and EcoRl.
  • the digested fragments were ligated into the Pstl and EcoRl sites of the vector pG ⁇ M-4 (the pGEM series of plasmids are available from Promega, Madison WI; see also. U.S. Patent No. 4,766.072.
  • This plasmid contains a regenerated colinear piece of DNA encompassing the entire mature HBEGF coding region (see, e.g., nucleotides 1-234 of SEQ ID No. 33).
  • the mature HBEGF encoding region was amplified using the primers set forth in SEQ ID NO. 25 that corresponds to the "sense" strand of mature HBEGF including an Noel restriction site adaptor just upstream of the codon for amino acid 73 of precursor HBEGF.
  • the other primer corresponds to the "antisense” strand of HBEGF spanning nucleotides encoding amino acids 143-149 of precursor HBEGF (see, e.g., SEQ. ID NO. 1). and introduces an Ala-Met-Mcfil restriction site just downstream of mature HBEGF-encoding DNA (SEQ ID NO. 32):
  • HBEGF sequences using these two primers and the above pGEM/HBEGF plasmid as a template, generates a mature HBEGF encoding fragment with an Ndel site at the 5' end and a unique Ncol site at the 3' end.
  • An aliquot of the amplification reaction was run on an agarose gel and the amplified product was purified using the Geneclean II kit.
  • the purified D ⁇ A was then digested with Noel and Ncol and ligated into the ⁇ del ⁇ col sites of pZlBl (digested to remove FGF-encoding D ⁇ A. as described in Example 4. B.).
  • pZ32Bl plasmid was sequenced starting within the vector sequence (using the T7 promoter primer) and extending through the HBEGF-coding sequence, the two amino acid Ala-Met linker and into the saporin sequence.
  • the plasmid pZ32Bl gave the desired sequence for the HBEGF-SAP fusion gene set forth in SEQ ID NO. 33.
  • the fusion protein encoded by the plasmid pZ32Bl includes 78 amino acids of HBEGF (including a methionine introduced by the N ⁇ cI restriction site), a two amino acid (Ala-Met) linker and 253 amino acids of saporin (SEQ ID NO. 33).
  • the fusion protein is therefore 333 amino acids long with a predicted molecular weight of about 37.6 kD and an isoelectric point of 9.6.
  • the resulting linker-amenable HBEGF-SAP plasmid pZ32Bl differs from the HBEGF-SAP encoding plasmid PZ31B1 described in Example 4.D. in the following ways: 1 ) Two Ncol sites within the coding region for mature HBEGF have been mutated (by amplification) so that the Ncol sites are destroyed without changing the reading frame or amino acid composition of HBEGF.
  • the two amino acid linker between HBEGF and SAP is Ala-Met in the new plasmid construct pZ32Bl .
  • This Ala-Met linker encompasses an Ncol site (the only Ncol site in the new plasmid). Therefore, the resulting HBEGF-SAP plasmid can be linearized by digestion with Ncol. Different linkers, which have Ncol sites at their ends, can then be inserted between the HBEGF and SAP sequences. The desired linker is then inserted into plasmid pZ32B 1.
  • the resulting plasmid is introduced into E. coli host cells, expressed and the fusion proteins isolated as described in Example 4.F. Fusion proteins may also be isolated using the same procedures as those described for HBEGF (see, e.g.. International PCT Application WO 92/06705, which is based on U.S. Application Serial No. 08/598.082).
  • MCS multiple cloning sites
  • SAP cassette plasmid (PETSAP-MCS) was made that would be amenable to insertion of any growth factor sequence downstream from the saporin- encoding DNA.
  • SAP encoding DNA was amplified (using PZ32B1 as a template) to give
  • SEQ ID NO. 81 The sense and antisense primers respectively, used to amplify this SAP fragment were:
  • SEQ ID NO. 55 5'TATATGAA ⁇ CCATGGCCT ⁇ GGTTTGCCCAAATAr.AT
  • the resulting SAP-encoding DNA has an EcoRl site at its 5' end followed by an Ndel site that encompasses the ATG codon.
