AU710309B2 - Compositions containing nucleic acids and ligands for therapeutic treatment - Google Patents

Compositions containing nucleic acids and ligands for therapeutic treatment Download PDF

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AU710309B2
AU710309B2 AU58628/96A AU5862896A AU710309B2 AU 710309 B2 AU710309 B2 AU 710309B2 AU 58628/96 A AU58628/96 A AU 58628/96A AU 5862896 A AU5862896 A AU 5862896A AU 710309 B2 AU710309 B2 AU 710309B2
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receptor
nucleic acid
fgf
dna
composition
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J. Andrew Baird
Lois A. Chandler
Barbara A Sosnowski
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Selective Genetics Inc
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Description

WO 96/36362 PCT/US96/07164 1 Description COMPOSITIONS CONTAINING NUCLEIC ACIDS AND LIGANDS FOR THERAPEUTIC TREATMENT Technical Field The present invention relates generally to the treatment of diseases, and more specifically, to the preparation and use of complexes containing receptor-binding internalized ligands NABD and cytocide-encoding agents to alter the function, gene expression, or viability of a cell in a therapeutic manner.
Background of the Invention A major goal of treatment of neoplastic diseases and hyperproliferative disorders is to ablate the abnormally growing cells while leaving normal cells untouched. Various methods are under development for providing treatment, but none provide the requisite degree of specificity.
One method of treatment is to provide toxins. Immunotoxins and cytotoxins are protein conjugates of toxin molecules with either antibodies or factors which bind to receptors on target cells. Three major problems may limit the usefulness of immunotoxins. First, the antibodies may react with more than one cell surface molecule, thereby effecting delivery to multiple cell types, possibly including normal cells. Second, even if the antibody is specific, the antibody reactive molecule may be present on normal cells. Third, the toxin molecule may be toxic to cells prior to delivery and internalization. Cytotoxins suffer from similar disadvantages of specificity and toxicity. Another limitation in the therapeutic use of immunotoxins and cytotoxins is the relatively low ratio of therapeutic to toxic dosage. Additionally, it may be difficult to direct sufficient concentrations of the toxin into the cytoplasm and intracellular compartments in which the agent can exert its desired activity.
Given these limitations, cytotoxic therapy has been attempted using viral vectors to deliver DNA encoding the toxins into cells. If eukaryotic viruses are used, such as the retroviruses currently in use, they may recombine with host DNA to produce infectious virus. Moreover, because retroviral vectors are often inactivated by the WO 96/36362 PCTIUS96/07164 2 complement system, use in vivo is limited. Retroviral vectors also lack specificity in delivery; receptors for most viral vectors are present on a large fraction, if not all, cells.
Thus, infection with such a viral vector will infect normal as well as abnormal cells.
Because of this general infection mechanism, it is not desirable for the viral vector to directly encode a cytotoxic molecule.
While delivery of nucleic acids offers advantages over delivery of cytotoxic proteins such as reduced toxicity prior to internalization, there is a need for high specificity of delivery, which is currently unavailable with the present systems.
In view of the problems associated with gene therapy, there is a compelling need for improved treatments which are more effective and are not associated with such disadvantages. The present invention exploits the use of conjugates which have increased specificity and deliver higher amounts of nucleic acids to targeted cells, while providing other related advantages.
Summary of the Invention The present invention generally provides therapeutic compositions. In one aspect, the composition has the formula: receptor-binding internalized ligand--nucleic acid binding domain-cytocide-encoding agent. The receptor-binding internalized ligand is a polypeptide reactive with a cell surface receptor, the nucleic acid binding domain binds to a nucleic acid, the cytocide-encoding agent is a nucleic acid molecule encoding a cytocide and which binds to the nucleic acid binding domain, and the composition binds to the cell surface receptor and internalizes the cytocide-encoding agent in cells bearing the receptor. In another aspect, the composition has the formula: receptor-binding internalized ligand-nucleic acid binding comain-prodrug-encoding agent.
In certain embodiments, the receptor-binding internalized ligand is a polypeptide reactive with an FGF receptor, VEGF receptor, HBEGF receptor, or a cytokine. In other embodiments, the cytocide-encoding agent encodes a protein that inhibits protein synthesis and is preferably a ribosome inactivating protein, most preferably saporin. The protein is gelonin or diphtheria toxin in other embodiments. In other embodiments, the prodrug-encoding agent encodes HSV-thymidine kinase.
WO 96/36362 PCTfUS96/07164 3 The nucleic acid binding domain is poly-L-lysine in one embodiment. In other embodiments, the nucleic acid binding domain is a transcription factor selected from the group consisting of helix-turn-helix motif proteins, homeodomain proteins, zinc finger motif proteins, steroid receptor proteins, leucine zipper motif proteins, helixloop-helix motif proteins, and P-sheet motif proteins. In other embodiments, the nucleic acid binding domain binds nonspecifically to nucleic acids and is selected from the group consisting of poly-L-lysine, protamine, histone and spermine. In a preferred embodiment, the nucleic acid binding domain binds the coding region of a ribosome inactivating protein such as saporin. In another preferred embodiment, FGF is conjugated to poly-L-lysine.
In yet other embodiments, the cytocide-encoding agent contains a tissuespecific promoter, such as alpha-crystalline, gamma-crystalline, a-fetoprotein, CEA, prostate-specific antigen, erbB-2, tyrosinase, a-actin, c-myc, VEGF receptor, FGF receptor or cyclin D.
In another aspect, the composition also contains a linker. In various embodiments, the linker increases the flexibility of the conjugate and is (GlymSerp), (Ala Ala Pro Ala),, wherein n is 1 to 6, m is 1 to 6 and p is 1 to 4, or the linker is a disulfide bond.
In yet another aspect, the composition has the formula: receptor-binding internalized ligand-cytocide encoding agent-nucleic acid binding domain, wherein the receptor-binding internalized ligand is conjugated to the cytocide-encoding agent, which is bound to the nucleic acid binding domain to form a complex.
In other aspects, the invention provides methods for preventing excessive cell proliferation in the anterior eye following surgery, treating corneal clouding following excimer laser surgery, preventing closure of a trabeculectomy, preventing pterygii recurrence, treating hyperproliferative diseases in the back of the eye, such as macular degeneration, diabetic retinopathy and proliferative virtreal retinopathy, treating smooth muscle cell hyperplasia after a wound healing response to a procedure, WO 96/36362 PCT/US96/07164 4 vein grafting, endarterectomies and arteriovenous shunts and treating cancer. In these aspects, an effective amount of the compositions described above are administered.
Brief Description of the Drawings Figure 1 is a photograph of an SDS-PAGE of FGF2-K152 under nonreducing (left) and reducing (right) conditions. Lane 1, FGF2-K152; lane 2, FGF2; lane 3, FGF2-K152: lane 4, FGF2. The open arrow identifies material unable to enter the gel. The closed arrow identifies a protein band corresponding to FGF2.
Figure 2 is a graph depicting the proliferation of bovine aortic endothelial cells in response to FGF2 (closed box) and FGF2-K152 (open circle) conjugate.
Figure 3 is a photograph of a gel showing the effects of various lengths of poly-L-lysine on the ability to interact with DNA. Thirty-five ng of labeled DNA were added to increasing concentrations of either FGF2 or FGF2-K: lanes 1, 0 ng; lanes 2, 0.1 ng; lanes 3, 1 ng; lanes 4, 10 ng; lanes 5, 20 ng; lanes 6, 35 ng; lanes 7, 100 ng. Panel A: FGF2; panel B, FGF2-K152; panel C, FGF2-K13; panel D, FGF2- K84; panel E, EGF2-K267; panel F, FGF2-K39. The lengths of the digested DNA are indicated.
Figure 4 is a chart depicting the activity of 0-gal following transfection of FGF2/poly-L-lysine/DNAp-gal into COS cells. Lane 1, 10:1 ratio of FGF2/poly-L-lysine conjugate to DNA; lane 2, 5:1 ratio; lane 3, 2:1 ratio; lane 4, 1:1 ratio; lane 5, 0.5:1 ratio. The five bars, from left to right, are FGF2, FGF2-K13, FGF2- K39, FGF2-K84, and FGF2-K152.
Figure 5 are photographs of toroid format observed by electron microscopy. The upper panel shows an example of a toroid; the lower panel shows an incomplete toroid.
Figure 6 is a graph depicting proliferation of bovine aortic-endothelial cells. In the upper panel, cells were treated with FGF2-K152-DNA; in the lower panel, cells were treated with a mixture of FGF2, K152, and DNA.
WO 96/36362 PCT/US96/07164 Figure 7A is a graph displaying p-gal activity after transfection of FGF2/poly-L-lysine/pSVp-gal into COS cells (lane B16 cells (lane NIH 3T3 cells (lane and BHK cells (lane 4).
Figure 7B is a graph depicting p-gal expression in COS cells, pSVp-gal (lanes 1, 3) or pNASSp-gal (lanes 2, 4) were incubated with (lanes 1, 2) or without (lanes 3, 4) FGF2-K84 and the complexes incubated on COS cells for 48 hrs.
Figure 7C is a graph showing activity of p-gal activity at various times following transfection with either plasmid alone or with complexes of FGF2/K84/pSV P-gal. DNA alone; FGF2-K84-DNA.
Figure 7D is a graph showing p-gal activity after transfection of various concentrations of FGF2/K84/pSVp-gal. Lane 1, Og; lane 2, 0,1 g; lane 3, lug; lane 4, g; lane 5, Figure 8A is a graph showing p-gal activity in COS cells following transfection of FGF2-K84-pSVp-gal (lane FGF2+K84+pSVp-gal (lane 2), FGF2+pSVp-gal (lane K84+pSVp-gal (lane pSVp-gal (lane FGF2-K84 (lane FGF2 (lane 7) and K84 (lane 8).
Figure 8B is a graph showing completion for cell bindings. Lane 1, FGF2-K84-pSVp-gal complex transfected into COS cells; lane 2, FGF2-K84-pSVp-gal plus 100 pg FGF2; lane 3, no complex.
Figure 8C is a graph showing the attenuation of p-gal activity upon the addition of heparin during transfection. Lane 1, FGF2-K84-pSVp-gal+l10g heparin; lane 2, FGF2-K84-pSVp-gal; lane 3, heparin alone; lane 4, pSVp-gal alone.
Figure 8D is a graph showing ligand targeting of DNA, pSVp-gal DNA alone (lane FGF2-K84 (lane histone HI-K84 (lane 3) and cytochrome C-K84 (lane 4) were condensed with pSVp-gal DNA and added to BHK cells. p-gal activity was measured 48 hr later.
Figure 9A is a graph showing the effect of chloroquine on p-gal expression, pSVp-gal and FGF2-K84 were mixed in the absence (lane 1) or presence (lane 2) of 100 pM chloroquine and incubated for 1 hr at room temperature prior to addition of the complexes to COS cells. Lane 3, chloroquine alone; lane 4, DNA alone.
WO 96/36362 PCT/US96/07164 6 Figure 9B is a graph showing the effect of endosome disruptive peptide on P-gal expression. Lane 1, control; lane 2, FGF2-K84-pSVp-gal; lane 3, FGF2-K84pSVp-gal+EDP.
Figure 9C are photographs of cells stained for p-gal activity following transfection of COS cells with (right panel) or without (left panel) endosome disruptive peptide and FGF2-K84-pSVp-gal.
Figure 10 is a photograph of a fluorograph analyzing cell-free translation products. Lane 1, no RNA; lane 2, saporin RNA; lane 3, luciferase RNA; lane 4, saporin RNA and luciferase RNA; lane 5, saporin RNA followed 30 min later with luciferase RNA.
Figure 11 is a graph depicting direct cytotoxicity of cells transfected by a CaPO 4 with an expression vector encoding saporin. Lane 1, mock transfection; lane 2, transfection with pSVp-gal; lane 3, transfection with saporin-containing vector.
Figure 12 is a pair of graphs showing cytotoxicity of cells transfected with FGF2-K84-pSVSAP. Left panel, BHK21 cells; right panel, NIH 3T3 cells. Lane 1, FGF2-K84-pSVp-gal; lane 2, FGF2-K84-pSVSAP.
Figure 13A is a graph showing p-gal activity with an endosome disruptive peptide in the complex.
Figure 13B is a graph showing p-gal activity with an endosome disruptive peptide in the complex.
Figure 13C is a graph showing p-gal activity with an endosome disruptive peptide in the complex.
Detailed Description of the Invention Definitions All U.S. patents and all publications mentioned herein are incorporated in their entirety by reference thereto.
The "amino acids," which occur in the various amino acid sequences appearing herein, are identified according to their well known, three letter or one letter WO 96/36362 PCTUS96/07164 7 abbreviations. The nucleotides, which occur in the various DNA fragments, are designated with the standard single letter designations used routinely in the art.
As used herein, to "bind to a receptor" refers to the ability of a ligand to specifically recognize and detectably bind to such receptors, as assayed by standard in vitro assays. For example, as used herein, binding measures the capacity of a VEGF conjugate, VEGF monomer, or VEGF dimer to recognize a VEGF receptor on a vascular endothelial cell, such as an aortic vascular endothelial cell line, using a procedure substantially as described in Moscatelli, J. Cell Physiol. 131:123-130, 1987.
As used herein, "biological activity" refers to the in vivo activities of a compound or physiological responses that result upon in vivo administration of a compound, composition or other mixture. Biological activity thus encompasses therapeutic effects and pharmaceutical activity of such compounds, compositions and mixtures. Such biological activity may be defined with reference to particular in vitro activities as measured in a defined assay. For example, reference herein to the biological activity of FGF, or fragments of FGF, refers to the ability of FGF to bind to cells bearing FGF receptors and internalize a linked agent. Such activity is typically assessed in vitro by linking the FGF to a cytotoxic agent, such as saporin, contacting cells bearing FGF receptors, such as fibroblasts, with the conjugate and assessing cell proliferation or growth. In vivo activity may be determined using recognized animal models, such as the mouse xenograft model for anti-tumor activity (see, Beitz et al., Cancer Research 52:227-230, 1992; Houghton et al., Cancer Res. 42:535-539, 1982; Bogden et al., Cancer (Philadelphia) 48:10-20, 1981; Hoogenhout et al., Int. J.
Radiat. Oncol., Biol. Phys. 9:871-879, 1983; Stastny et al., Cancer Res. 53:5740-5744, 1993).
As used herein, reference to the "biological activity of a cytocideencoding agent," such as DNA encoding saporin, refers to the ability of such agent to interfere with the metabolism of the cell by inhibiting protein synthesis. Such biological or cytotoxic activity may be assayed by any method known to those of skill in the art including, but not limited to, in vitro assays that measure protein synthesis and in vivo assays that assess cytotoxicity by measuring the effect of a test compound on WO 96/36362 PCTIUS96/07164 8 cell proliferation or on protein synthesis. Assays that assess cytotoxicity in targeted cells are particularly preferred.
As used herein, a "conjugate" refers to a molecule that contains at least one receptor-internalized binding ligand and at least one nucleic acid binding domain that are linked directly or via a linker and that are produced by chemical coupling methods or by recombinant expression of chimeric DNA molecules to produce fusion proteins.
A "cytocide-encoding agent" is a nucleic acid molecule that encodes a protein that inhibits protein synthesis. Such a protein may act by cleaving rRNA or ribonucloprotein, inhibiting an elongation factor, cleaving mRNA, or other mechanism that reduces protein synthesis to a level such that the cell cannot survive. The cytocideencoding agent may contain additional elements besides the cytocide gene. Such elements include a promoter, enhancer, splice sites, transcription terminator, poly(A) signal sequence, bacterial or mammalian origins of replication, selection markers, and the like.
As used herein, the term "cytotoxic agent" refers to a molecule capable of inhibiting cell function. The agent may inhibit proliferation or may be toxic to cells.
A variety of cytotoxic agents can be used and include those that inhibit protein synthesis and those that inhibit expression of certain genes essential for cellular growth or survival. Cytotoxic agents include those that result in cell death and those that inhibit cell growth, proliferation and/or differentiation.
As used herein, cytotoxic agents include, but are not limited to, saporin, the ricins, abrin and other ribosome inactivating proteins (RIPs), aquatic-derived cytotoxins, Pseudomonas exotoxin, inhibitors of DNA, RNA or protein synthesis, such as antisense nucleic acids, other metabolic inhibitors, such as DNA cleaving molecules, prodrugs, such as thymidine kinase from HSV and bacterial cytosine deaminase, and light activated porphyrin. While saporin is the preferred RIP, other suitable RIPs include ricin, ricin A chain, maize RIP, gelonin, diphtheria toxin, diphtheria toxin A chain, trichosanthin, tritin, pokeweed antiviral protein (PAP), mirabilis antiviral protein (MAP), Dianthins 32 and 30, abrin, monordin, bryodin, shiga, a catalytic inhibitor of WO 96/36362 PCTIS96/07164 9 protein biosynthesis from cucumber seeds (see, WO 93/24620), Pseudomonas exotoxin, biologically active fragments of cytotoxins and others known to those of skill in this art. Suitable cytotoxic agents also include cytotoxic molecules that inhibit cellular metabolic processes, including transcription, translation, biosynthetic or degradative pathways, DNA synthesis, and other such processes that kill cells or inhibit cell proliferation.
"Heparin-binding growth factor" refers to any member of a family of heparin-binding growth factor proteins, in which at least one member of the family binds heparin. Preferred growth factors in this regard include FGF, VEGF, and HBEGF. Such growth factors encompass isoforms, peptide fragments derived from a family member, splice variants, and single or multiple exons, some forms of which may not bind heparin.
As used herein, to "hybridize" under conditions of a specified stringency is used to describe the stability of hybrids formed between two single-stranded nucleic acid molecules. Stringency of hybridization is typically expressed in conditions of ionic strength and temperature at which such hybrids are annealed and washed. Typically high, medium and low stringency encompass the following conditions or equivalent conditions thereto: 1) high stringency: 0.1 x SSPE or SSC, 0.1% SDS, 65 0
C
2) medium stringency: 0.2 x SSPE or SSC, 0.1% SDS, 3) low stringency: 1.0 x SSPE or SSC, 0.1% SDS, 50 0
C.
"Nucleic acid binding domain" (NABD) refers to a molecule, usually a protein, polypeptide, or peptide (but may also be a polycation) that binds nucleic acids, such as DNA or RNA. The NABD may bind to single or double strands of RNA or DNA or mixed RNA/DNA hybrids. The nucleic acid binding domain may bind to a specific sequence or bind irrespective of the sequence.
As used herein, "nucleic acids" refer to RNA or DNA that are intended for internalization into a cell and includes, but are not limited to, DNA encoding a therapeutic protein, DNA encoding a cytotoxic protein, DNA encoding a prodrug, DNA encoding a cytocide, the complement of these DNAs, an antisense nucleic acid and WO 96/36362 PCTIUS96/07164 other such molecules. Reference to nucleic acids includes duplex DNA, single-stranded DNA, RNA in any form, including triplex, duplex or single-stranded RNA, anti-sense RNA, polynucleotides, oligonucleotides, single nucleotides, chimeras, and derivatives thereof.