  • the 3' end of the SAP fragment has no stop codon. and has an Ncol site followed by an EcoRl restriction site.
  • the amplified SAP fragment was then digested with EcoRl and subcloned into the EcoRl site of the plasmid pG ⁇ M-4 (pGEM-4 serves as the source of the MCS.
  • pGEM-4 serves as the source of the MCS.
  • the pGEM series of plasmids are available from Promega, Madison WI; see also. U.S. Patent No. 4,766,072, which describes construction of the pGEM plasmids) in such an orientation that the multicloning site (MCS) of pGEM-4 lies downstream (3' of) from the SAP -encoding DNA.
  • Plasmid pGEMSAP was digested with Pstl and the ends of the fragment were blunt-ended. The fragment was then digested with No l, thereby generating a fragment that contains all of the saporin-encoding D ⁇ A and most of the MCS of pGEM-4. This fragment was then cloned into the NdellBamHl sites of pET 1 1 a. in which the BamHl site had been blunt-ended by filling in with Klenow polymerase. The resulting plasmid was designated PETSAP-MCS. It has unique S ⁇ cl. Smal. and Sail sites in the MCS for insertion of D ⁇ A encoding a desired linker. HBEGF, or combination of HBEGF and linker downstream from (3' of) the saporin-encoding D ⁇ A. EXAMPLE 7
  • Complementary single-stranded oligos in which the sense strand encodes a protease substrate or flexible linker have been synthesized either using a Cyclone machine (MILLIPORE, Bedford. MA) according the instructions provided by the manufacturer or, if greater than 80 bases, were made by Midland Certified Reagent Co.
  • oligos have been synthesized and can be introduced into constructs encoding HBEGF-SAP or SAP-HBEGF.
  • HBEGF-encoding DNA was amplified (using PZ32B1 as a template) to produce a Ncol site at the 5' end and a stop codon followed by a Sail site at the 3' end.
  • the sense and antisense primers, respectively, used in the amplification reaction were: SEQ ID No . 52 : 5 ' -TATATGCXATJ ⁇ CCAGAGTCACT ⁇ ATCCTCCAAG H ⁇ l
  • the resulting amplified product was digested with Ncol and Sail and ligated into Ncol/Sall digested PETSAP-MCS.
  • the resulting plasmid (PZ36B1 ) encodes a protein with an Ala-Met-Ala linker between the SAP and HBEGF moieties (SEQ ID No. 82).
  • Plasmid PZ36B 1 was digested with Ncol and a linker encoding Ala-Met- (Gly4Ser)4-Ala-Met-Ala was inserted. The resulting plasmid was designated PZ37B1.
  • Table 3 are expressed above for PZ31 B 1 using plasmids prepared as described above and summarized in TABLE 3.
  • HBEGF constructs have demonstrated inducible expression of proteins of the expected size when analyzed by Western blotting.
  • protocol set forth in Example 4. F. purification (to greater than 95%) of HBEGF-Ala-Met- Gly4SerGly2Ser (Gly4Ser)2-Ala-Met-SAP and SAP-Ala-Met-(Gly4Ser) x 4-Ala-Met- Ala-HBEGF has been achieved.
  • the fusion protein HBEGF-Val-Met-SAP (encoded by plasmid PZ31 B 1 ) was active in the cell-free RIP assay
  • Insertion of the (Gly4Ser) x 4 linker into HBEGF-SAP has generated a fusion protein with cytotoxicity to A431 cells (ID50 on the order of 10 ' 1 -10 M).
  • the purified fusion protein SAP-Ala-Met-(Gly4Ser) x 4-Ala-Met-Ala-HBEGF exhibits similar, perhaps somewhat higher, cytotoxicity.
  • These two HBEGF fusion proteins have also been tested for their cytotoxicity (relative to FGF-SAP) using other cell lines including aortic smooth muscle cells (active), glioblastoma and medulloblastoma cells (active), SK-MEL melanoma cells (somewhat active), and small cell lung carcinoma cells (inactive). Therefore, there are cell-type differences in the cytotoxicity of these proteins.