Nucleic acids may be composed of the well-known deoxyribonucleotides and ribonucleotides composed of the bases adenosine, cytosine, guanine, thymidine, and uridine. As well, various other nucleotide derivatives and non-phosphate backbones or phosphate-derivative backbones may be used. For example, because normal phosphodiester oligonucleotides (referred to as PO oligonucleotides) are sensitive to DNA- and RNA-specific nucleases, several resistant types of oligonucleotides have been developed in which the phosphate group has been altered to a phosphotriester, methylphosphonate, or phosphorothioate (see U.S. Patent No. 5,218,088).
As used herein, "operative linkage" or operative association of heterologous DNA to regulatory and effector sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, refers to the functional relationship between such DNA and such sequences of nucleotides. For example, operative linkage of heterologous DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA in reading frame.
As used herein, the term "polypeptide reactive with an FGF receptor" refers to any polypeptide that specifically interacts with an FGF receptor, preferably the high-affinity FGF receptor and that is transported into the cell by virtue of its interaction with the FGF receptor. Polypeptides reactive with an FGF receptor are also called FGF proteins. Such polypeptides include, but are not limited to, FGF-1 to FGF- 9. For example, bFGF (FGF-2) should be generally understood to refer to polypeptides having substantially the same amino acid sequences and receptor-targeting activity as that of bovine bFGF or human bFGF. It is understood that differences in amino acid sequences can occur among FGFs of different species as well as among FGFs from individual organisms or species. In addition, chimeras or hybrids of any of FGF-1 WO 96/36362 PCTIUS96/07164 11 through FGF-9, or FGFs that have deletions (see, PCT Application No. WO 90/02800), insertions or substitutions of amino acids are within the scope of FGF proteins, as long as the resulting peptide or protein specifically interacts with an FGF receptor and is internalized by virtue of this interaction.
As used herein, a "prodrug" is a compound that metabolizes or otherwise converts an inactive, nontoxic compound to a biologically, pharmaceutically, therapeutically, of toxic active form of the compound. A prodrug may also be a pharmaceutically inactive compound that is modified upon administration to yield an active compound through metabolic or other processes. The prodrug may alter the metabolic stability or the transport characteristics of a drug, mask side effects or toxicity, improve or alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design inactive forms of the compound (see, Nogrady, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, 1985).
As used herein, "receptor-binding internalized ligand" or "ligand" refers to any peptide, polypeptide, protein or non-protein, such as a peptidomimetic, that is capable of binding to a cell-surface molecule and is internalized. Within the context of this invention, the receptor-binding internalized ligand is conjugated to a nucleic acid binding domain, either as a fusion protein or through chemical conjugation, and is used to deliver a cytocide-encoding or pro-drug encoding agent to a cell. In one aspect, the ligand is directly conjugated to a nucleic acid molecule, which may be further complexed with a nucleic acid binding domain. Such ligands include growth factors, cytokines, antibodies or fragments thereof, hormones, and the like.
As used herein, "saporin" (abbreviated herein as SAP) refers to polypeptides that are isolated from the leaves or seeds of Saponaria officinalis, as well as modified forms that have amino acid substitutions, deletions, insertions or additions, which still express substantial ribosome inactivating activity. Purified preparations of saporin are frequently observed to include several molecular isoforms of the protein. It is understood that differences in amino acid sequences can occur in saporin from WO 96/36362 PCT/US96/07164 12 different species as well as between saporin molecules from individual organisms of the same species. Saporin for use herein may be purified from leaves, chemically synthesized, or synthesized by expression of DNA encoding a saporin polypeptide.
As used herein, a "targeted agent" is a nucleic acid molecule that is intended for internalization by complexing or linkage to a receptor-binding internalized ligand, and nucleic acid binding domain, and that upon internalization in some manner alters or affects cellular metabolism, growth, activity, viability or other property or characteristic of the cell.
As used herein, a "therapeutic nucleic acid" describes any nucleic acid molecule used in the context of the invention that modifies gene transcription or translation. This term also includes nucleic acids that bind to sites on proteins. It includes, but is not limited to, the following types of nucleic acids: nucleic acids encoding a protein, antisense RNA, DNA intended to form triplex molecules, extracellular protein binding oligonucleotides, and small nucleotide molecules. A therapeutic nucleic acid may be used to effect genetic therapy by serving as a replacement for a defective gene, by encoding a therapeutic product, such as TNF, or by encoding a cytotoxic molecule, especially an enzyme, such as saporin. The therapeutic nucleic acid may encode all or a portion of a gene, and may function by recombining with DNA already present in a cell, thereby replacing a defective portion of a gene. It may also encode a portion of a protein and exert its effect by virtue of co-suppression of a gene product.
PREPARATION OF RECEPTOR-BINDING INTERNALIZED LIGAND, NUCLEIC ACID BINDING DOMAIN AND CYTOCIDE-ENCODING AGENT COMPLEXES As noted above, the present invention provides cytocide-encoding agents complexed with a conjugate of a receptor-binding internalized ligand and a nucleic acid binding domain. Upon binding to an appropriate receptor, the complex is internalized by the cell and is trafficked through the cell via the endosomal compartment, where at least a portion of the complex may be cleaved.
WO 96/36362 PCTJUS96/07164 13 A. Receptor-binding internalized ligands As noted above, receptor-binding internalized ligands are used to deliver a cytocide-encoding agent to a cell expressing an appropriate receptor on its cell surface. Numerous molecules that bind specific receptors have been identified and are suitable for use in the present invention. Such molecules include growth factors, cytokines, and antibodies. Many growth factors and families of growth factors share structural and functional features and may be used in the present invention. One such family of growth factors specifically binds to heparin. The ability of heparin-binding growth factors to interact with heparin appears in general to be a reflection of a physiologically more relevant interaction occurring in vivo between these factors and heparin sulfate proteoglycan molecules, which are found on the surface of cells and in extracellular matrix. Heparin-binding growth factors include the fibroblast growth factors FGF-1 through FGF-9, vascular endothelial growth factor (VEGF), and heparin binding-epidermal growth factor (HBEGF). Antibodies that are specific to cell surface molecules expressed by a selected cell type are readily generated as monoclonals or polyclonal antisera. Many such antibodies are available American Type Culture Collection, Rockville, MD). Other growth factors, such as PDGF (platelet-derived growth factor), EGF (epidermal growth factor), TGF-a (tumor growth factor), TGF-p, IGF-I (insulin-like growth factor), and IGF-II also bind to specific identified receptors on cell surfaces and may be used in the present invention. Cytokines, including interleukins, CSFs (colony stimulating factors), and interferons, have specific receptors, which are mostly found on hematopoeitic cells, and may be used as described herein.
These ligands are discussed in more detail below.
Fragments of these ligands may be used within the present invention, so long as the fragment retains the ability to bind to the appropriate cell surface molecule.
Likewise, ligands with substitutions or other alterations, but which retain binding ability, may also be used.
1. Fibroblast growth factors One family of growth factors that has a broad spectrum of activities is the fibroblast growth factor (FGF) family. These proteins share the ability to bind to WO 96/36362 PCT/US96/07164 14 heparin, induce intracellular receptor-mediated tyrosine phosphorylation and the expression of the c-fos mRNA transcript, and stimulate DNA synthesis and cell proliferation. This family of proteins includes FGFs designated FGF-1 (acidic FGF (aFGF)), FGF-2 (basic FGF (bFGF)), FGF-3 (int-2) (see, Moore et al., EMBO J.
5:919-924, 1986), FGF-4 (hst-1/K-FGF) (see, Sakamoto et al., Proc. Natl. Acad.
Sci. USA 86:1836-1840, 1986; U.S. Patent No. 5,126,323), FGF-5 (see, U.S. Patent No. 5,155,217), FGF-6 (hst-2) (see, published European Application EP 0 488 196 A2; Uda et al., Oncogene 7:303-309, 1992), FGF-7 (keratinocyte growth factor) (KGF) (see, Finch et al., Science 245:752-755, 1985; Rubin et al., Proc. Natl. Acad. Sci.
USA 86:802-806, 1989; and International Application WO 90/08771), FGF-8 (see, e.g., Tanaka et al., Proc Natl. Acad. Sci. USA 89:8528-8532, 1992); and FGF-9 (see, Miyamoto et al., Mol. Cell. Biol. 13:4251-4259, 1993).
DNA encoding FGF peptides and/or the amino acid sequences of FGFs are known to those of skill in the art. DNA encoding an FGF may be prepared synthetically based on a known amino acid or DNA sequence, isolated using methods known to those of skill in the art, or obtained from commercial or other sources. DNA encoding virtually all of the FGF family of peptides is known. For example, DNA encoding human FGF-1 (Jaye et al., Science 233:541-545, 1986; U.S. Patent No. 5,223,483), bovine FGF-2 (Abraham et al., Science 233:545-548, 1986; Esch et al., Proc. Natl. Acad Sci. USA 82:6507-6511, 1985; and U.S. Patent No. 4,956,455), human FGF-2 (Abraham et al., EMBO J. 5:2523-2528, 1986; U.S. Patent No. 4,994,559; U.S. Patent No. 5,155,214; EP 470,183B; and Abraham et al., Quant.
Biol. 51:657-668, 1986) and rat FGF-2 (see Shimasaki et al., Biochem. Biophys. Res.
Comm., 1988, and Kurokawa et al., Nucleic Acids Res. 16:5201, 1988), FGF-3, FGF-6, FGF-7 and FGF-9 are known (see also U.S. Patent No. 5,155,214; U.S. Patent No. 4,956,455; U.S. Patent No. 5,026,839; U.S. Patent No. 4,994,559, EP 0,488,196 A2, DNASTAR, EMBL or GenBank databases, and references discussed above and below). DNA encoding an FGF may be produced from any of the preceding DNA fragments by substitution of degenerate codons. It is understood that once the complete amino acid sequence of a peptide, such as an FGF peptide, and the DNA WO 96/36362 PCTUS96/07164 fragment encoding such peptide are available to those of skill in this art, it is routine to substitute degenerate codons and produce any of the possible DNA fragments that encode such peptide. It is also generally possible to synthesize DNA encoding such peptide based on the amino acid sequence.
Thus, as used herein, "FGF" refers to polypeptides having amino acid sequences of native FGF proteins, as well as modified sequences, having amino acid substitutions, deletions, insertions or additions in the native protein but retaining the ability to bind to FGF receptors and to be internalized. It is understood that differences in amino acid sequences can occur among FGFs of different species as well as among FGFs from individual organisms or species.
Reference to FGFs is intended to encompass proteins isolated from natural sources as well as those made synthetically, as by recombinant means or possibly by chemical synthesis. FGF also encompasses muteins that possess the ability to bind to FGF-receptor expressing cells. Such muteins include, but are not limited to, those produced by replacing one or more of the cysteines with serine as described herein or that have any other amino acids deleted or replaced as long as the resulting protein has the ability to bind to FGF-receptor bearing cells and internalize the linked targeted agent. Typically, such muteins will have conservative amino acid changes, such as those set forth below in Table 1. DNA encoding such muteins will, unless modified by replacement of degenerate codons, hybridize under conditions of at least low stringency to native DNA sequence encoding the starting FGF.
Acidic and basic FGF are about 55% identical at the amino acid level and are highly conserved among species. The other members of the FGF family have a high degree of amino acid sequence similarities and common physical and biological properties with FGF-1 and FGF-2, including the ability to bind to one or more FGF receptors. Basic FGF, int-2, hst-1/K-FGF, FGF-5, hst-2/FGF-6 and FGF-8 may have oncogenic potential; bFGF is expressed in melanomas, int-2 is expressed in mammary tumor virus and hst-1/K-FGF is expressed in angiogenic tumors. Acidic FGF, bFGF, KGF and FGF-9 are expressed in normal cells and tissues.
WO 96/36362 PCT/US96/07164 16 FGFs exhibit a mitogenic effect on a wide variety of mesenchymal, endocrine and neural cells and are also important in differentiation and development.
Of particular interest is their stimulatory effect on collateral vascularization and angiogenesis. In some instances, FGF-induced mitogenic stimulation may be detrimental. For example, cell proliferation and angiogenesis are an integral aspect of tumor growth. Members of the FGF family, including bFGF, are thought to play a pathophysiological role, for example, in tumor development, rheumatoid arthritis, proliferative diabetic retinopathies and other complications of diabetes.
The effects of FGFs are mediated by high affinity receptor tyrosine kinases present on the cell surface of FGF-responsive cells (see, PCT WO 91/00916, WO 90/05522, PCT WO 92/12948; Imamura et al., Biochem. Biophys. Res.
Comm. 155:583-590, 1988; Huang et al., J. Biol. Chem. 261:9568-9571, 1986; Partanen et al., EMBOJ. 10:1347, 1991; and Moscatelli, J. Cell. Physiol. 131:123, 1987). Lower affinity receptors also appear to play a role in mediating FGF activities. The high affinity receptor proteins are single chain polypeptides with molecular weights ranging from 110 to 150 kD, depending on cell type that constitute a family of structurally related FGF receptors. Four FGF receptor genes have been identified, and three of these genes generate multiple mRNA transcripts via alternative splicing of the primary transcript.
2. Vascular endothelial growth factors Vascular endothelial growth factors (VEGFs) were identified by their ability to directly stimulate endothelial cell growth, but do not appear to have mitogenic effects on other types of cells. VEGFs also cause a rapid and reversible increase in blood vessel permeability. The members of this family have been referred to variously as vascular endothelial growth factor (VEGF), vascular permeability factor (VPF) and vasculotropin (see, Plouet et al., EMBO J. 8:3801-3806, 1989). Herein, they are collectively referred to as VEGF.
VEGF was originally isolated from a guinea pig heptocarcinoma cell line, line 10 (see, U.S. Patent No. 4,456,550), and has subsequently been identified in humans and in normal cells. It is expressed during normal development and in c WO 96/36362 PCTIUS96/07164 17 certain normal adult organs. Purified VEGF is a basic, heparin-binding, homodimeric glycoprotein that is heat-stable, acid-stable and may be inactivated by reducing agents.
DNA sequences encoding VEGF and methods to isolate these sequences may be found primarily in U.S. Patent No. 5,240,848, U.S. Patent No. 5,332,671, U.S.
Patent No. 5,219,739, U.S. Patent No. 5,194,596, and Houch etal., Mol. Endocrin.
5:180, 1991. As used herein, "DNA encoding a VEGF peptide or polypeptide" refers to any of the DNA fragments set forth herein as coding such peptides, to any such DNA fragments known to those of skill in the art, any DNA fragment that encodes a VEGF that binds to a VEGF receptor and is internalized thereby. VEGF DNA may be isolated from a human cell library, for example, using any of the preceding DNA fragments as a probe or any DNA fragment that encodes any of the VEGF peptides set forth in SEQ ID NOs. 1-4. It is understood that once the complete amino acid sequence of a peptide, such as a VEGF peptide, and the DNA fragment encoding such peptide are available to those of skill in this art, it is routine to substitute degenerate codons and produce any of the possible DNA fragments that encode such peptide. It is also generally possible to synthesize DNA encoding such peptide based on the amino acid sequence.
VEGF family members arise from a single gene organized as eight exons and spanning approximately 14 kb in the human genome. Four molecular species of VEGF result from alternative splicing of mRNA and contain 121, 165, 189 and 206 amino acids. The four species have similar biological activities, but differ markedly in their secretion patterns. The predominant isoform secreted by a variety of normal and transformed cells is VEGF, 65 Transcripts encoding VEGF 1 21 and VEGF, 89 are detectable in most cells and tissues that express the VEGF gene. In contrast, VEGF 2 0 6 is less abundant and has been identified only in a human fetal liver cDNA library.
VEGF
2 1 is a weakly acidic polypeptide that lacks the heparin binding domain and, consequently, does not bind to heparin. VEGFi,, and VEGF20 6 are more basic than
VEGF,
65 and bind to heparin with greater affinity. Although not every identified VEGF isoform binds heparin, all isoforms are considered to be heparin-binding growth factors within the context of this invention.
WO 96/36362 PCT/US96/07164 18 The secreted isoforms, VEGF 2 and VEGF 6 5 are preferred VEGF proteins. The longer isoforms, VEGF, 89 and VEGF20 6 are almost completely bound to the extracellular matrix and need to be released by an agent, such as suramin, heparin or heparinase, or plasmin. Other preferred VEGF proteins contain various combinations of VEGF exons, such that the protein still binds VEGF receptor and is internalized. It is not necessary that a VEGF protein used in the context of this invention either retain any of its in vivo biological activities, such as stimulating endothelial cell growth, or bind heparin. It is only necessary that the VEGF protein or fragment thereof bind the VEGF receptor and be internalized into the cell bearing the receptor. However, it may be desirable in certain contexts for VEGF to manifest certain of its biological activities.
For example, if VEGF is used as a carrier for DNA encoding a molecule useful in wound healing, it would be desirable that VEGF exhibit vessel permeability activity and promotion of fibroblast migration and angiogenesis. It will be apparent from the teachings provided within the subject application which of the activities of VEGF are desirable to maintain.
VEGF promotes an array of responses in endothelium, including blood vessel hyperpermeability, endothelial cell growth, angiogenesis, and enhanced glucose transport. VEGF stimulates the growth of endothelial cells from a variety of sources (including brain capillaries, fetal and adult aortas, and umbilical veins) at low concentrations, but is reported to have no effect on the growth of vascular smooth muscle cells, adrenal cortex cells, keratinocytes, lens epithelial cells, or BHK-21 fibroblasts. VEGF also is a potent polypeptide regulator of blood vessel function; it causes a rapid but transient increase in microvascular permeability without causing endothelial cell damage or mast cell degranulation, and its action is not blocked by antihistamines. VEGF has also been reported to induce monocyte migration and activation and has been implicated as a tumor angiogenesis factor in some human gliomas. Also, VEGF is a chemoattractant for monocytes and VEGF has been shown to enhance the activity of the inflammatory mediator tumor necrosis factor (TNF).
Quiescent and proliferating endothelial cells display high-affinity binding to VEGF, and endothelial cell responses to VEGF appear to be mediated by WO 96/36362 PCTIUS96/07164 19 high affinity cell surface receptors (see, International Application WO 92/14748, which is based on U.S. Applications Serial No. 08/657,236, de Vries et al., Science 255:989-91, 1992; Terman et al., Biochem. Biophys. Res. Commun. 187:1579-1586, 1992; Kendall et al., Proc. Natl. Acad. Sci. USA 90:10705-10709, 1993; and Peters et al., Proc. Natl. Acad. Sci. USA 90:8915-8919, 1993). Two tyrosine kinases have been identified as VEGF receptors. The first, known as fins-like tyrosine kinase or FLT is a receptor tyrosine kinase that is specific for VEGF. In adult and embryonic tissues, expression of FLT mRNA is localized to the endothelium and to populations of cells that give rise to endothelium. The second receptor, KDR (human kinase insert domaincontaining receptor), and its mouse homologue FLK-1, are closely related to FLT. The KDR/FLK-1 receptor is expressed in endothelium during the fetal growth stage, during earlier embryonic development, and in adult tissues. In addition, messenger RNA encoding FLT and KDR have been identified in tumor blood vessels and specifically by endothelial cells of blood vessels supplying glioblastomas. Similarly, FLT and KDR mRNAs are upregulated in tumor blood vessels in invasive human colon adenocarcinoma, but not in the blood vessels of adjacent normal tissues.
3. Heparin-binding epidermal growth factors Several new mitogens in the epidermal growth factor protein family have recently been identified that display the ability to bind the glycosaminoglycan heparin.
Among these is the mitogen known as heparin-binding EGF-like growth factor (HBEGF), which elutes from heparin-Sepharose T M columns at about 1.0 1.2 M NaCl and which was first identified as a secreted product of cultured human monocytes, macrophages, and the macrophage-like U-937 cell line (Higashiyama et al., Science 251:936-939, 1991; Besner et al., Cell Regul. 1:811-19, 1990). HBEGF has been shown to interact with the same high affinity receptors as EGF on bovine aortic smooth muscle cells and human A431 epidermoid carcinoma cells (Higashiyama, Science 251:936-939, 1991).
HBEGFs exhibit a mitogenic effect on a wide variety of cells including BALB/c 3T3 fibroblast cells and smooth muscle cells, but unlike VEGFs, are not mitogenic for endothelial cells (Higashiyama et al., Science 251:936-939, 1991).
WO 96/36362 PCT/US96/07164 HBEGF also has a stimulatory effect on collateral vascularization and angiogenesis.
Members of the HBEGF family are thought to play a pathophysiological role, for example, in a variety of tumors, such as bladder carcinomas, breast tumors and nonsmall cell lung tumors. Thus, these cell types are likely candidates for delivery of cytocide-encoded agents.
HBEGF isolated from U-937 cells is heterogeneous in structure and contains at least 86 amino acids and two sites of O-linked glycosyl groups (Higashiyama et al., J. Biol. Chem. 267:6205-6212, 1992). The carboxyl-terminal half of the secreted HBEGF shares approximately 35% sequence identity with human EGF, including six cysteines spaced in the pattern characteristic of members of the EGF protein family. In contrast, the amino-terminal portion of the mature factor is characterized by stretches of hydrophilic residues and has no structural equivalent in EGF. Site-directed mutagenesis of HBEGF and studies with peptide fragments have indicated that the heparin-binding sequences of HBEGF reside primarily in a 21 amino acid stretch upstream of and slightly overlapping the EGF-like domain.
The effects of HBEGFs are mediated by EGF receptor tyrosine kinases expressed on cell surfaces of HBEGF-responsive cells (see, U.S. Patent Nos.
5,183,884 and 5,218,090; and Ullrich et al., Nature 309:4113-425, 1984). The EGF receptor proteins, which are single chain polypeptides with molecular weights 170 kD, constitute a family of structurally related EGF receptors. Cells known to express the EGF receptors include smooth muscle cells, fibroblasts, keratinocytes, and numerous human cancer cell lines, such as the: A431 (epidermoid); KB3-1 (epidermoid);
COLO
205 (colon); CRL 1739 (gastric); HEP G2 (hepatoma); LNCAP (prostate); MCF-7 (breast); MDA-MB-468 (breast); NCI 417D (lung); MG63 (osteosarcoma); U-251 (glioblastoma); D-54MB (glioma); and SW-13 (adrenal).
For the purposes of this invention, HBEGF need only bind a specific HBEGF receptor and be internalized. Any member of the HBEGF family, whether or not it binds heparin, is useful within the context of this invention as long as it meets the requirements set forth above. Members of the HBEGF family are those that have sufficient nucleotide identity to hybridize under normal stringency conditions (typically WO 96/36362 PCT/US96/07164 21 greater than 75% nucleotide identity). Subfragments or subportions of a full-length HBEGF may also be desirable. One skilled in the art may find from the teachings provided within that certain biological activities are more or less desirable, depending upon the application.
DNA encoding an HBEGF peptide or polypeptide refers to any DNA fragment encoding an HBEGF, as defined above. Exemplary DNA fragments include: any such DNA fragments known to those of skill in the art; any DNA fragment that encodes an HBEGF or fragment that binds to an HBEGF receptor and is internalized thereby; and any DNA fragment that encodes any of the HBEGF polypeptides set forth in SEQ ID NOs. 5-8. Such DNA sequences encoding HBEGF fragments are available from publicly accessible databases, such as: EMBL, GenBank (Accession Nos. M93012 (monkey) and M60278 (human)); the plasmid pMTN-HBEGF (ATCC #40900) and pAX-HBEGF (ATCC #40899) (described in PCT Application WO/92/06705); and Abraham et al., Biochem. Biophys. Res. Comm. 190:125-133, 1993). Unless modified by replacement of degenerate codons, DNA encoding HBEGF polypeptides will hybridize under conditions of at least low stringency to DNA encoding a native human HBEGF SEQ ID NO. In addition, any DNA fragment that may be produced from any of the preceding DNA fragments by substitution of degenerate codons is also contemplated for use herein. It is understood that since the complete amino acid sequence of HBEGF polypeptides, and DNA fragments encoding such peptides, are available to those of skill in this art, it is routine to substitute degenerate codons and produce any of the possible DNA fragments that encode such HBEGF polypeptides. It is also generally possible to synthesize DNA encoding such peptides based on the amino acid sequence.
4. Other receptor-binding internalized ligands Other receptor-binding ligands may be used in the present invention.
Any protein, polypeptide, analogue, or fragment that binds to a cell-surface receptor and is internalized may be used. In general, in addition to the specific heparin-binding growth factors discussed above, other growth factors and cytokines are especially well suited for use. These ligands may be produced by recombinant or other means in WO 96/36362 PCTUS961/07164 22 preparation for conjugation to the nucleic acid binding domain. The DNA sequences and methods to obtain the sequences of these receptor-binding internalized ligands are well known. For example, these ligands include CSF-1 (GenBank Accession Nos.
M11038, M37435; Kawasaki et al., Science 230:291-296, 1985; Wong et al., Science 235:1504-1508, 1987); GM-CSF (GenBank Accession No. X03021; Miyatake et al., EMBO J. 4:2561-2568, 1985); IFN-a (interferon) (GenBank Accession No. A02076; Patent No. WO 8502862-A, July 4, 1985); IFN-y (GenBank Accession No. A02137; Patent No. WO 8502624-A, June 20, 1985); hepatocyte growth factor (GenBank Accession No. X16323, S80567, X57574; Nakamura, et al., Nature 342:440-443, 1989; Nakamura et al., Prog. Growth Factor Res. 3:67-85, 1991; Miyazawa et al., Eur. J.
Biochem. 197:15-22, 1991); IGF-Ia (Insulin-like growth factor Ia) (GenBank Accession No. X56773, S61841; Sandberg-Nordqvist et al., Brain Res. Mol. Brain Res. 12:275- 277, 1992; Sandberg, Sandberg-Nordqvist et al., Cancer Res. 53:2475-2478, 1993); IGF-Ib (GenBank Accession No. X56774 S61860; Sandberg-Nordqvist et al., Brain Res. Mol. Brain Res. 12:275-277, 1992; Sandberg-Nordqvist, Cancer Res.
53:2475-2478, 1993); IGF-I (GenBank Accession No. X03563, M29644; Dull et al., Nature 310:771-781, 1984; Rail et al., Meth. Enzymol. 146:239-248, 1987); IGF-II (GenBank Accession No. J03242; Shen et al., Proc. Natl. Acad. Sci. USA 85:1947- 1951, 1988); IL--a (interleukin 1 alpha) (GenBank Accession No. X02531, M15329; March et al., Nature 315:641-647, 1985; Nishida et al., Biochem. Biophys. Res.
Commun. 143:345-352, 1987); IL-1-p (interleukin 1 beta) (GenBank Accession No. X02532, M15330, M15840; March et al., Nature 315:641-647, 1985; Nishida et al., Biochem. Biophys. Res. Commun. 143:345-352, 1987; Bensi et al., Gene 52:95-101, 1987); IL-1 (GenBank Accession No. K02770, M54933, M38756; Auron et al., Proc.
Natl. Acad Sci. USA 81:7907-7911, 1984; Webb et al., Adv. Gene Technol. 22:339-340, 1985); IL-2 (GenBank Accession No. A14844, A21785, X00695, X00200, X00201, X00202; Lupker et al., Patent No. EP 0307285-A, March 15, 1989; Perez et al., Patent No. EP 0416673-A, March 13, 1991; Holbrook et al., Nucleic Acids Res. 12:5005-5013, 1984; Degrave et al., EMBO J. 2:2349-2353, 1983; Taniguchi et al., Nature 302:305- 310, 1983); IL-3 (GenBank Accession No. M14743, M20137; Yang et al., Cell 47:3-10, WO 96/36362 PCT/US96/07164 23 1986; Otsuka et al., J. Immunol. 140:2288-2295, 1988); IL-4 (GenBank Accession No. M13982; Yokota et al., Proc. Natl. Acad. Sci. USA 83:5894-5898, 1986); (GenBank Accession No. X04688, J03478; Azuma et al., Nucleic Acids Res. 14:9149- 9158, 1986; Tanabe et al., J. Biol. Chem. 262:16580-16584, 1987); IL-6 (GenBank Accession No. Y00081, X04602, M54894, M38669, M14584; Yasukawa et al., EMBO J. 6:2939-2945, 1987; Hirano et al., Nature 324:73-76, 1986; Wong et al., Behring Inst.
Mitt. 83:40-47, 1988; May et al., Proc. Natl. Acad. Sci. USA 83:8957-8961, 1986); IL-7 (GenBank Accession No. J04156; Goodwin et al., Proc. Natl. Acad Sci. USA 86:302- 306, 1989); IL-8 (GenBank Accession No. Z 1686; Kusner et al., Kidney Int. 39:1240- 1248, 1991); IL-10 (GenBank Accession No. X78437, M57627; Vieira et al., Proc.
Natl. Acad Sci. USA 88:1172-1176, 1991); IL-11 (GenBank Accession No. M57765 M37006; Paul et al., Proc. Natl. Acad. Sci. USA 87:7512-7516, 1990); IL-13 (GenBank Accession No. X69079, U10307; Minty et al., Nature 362:248-250, 1993; Smirnov, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, June 2, 1994); TNF-a (Tumor necrosis factor) (GenBank Accession No. A21522; Patent No. GB 2246569-A1, February 5, 1992); TNF-p (GenBank Accession No. D12614; Matsuyama et al., FEBS LETTERS 302:141-144, 1992). DNA sequences of other suitable receptor-binding internalized ligands may be obtained from GenBank or EMBL DNA databases, reversesynthesized from protein sequence obtained from PIR database or isolated by standard methods (Sambrook et al., supra) from cDNA or genomic libraries.
Modifications of receptor-binding internalized ligands These ligands may be customized for a particular application. Means for modifying proteins is provided below. Briefly, additions, substitutions and deletions of amino acids may be produced by any commonly employed recombinant DNA method.
An amino acid residue of FGF, VEGF, HBEGF or other receptorbinding internalized ligand is non-essential if the polypeptide that has been modified by deletion of the residue possesses substantially the same ability to bind to its receptor and internalize a linked agent as the unmodified polypeptide.
As noted above, any polypeptide or peptide analogue, including peptidomimetics, that is reactive with an FGF receptor, a VEGF receptor, an HBEGF WO 96/36362 PCTIUS96/07164 24 receptor, other growth factor receptor PDGF receptor), cytokine receptor or other cell surface molecule including members of the families and fragments thereof, or constrained analogs of such peptides that bind to the receptor and internalize a linked targeted agent may be used in the context of this invention. Members of the FGF peptide family, including FGF- 1 to FGF-9, are preferred. Modified peptides, especially those lacking proliferative function, and chimeric peptides, which retain the specific binding and internalizing activities are also contemplated for use herein.
A modification that is effected substantially near the N-terminus of a polypeptide is generally effected within the first about ten residues of the protein. Such modifications include the addition or deletion of residues, such as the addition of a cysteine to facilitate conjugation and form conjugates that contain a defined molar ratio, preferably a ratio of 1:1 of the polypeptides.
DNA encoding one of the receptor-binding internalized ligands discussed above may be mutagenized using standard methodologies to delete or replace any cysteine residues that are responsible for aggregate formation. If necessary, the identity of cysteine residues that contribute to aggregate formation may be determined empirically, by deleting and/or replacing a cysteine residue and ascertaining whether the resulting protein aggregates in solutions containing physiologically acceptable buffers and salts. In addition, fragments of these receptor-binding internalized ligands may be constructed and used. The binding region of many of these ligands have been delineated. Fragments may also be shown to bind and internalize by any one of the tests described herein.
Modification of the polypeptide may be effected by any means known to those of skill in this art. The preferred methods herein rely on modification of DNA encoding the polypeptide and expression of the modified DNA.
Merely by way of example, DNA encoding the FGF polypeptide may be isolated, synthesized or obtained from commercial sources (the amino acid sequences of FGF-1 FGF-9 are set forth in SEQ ID NOs. 10-18; DNA sequences may be based on these amino acid sequences or may be obtained from public DNA databases and references (see, GenBank, see also U.S. Patent No. 4,956,455, U.S. Patent WO 96/36362 PCT/US96/07164 No. 5,126,323, U.S. Patent No. 5,155,217, U.S. Patent No. 4,868,113, PCT Application WO 90/08771, EP Application 0 488 196 A2, and Miyamoto et al., Mol. Cell. Biol.
13:4251-4259, 1993). Expression of a recombinant FGF-2 protein in yeast and E. coli is described in Barr et al., J. Biol. Chem. 263:16471-16478, 1988, in PCT Application Serial No. PCT/US93/05702 and United States Application Serial No. 07/901,718.
Expression of recombinant FGF proteins may be performed as described herein or using methods known to those of skill in the art.
Similarly, DNA encoding any of the other receptor-binding internalized ligands, including VEGF, HBEGF, IL-1, IL-2, and other cytokines and growth factors may also be isolated, synthesized, or obtained from commercial sources. As noted above, DNA sequences are available in public databases, such as GenBank. Based on these sequences, oligonucleotide primers may be designed and used to amplify the gene from cDNA or mRNA by polymerase chain reaction technique as one means of obtaining DNA.
Mutations may be made by any method known to those of skill in the art, including site-specific or site-directed mutagenesis of DNA encoding the protein and the use of DNA amplification methods using primers to introduce and amplify alterations in the DNA template, such as PCR splicing by overlap extension
(SOE).
Site-directed mutagenesis is typically effected using a phage vector that has single- and double-stranded forms, such as M13 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, Veira et al., Meth. Enzymol. 15:3, 1987). In general, site-directed mutagenesis is performed by preparing a single-stranded vector that encodes the protein of interest a member of the FGF family or a cytotoxic molecule, such as a saporin). An oligonucleotide primer that contains the desired mutation within a region of homology to the DNA in the single-stranded vector is annealed to the vector followed by addition of a DNA polymerase, such as E. coli DNA polymerase I (Klenow fragment), which uses the double stranded region as a primer to produce a heteroduplex in which one strand encodes the altered sequence and the other the original sequence. The heteroduplex is introduced into appropriate bacterial cells WO 96/36362 PCTfUS96/07164 26 and clones that include the desired mutation are selected. The resulting altered DNA molecules may be expressed recombinantly in appropriate host cells to produce the modified protein.
Suitable conservative substitutions of amino acids are well-known and may be made generally without altering the biological activity of the resulting molecule.
For example, such substitutions may be made in accordance with those set forth in TABLE 1 as follows: TABLE 1 Conservative Original residue substitution Ala Gly; Ser Arg Lys Asn Gin; His Cys Ser Gin Asn Glu Asp Gly Ala; Pro His Asn; Gin Ile Leu; Val Leu lie; Val Lys Arg; Gin; Glu Met Leu; Tyr; Ile Phe Met; Leu; Tyr Ser Thr Thr(T) Ser Trp Tyr Tyr(Y) Trp;Phe Val lie; Leu Other similarly conservative substitutions may be made. If necessary, such substitutions may be determined empirically merely by testing the resulting modified protein for the ability to bind to and internalize upon binding to the appropriate WO 96/36362 PCT/US96/07164 27 receptors. Those that retain this ability are suitable for use in the conjugates and methods herein. In addition, muteins of the FGFs are known to those of skill in the art (see, U.S. Patent No. 5,175,147; PCT Application No. WO 89/00198, U.S. Serial No. 07/070,797; PCT Application No. WO 91/15229; and U.S. Serial No. 07/505,124).
B. Nucleic acid binding domains As previously noted, nucleic acid binding domains (NABD) interact with the target nucleic acid either in a sequence-specific manner or a sequence-nonspecific manner. When the interaction is non-specific, the nucleic acid binding domain binds nucleic acid regardless of the sequence. For example, poly-L-lysine is a basic polypeptide that binds to oppositely charged DNA. Other highly basic proteins or polycationic compounds, such as histones, protamines, and spermidine, also bind to nucleic acids in a nonspecific manner.
Many proteins have been identified that bind specific sequences of DNA.
These proteins are responsible for genome replication, transcription and repair of damaged DNA. The transcription factors regulate gene expression and are a diverse group of proteins. These factors are especially well suited for purposes of the subject invention because of their sequence-specific recognition. Host transcription factors have been grouped into seven well-established classes based upon the structural motif used for recognition. The major families include helix-turn-helix (HTH) proteins, homeodomains, zinc finger proteins, steroid receptors, leucine zipper proteins, the helix-loop-helix (HLH) proteins, and P-sheets. Other classes or subclasses may eventually be delineated as more factors are discovered and defined. Proteins from those classes or proteins that do not fit within one of these classes but bind nucleic acid in a sequence-specific manner, such as SV40 T antigen and p53 may also be used.
These families of transcription factors are generally well-known (see GenBank; Pabo and Sauer, Ann. Rev. Biochem. 61:1053-1095, 1992; and references below). Many of these factors are cloned and the precise DNA-binding region delineated in certain instances. When the sequence of the DNA-binding domain is known, a gene encoding it may be synthesized if the region is short. Alternatively, the genes may be cloned from the host genomic libraries or from cDNA libraries using WO 96/36362 PCT/US96/07164 28 oligonucleotides as probes or from genomic DNA or cDNA by polymerase chain reaction methods. Such methods may be found in Sambrook et al., supra.
Helix-turn-helix proteins include the well studied X Cro protein, XcI, and E. coli CAP proteins (see Steitz et al., Proc. Natl. Acad Sci. USA 79:3097-3100, 1982; Ohlendorf et al., J. Mol. Biol. 169:757-769, 1983). In addition, the lac repressor (Kaptein et al., J. Mol. Biol. 182:179-182, 1985) and Trp repressor (Scheritz et al., Nature 317:782-786, 1985) belong to this family. Members of the homeodomain family include the Drosophila protein Antennapaedia (Qian et al., Cell. 59:573-580, 1989) and yeast MATa2 (Wolberger et al., Cell. 67:517-528, 1991). Zinc finger proteins include TFIIIA (Miller et al., EMBO J. 4:1609-1614, 1985), Sp-1, zif 268, and many others (see generally Krizek et al., J. Am. Chem. Soc. 113:4518-4523, 1991).
Steroid receptor proteins include receptors for steroid hormones, retinoids, vitamin D, thyroid hormones, as well as other compounds. Specific examples include retinoic acid, knirps, progesterone, androgen, glucocosteroid and estrogen receptor proteins. The leucine zipper family was defined by a heptad repeat of leucines over a region of 30 to residues. Specific members of this family include C/EBP, c-fos, c-jun, GCN4, sis-A, and CREB (see generally O'Shea et al., Science 254:539-544, 1991). The helix-loophelix (HLH) family of proteins appears to have some similarities to the leucine zipper family. Well-known members of this family include myoD (Weintraub et al., Science 251:761-766, 1991); c-myc; and AP-2 (Williams and Tijan, Science 251:1067-1071, 1991). The p-sheet family uses an antiparallel p-sheet for DNA binding, rather than the more common a-helix. The family contains the MetJ (Phillips, Curr. Opin. Struc. Biol.