  • Met-Cys-HBEGF Met-Cys-Ala-Met-Ala-HBEGF (linker amenable), Met-Cys-Ala- Met-(Gly4Ser)2-Ala-Met-Ala-HBEGF (prepared by insertion of linker into the Ncol site of Met-Cys-Ala-Met-Ala-HBEGF), and Met-Cys-Ala-Met-(Gly4Ser) x 4-Ala-Met- Ala-HBEGF (prepared by insertion of linker into the Ncol site of Met-Cys-Ala-Met- Ala-HBEGF).
  • a linker-amenable HBEGF/BlueBac clone was made by amplifying HBEGF sequences as above using a different sense primer (SEQ ID NO. 86):
  • the resulting amplified fragment (SEQ ID No. 84) has a BamHl site at the 5' end followed by Met-Cys-Ala-Met-Ala (SEQ ID NO. 85) codons that encompass a Ncol site.
  • Balloon catheter denudation is performed on the left carotid artery of 5-6 month old male Sprague-Dawley rats by intraluminal passage of a 2F Fogarty balloon. Body weights range from 300-350 g the day prior to surgery. At 0. 3. 6. 9 days after balloon injury, wild-type chemical conjugate HBEGF-SAP ( 1-10 ⁇ g/kg/dose). fusion protein HBEGF-SAP (1 -10 ⁇ g/kg/dose). or vehicle (0.9% NaCl, 0.1% human serum albumin (HSA)) is injected via the tail vein. The therapeutic composition is prepared by mixing the test materials with appropriate volumes of 0.9% NaCl, 0.1% HSA.
  • HSA human serum albumin
  • the wild-type chemical conjugate is supplied in l O mM citrate, 0.14 m NaCl. 0.1 mM EDTA, pH 6 at a concentration of 1.0 mg/ml prior to being prepared in appropriate dosages.
  • the fusion protein is supplied in lO mM citrate. 0.14 m NaCl. 0.1 M EDTA, pH 6 at a concentration of 0.256 mg/ml prior to being prepared in appropriate dosages.
  • animals are sacrificed with an overdose of intravenous KCl under deep anesthesia.
  • animals are injected intravenously with Evans blue dye (0.5 ml.
  • Tissue samples are dehydrated and embedded in paraffin, cut into 4 ⁇ sections, and stained with hematoxylin-eosin and Movat pentachrome stain. Vessels are then measured for intimal, medial, and neointimal areas by computerized planimetry.
  • Anti- BrdU antibody is used for detection of BrdU positive cells; smooth muscle cell proliferation is quantitated by counting BrdU positive cells as a percent of total smooth muscle cells.
  • the in vivo mouse solid tumor xenograft model which assays for a compound's ability to inhibit tumor cell proliferation, has been described, in Beitz et al. (1992) Cancer Res. 52.227-230.
  • wild-type chemical conjugate and fusion protein HBEGF-SAP are evaluated for anti-tumor activity against any EGF-receptor expressing tumor subtype, e.g., bladder carcinoma, in a mouse tumor xenograft model.
  • HBEGF-SAP wild-type chemical conjugate HBEGF-SAP
  • fusion protein HBEGF -SAP 0.5, 5.0, and 50 ⁇ g/kg
  • SAP only 85 ⁇ g/kg
  • HBEGF only 50 ⁇ g/kg
  • SAP with HBEGF 85 and 50 ⁇ g/kg. respectively
  • Dosing material is prepared by mixing the test material with appropriate volumes of PBS/0.1% BSA to achieve the desired doses. Individual syringes are prepared for each animal. Mice receive four weekly IV injections (250-300 ul) into the tail vein on days 5, 12, 19 and 26 with day 1 designated as the day that the tumor cells are injected into the mice. Doses are individualized for differences in body weight. Tumor volume is measured twice weekly for a period of 61 days.