1:89-98, 1991), Arc (Breg et al., Nature 346:586-589, 1990) and Mnt repressors. In addition, other motifs are used for DNA binding, such as the cysteine-rich motif in yeast GAL4 repressor, and the GATA factor. Viruses also contain gene products that bind specific sequences. One of the most-studied such viral genes is the rev gene from HIV.
The rev gene product binds a sequence called RRE (rev responsive element) found in the env gene. Other proteins or peptides that bind DNA may be discovered on the basis of sequence similarity to the known classes or functionally by selection.
WO 96/36362 PCTIUS96/07164 29 Several techniques may be used to select other nucleic acid binding domains (see U.S. Patent No. 5,270,170; PCT Application WO 93/14108; and U.S.
Patent No. 5,223,409). One of these techniques is phage display. (See, for example, U.S. Patent No. 5,223,409.) In this method, DNA sequences are inserted into the gene III or gene VIII gene of a filamentous phage, such as M13. Several vectors with multicloning sites have been developed for insertion (McLafferty et al., Gene 128:29- 36, 1993; Scott and Smith, Science 249:386-390, 1990; Smith and Scott, Methods Enzymol. 217:228-257, 1993). The inserted DNA sequences may be randomly generated or variants of a known DNA-binding domain. Generally, the inserts encode from 6 to 20 amino acids. The peptide encoded by the inserted sequence is displayed on the surface of the bacteriophage. Bacteriophage expressing a desired nucleic acidbinding domain are selected for by binding to the cytocide-encoding agent. This target molecule may be single stranded or double stranded DNA or RNA. When the cytocideencoding agent to be delivered is single-stranded, such as RNA, the appropriate target is single-stranded. When the molecule to be delivered is double-stranded, the target molecule is preferably double-stranded. Preferably, the entire coding region of the cytocide-encoding agent is used as the target. In addition, elements necessary for transcription that are included for in vivo or in vitro delivery may be present in the target DNA molecule. Bacteriophage that bind the target are recovered and propagated.
Subsequent rounds of selection may be performed. The final selected bacteriophage are propagated and the DNA sequence of the insert is determined. Once the predicted amino acid sequence of the binding peptide is known, sufficient peptide for use herein as an nucleic acid binding domain may be made either by recombinant means or synthetically. Recombinant means is used when the receptor-binding internalized ligand/nucleic acid binding domain is produced as a fusion protein. In addition, the peptide may be generated as a tandem array of two or more peptides, in order to maximize affinity or binding of multiple DNA molecules to a single polypeptide.
As an example of the phage display selection technique, a DNA-binding domain/peptide that recognizes the coding region of saporin is isolated. Briefly, DNA fragments encoding saporin may be isolated from a plasmid containing these sequences.
WO 96/36362 PCTUS96/07164 The plasmid FPFS1 contains the entire coding region of saporin. Digestion of the plasmid with NcoI and EcoRI restriction enzymes liberates the saporin specific sequence as a single fragment of approximately 780 bp. This fragment may be purified by any one of a number of methods, such as agarose gel electrophoresis and subsequent elution from the gel. The saporin fragment is fixed to a solid support, such as in the wells of a 96-well plate. If the double-stranded fragment does not bind well to the plate, a coating such as a positively charged molecule, may be used to promote DNA adherence. The phage library is added to the wells and an incubation period allows for binding of the phage to the DNA. Unbound phage are removed by a wash, typically containing 10 mM Tris, 1 mM EDTA, and without salt or with a low salt concentration.
Bound phage are eluted starting at a 0.1 M NaCI containing buffer. The NaCI concentration is increased in a step-wise fashion until all the phage are eluted.
Typically, phage binding with higher affinity will only be released by higher salt concentrations.
Eluted phage are propagated in the bacteria host. Further rounds of selection may be performed to select for a few phage binding with high affinity. The DNA sequence of the insert in the binding phage is then determined. In addition, peptides having a higher affinity may be isolated by making variants of the insert sequence and subjecting these variants to further rounds of selection.
C. Cytocide-encoding agents A cytocide-encoding agent is a nucleic acid molecule (DNA or RNA) that, upon internalization by a cell, and subsequent transcription (if DNA) and[/or] translation into a cytocidal agent, is cytotoxic to a cell or inhibits cell growth by inhibiting protein synthesis.
Cytocides include saporin, the ricins, abrin and other ribosome inactivating proteins, Pseudomonas exotoxin, diphtheria toxin, angiogenin, tritin, dianthins 32 and 30, momordin, pokeweed antiviral protein, mirabilis antiviral protein, bryodin, angiogenin, and shiga exotoxin, as well as other cytocides that are known to those of skill in the art. Alternatively, cytocide gene products may be noncytotoxic but WO 96/36362 PCTIUS96/07164 31 activate a compound, which is endogenously produced or exogenously applied, from a nontoxic form to a toxic product that inhibits protein synthesis.
Especially of interest are DNA molecules that encode an enzyme that results in cell death or renders a cell susceptible to cell death upon the addition of another product. For example, saporin is an enzyme that cleaves rRNA and inhibits protein synthesis. Other enzymes that inhibit protein synthesis are especially well suited for use in the present invention. In addition, enzymes may be used where the enzyme activates a compound with little or no cytotoxicity into a toxic product that inhibits protein synthesis.
1. Ribosome inactivating proteins Ribosome-inactivating proteins (RIPs), which include ricin, abrin, and saporin, are plant proteins that catalytically inactivate eukaryotic ribosomes. Ribosomeinactivating proteins inactivate ribosomes by interfering with the protein elongation step of protein synthesis. For example, the ribosome-inactivating protein saporin (hereinafter also referred to as SAP) has been shown to inactivate 60S ribosomes by cleavage of the N-glycosidic bond of the adenine at position 4324 in the rat 28S ribosomal RNA (rRNA). The particular region in which A 432 4 is located in the rRNA is highly conserved among prokaryotes and eukaryotes; A4324 in 28S rRNA corresponds to
A
2 66 0 in E. coli 23S rRNA. Several of the ribosome inactivating proteins also appear to interfere with protein synthesis in prokaryotes, such as E. coli.
Saporin is preferred as a cytocide, but other suitable ribosome inactivating proteins (RIPs) and toxins may be used. Other suitable RIPs include, but are not limited to, ricin, ricin A chain, maize ribosome inactivating protein, gelonin, diphtheria toxin, diphtheria toxin A chain, trichosanthin, tritin, pokeweed antiviral protein (PAP), mirabilis antiviral protein (MAP), Dianthins 32 and 30, abrin, monordin, bryodin, shiga (see, WO 93/24620) and others (see, Barbieri et al., Cancer Surveys 1:489-520, 1982, and European patent application No. 0466 222, incorporated herein by reference, which provide lists of numerous ribosome inactivating proteins and their sources; see also U.S. Patent No. 5,248,608 to Walsh et Some ribosome inactivating proteins, such as abrin and ricin, contain two constituent chains: a cell- WO 96/36362 PCTIUS96/07164 32 binding chain that mediates binding to cell surface receptors and internalization of the molecule and a chain responsible for toxicity. Single chain ribosome inactivating proteins (type I RIPS), such as the saporins, do not have a cell-binding chain. As a result, unless internalized, they are substantially less toxic to whole cells than the ribosome inactivating proteins that have two chains.
Several structurally related ribosome inactivating proteins have been isolated from seeds and leaves of the plant Saponaria officinalis (soapwort) (GB Patent 2,194,241 B; GP Patent 2,216,891; EP Patent 89306016). Saporin proteins for use in this invention have amino acid sequences found in the natural plant host Saponaria officinalis or modified sequences, having amino acid substitutions, deletions, insertions or additions, but which still express substantial ribosome inactivating activity. Purified preparations of saporin are frequently observed to include several molecular isoforms of the protein. It is understood that differences in amino acid sequences can occur in saporin from different species as well as between saporin molecules from individual organisms of the same species. Among these, SO-6 is the most active and abundant, representing 7% of total seed proteins. Saporin is very stable, has a high isoelectric point, does not contain carbohydrates, and is resistant to denaturing agents, such as sodium dodecyl sulfate (SDS), and a variety of proteases. The amino acid sequences of several saporin-6 isoforms from seeds are known, and there appear to be families of saporin ribosome inactivating proteins differing in few amino acid residues. Any of these saporin proteins or modified proteins that are cytotoxic may be used in the present invention.
a. Isolation of DNA encoding saporin Some of the DNA molecules provided herein encode saporin that has substantially the same amino acid sequence and ribosome inactivating activity as that of saporin-6 including any of four isoforms, which have heterogeneity at amino acid positions 48 and 91 (see, Maras et al., Biochem. Internat. 21:631-638, 1990, and Barra et al., Biotechnol. Appl. Biochem. 13:48-53, 1991; GB Patent 2,216,891 B and EP Patent 89306106; and SEQ ID NOs. 19-23). Other suitable saporin polypeptides include other members of the multi-gene family coding for isoforms of WO 96/36362 PCT/US96/07164 33 saporin-type ribosome inactivating proteins including SO-1 and SO-3 (Fordham- Skelton et al., Mol. Gen. Genet. 221:134-138, 1990), SO-2 (see, U.S. Application Serial No. 07/885,242; GB 2,216,891; see also Fordham-Skelton et al., Mol. Gen.
Genet. 229:460-466, 1991), SO-4 (see, GB 2,194,241 B; see also Lappi et al., Biochem. Biophys. Res. Commun. 129:934-942, 1985) and SO-5 (see,
GB
2,194,241 B; see also Montecucchi et al., Int. J. Peptide Protein Res. 33:263-267, 1989).
The saporin polypeptides for use in this invention include any of the isoforms of saporin that may be isolated from Saponaria officinalis or related species or modified forms that retain cytotoxic activity. In particular, such modified saporin may be produced by modifying the DNA encoding the protein (see, International
PCT
Application Serial No. PCT/US93/05702, and United States Application Serial No. 07/901,718; see also U.S. Patent Application No. 07/885,242, and Italian Patent No. 1,231,914) by altering one or more amino acids or deleting or inserting one or more amino acids. Any such protein, or portion thereof, that exhibits cytotoxicity in standard in vitro or in vivo assays within at least about an order of magnitude of the saporin conjugates described herein is contemplated for use herein.
Preferably, the saporin DNA sequence contains mammalian-preferred codons (SEQ. ID NO. 79). Preferred codon usage as exemplified in Current Protocols in Molecular Biology, infra, and Zhang et al. (Gene 105:61, 1991) for mammals, yeast, Drosophila, E. coli, and primates is established for saporin sequence.
The cytocide-encoding agent, such as saporin DNA sequence, is introduced into a plasmid in operative linkage to an appropriate promoter for expression of polypeptides in the organism. The presently preferred saporin proteins are SO-6 and SO-4. The DNA can optionally include sequences, such as origins of replication that allow for the extrachromosomal maintenance of the saporin-containing plasmid, or can be designed to integrate into the genome of the host (as an alternative means to ensure stable maintenance in the host).
WO 96/36362 PCT/US96/07164 34 b. Nucleic acids encoding other ribosome inactivating proteins and cvtocides In addition to saporin discussed above, other cytocides that inhibit protein synthesis are useful in the present invention. The gene sequences for these cytocides may be isolated by standard methods, such as PCR, probe hybridization of genomic or cDNA libraries, antibody screenings of expression libraries, or clones may be obtained from commercial or other sources. The DNA sequences of many of these cytocides are well known, including ricin A chain (GenBank Accession No. X02388); maize ribosome inactivating protein (GenBank Accession No. L26305); gelonin (GenBank Accession No. L12243; PCT Application WO 92/03155; U.S. Patent No. 5,376,546; diphtheria toxin (GenBank Accession No. K01722); trichosanthin (GenBank Accession No. M34858); tritin (GenBank Accession No. 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. K01397, M23348). When DNA sequences or amino acid sequences are known, DNA molecules encoding these proteins may be synthesized, and preferably contain mammalianpreferred codons.
D. Prodrug-encoding agent A nucleic acid molecule encoding a prodrug may alternatively be used within the context of the present invention. Prodrugs are inactive in the host cell until either a substrate is provided or an activating molecule is provided. Most typically, a prodrug activates a compound with little or no cytotoxicity into a toxic product. Two of the more often used prodrug molecules, both of which may be used in the present invention, are HSV thymidine kinase and E. coli cytosine deaminase.
Briefly, a wide variety of gene products which either directly or indirectly activate a compound with little or no cytotoxicity into a toxic product may be utilized within the context of the present invention. Representative examples of such gene products include HSVTK (herpes simplex virus thymidine kinase) and VZVTK WO 96/36362 PCT/US96/07164 (varicella zoster virus thymidine kinase), which selectively phosphorylate certain purine arabinosides and substituted pyrimidine compounds. Phosphoryation converts these compounds to metabolites that are cytotoxic or cytostatic. For example, exposure of the drugs ganciclovir, acyclovir, or any of their analogues FIAU, FIAC, DHPG) to cells expressing HSVTK allows conversion of the drug into its corresponding active nucleotide triphosphate form.
Other gene products that may be utilized within the context of the present invention include E. coli guanine phosphoribosyl transferase, which converts thioxanthine into toxic thioxanthine monophosphate (Besnard et al., Mol. Cell. Biol.
7:4139-4141, 1987); alkaline phosphatase, which converts inactive phosphorylated compounds such as mitomycin phosphate and doxorubicin-phosphate to toxic dephosphorylated compounds; fungal Fusarium oxysporum) or bacterial cytosine deaminase, which converts 5-fluorocytosine to the toxic compound (Mullen, PNAS 89:33, 1992); carboxypeptidase G2, which cleaves glutamic acid from para-N-bis (2-chloroethyl) aminobenzoyl glutamic acid, thereby creating a toxic benzoic acid mustard; and Penicillin-V amidase, which converts phenoxyacetabide derivatives of doxorubicin and melphalan to toxic compounds (see generally, Vrudhula et al., J. of Med Chem. 36(7):919-923, 1993; Kern et al., Cane. Immun. Immunother.
31(4):202-206, 1990). Moreover, a wide variety of Herpesviridae thymidine kinases, including both primate and non-primate herpesviruses, are suitable. Such herpesviruses include Herpes Simplex Virus Type 1 (McKnight et al., Nuc. Acids Res 8:5949-5964, 1980), Herpes Simplex Virus Type 2 (Swain and Galloway, J. Virol. 46:1045-1050, 1983), Varicella Zoster Virus (Davison and Scott, J. Gen. Virol. 67:1759-1816, 1986), marmoset herpesvirus (Otsuka and Kit, Virology 135:316-330, 1984), feline herpesvirus type 1 (Nunberg et al., J. Virol. 63:3240-3249, 1989), pseudorabies virus (Kit and Kit, U.S. Patent No. 4,514,497, 1985), equine herpesvirus type 1 (Robertson and Whalley, Nuc. Acids Res. 16:11303-11317, 1988), bovine herpesvirus type 1 (Mittal and Field, J.
Virol 70:2901-2918, 1989), turkey herpesvirus (Martin et al., J. Virol. 63:2847-2852, 1989), Marek's disease virus (Scott et al., J. Gen. Virol. 70:3055-3065, 1989), herpesvirus saimiri (Honess et al., J Gen. Virol. 70:3003-3013, 1989) and Epstein-Barr WO 96/36362 PCT/US96/07164 36 virus (Baer et al., Nature (London) 310:207-311, 1984). Such herpesviruses may be readily obtained from commercial sources such as the American Type Culture Collection ("ATCC", Rockville, Maryland).
Furthermore, as indicated above, a wide variety of inactive precursors may be converted into active inhibitors. For example, thymidine kinase can phosphorylate nucleosides dT) and nucleoside analogues such as ganciclovir (9- {[2-hydroxy-l-(hydroxymethyl)ethoxyl methyl} guanosine), famciclovir, buciclovir, penciclovir, valciclovir, acyclovir (9-[2-hydroxy ethoxy)methyl] guanosine), trifluorothymidine, 1-[2-deoxy, 2-fluoro, beta-D-arabino furanosyl]-5-iodouracil, ara-A (adenosine arabinoside, vivarabine), 1-beta-D-arabinofuranoxyl thymine, 5-ethyl-2'deoxyuridine, 5-iodo-5'-amino-2,5'-dideoxyuridine, idoxuridine (5-iodo-2'deoxyuridine), AZT azido-3' thymidine), ddC (dideoxycytidine), AIU amino 5'-dideoxyuridine) and AraC (cytidine arabinoside).
E. Other nucleic acid molecules The conjugates provided herein may also be used to deliver other types of nucleic acids to targeted cells. Such other nucleic acids include antisense RNA, antisense DNA, ribozymes, triplex-forming oligonucleotides, and oligonucleotides that bind proteins. The nucleic acids can also include RNA trafficking signals, such as viral packaging sequences (see, Sullenger et al. (1994) Science 262:1566-1569). The nucleic acids also include DNA molecules that encode proteins that replace defective genes, such as the gene associated with cystic fibrosis (see, PCT Application WO 93/03709, U.S. Application Serial No. 07/745,900; and Riordan et al. (1989) Science 245:1066-1073). Other DNA molecules may encode tumor-specific cytotoxic molecules, such as tumor necrosis factor, viral antigens and other proteins to render a cell susceptible to anti-cancer agents.
Nucleic acids and oligonucleotides for use as described herein can be synthesized by any method known to those of skill in this art (see, WO 93/01286, U.S. Application Serial No. 07/723,454; Patent No. 5,218,088; U.S. Patent No.
5,175,269; U.S. Patent No. 5,109,124). Identification of oligonucleotides and ribozymes for use as antisense agents and DNA encoding genes for targeted delivery for WO 96/36362 PCT/US96/07164 37 genetic therapy involve methods well known in the art. For example, the desirable properties, lengths and other characteristics of such oligonucleotides are well known.
Antisense oligonucleotides are typically designed to resist degradation by endogenous nucleolytic enzymes and include, but are not limited to: phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and other such linkages (see, Agrwal et al., Tetrehedron Lett.
28:3539-3542 (1987); Miller et al., J. Am. Chem. Soc. 93:6657-6665 (1971); Stec et al., Tetrehedron Lett. 26:2191-2194 (1985); Moody et al., Nucl. Acids Res. 12:4769-4782 (1989); Uznanski et al., Nucl. Acids Res. (1989); Letsinger et al., Tetrahedron 40:137- 143 (1984); Eckstein, Annu. Rev. Biochem. 54:367-402 (1985); Eckstein, Trends Biol.
Sci. 14:97-100 (1989); Stein In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, Ed, Macmillan Press, London, pp. 97-117 (1989); Jager et al., Biochemistry 27:7237-7246 (1988)).
Antisense nucleotides are oligonucleotides that bind in a sequencespecific manner to nucleic acids, such as mRNA or DNA. When bound to mRNA that has complementary sequences, antisense prevents translation of the mRNA (see, e.g., U.S. Patent No. 5,168,053 to Altman et al.; U.S. Patent No. 5,190,931 to Inouye, U.S.