  • mice Female Balb/c nu/nu athymic mice (Roger Williams Hospital Animal
  • Sixty-three animals are selected for the study such that body weights range from 25-30 grams the day prior to dosing. Animals are maintained in a quarantined room and handled under aseptic conditions. Food and water are supplied ad libitum throughout the experiment.
  • mice receive a subcutaneous injection of tumor cells (approximately 2 x 10 cells/mouse) in the right rear flank.
  • Calipers are used to measure the dimensions of each tumor.
  • Purification steps 1 and 2 are performed with crude material being incubated on ice and all other steps are performed at room temperature using FPLC units/Biopilot (Pharmacia) equipped with P6000 pumps. Fractions/pools of the recombinant mitotoxins from the various chromatography steps are analyzed by SDS-
  • Step 1 Preparation of cell extract.
  • Cell paste (900 - 1200 g wet weight) is suspended in 3-4 volumes of ice-cold cell lysis buffer. (lOmM sodium citrate. pH 6.0, containing 1 M urea, 5 mM EDTA. 5 mM EGTA and 50 mM NaCl ) and passaged 3 times through a microfluidizer (Microfluidics Corp., Newton, MA, U.S.A.) at 18,000 lb/in ' . The resultant mixture was diluted to 10 volumes with lysis buffer.
  • a microfluidizer Microfluidics Corp., Newton, MA, U.S.A.
  • Step 2 Expanded Bed Adsorption Chromatography (EBAC). Crude cell extract is loaded onto an expanded bed (300 ml of resin in a 5 x 100 cm column) of Streamline SP cation - exchange resin which is previously equilibrated with lysis buffer at 70 ml/min upwards flow. After loading, the resin is washed with the same buffer until the resin appears clear. The plunger is then slowly (1 - 2 cm/min) lowered. When the plunger nears the expanded bed and the A 280 decreases to zero, the flow is stopped and the resin is allowed to settle. Once the plunger is 0.5 cm from the packed bed. proteins are eluted using 2 buffers containing increasing NaCl concentrations.
  • the column is first washed with buffer A (10 mM sodium phosphate, pH 8.0. containing 5 mM EDTA. 5 mM EGTA and 0.25 M NaCl ). After A 280 reaches zero, buffer B (buffer A with 0.8 M NaCl ) is applied. This eluate contains the conjugate which is subsequently diluted 1 :3 (v/v) with buffer C (buffer A containing no NaCl ) before being subjected to anion-exchange chromatography.
  • buffer A 10 mM sodium phosphate, pH 8.0. containing 5 mM EDTA. 5 mM EGTA and 0.25 M NaCl .
  • buffer B buffer A with 0.8 M NaCl
  • This eluate contains the conjugate which is subsequently diluted 1 :3 (v/v) with buffer C (buffer A containing no NaCl ) before being subjected to anion-exchange chromatography.
  • Step 3 Q-Sepharose anion-exchange chromatography and SP- Sepharose cation-exchange chromatography.
  • Q-Sepharose removes contaminating E. coli proteins and DNA as well as endotoxin.
  • the diluted HB-EGF-SAP pool from the previous step is loaded onto a column (2.6 x 7 cm) of Q-Sepharose FF directly connected to a column (2.6 x 13) of SP-sepharose HP. Both columns are previously equilibrated in tandem with buffer A containing 0.2 M NaCl .
  • the pi of the conjugate is above 9.5, it does not bind to the Q-Sepharose resin, but directly flows through and binds to S-Sepharose resin.
  • the anion- exchange column is disconnected from the cation-exchange column. Proteins bound to the S-Sepharose column are eluted with a gradient (10 column volumes) of 0.25 to 1 M NaCl in buffer A.