Patent No. 5,135,917 to Burch; U.S. Patent No. 5,087,617 to Smith and Clusel et al.
(1993) Nucl. Acids Res. 21:3405-3411, which describes dumbbell antisense oligonucleotides). Triplex molecules refer to single DNA strands that bind duplex DNA forming a colinear triplex molecule and thereby prevent transcription (see, e.g., U.S. Patent No. 5,176,996 to Hogan et al., which describes methods for making synthetic oligonucleotides that bind to target sites on duplex DNA).
Particularly useful antisense nucleotides and triplex molecules are molecules that are complementary or bind to the sense strand of DNA or mRNA that encodes an oncogene, such as bFGF, int-2, hst-1/K-FGF, FGF-5, hst-2/FGF-6, FGF-8.
Other useful antisense oligonucleotides include those that are specific for IL-8 (see, e.g., U.S. Patent No. 5,241,049; and PCT Applications WO 89/004836; WO 90/06321; WO 89/10962; WO 90/00563; and WO 91/08483), which can be linked to bFGF for the treatment of psoriasis, anti-sense oligonucleotides that are specific for nonmuscle WO 96/36362 PCTIUS96/07164 38 myosin heavy chain and/or c-myb (see, Simons et al. (1992) Circ. Res. 70:835-843; PCT Application WO 93/01286, U.S. application Serial No. 07/723,454: LeClerc et al.
(1991) J. Am. Coll. Cardiol. 17 (2 Suppl. A):105A; Ebbecke et al. (1992) Basic Res.
Cardiol. 87:585-591), which can be targeted by an FGF to inhibit smooth muscle cell proliferation, such as that following angioplasty and thereby prevent restenosis or inhibit viral gene expression in transformed or infected cells.
A ribozyme is an RNA molecule that specifically cleaves RNA substrates, such mRNA, and thus inhibits or interferes with cell growth or expression.
There are at least five classes of ribozymes that are known that are involved in the cleavage and/or ligation of RNA chains. Ribozymes can be targeted to any RNA transcript and can catalytically cleave such transcript (see, U.S. Patent No.
5,272,262; U.S. Patent No. 5,144,019; and U.S. Patent Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246 to Cech et al., which described ribozymes and methods for production thereof). Any such ribosome may be linked to the growth factor for delivery to a cell bearing a receptor for a receptor-internalized binding ligand.
The ribozymes may be delivered to the targeted cells by DNA encoding the ribozyme linked to a eukaryotic promoter, such as a eukaryotic viral promoter, such that upon introduction into the nucleus, the ribozyme will be directly transcribed. In such instances, the construct will also include a nuclear translocation sequence, generally as part of the ligand or as part of a linker between the ligand and nucleic acid binding domain.
DNA that encodes a therapeutic product contemplated for use includes DNA encoding correct copies of defective genes, such as the defective gene (CFTR) associated with cystic fibrosis (see, International Application WO 93/03709, U.S.
Application Serial No. 07/745,900; and Riordan et al. (1989) Science 245:1066-1073), and anticancer agents, such as tumor necrosis factors. The conjugate preferably includes an NTS. If the conjugate is designed such that the ligand and nucleic acid binding domain are cleaved in the cytoplasm, then the NTS should be included in a portion of the conjugate or linker that remains bound to the DNA. The nuclear WO 96/36362 PCT/US96/07164 39 translocation sequence (NTS) may be a heterologous sequence or a may be derived from the selected growth factor.
F. Construct containing cytocidal-encoding agent In the case of cytotocide molecules such as the ribosome inactivating proteins, very few molecules may need to be expressed to effect cell killing. Indeed, only a single molecule of diphtheria toxoid introduced into a cell was sufficient to kill the cell. With other cytocides or prodrugs, it may be that propagation or stable maintenance of the construct is necessary to attain a sufficient amount or concentration of the gene product for effective gene therapy. Examples of replicating and stable eukaryotic plasmids may be found in the scientific literature.
In general, constructs will also contain elements necessary for transcription and translation. If the cytocide-encoding agent is DNA, then it must contain a promoter. The choice of the promoter will depend upon the cell type to be transformed and the degree or type of control desired. Promoters can be constitutive or active in any cell type, tissue specific, cell specific, event specific temporal-specific or inducible. Cell-type specific promoters and event type specific promoters are preferred.
Examples of constitutive or nonspecific promoters include the SV40 early promoter Patent No. 5,118,627), the SV40 late promoter Patent No. 5,118,627), CMV early gene promoter Patent No. 5,168,062), and adenovirus promoter. In addition to viral promoters, cellular promoters are also amenable within the context of this invention. In particular, cellular promoters for the so-called housekeeping genes are useful. Viral promoters are preferred, because generally they are stronger promoters than cellular promoters.
Tissue specific promoters are particularly useful when a certain tissue type is to be targeted for transformation. By using one of this class of promoters, an extra margin of specificity can be attained. For example, when the indication to be treated is ophthalmological secondary lens clouding), either the alpha-crystalline promoter or gamma-crystalline promoter is preferred. When a tumor is the target of gene delivery, cellular promoters for specific tumor markers or promoters more active in tumor cells should be chosen. Thus, to treat prostate tumor, the prostate-specific WO 96/36362 PCT/US96/07164 antigen promoter is especially useful. Similarly, the tyrosinase promoter or tyrosinaserelated protein promoter is a preferred promoter for melanoma treatment. For treatment of diseases that are angiogenic or exacerbated by angiogenesis, the VEGF receptor promoter is preferred. The VEGF receptor is expressed in developing capillaries. For treatment of breast cancer, the promoter from heat shock protein 27 is preferred; for treatment of colon or lung cancer, the promoter from carcinoembryonic antigen is preferred; for treatment of restenosis or other diseases involving smooth muscle cells, the promoter from a-actin or myosin heavy chain is preferred. For B lymphocytes, the immunoglobulin variable region gene promoter; for T lymphocytes, the TCR receptor variable region promoter; for helper T lymphocytes, the CD4 promoter; for liver, the albumin or a-fetoprotein promoter, are a few additional examples of tissue specific promoters. Many other examples of tissue specific promoters are readily available to one skilled in the art. Some of these promoters are temporally regulated, such as c-myc and cyclin D.
Inducible promoters may also be used. These promoters include the MMTV LTR (PCT WO 91/13160), which is inducible by dexamethasone, metallothionein, which is inducible by heavy metals, and promoters with cAMP response elements, which are inducible by cAMP. By using an inducible promoter, the nucleic acid may be delivered to a cell and will remain quiescent until the addition of the inducer. This allows further control on the timing of production of the therapeutic gene.
Event-type specific promoters are active or up-regulated only upon the occurrence of an event, such as tumorigenecity or viral infection. The HIV LTR is a well known example of an event-specific promoter. The promoter is inactive unless the tat gene product is present, which occurs upon viral infection. Another promoter is c-myc.
Additionally, promoters that are coordinately regulated with a particular cellular gene may be used. For example, promoters of genes that are coordinately expressed when a particular FGF receptor gene is expressed may be used. Then, the nucleic acid will be transcribed when the FGF receptor, such as FGFR1, is expressed, WO 96/36362 PCT/US96/07164 41 and not when FGFR2 is expressed. This type of promoter is especially useful when one knows the pattern of FGF receptor expression in a particular tissue, so that specific cells within that tissue may be killed upon transcription of a cytotoxic agent gene without affecting the surrounding tissues.
If the domain binds in a sequence specific manner, the construct must contain the sequence that binds to the nucleic acid binding domain. As described below, the target nucleotide sequence may be contained within the coding region of the cytocide, in which case, no additional sequence need be incorporated. Additionally, it may be desirable to have multiple copies of target sequence. If the target sequence is coding sequence, the additional copies must be located in non-coding regions of the cytocide-encoding agent. The target sequences of the nucleic acid binding domains are typically generally known. If unknown, the target sequence may be readily determined.
Techniques are generally available for establishing the target sequence see PCT Application WO 92/05285 and U.S. Serial No. 586,769).
G. Other Elements 1. Nuclear translocation signal As used herein, a "nuclear translocation or targeting sequence" (NTS) is a sequence of amino acids in a protein that are required for translocation of the protein into a cell nucleus. Examples of NTSs are set forth in Table 2 below. Comparison with known NTSs, and if necessary testing of candidate sequences, should permit those of skill in the art to readily identify other amino acid sequences that function as NTSs. A heterologous NTS refers to an NTS that is different from the NTS that occurs in the wild-type peptide, polypeptide, or protein. For example, the NTS may be derived from another polypeptide, it may be synthesized, or it may be derived from another region in the same polypeptide.
WO 96/36362 PCT/fUS96/07164 42 TABLE 2 Source Sequence SEQ ID
NO.
large T Pr 26 LysLysArgLysValGlu 24 Polyoma large T Pro 279 ProLysLysAlaArgGluVal Human c-Myc: Pro 120AalysralseuP 26 Adenovirus ElA Lys 28 1 ArgProArgPro 27 Yeast mat aC 2 Lys 3IleProlleLys 28 cEbAA. Gl 2LysArgLysArgLysSer 29 B. Ser'2 LysArgValAlaLysArgLysLeu C. Ser 1 81 HisTrpLysGlnLysArgLysPhe 31 LeuLeuLysLysileLysGin 32 p 5 3 Po 316 GlnProLysLysLysPro 33 Pro_27 GlyLysArgLysLysGluMetThrLyscjlnLysGluValPro 34 HIV Tat Gly 48 ArgLysLysArgArgGlnArgArgArgAlaPro FGF- I AsnTyrLysLysProLysLeu 36 FGF-2 HisPheLysAspProLysArg 37 FGF-3 AlaProArgArgArgLysLeu 38 FGF-4 leLysArgLeuArg~rg 39 GlyArgArg FGF-6 IleLysArgGlnArgArg [FGF-7 IleArgValArgArg 41 *Superscript indicates position in protein In order to deliver the nucleic acid to the nucleus, the conjugate should include an NTS. If the conjugate is designed such that the receptor-binding internalized ligand and linked nucleic acid binding domain is cleaved or dissociated in the cytoplasm, then the NTS should be included in a portion of the complex that remains bound to the nucleic acid, so that, upon internalization, the conjugate will be trafficked to the nucleus. Thus, the NTS is preferably included in the nucleic acid binding domain, but may additionally be included in the ligand. An NTS is preferred if the cytocide-encoding agent is DNA. If the cytocide-encoding agent is mRNA, an NTS WO 96/36362 PCT/US96/07164 43 may be omitted. The nuclear translocation sequence (NTS) may be a heterologous sequence or a may be derived from the selected growth factor. All presently identified members of the FGF family of peptides contain an NTS (see, International Application WO 91/15229 and Table 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, Dang et al., J. Biol. Chem. 264:18019-18023, 1989; Dang et al., Mol. Cell. Biol. 8:4049-4058, 1988, and Table 2).
2. Cvtoplasm-translocation signal Cytoplasm-translocation signal sequence is a sequence of amino acids in a protein that cause retention of proteins in the lumen of the endoplasmic reticulum and/or translocate proteins to the cytosol. The signal sequence in mammalian cells is KDEL (Lys-Asp-Glu-Leu) (SEQ ID NO. 42) (Munro and Pelham, Cell 48:899-907, 1987). Some modifications of this sequence have been made without loss of activity.
For example, the sequences RDEL (Arg-Asp-Glu-Leu) (SEQ ID NO. 43) and KEEL (Lys-Glu-Glu-Leu) (SEQ ID NO. 44) confer efficient or partial retention, respectively, in plants (Denecke et al., Embo. J 11:2345-2355, 1992).
A cytoplasm-translocation signal sequence may be included in either the receptor-internalized binding ligand or the nucleic acid binding domain part or both. If cleavable linkers are used to link the ligand with the nucleic acid binding domain, the cytoplasm-translocation signal is preferably included in the nucleic acid binding domain, which will stay bound to the cytocide-encoding agent. Additionally, a cytoplasmic-translocation signal sequence may be included in the receptor-internalized binding ligand, as long as it does not interfere with receptor binding. Similarly, the signal sequence placed in the nucleic acid binding domain should not interfere with binding to the cytocide-encoding agent.
3. Endosome-disruptive peptides In addition, or alternatively, membrane-disruptive peptides may be incorporated into the complexes. For example, adenoviruses are known to enhance WO 96/36362 PCT/US96/07164 44 disruption of endosomes. Virus-free viral proteins, such as influenza virus hemagglutinin HA-2, also disrupt endosomes and are useful in the present invention.
Other proteins may be tested in the assays described herein to find specific endosome disrupting agents that enhance gene delivery. In general, these proteins and peptides are amphipathic (see Wagner et al., Adv. Drug. Del. Rev. 14:113-135, 1994).
Endosome-disruptive peptides, sometimes called fusogenic peptides, may be incorporated into the complex of receptor-internalized binding ligand, nucleic acid binding domain, and cytocide-encoding agent. Two such peptides derived from influenza virus are: GLFEAIEGFIENGWEGMIDGGGC (SEQ. ID NO. 45) and GLFEAIEGFIENGWEGMIDGWYGC (SEQ. ID NO. 46). Other peptides useful for disrupting endosomes may be identified by general characteristics: 25-30 residues in length, contain an alternating pattern of hydrophobic domains and acidic domains, and at low pH pH 5) from amphipathic a-helices. A candidate endosome-disrupting peptide is tested by incorporating it into the complex and determining whether it increases the total number of cells expressing the target gene. The peptides are added to a complex having excess negative charge. For example, a DNA construct is complexed with an FGF-poly-L-lysine chemical conjugate so that only a portion of the negative charge of the DNA is neutralized. Poly-L-lysine is added to further bind the DNA and a fusogenic peptide is then added. Optional ratios of DNA, poly-L-lysine and fusogenic peptide are determined using assays, such as gene expression and cell viability.
The fusogenic peptides may alternatively be incorporated into the complex as a fusion protein with either the ligand or the nucleic acid binding domain or both. The endosome-disruptive peptide may be present as single or multiple copies at the N- or C- terminus of the ligand. A single fusion protein of the endosome-disruptive peptide, nucleic acid binding domain, and receptor-interalized binding ligand may be constructed and expressed. For insertion into a construct, DNA encoding the endosome-disruptive peptide may be synthesized by PCR using overlapping oligonucleotides and incorporating a restriction site at the 5' and 3' end to facilitate cloning. The sequence may be verified by sequence analysis.
WO 96/36362 PCT/US96/07164 4. Linkers As used herein, a "linker" is an extension that links the receptor-binding internalized ligand or fragment thereof and the nucleic acid binding domain. In certain instances, the linker is used to conjugate the ligand directly to the nucleic acid. The linkers provided herein confer specificity, enhance intracellular availability, serum stability and/or solubility on the conjugate and may serve to promote condensation of the nucleic acid.
The linkers provided herein confer specificity and serum stability on the cytotoxic conjugate, for example, by conferring specificity for certain proteases, particularly proteases that are present in only certain subcellular compartments or that are present at higher levels in tumor cells than normal cells. Specificity for proteases present in intracellular compartments and absent in blood is particularly preferred. The linkers may also include sorting signals that direct the conjugate to particular intracellular loci or compartments. Additionally, the linkers may reduce steric hindrance between the growth factor and other protein or linked nucleic acid by distancing the components of the conjugate. Linkers may also condense the nucleic acid. For this purpose, the linker comprises highly basic amino acids Lys, Arg) and may even by poly-L-lysine.
In order to increase the serum stability, solubility and/or intracellular concentration or condense the targeted agent, one or more linkers (are) inserted between the receptor-binding internalized ligand and the nucleic acid binding domain. These linkers include peptide linkers, such as intracellular protease substrates, and chemical linkers, such as acid labile linkers, ribozyme substrate linkers and others. Peptides linkers may be inserted using heterobifunctional reagents, described below, or, preferably, are linked to FGF, other growth factors, including heparin-binding growth factors, or cytokines by linking DNA encoding the ligand to the DNA encoding the nucleic acid binding domain.
Chemical linkers may be inserted by covalently coupling the linker to the FGF, other growth factor protein, or cytokine and the nucleic acid binding domain. The linker may be bound via the N- or C-terminus or an internal residue. The WO 96/36362 PCTIUS96/07164 46 heterobifunctional agents, described below, may be used to effect such covalent coupling.
a. Protease substrates Peptides encoding protease-specific substrates may be introduced between the ligand and the nucleic acid binding domain. The peptides may be inserted using heterobifunctional reagents, as described below, or preferably inserted by recombinant means and expression of the resulting chimera.
Any protease specific substrate (see, O'Hare et al., FEBS 273:200- 204, 1990; Forsberg et al., J. Protein Chem. 10:517-526, 1991; Westby et al., Bioconjugate Chem. 3:375-381, 1992) may be introduced as a linker as long as the substrate is cleaved in an intracellular compartment. Preferred substrates include those that are specific for proteases that are expressed at higher levels in tumor cells, that are preferentially expressed in the endosome, or that are absent in blood. The following substrates are among those contemplated for use in accord with the methods herein: cathepsin B substrate, cathepsin D substrate, trypsin substrate, thrombin substrate, and recombinant subtilisin substrate.
b. Flexible linkers and linkers that increase the solubility of the conjugates Flexible linkers, which reduce steric hindrance, and linkers that increase solubility of the conjugates are contemplated for use, either alone or with other linkers, such as the protease specific substrate linkers. Typically, these linkers are simple polymers of small amino acids small side groups) with uncharged polar side groups. These amino acids (Gly, Ser, Thr, Cys, Tyr, Asn, Gin) are more soluble in water. Of these amino acids, Gly and Ser are preferred. Such linkers include, but are not limited to, (Gly 4 Ser),, (Ser 4 Gly), and (AlaAlaProAla), in which n is 1 to 6, preferably 1-4, such as: a. Gly 4 Ser SEQ ID NO: 47 CCATGGGCGG CGGCGGCTCT GCCATGG WO 96/36362 PCT/US96Io7164 47 b. (GlySer), SEQ ID NO: 48 CCATGGGCGG CGGCGGCTCT GGCGGCGGCG GCTCTGCCAT
GG
c. (SerGIY) 4 SEQ ID NO: 49 CCATGGCCTC GTCGTCGTCG GGCTCGTCGT CGTCGGGCTC GTCGTCGTCG
GGCTCGTCGT
CGTCGGGCGC CATGG d. (SerGly), SEQ ID NO: CCATGGCCTC GTCGTCGTCG GGCTCGTCGT CGTCGGGCGC
CATGG
e. (AlaProAla), where n is 1 to 4. preferably 2 (see SEQ ID NO: 51) C. Heterobifunctional cross-linking reagents Numerous heterobifunctional cross-linking reagents that are used to form covalent bonds between amino groups and thiol groups and to introduce thiol groups into proteins, are known to those of skill in this art (see, e.g, the PIERCE CATALOG, ImmunoTechnology Catalog Handbook, 1992-1993, which describes the preparation of and use of such reagents and provides a commercial source for such reagents; see also, Cumber et al., Bioconjugate Chem. 3:397-401, 1992; Thorpe et al., Cancer Res. 47:5924-5931, 1987; Gordon et al., Proc. Nat. Acad Sci. 84:308-312, 1987; Walden et al., J1. Mol Cell Immunol. 2:191-197, 1986; Carlsson et al., Biochem. J 173:723-737, 1978; Mahan et al., Anal Biochem. 162:163-170, 1987; Wawryznaczak et al., Br. J Cancer 66:361-366, 1992; Fattomn et al., Infection Immun. 60:584-5 89, 1992). These reagents may be used to form covalent bonds between the receptorbinding internalized ligands with protease substrate peptide linkers and nucleic acid binding domain. These reagents include, but are not limited to: N-succinimnidyl-3-(2pyridyldithio)propionate (SPDP; disulfide linker); sulfosuccinirnidyl pyridyldithio)propionamnido]hexanoate (sulfo-LC-SPDP); succinimidyloxycarbonyl-cmethyl benzyl thiosulfate (SMBT, hindered disulfate linker); succinimidyl pyridyldithio) propionamido]hexanoate (LC-SPDP); sulfosuccinimidyl 4 -(N-maleimidomethyl)cyclohexane- 1 -carboxylate (sulfo-SMCC); succinimidyl 3 -(2-pyridvldithio)butyrate (SPDB; hindered disulfide bond linker); sulfosuccinimidyl 2 7 -azido-4-methylcoumarin-3-acetaniide) ethyl- 1 ,3-dithiopropionate
(SAED);
sulfosuccinimidyl 7-azido-4-methylcoumarin-3 -acetate (SAMCA); sulfosuccinirnidyl WO 96/36362 PCT/US96/07164 48 6-alpha-methyl-alpha-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-SMPT); 1, 4 -di-[ 3 2 '-pyridyldithio)propionamido]butane
(DPDPB);
4 -succinimidyloxycarbonyl- -methyl- 2 -pyridylthio)toluene (SMPT, hindered disulfate linker); sulfosuccinimidyl6[ -methyl- 2 -pyridyldithio)toluamido]hexanoate (sulfo-LC- SMPT); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); m-maleimidobenzoyl- N-hydroxysulfosuccinimide ester (sulfo-MBS); N-succinimidyl(4iodoacetyl)aminobenzoate (SIAB; thioether linker); sulfosuccinimidyl(4-iodoacetyl)amino benzoate (sulfo-SIAB); succinimidyl 4 (p-maleimidophenyl)butyrate (SMPB); sulfosuccinimidyl 4 -(p-maleimidophenyl)butyte (sulfo-SMPB); azidobenzoyl hydrazide
(ABH).