  • Step 4 Hydrophobic Interactions Chromatography (H1C). Fractions containing the conjugate are pooled, and solid ammonium sulfate is added (on ice and stirring) over a period of 15-20 min to a final concentration of 2 M. The pool is passed through a 0.8 ⁇ filter and loaded onto a Phenyl-Sepharose HP column (2.6 x 10 cm) previously equilibrated with buffer D (10 mM sodium citrate, pH 6.0, containing 1 mM EGTA. 1 mM EGTA. 1 mM EDTA and 2 M (NH 4 ) 2 S0 4 ).
  • buffer D (10 mM sodium citrate, pH 6.0, containing 1 mM EGTA. 1 mM EGTA. 1 mM EDTA and 2 M (NH 4 ) 2 S0 4 ).
  • Pool A is dialyzed against formulation buffer (10 mM sodium citrate, pH 6.0, containing 0.1 mM EDTA and 0.14 M NaCl ) and subjected to size-exclusion chromatography (Step 5).
  • Pool B is dialyzed against buffer E (10 mM sodium phosphate buffer, pH 8.5, containing 5 mM EDTA and 5 mM EGTA) and subjected to another cation-exchange fractionation (Step 4).
  • Step 4B S-Source cation-exchange chromatography.
  • Pool B is applied to an S-Source column (2.6 x 5.7) previously equilibrated with buffer E and bound proteins are eluted with a gradient (10 column volumes) of 0 lo 1 M NaCl in buffer E.
  • Two pools (C and D) are made on the basis of SDS-PAGE analysis.
  • Pool C contained an E. coli a major contaminant ( ⁇ 25-27 KDa). which is difficult to remove.
  • Pool D is then subjected to size-exclusion chromatography (Step 5).
  • Step 5 Size exclusion chromatography. Both pools are passed separately through a suitably sized (i.e., the sample load volume is 10-15% of the total column volume) column containing SI 00 resin previously equilibrated with formulation buffer. From 1 kg. of wet weight paste, approximately 50 mg of purified FPHS5 (Pool A) and 10 mg of Pool D are recovered. At least 3 isoforms are apparent. Various analytical methods reveal the conjugates to be over 95% pure.
  • GAA TGC AAA TAT GTG AAG GAG CTC CGG GCT CCC TCC TGC ATC TGC CAC 192 Glu Cys Lys Tyr Val Lys Glu Leu Arg Ala Pro Ser Cys He Cys His 50 55 60
  • AAA AAC GAA GCT AGG TTT CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG 768 Lys Asn Glu Ala Arg Phe Leu Leu He Ala He Gin Met Thr Ala Glu 245 250 255

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Abstract

Conjugués d'un facteur de croissance (HBEGF) semblable au facteur de croissance épidermique de type HBGF, lié, directement ou par l'intermédiaire d'un agent de liaison, à un agent à cible définie. L'agent à cible définie est un agent cytotoxique tel qu'une protéine d'inactivation de ribosomes (RIP) et un acide nucléique anti-sens, ou bien un acide nucléique thérapeutique adapté pour être administré au niveau d'une cible définie. L'agent à cible définie est fixé au HBEGF, ou par l'intermédiaire d'un agent de liaison, au moyen d'une liaison chimique, ou bien le conjugué est préparé sous forme de chimère à l'aide des techniques de l'ADN recombiné. Les conjugués servent à diriger les agents cytotoxiques ou les nucléotides thérapeutiques vers des cellules portant des récepteurs du facteur de croissance épidermique, et trouvent une application notamment dans le traitement des tumeurs solides telles que les tumeurs du sein et de la vessie, et dans le traitement des affections impliquant une prolifération pathophysiologique des cellules musculaires lisses, par exemple la resténose.
PCT/US1995/012205 1994-09-13 1995-09-13 Conjugues d'un facteur de croissance semblable au facteur de croissance epidermique de type hbgf, et d'agents a cible definie WO1996008274A2 (fr)

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US7179608B2 (en) 1995-10-23 2007-02-20 The Children's Medical Center Corporation Therapeutic antiangiogenic compositions and methods
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US6838238B1 (en) 1996-10-17 2005-01-04 Invitrogen Corporation Morphatides: novel shape and structure libraries
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