These linkers should be particularly useful when used in combination with peptide linkers, such as those that increase flexibility.
d. Acid cleavable, photocleavable and heat sensitive linkers Acid cleavable linkers include, but are not limited to, bismaleimideothoxy propane, adipic acid dihydrazide linkers (see, Fattom et al., Infection Immun. 60:584-589, 1992) and acid labile transferrin conjugates that contain a sufficient portion of transferrin to permit entry into the intracellular transferrin cycling pathway (see, Welh6ner et al., J. Biol. Chem. 266:4309-4314, 1991).
Conjugates linked via acid cleavable linkers should be preferentially cleaved in acidic intracellular compartments, such as the endosome.
Photocleavable linkers are linkers that are cleaved upon exposure to light (see, Goldmacher et al., Bioconj. Chem. 3:104-107, 1992), thereby releasing the targeted agent upon exposure to light. (Hazum et al., Proc. Eur. Pept. Symp., 16th, Brunfeldt, K pp. 105-110, 1981; nitrobenzyl group as a photocleavable protective group for cysteine; Yen et al., Makromol. Chem 190:69-82, 1989; water soluble photocleavable copolymers, including hydroxypropylmethacrylamide copolymer, glycine copolymer, fluorescein copolymer and methylrhodamine copolymer; and Senter et al., Photochem. Photobiol. 42:231-237, 1985; nitrobenzyloxycarbonyl chloride cross linking reagents that produce photocleavable linkages). Such linkers are particularly useful in treating dermatological or ophthalmic conditions and other tissues, such as WO 96/36362 PCT/US96/07164 49 blood vessels during angioplasty in the prevention or treatment of restenosis, that can be exposed to light using fiber optics. After administration of the conjugate, the eye or skin or other body part can be exposed to light, resulting in release of the targeted moiety from the conjugate. This should permit administration of higher dosages of such conjugates compared to conjugates that release a cytotoxic agent upon internalization.
Heat sensitive linkers would also have similar applicability.
H. Expression vectors and host cells for expression of receptor-binding internalized ligands and nucleic acid binding domains Host organisms include those organisms in which recombinant production of heterologous proteins have been carried out, such as bacteria (for example, E. coli), yeast (for example, Saccharomyces cerevisiae and Pichia pastoris), mammalian cells, and insect cells. Presently preferred host organisms are E. coli bacterial strains.
The DNA construct encoding the desired protein is introduced into a plasmid for expression in an appropriate host. In preferred embodiments, the host is a bacterial host. The sequence encoding the ligand or nucleic acid binding domain is preferably codon-optimized for expression in the particular host. Thus, for example, if human FGF-2 is expressed in bacteria, the codons would be optimized for bacterial usage. For small coding regions the gene can be synthesized as a single oligonucleotide. For larger proteins, splicing of multiple oligonucleotides, mutagenesis, or other techniques known to those in the art may be used. For example, the sequence of a bacterial-codon preferred FGF-SAP fusion is shown in SEQ. ID NO. 80. The sequences of nucleotides in the plasmids that are regulatory regions, such as promoters and operators, are operationally associated with one another for transcription. The sequence of nucleotides encoding the growth factor or growth factor-chimera may also include DNA encoding a secretion signal, whereby the resulting peptide is a precursor protein. The resulting processed protein may be recovered from the periplasmic space or the fermentation medium.
In preferred embodiments, the DNA plasmids also include a transcription terminator sequence. As used herein, a "transcription terminator region" has either a WO 96/36362 PCT/US96/07164 subsegment that encodes a polyadenylation signal and polyadenylation site in the transcript, and/or a subsegment that provides a transcription termination signal that terminates transcription by the polymerase that recognizes the selected promoter. The entire transcription terminator may be obtained from a protein-encoding gene, which may be the same or different from the inserted gene or the source of the promoter.
Transcription terminators are optional components of the expression systems herein, but are employed in preferred embodiments.
The plasmids used herein include a promoter in operable association with the DNA encoding the protein or polypeptide of interest and are designed for expression of proteins in a bacterial host. It has been found that tightly regulatable promoters are preferred for expression of saporin. Suitable promoters for expression of proteins and polypeptides herein are widely available and are well known in the art.
Inducible promoters or constitutive promoters that are linked to regulatory regions are preferred. Such promoters include, but are not limited to, the T7 phage promoter and other T7-like phage promoters, such as the T3, T5 and SP6 promoters, the trp, Ipp, and lac promoters, such as the lacUV5, from E. coli; the P10 or polyhedron gene promoter of baculovirus/insect cell expression systems (see, U.S. Patent Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784) and inducible promoters from other eukaryotic expression systems. For expression of the proteins such promoters are inserted in a plasmid in operative linkage with a control region such as the lac operon.
Preferred promoter regions are those that are inducible and functional in E. coli. Examples of suitable inducible promoters and promoter regions include, but are not limited to: the E. coli lac operator responsive to isopropyl p -D-thiogalactopyranoside (IPTG; see, et al. Nakamura et al., Cell 18:1109-1117, 1979); the metallothionein promoter metal-regulatory-elements responsive to heavy-metal zinc) induction (see, U.S. Patent No. 4,870,009 to Evans et the phage T71ac promoter responsive to IPTG (see, U.S. Patent No. 4,952,496; and Studier et al., Meth. Enzymol. 185:60-89, 1990) and the TAC promoter.
The plasmids also preferably include a selectable marker gene or genes that are functional in the host. A selectable marker gene includes any gene that confers WO 96/36362 PCT/US96/07164 51 a phenotype on bacteria that allows transformed bacterial cells to be identified and selectively grown from among a vast majority of untransformed cells. Suitable selectable marker genes for bacterial hosts, for example, include the ampicillin resistance gene (Ampr), tetracycline resistance gene (Tcr) and the kanamycin resistance gene (Kanr). The kanamycin resistance gene is presently preferred.
The plasmids may also include DNA encoding a signal for secretion of the operably linked protein. Secretion signals suitable for use are widely available and are well known in the art. Prokaryotic and eukaryotic secretion signals functional in E. coli may be employed. The presently preferred secretion signals include, but are not limited to, those encoded by the following E. coli genes: ompA, ompT, ompF, ompC, beta-lactamase, and alkaline phosphatase, and the like (von Heijne, J. Mol. Biol.
184:99-105, 1985). In addition, the bacterial pelB gene secretion signal (Lei et al., J Bacteriol. 169:4379, 1987), the phoA secretion signal, and the cek2 functional in insect cell may be employed. The most preferred secretion signal is the E. coli ompA secretion signal. Other prokaryotic and eukaryotic secretion signals known to those of skill in the art may also be employed (see, von Heijne, J. Mol. Biol. 184:99-105, 1985). Using the methods described herein, one of skill in the art can substitute secretion signals that are functional in either yeast, insect or mammalian cells to secrete proteins from those cells.
Particularly preferred plasmids for transformation of E. coli cells include the pET expression vectors (see U.S patent 4,952,496; available from Novagen, Madison, WI; see also literature published by Novagen describing the system). Such plasmids include pET 11 a, 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 (Novagen, Madison, WI), which contains a His-TagTM leader sequence for use in purification with a His column and a thrombin cleavage site that permits cleavage following purification over the column, the T7-lac promoter region and the T7 terminator.
WO 96/36362 PCT/US96/07164 52 Other preferred plasmids include the pKK plasmids, particularly pKK 223-3, which contains the tac promoter, (available from Pharmacia; see also Brosius et al., Proc. Natl. Acad. Sci. 81:6929, 1984; Ausubel et al., Current Protocols in Molecular Biology; U.S. Patent Nos. 5,122,463, 5,173,403, 5,187,153, 5,204,254, 5,212,058, 5,212,286, 5,215,907, 5,220,013, 5,223,483, and 5,229,279). Plasmid pKK has been modified by replacement of the ampicillin resistance marker gene, by digestion with EcoRI, with a kanamycin resistance cassette with EcoRI sticky ends (purchased from Pharmacia; obtained from pUC4K, see, Vieira et al. (Gene 19:259-268, 1982; and U.S. Patent No. 4,719,179). Baculovirus vectors, such as pBlueBac (also called pJVETL and derivatives thereof), particularly pBlueBac III, (see, U.S. Patent Nos. 5,278,050, 5,244,805, 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784; available from Invitrogen, San Diego) may also be used for expression of the polypeptides in insect cells. The pBlueBacIII vector is a dual promoter vector and provides for the selection of recombinants by blue/white screening as this plasmid contains the P-galactosidase gene (lacZ) under the control of the insect recognizable ETL promoter and is inducible with IPTG. A DNA construct may be made in baculovirus vector pBluebac III and then co-transfected with wild type virus into insect cells Spodoptera frugiperda (sf9 cells; see, Luckow et al., Bio/technology 6:47-55, 1988, and U.S. Patent No. 4,745,051).
Other plasmids include the plN-IIIompA plasmids (see U.S. Patent No. 4,575,013; see also Duffaud et al., Meth. Enz. 153:492-507, 1987), such as pIN- IIIompA2. The pIN-IIIompA plasmids include an insertion site for heterologous
DNA
linked in transcriptional reading frame with four functional fragments derived from the lipoprotein gene of E. coli. The plasmids also include a DNA fragment coding for the signal peptide of the ompA protein of E. coli, positioned such that the desired polypeptide is expressed with the ompA signal peptide at its amino terminus, thereby allowing efficient secretion across the cytoplasmic membrane. The plasmids further include DNA encoding a specific segment of the E. coli lac promoter-operator, which is positioned in the proper orientation for transcriptional expression of the desired polypeptide, as well as a separate functional E. coli lacI gene encoding the associated WO 96/36362 PCT/US96/071 6 4 53 repressor molecule that, in the absence of lac operon inducer, interacts with the lac promoter-operator to prevent transcription therefrom. Expression of the desired polypeptide is under the control of the lipoprotein (lpp) promoter and the lac promoter-operator, although transcription from either promoter is normally blocked by the repressor molecule. The repressor is selectively inactivated by means of an inducer molecule thereby inducing transcriptional expression of the desired polypeptide from both promoters.
Preferably, the DNA fragment is replicated in bacterial cells, preferably in E. coli. The preferred DNA fragment also includes a bacterial origin of replication, to ensure the maintenance of the DNA fragment from generation to generation of the bacteria. In this way, large quantities of the DNA fragment can be produced by replication in bacteria. Preferred bacterial origins of replication include, but are not limited to, the fl-ori and col El origins of replication. Preferred hosts contain chromosomal copies of DNA encoding T7 RNA polymerase operably linked to an inducible promoter, such as the lacUV promoter (see U.S. Patent No. 4,952,496). Such hosts include, but are not limited to, lysogens E. coli strains HMS17 4 (DE3)pLysS, BL21(DE3)pLysS, HMS174(DE3) and BL21(DE3). Strain BL21(DE3) is preferred.
The pLys strains provide low levels of T7 lysozyme, a natural inhibitor of T7 RNA polymerase.
The DNA fragments provided may also contain a gene coding for a repressor protein. The repressor protein is capable of repressing the transcription of a promoter that contains sequences of nucleotides to which the repressor protein binds.
The promoter can be derepressed by altering the physiological conditions of the cell.
For example, the alteration can be accomplished by adding to the growth medium a molecule that inhibits the ability to interact with the operator or with regulatory proteins or other regions of the DNA or by altering the temperature of the growth media.
Preferred repressor proteins include, but are not limited to the E. coli lad repressor responsive to IPTG induction, the temperature sensitive k c1857 repressor, and the like.
The E. coli lad repressor is preferred.
WO 96/36362 PCT/US96/07164 54 DNA encoding full-length FGF-2 or FGF-2 mutein is linked to DNA encoding an nucleic acid binding domain, such as protamine, and introduced into the pET vectors, including pET-I la and pET-12a expression vectors (Novagen, Madison, WI), for intracellular and periplasmic expression, respectively, of FGF-protamine fusion proteins.
I. Preparation of complexes containing receptor-binding internalized ligands/nucleic acid binding domain conuates and ctocide-encoding aents Within the context of this invention, specificity of delivery is achieved through the ligand. Typically, a nucleic acid binding domain is coupled to a receptorbinding internalized ligand, either by chemical conjugation or as a fusion protein. As described below, the ligand may alternatively be coupled directly to the nucleic acid and then complexed with a nucleic acid binding protein, such as poly-lysine, which serves to condense the nucleic acid. Linkers as described above may optionally be used. The receptor-binding internalized ligand confers specificity of delivery in a cell-specific manner. The choice of the receptor-binding internalized ligand to use will depend upon the receptor expressed by the target cells. The receptor type of the target cell population may be determined by conventional techniques such as antibody staining, PCR of cDNA using receptor-specific primers, and biochemical or functional receptor binding assays. It is preferable that the receptor be cell type-specific or have increased expression or activity higher rate of internalization) within the target cell population.
As described herein, the nucleic acid binding domain can be of two types, non-specific in its ability to bind nucleic acid, or highly specific so that the amino acid residues bind only the desired nucleic acid sequence. Nonspecific binding proteins, polypeptides, or compounds are generally polycationic or highly basic. Lys and Arg are the most basic of the 20 common amino acids; proteins enriched for these residues are candidates for nucleic acid binding domains. Examples of basic proteins include histones, protamines, and repeating units of lysine and arginine. Poly-L-lysine is an often-used nucleic acid binding domain (see U.S. Patent Nos. 5,166,320 and 5,354,844). Poly-L-lysine and protamine are preferred. Other polycations, such as WO 96/36362 PCT/US96/07164 spermine and spermidine, may also be used to bind nucleic acids. By way of example, the sequence-specific proteins, including gal4, Sp-1, AP-1, myoD and the rev gene product from HIV, may be used. Specific nucleic acid binding domains can be cloned in tandem, individually, or multiply to a desired region of the receptor-binding internalized ligand of interest. Alternatively, the ligand and binding domain can be chemically conjugated to each other.
The corresponding sequence that binds a sequence-specific domain is 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 to the nucleic acid binding domain. Even greater specificity of binding may be achieved by identifying and using the minimal amino acid sequence that binds to the cytocidal-encoding agent of interest. For example, phage display methods can be used to identify amino acids residues of varying length that will bind to specific nucleic acid sequences with high affinity. (See U.S. Patent No. 5,223,409.) The peptide sequence can then be cloned into the receptor-binding internalized ligand as a single copy or multiple copies. Alternatively, the peptide may be chemically conjugated to the receptor-binding internalized ligand. Incubation of the cytocide-encoding agent with the conjugated proteins will result in a specific binding between the two.
These complexes may be used to deliver nucleic acids that encode saporin, other cytocidal proteins, or prodrugs into cells with appropriate receptors that are expressed, over-expressed or more active in internalization upon binding. The cytocide gene is cloned downstream of a mammalian promoter such as c-myc, early or late gene, CMV-IE, TK or adenovirus promoter. As described above, promoters of interest may be active in any cell type, active only in a tissue-specific manner, such as a-crystalline or tyrosinase, event specific, or inducible, such as the MMTV LTR.
WO 96/36362 PCTfUS96/07164 56 1. Chemical conjugation a. Preparation of receptor-binding internalized ligands Receptor-binding internalized ligands are prepared as discussed by any suitable method, including recombinant DNA technology, isolation from a suitable source, purchase from a commercial source, or chemical synthesis. The selected linker or linkers is (are) linked to the receptor-binding internalized ligands by chemical reaction, generally relying on an available thiol or amine group on the receptor-binding internalized ligands. Heterobifunctional linkers are particularly suited for chemical conjugation. Alternatively, if the linker is a peptide linker, then the receptor-binding internalized ligands, linker and nucleic acid binding domain can be expressed recombinantly as a fusion protein.
Any protein that binds and internalizes through a receptor interaction may be used herein. In particular, any member of the FGF family of peptides or portion thereof that binds to an FGF receptor and internalizes a linked agent may be used herein. For the chemical conjugation methods the protein may be produced recombinantly, produced synthetically or obtained from commercial or other sources.
For the preparation of fusion proteins, the DNA encoding the FGF may be obtained from any known source or synthesized according to its DNA or amino acid sequences (see discussion above).
Although any of the growth factors may be conjugated in this manner, FGF, VEGF, and HBEGF conjugation are discussed merely by way of example and not by way of limitation.
If necessary or desired, the heterogeneity of preparations of ligand FGF) containing chemical conjugates and fusion proteins can be reduced by modifying the ligand by deleting or replacing a site(s) that causes the heterogeneity. Such sites in FGF are typically cysteine residues that upon folding of the protein remain available for interaction with other cysteines or for interaction with more than one cytotoxic molecule per molecule of FGF peptide. Thus, such cysteine residues do not include any cysteine residue that is required for proper folding of the FGF peptide or for binding to an FGF receptor and internalization. For chemical conjugation, one cysteine residue WO 96/36362 PCTIUS96/07164 57 that in physiological conditions is available for interaction is not replaced but is used as the site for linking the cytotoxic moiety. The resulting modified FGF is thus conjugated with a single species of nucleic acid binding domain (or nucleic acid).
The polypeptide reactive with an FGF receptor may be modified by removing one or more reactive cysteines that are not required for receptor binding, but that are available for reaction with appropriately derivatized cytotoxic agent, so that the resulting FGF protein has only one cysteine residue available for conjugation with the cytotoxic agent. If necessary, the contribution of each cysteine to the ability to bind to FGF receptors may be determined empirically. Each cysteine residue may be systematically replaced with a conservative amino acid change (see Table 1, above) or deleted. The resulting mutein is tested for the requisite biological activity, the ability to bind to FGF receptors and internalize linked cytotoxic moieties. If the mutein retains at least 50% of wild-type activity, then the cysteine residue is not required. Additional cysteines are systematically deleted and replaced and the resulting muteins are tested for activity. In this manner the minimum number and identity of the cysteines needed to retain the ability to bind to an FGF receptor and internalize may be determined. The resulting mutant FGF is then tested for retention of the ability to target a cytotoxic agent to a cell that expresses an FGF receptor and to internalize the cytotoxic agent into such cells. Retention of proliferative activity is indicative, though not definitive, of the retention of such activities. Proliferative activity may be measured by any suitable proliferation assay, such as the assay, exemplified below, that measures the increase in cell number of bovine aortic endothelial cells.
It is noted, however, that modified or mutant FGFs may exhibit reduced or no proliferative activity, but may be suitable for use herein, if they retain the ability to target cytocide-encoding agent to cells bearing FGF receptors and result in internalization. Certain residues of FGF-2 have been associated with proliferative activity. Modification of these residues arg 116, lys 119, tyr 120, trp 123 to ile 116, glu 119, ala 120, ala 123 may be made individually (see SEQ ID NOs. 81-84) to remove this function. The resulting protein is tested for proliferative activity by a standard assay.
WO 96/36362 PCT/US96/07164 58 Any ofFGF-1 FGF-9 may be used. The complete amino acid sequence of each of FGF-1 FGF- 9 is known (see, SEQ ID NO. 10 (FGF-1) and SEQ ID NOs. 12-18 (FGF-3 FGF-9, respectively)). Comparison among the amino acid sequences of FGF-1 -FGF-9 reveals that one Cys is conserved among FGF family of peptides (see Table These cysteine residues may be required for secondary structure and are not preferred residues to be altered. Each of the remaining cysteine residues may be systematically deleted and/or replaced by a serine residue or other residue that would not be expected to alter the structure of the protein. The resulting peptide is tested for biological activity. If the cysteine residue is necessary for retention of biological activity it is not deleted; if it not necessary, then it is preferably replaced with a serine or other residue that should not alter the secondary structure of the resulting protein.
The cysteine residues from each of FGF-1 FGF-9 that appear to be essential for retention of biological activity and that are not preferred residues for deletion or replacement are as follows: TABLE 3 FGF-1 cys 9 8 FGF-2 cys' 01 FGF-3 cys" FGF-4 cys 1 5 cys' 60 FGF-6 cys 147 FGF-7 cys' 37 FGF-8 cys' 27 FGF-9 cys' 34 For example, FGF-1 has cysteines at positions 31, 98 and 132; FGF-2 has cysteines at positions 34, 78, 96 and 101; FGF-3 has cysteines at positions 50 and 115; FGF-4 has cysteines at positions 88 and 155; FGF-5 has cysteines at positions 19, WO 96/36362 PCT/US96/07164 59 93, 160 and 202; FGF-6 has cysteines at positions 80 and 147; FGF-7 has cysteines at positions 18, 23, 32, 46, 71, 133 and 137; FGF-8 has cysteines at positions 10, 19, 109 and 127; and FGF-9 has cysteines at positions 68 and 134.
Since FGF-3, FGF-4 and FGF-6 have only two cysteines, for purposes of chemical conjugation, preferably neither cysteine is deleted or replaced, unless another residue, preferably one near either terminus, is replaced with a cysteine. With respect to the other FGF family members, at least one cysteine must remain available for conjugation with the cytotoxic conjugate and probably two cysteines, but at least the cysteine residues set forth in Table 3. A second cysteine may be required to form a disulfide bond. Thus, any FGF peptide that has more than three cysteines is be modified for chemical conjugation by deleting or replacing the other cysteine residues.
FGF peptides that have three cysteine residues are modified by elimination of one cysteine, conjugated to a cytotoxic moiety and tested for the ability to bind to FGF receptors and internalize the cytotoxic moiety.
In accord with the methods herein, several muteins of basic FGF for chemical conjugation have been produced (preparation of muteins for recombinant expression of the conjugate is described below). DNA, obtained from pFC80 (see PCT Application Serial No. PCT/US93/05702; United States Application Serial No. 07/901,718; see also SEQ ID NO. 52) encoding basic FGF has been mutagenized.
Mutagenesis of cysteine 78 of basic FGF (FGF-2) to serine ([C78S]FGF) or cysteine 96 to serine ([C96S]FGF) produced two mutants that retain virtually complete proliferative activity of native basic FGF as judged by the ability to stimulate endothelial cell proliferation in culture. The activities of the two mutants and the native protein do not significantly differ as assessed by efficacy or maximal response. Sequence analysis of the modified DNA verified that each of the mutants has one codon for cysteine converted to that for serine. The construction and biological activity of FGF-1 with cysteine substitutions of one, two or all three cysteines has been disclosed Patent No. 5,223,483). The mitogenic activity of the mutants was similar to or increased over the native protein. Thus, any of the cysteines may be mutated and FGF-1 will still bind and internalize.
WO 96/36362 PCT/US96/07164 The resulting mutein FGF or unmodified FGF is reacted with a nucleic acid binding domain. The bFGF muteins may react with a single species of derivatized nucleic acid binding domain (mono-derivatized nucleic acid binding domain), thereby resulting in monogenous preparations of FGF-nucleic acid binding domain conjugates and homogeneous compositions of FGF-nucleic acid binding domain chemical conjugates. The resulting chemical conjugates do not aggregate and retain the requisite biological activities.
VEGF or HBEGF may be isolated from a suitable source or may be produced using recombinant DNA methodology, discussed below. To effect chemical conjugation herein, the growth factor protein is conjugated generally via a reactive amine group or thiol group to the nucleic acid binding domain directly or through a linker to the nucleic acid binding domain. The growth factor protein is conjugated either via its N-terminus, C-terminus, or elsewhere in the polypeptide. In preferred embodiments, the growth factor protein is conjugated via a reactive cysteine residue to the linker or to the nucleic acid binding domain. The growth factor can also be modified by addition of a cysteine residue, either by replacing a residue or by inserting the cysteine, at or near the amino or carboxyl terminus, within about 20, preferably residues from either end, and preferably at or near the amino terminus.
In certain embodiments, the heterogeneity of preparations may be reduced by mutagenizing the growth factor protein to replace reactive cysteines, leaving, preferably, only one available cysteine for reaction. The growth factor protein is modified by deleting or replacing a site(s) on the growth factor that causes the heterogeneity. Such sites are typically cysteine residues that, upon folding of the protein, remain available for interaction with other cysteines or for interaction with more than one cytotoxic molecule per molecule of heparin-binding growth factor peptide. Thus, such cysteine residues do not include any cysteine residue that are required for proper folding of the growth factor or for retention of the ability to bind to a growth factor receptor and internalize. For chemical conjugation, one cysteine residue that, in physiological conditions, is available for interaction, is not replaced because it is WO 96/36362 PCT/US96/07164 61 used as the site for linking the cytotoxic moiety. The resulting modified heparin-binding growth factor is conjugated with a single species of cytotoxic conjugate.
Alternatively, the contribution of each cysteine to the ability to bind to VEGF, HBEGF or other heparin-binding growth factor receptors may be determined empirically as described herein. Each cysteine residue may be systematically replaced with a conservative amino acid change (see Table 1, above) or deleted. The resulting mutein is tested for the requisite biological activity: the ability to bind to growth factor receptors and internalize linked nucleic acid binding domain and agents. If the mutein retains this activity, then the cysteine residue is not required. Additional cysteines are systematically deleted and replaced and the resulting muteins are tested for activity.
Each of the remaining cysteine residues may be systematically deleted and/or replaced by a serine residue or other residue that would not be expected to alter the structure of the protein. The resulting peptide is tested for biological activity. If the cysteine residue is necessary for retention of biological activity it is not deleted; if it not necessary, then it is preferably replaced with a serine or other residue that should not alter the secondary structure of the resulting protein. In this manner the minimum number and identity of the cysteines needed to retain the ability to bind to a heparin-binding growth factor receptor and internalize may be determined. It is noted, however, that modified or mutant heparin-binding growth factors may exhibit reduced or no proliferative activity, but may be suitable for use herein, if they retain the ability to target a linked cytotoxic agent to cells bearing receptors to which the unmodified heparin-binding growth factor binds and result in internalization of the cytotoxic moiety. In the case of VEGF,
VEGF
2 contains 9 cysteines and each of VEGF 5
VEGF,
8 9 and VEGF 2 06 contain 7 additional residues in the region not present in VEGF 2 Any of the 7 are likely to be non-essential for targeting and internalization of linked cytotoxic agents. Recently, the role of Cys-25, Cys-56, Cys-67, Cys-101, and Cys-145 in dimerization and biological activity was assessed (Claffery et al., Biochem. Biophys. Acta 1246:1-9, 1995).
Dimerization requires Cys-25, Cys-56, and Cys-67. Substitution of any one of these cysteine residues resulted in secretion of a monomeric VEGF, which was inactive in both vascular permeability and endothelial cell mitotic assays. In contrast, substitution WO 96/36362 PCT/US96/07164 62 of Cys 145 had no effect on dimerization, although biological activities were somewhat reduced. Substitution of Cys-101 did not result in the production of a secreted or cytoplasmic protein. Thus, substitution of Cys-145 is preferred.
The VEGF monomers are preferably linked via non-essential cysteine residues to the linkers or to the targeted agent. VEGF that has been modified by introduction of a Cys residue at or near one terminus, preferably the N-terminus is preferred for use in chemical conjugation. For use herein, preferably the VEGF is dimerized prior to linkage to the linker and/or targeted agent. Methods for coupling proteins to the linkers, such as the heterobifunctional agents, or to nucleic acids, or to proteins are known to those of skill in the art and are also described herein.
For recombinant expression using the methods described herein, up to all cysteines in the HBEGF polypeptide that are not required for biological activity can be deleted or replaced. Alternatively, for use in the chemical conjugation methods herein, all except one of these cysteines, which will be used for chemical conjugation to the cytotoxic agent, can be deleted or replaced. Each of the HBEGF polypeptides described herein have six cysteine residues. Each of the six cysteines may independently be replaced and the resulting mutein tested for the ability to bind to HBEGF receptors and to be internalized. Alternatively, the resulting mutein-encoding DNA is used as part of a construct containing DNA encoding the nucleic acid binding domain linked to the HBEGF-encoding DNA. The construct is expressed in a suitable host cell and the resulting protein tested for the ability to bind to HBEGF receptors and internalize. As long as this ability is retained the mutein is suitable for use herein.
Methods for chemical conjugation of proteins are known to those of skill in the art. The preferred methods for chemical conjugation depend on the selected components, but preferably rely on disulfide bond formation. For example, if the targeted agent is SPDP-derivatized saporin, then it is advantageous to dimerize the VEGF moiety prior coupling or conjugating to the derivatized saporin. If VEGF is modified to include a cysteine residue at or near the preferably, or C- terminus, then dimerization should follow coupling to the nucleic acid binding domain. To effect WO 96/36362 PCT/US96/071 6 4 63 chemical conjugation herein, the HBEGF polypeptide is linked via one or more selected linkers or directly to the nucleic acid binding domain.
b. Preparation of nucleic acid bindin domins for chemical conjugation A nucleic acid binding domain is prepared for chemical conjugation. For chemical conjugation, a nucleic acid binding domain may be derivatized with SPDP or other suitable chemicals. If the binding domain does not have a Cys residue available for reaction, one can be either inserted or substituted for another amino acid. If desired, mono-derivatized species may be isolated, essentially as described.
For chemical conjugation, the nucleic acid binding domain may be derivatized or modified such that it includes a cysteine residue for conjugation to the receptor-binding internalized ligand. Typically, derivatization proceeds by reaction with SPDP. This results in a heterogeneous population. For example, nucleic acid binding domain that is derivatized by SPDP to a level of 0.9 moles pyridine-disulfide per mole of nucleic acid binding domain includes a population of non-derivatized mono-derivatized and di-derivatized SAP. nucleic acid binding domain proteins, which are overly derivatized with SPDP, may lose ability to bind nucleic acid because of reaction with sensitive lysines (Lambert et al., Cancer Treat. Res. 37:175-209, 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 monoderivatized 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 WO 96/36362 PCT/US96/07 16 4 64 produced. There may still be sources of heterogeneity with the mono-derivatized nucleic acid binding domain used here but is acceptable as long as binding to the cytocide-encoding agent is not impacted.
Because more than one amino group on the nucleic acid binding domain may react with the succinimidyl moiety, it is possible that more than one amino group on the surface of the protein is reactive. This creates potential for heterogeneity in the mono-derivatized nucleic acid binding domain.
As an alternative to derivatizing to introduce a sulfhydryl, the nucleic acid binding domain can be modified by the introduction of a cysteine residue.
Preferred loci for introduction of a cysteine residue include the N-terminus region, preferably within about one to twenty residues from the N-terminus of the nucleic acid binding domain.
Using either methodology (reacting mono-derivatized nucleic acid binding domain or introducing a Cys residue into nucleic acid binding domain), the resulting preparations of chemical conjugates are monogenous; compositions containing the conjugates also appear to be free of aggregates.
2. Fusion protein of recetor-bindin internalized liands and nucleic acid binding domain As a preferred alternative, heterogeneity can be avoided by producing a fusion protein of receptor-binding internalized ligand and nucleic acid binding domain, as described below. Expression of DNA encoding a fusion of a receptor-binding internalized ligand polypeptide linked to the nucleic acid binding domain results in a more homogeneous preparation of cytotoxic conjugates. Aggregate formation can be reduced in preparations containing the fusion proteins by modifying the receptorbinding internalized ligand, such as by removal of nonessential cysteines, and/or the nucleic acid binding domain to prevent interactions between conjugates via free cysteines. Optionally, one or more coding regions for endosome-disruptive peptide may be constructed as part of the fusion protein.
DNA encoding the polypeptides may be isolated, synthesized or obtained from commercial sources or prepared as described herein. Expression of WO 96/36362 PCT/US96/0 7 16 4 recombinant polypeptides may be performed as described herein; and DNA encoding these polypeptides may be used as the starting materials for the methods herein.
As described above, DNA encoding FGF, VEGF, HBEGF hepatocyte growth factor, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-11, IL-13, TNF,
GM-
CSF, IFN and IGF polypeptides and/or the amino acid sequences of these factors are described above. DNA may be prepared synthetically based on the amino acid or DNA sequence or may be isolated using methods known to those of skill in the art, such as PCR, probe hybridization of libraries, and the like or obtained from commercial or other sources. For example, suitable methods are described in the Examples for amplifying FGF encoding cDNA from plasmids containing FGF encoding cDNA.
As described herein, such DNA may then be mutagenized using standard methodologies to delete or replace any cysteine residues that are responsible for aggregate formation. If necessary, the identity of cysteine residues that contribute to aggregate formation may be determined empirically, by deleting and/or replacing a cysteine residue and ascertaining whether the resulting growth factor with the deleted cysteine forms aggregates in solutions containing physiologically acceptable buffers and salts. Loci for insertion of cysteine residues may also be determined empirically.
Generally, regions at or near (within 20, preferably 10 amino acids) the C- or, preferably, the N-terminus are preferred.
The DNA construct encoding the fusion protein can be inserted into a plasmid and expressed in a selected host, as described above, to produce a recombinant receptor-binding internalized 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 one plasmid.
a. Prearation of muteins for recombinant production of the fusion protein Removal of cysteines not required for binding and internalization is preferred for both chemical conjugation and recombinant methods in the chemical WO 96/36362 PCT/US96/07164 66 conjugation methods, all except one cysteine, which is necessary for chemical conjugation are deleted or replaced. In practice, it appears that for FGF polypeptides only two cysteines (including each of the cysteine residues set forth in Table and perhaps only the cysteines set forth in Table 3, are required for retention of the requisite biological activity of the FGF peptide. Thus, FGF peptides that have more than two cysteines are modified by replacing the remaining cysteines with serines. The resulting muteins may be tested for the requisite biological activity.
FGF peptides, such as FGF-3, FGF-4 and FGF-6, that have two cysteines can be modified by replacing the second cysteine, which is not listed in Table 3, and the resulting mutein used as part of a construct containing DNA encoding the cytotoxic agent linked to the FGF-encoding DNA. The construct is expressed in a suitable host cell and the resulting protein tested for the ability to bind to FGF receptors and internalize the cytotoxic agent. As exemplified herein, conjugates containing bFGF muteins in which Cys 78 and Cys 96 have been replaced with serine residues have been prepared.
b. DNA constructs and expression of the DNA constructs To produce monogenous preparations of fusion protein, DNA encoding the FGF protein or other receptor-binding internalized ligand is modified so that, upon expression, the resulting FGF portion of the fusion protein does not include any cysteines available for reaction. In preferred embodiments, DNA encoding an FGF polypeptide is linked to DNA encoding a nucleic acid binding domain. The DNA encoding the FGF polypeptide or other receptor-binding internalized ligand is modified in order to remove the translation stop codon and other transcriptional or translational stop signals that may be present and to remove or replace DNA encoding the available cysteines. The DNA is then ligated to the DNA encoding the nucleic acid binding domain polypeptide directly or via a linker region of one or more codons between the first codon of the nucleic acid binding domain and the last codon of the FGF. The size of the linker region may be any length as long as the resulting conjugate binds and is internalized by a target cell. Presently, spacer regions of from about one to about WO 96/36362 PCT/US96/07164 67 seventy-five to ninety codons are preferred. The order of the receptor-binding internalized ligand and nucleic acid binding domain in the fusion protein may be reversed. If the nucleic acid binding domain is N-terminal, then it is modified to remove the stop codon and any stop signals.
As discussed above, any heparin-binding protein, including FGF, VEGF, HBEGF, cytokine, growth factor and the like may be modified and expressed in accord with the methods herein. Binding to an FGF receptor followed by internalization are the only activities required for an FGF protein to be suitable for use herein. All of the FGF proteins induce mitogenic activity in a wide variety of normal diploid mesodermderived and neural crest-derived cells and this activity is mediated by binding to an FGF cell surface receptor followed by internalization. A test of such "FGF mitogenic activity", which reflects the ability to bind to FGF receptors and to be internalized, is the ability to stimulate proliferation of cultured bovine aortic endothelial cells (see, e.g., Gospodarowicz et al., J Biol. Chem. 257:12266-12278, 1982; Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 73:4120-4124, 1976).
If the FGF or other ligand has been modified so as to lack mitogenic activity or other biological activities, binding and internalization may still be readily assayed by any one of the following tests or other equivalent tests. Generally, these tests involve labeling the ligand, incubating it with target cells, and visualizing or measuring intracellular label. For example, briefly, FGF may be fluorescently labeled with FITC or radiolabeled with 125I. Fluorescein-conjugated FGF is incubated with cells and examined microscopically by fluorescence microscopy or confocal microscopy for internalization. When FGF is labeled with 1251, the labeled FGF is incubated with cells at 4 0 C. Cells are temperature shifted to 37 0 C and washed with 2 M NaCl at low pH to remove any cell-bound FGF. Label is then counted and thereby measuring internalization of FGF. Alternatively, the ligand can be conjugated with an nucleic acid binding domain by any of the methods described herein and complexed with a plasmid encoding saporin. As discussed below, the complex may be used to transfect cells and cytotoxicity measured.
WO 96/36362 PCTUS96/07 16 4 68 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. Fusion proteins of FGF-2 and protamine are especially suitable for use in the present invention.
Multiple copies of the modified receptor-binding internalized ligand/nucleic acid binding domain chimera can be inserted into a single plasmid in operative linkage with one promoter. When expressed, the resulting protein will be a multimer. Typically two to six copies of the chimera are inserted, preferably in a head to tail fashion, into one plasmid.
Merely by way of example, DNA encoding human bFGF-SAP having SEQ ID NO. 52 has been mutagenized as described in the Examples using splicing by overlap extension (SOE). Another preferred coding region is set forth in SEQ ID NO. 53. In both instances, in preferred embodiments, the DNA is modified by replacing the cysteines at positions 78 and 96 with serine. The codons encoding cysteine residues at positions 78 and 96 of FGF were converted to serine codons by SOE. Each application of the SOE method uses two amplified oligonucleotide products, which have complementary ends as primers and which include an altered codon at the locus at which the mutation is desired, to produce a hybrid product.
A
second amplification reaction that uses two primers that anneal at the non-overlapping ends amplify the hybrid to produce DNA that has the desired alteration.
3. Bindine of the receptor-bindin internalized lian/nucle acid bindin domain conjugate to cvtocide-encoding agents The receptor-binding internalized ligand/nucleic acid binding domain is incubated with the cytocide-encoding agent, preferably a linear DNA molecule, to be delivered under conditions that allow binding of the nucleic acid binding domain to the agent. Conditions will vary somewhat depending on the nature of the nucleic acid binding domain, but will typically occur in 0.1M NaCI and 20 mM HEPES or other similar buffer. Alternatively, salt conditions can be varied to increase the packing or condensation of DNA. The extent of binding is preferably tested for each preparation.
WO 96/36362 PCT/US96/07 16 4 69 After complexing, additional nucleic acid binding domain, such as poly-L-lysine, may be added to further condense the nucleic acid.
Merely by way of example, test constructs have been made and tested.
One construct is a chemical conjugate of bFGF and poly-L-lysine. The bFGF molecule is a variant in which the Cys residue at position 96 has been changed to a serine; thus, only the Cys at position 78 is available for conjugation. This bFGF is called FGF2-3.
The poly-L-lysine was derivatized with SPDP and coupled to FGF2-3. This FGF2- 3 /poly-L-lysine conjugate was used to deliver a plasmid able to express the P-galactosidase gene.
The ability of a construct to bind nucleic acid molecules may be conveniently assessed by agarose gel electrophoresis. Briefly, a plasmid, such as pSV3, is digested with restriction enzymes to yield a variety of fragment sizes. For ease of detection, the fragments may be labeled with 32 P either by filling in of the ends with DNA polymerase I or by phosphorylation of the 5'-end with polynucleotide kinase following dephosphorylation by alkaline phosphatase. The plasmid fragments are then incubated with the receptor-binding internalized ligand/nucleic acid binding domain in this case, FGF 2 -3/poly-L-lysine in a buffered saline solution, such as 20 mM HEPES, pH 7.3, 0.1M NaC1. 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 FGF 2 -3/poly-L-lysine conjugate. If there is insufficient binding, poly-L-lysine may be additionally added until binding is observed.
Further testing of the conjugate is performed to show that it binds to the cell surface receptor and is internalized into the cell. It is not necessary that the receptor-binding internalized ligand part of the conjugate retain complete biological activity. For example, FGF is mitogenic on certain cell types. As discussed above, this WO 96/36362 PCT/US96/07164 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 expression of a reporter gene, such as P-gal enzymatic activity), in cells that have been transformed with a complex of a plasmid encoding a reporter gene and a conjugate of a receptor-binding internalized ligand and nucleic acid binding domain. This assay is particularly useful for optimizing conditions to give maximal transformation. Thus, the optimum ratio of receptorbinding 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 P-gal. As such, these first two assays are useful for preliminary analysis and failure to show receptor binding or p-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. While, in general, any cytocidal molecule may be used, ribosome inactivating proteins are preferred and saporin, or another type I ribosome inactivating protein, is particularly preferred. A statistically significant reduction in cell number demonstrates the ability of the receptor-binding internalized ligand/nucleic acid binding domain conjugate or fusion to deliver nucleic acids into a cell.
4. Conugation ofli and to nucleic acid and bindin to nucleic acid bindin domain As an alternative, the receptor-internalized binding ligand may be conjugated to the nucleic acid, either directly or through a linker. Methods for conjugating nucleic acids, at the 5' ends, 3' ends and elsewhere, to the amino and WO 96/36362 PCT/US96/07164 71 carboxyl termini and other sites in proteins are known to those of skill in the art (for a review see, Goodchild, (1993) In: Perspectives in Bioconjugate Chemistry, Mears, Ed., American Chemical Society, Washington, D.C. pp. 77-99). For example, proteins have been linked to nucleic acids using ultraviolet irradiation (Sperling et al. (1978) Nucleic Acids Res. 5:2755-2773; Fiser et al. (1975) FEBS Lett. 52:281-283), bifunctional chemicals (Biumert et al. (1978) Eur. J. Biochem. 89.353-359; and Oste et al. (1979) Mol. Gen. Genet. 168:81-86) and photochemical cross-linking (Vanin et al.
(1981) FEBSLett. 124:89-92; Rinke et al. (1980) Mol. Biol. 137:301-314; Millon et al. (1980) Eur. J Biochem. 110:485-454).
In particular, the reagents (N-acetyl-N'-(p-glyoxylylbenzolyl)cystamine and 2 -iminothiolane have been used to couple DNA to proteins, such as a-macroglobulin (a2M) via mixed disulfide formation (see Cheng et al., Nucleic Acids Res. 11:659-669, 1983). N-acety-N-(pglyoxylylbenzolyl)cystamine reacts specifically with nonpaired guaninine residues and, upon reduction, generates a free sulfhydryl group. 2 -iminothiolane reacts with proteins to generate sulfhydryl groups that are then conjugated to the derivatized DNA by an intermolecular disulfide interchange reaction. Any linkage may be used provided that the targeted nucleic acid is active upon internalization of the conjugate. Thus, it is expected that cleavage of the linkage may be necessary, although it is contemplated that for some reagents, such as DNA encoding ribozymes linked to promoters or DNA encoding therapeutic agents for delivery to the nucleus, such cleavage may not be necessary.
Thiol linkages, which are preferred, can be readily formed using heterbiofunctional reagents. Amines have also been attached to the terminal phosphate of unprotected oligonucleotides or nucleic acids in aqueous solutions by reacting the nucleic acid with a water-soluble carbodiimide, such as 1-ethyl-3'[3dimethylaminopropyl]carbodiimide (EDC) or N-ethyl-N'(3-dimethylaminopropylcarbodiimidehydrochloride (EDCI), in imidazole buffer at pH 6 to produce the Contacting the 5'phosphorimidazolide with amine-containing molecules, such as an FGF, and ethylenediamine, results in stable phosphoramidates (see, Chu et al., Nucleic Acids Res. 11:6513-6529, 1983; and WO 88/05077). In WO 96/36362 PCT/US96/07 16 4 72 particular, a solution of DNA is saturated with EDC, at pH 6 and incubated with agitation at 4 0 C overnight. The resulting solution is then buffered to pH 8.5 by adding, for example about 3 volutes of 100 mM citrate buffer, and adding about 5 tg about pg of an FGF, and agitating the resulting mixture at 4 0 C for about 48 hours. The unreacted protein may be removed from the mixture by column chromatography using, for example, Sephadex G75 (Pharmacia) using 0.1 M ammonium carbonate solution, pH 7.0 as an eluting buffer. The isolated conjugate may be lyophilized and stored until used.
U.S. Patent No. 5,237,016 provides methods for preparing nucleotides that are bromacetylated at their 5' termini and reacting the resulting oligonucleotides with thiol groups. Oligonucleotides derivatized at their 5'-termini bromoacetyl groups can be prepared by reacting 5'-aminohexyl-phosphoramidate oligonucleotides with bromoacetic acid-N-hydroxysuccinimide ester as described in U.S. Patent No. 5,237,016. This patent also describes methods for preparing thiol-derivatized nucleotides, which can then be reacted with thiol groups on the selected growth factor.
Briefly, thiol-derivatized nucleotides are prepared using a 5'-phosphorylated nucleotide in two steps: 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. 14:651, which describes a similar procedure).
The 5'-phosphorylated starting oligonucleotides can be prepared by methods known to those of skill in the art (see, Maniatis et al. (1982) Molecular Cloning.
A
Laboratory Manual, Cold Spring Harbor Laboratory, New York, p. 122).
The nucleic acid, such as a methylphosphonate oligonucleotide
(MP-
oligomer), may be derivatized by reaction with SPDP or SMPB. The resulting
MP-
oligomer may be purified by HPLC and then coupled to an FGF, such as an FGF or FGF mutein, modified by replacement of one or more cysteine residues, as described above. The MP-oligomer (about 0.1 pM) is dissolved in about 40-50 pl of 1:1 acetonitrile/water to which phosphate buffer (pH 7.5, final concentration 0.1 M) and a 1 mg MP-oligomer in about 1 ml phosphate buffered saline is added. The reaction is WO 96/36362 PCT/US96/07164 73 allowed to proceed for about 5-10 hours at room temperature and is then quenched with about 15 pL 0.1 iodoacetamide. FGF-oligonucleotide conjugates can be purified on heparin sepharose Hi Trap columns (1 ml, Pharmacia) and eluted with a linear or step gradient. The conjugate should elute in 0.6 M NaCI.
The ligand may be conjugated to the nucleic acid construct encoding the cytocide or cytotoxic agent or may be conjugated to a mixture of oligonucleotides complementary to one strand of the construct. The oligonucleotides are then added to single stranded construct produced by melting a double-stranded construct or grown and isolated as single-stranded. As a general guideline, the oligonucleotides should hybridize at a higher temperature than the construct alone, if a double-stranded construct is used as the starting material. The gaps are filled in by DNA polymerase I to generate a construct with one strand conjugated to ligand and one strand unconjugated.
Oligonucleotides conjugated to ligand and complementary to the other strand may be used in addition to generate a mixture of constructs with different strands linked to ligand. Any remaining single stranded plasmid may be digested with a single strand specific endonuclease. The ligand-conjugated constructs are then mixed with a nucleic acid binding domain, such as protamine or polylysine, to effect condensation of the construct for delivery. Optimal ratios of ligand to DNA may be determined experimentally by receptor-mediated transfection of a construct containing a reporter gene.
J. Formulation and administration of pharmaceutical compositions The conjugates and complexes provided herein are useful in the treatment and prevention of various diseases, syndromes, and hyperproliferative disorders. As used herein, "treatment" means any manner in which the symptoms of a conditions, disorder or disease are ameliorated or otherwise beneficially altered.
Treatment also encompasses any pharmaceutical use of the compositions herein. As used herein, "amelioration" of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with WO 96/36362 PCT/US96/07164 74 administration of the composition. For example, these conjugates and complexes may be used to treat complications of the eye following laser surgery, glaucoma surgery, and removal of pterygii. Following these treatments, reoccurrence of the problem often ensues due to proliferation of cells in the cornea or eye. The conjugates and complexes inhibit the proliferation of these cells. The conjugates and complexes may be used in general to treat pathophysiological conditions, especially FGF-, VEGF-, or HBEGF-mediated pathophysiological conditions by specifically targeting to cells having corresponding receptors.
As used herein, "FGF-mediated pathophysiological condition" refers to a deleterious condition characterized by or caused by proliferation of cells that are sensitive to FGF mitogenic stimulation. Basic FGF-mediated pathophysiological conditions include, but are not limited to, melanoma, other tumors, rheumatoid arthritis, restenosis, Dupuytren's Contracture and certain complications of diabetes, such as proliferative retinopathy.
As used herein, "HBEGF-mediated pathophysiological condition" refers to a deleterious condition characterized by or caused by proliferation of cells that are sensitive to HBEGF mitogenic stimulation. HBEGF-mediated pathophysiological conditions include conditions involving pathophysiological proliferation of smooth muscle cells, such as restenosis, certain tumors, such as solid tumors including breast and bladder tumors, tumors involving pathophysiological expression of EGF receptors, dermatological disorders, such as psoriasis, and ophthalmic disorders involving epithelial cells, such as recurrence ofpterygii and secondary lens clouding.
Similarly, tumors and hyperproliferating cells expressing cytokine receptors or growth factor receptors may be eliminated. Such diseases include restenosis, Dupuytren's Contracture, diabetic retinopathies, rheumatoid arthritis, Kaposi's sarcoma, lymphomas, leukemias, tumors such as renal cell carcinoma, colon carcinoma, breast cancer, bladder cancer, disorders with underlying vascular proliferation, such as diseases in the back of the eye proliferative vitreoritinopathy, inacular degeneration and diabetic retinopathy). For treatment of the back of the eye especially, use of the VEGF-receptor promoter to control expression of WO 96/36362 PCT/US96/07164 the cytocide or cytotoxic agent is preferred. The conjugates may be used to prevent corneal haze or clouding that results from exposure of the cornea to laser radiation during eye surgery, particularly LRK. The haze or clouding appears to result from fibroblastic keratocyte proliferation in the subepithelial zone following photoablation of the cornea.
The conjugates may be used to treat a "hyperproliferative skin disorder." As used herein, it is a disorder that is manifested by a proliferation of endothelial cells of the skin coupled with an underlying vascular proliferation, resulting in a localized patch of scaly or horny or thickened skin or a tumor of endothelial origin. Such disorders include actinic and atopic dermatitis, toxic eczema, allergic eczema, psoriasis, skin cancers and other tumors, such as Kaposi's sarcoma, angiosarcoma, hemangiomas, and other highly vascularized tumors, and vascular proliferative responses, such as varicose veins.
As well, the conjugates may be used to treat or prevent restenosis, a process and the resulting condition that occurs following angioplasty in which the arteries become reclogged. After treatment of arteries by balloon catheter or other such device, denudation of the interior wall of the vessel occurs, including removal of the endothelial cells that constitute the lining of the blood vessels. As a result of this removal and the concomitant vascular injury, smooth muscle cells (SMCs), which form the blood vessel structure, proliferate and fill the interior of the blood vessel. This process and the resulting condition is restenosis.
Pharmaceutical carriers or vehicles suitable for administration of the conjugates and complexes provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. In addition, the conjugates and complexes may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
The conjugates and complexes can be administered by any appropriate route, for example, orally, parenterally, including intravenously, intradermally, subcutaneously, or topically, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration. Preferred modes of administration WO 96/36362 PCT/US96/0 71 6 4 76 depend upon the indication treated. Dermatological and ophthalmologic indications will typically be treated locally; whereas, tumors and restenosis, will typically be treated by systemic, intradermal, or intramuscular modes of administration.
The conjugates and complexes herein may be formulated into pharmaceutical compositions suitable for topical, local, intravenous and systemic application. For the ophthalmic uses herein, local administration, either by topical administration or by injection is preferred. Time release formulations are also desirable.
Effective concentrations of one or more of the conjugates and complexes are mixed with a suitable pharmaceutical carrier or vehicle. As used herein an "effective amount" of a compound for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Repeated administration may be required to achieve the desired amelioration of symptoms.
As used herein, "an ophthalmically effective amount" is that amount which, in the composition administered and by the technique administered, provides an amount of therapeutic agent to the involved eye tissues sufficient to prevent or reduce corneal haze following excimer laser surgery, prevent closure of a trabeculectomy, prevent or substantially slow the recurrence of pterygii, and other conditions.
The concentrations or amounts of the conjugates and complexes that are effective requires delivery of an amount, upon administration, that ameliorates the symptoms or treats the disease. Typically, the compositions are formulated for single dosage administration. Therapeutically effective concentrations and amounts may be determined empirically by testing the conjugates and complexes in known in vitro and in vivo systems, such as those described here; dosages for humans or other animals may then be extrapolated therefrom.
The conjugate is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The conjugates may be delivered as WO 96/36362 PCT/US96/07 16 4 77 pharmaceutically acceptable salts, esters or other derivatives of the conjugates include any salts, esters or derivatives that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects. It is understood that number and degree of side effects depends upon the condition for which the conjugates and complexes are administered. For example, certain toxic and undesirable side effects are tolerated when treating life-threatening illnesses, such as tumors, that would not be tolerated when treating disorders of lesser consequence. The concentration of conjugate in the composition will depend on 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.
Preferably, the conjugate and complex are substantially pure. As used herein, "substantially pure" means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.
Typically a therapeutically effective dosage should produce a serum concentration of active ingredient of from about 0.1 ng/ml to about 50-100 gg/ml. The pharmaceutical compositions typically should provide a dosage of from about 0.01 mg to about 100 2000 mg of conjugate, depending upon the conjugate selected, per kilogram of body weight per day. For example, for treatment of restenosis a daily dosage of about between 0.05 and 0.5 mg/kg (based on FGF-SAP chemical conjugate or an amount of conjugate provided herein equivalent on a molar basis thereto) should be sufficient. Local application for ophthalmic disorders and dermatological disorders WO 96/36362 PCT/US96/0716 4 78 should provide about 1 ng up to 100 j g, preferably about 1 ng to about 10 jig, per single dosage administration. It is understood that the amount to administer will be a function of the conjugate selected, the indication treated, and possibly the side effects that will be tolerated.
Therapeutically effective concentrations and amounts may be determined for each application herein empirically by testing the conjugates and complexes in known in vitro and in vivo systems murine, rat, rabbit, or baboon models), such as those described herein; dosages for humans or other animals may then be extrapolated therefrom. Demonstration that the conjugates and complexes prevent or inhibit proliferation of serum stimulated corneal keratocytes or fibroblasts explanted from eyes, as shown herein, and demonstration of any inhibition of proliferation of such tissues in rabbits should establish human efficacy. The rabbit eye model is a recognized model for studying the effects of topically and locally applied drugs (see, U.S. Patent Nos.
5,288,735, 5,263,992, 5,262,178, 5,256,408, 5,252,319, 5,238,925, 5,165,952; see also Mirate et al., Curr. Eye Res. 1:491-493, 1981).
The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
The conjugates and complexes may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracistemrnal or intraspinal application. Such solutions, particularly those intended for WO 96/36362 PCT/US96/07 1 6 4 79 ophthalmic use, may be formulated as 0.01% -10% isotonic solutions, pH about 5-7, with appropriate salts. The ophthalmic compositions may also include additional components, such as hyaluronic acid. The conjugates and complexes may be formulated as aerosols for topical application (see, U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923).
Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of toxicity such as sodium chloride or dextrose. Parental preparations can be enclosed in ampules, disposable syringes or multiple dose vials made of glass, plastic or other suitable material.
If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art.
Upon mixing or addition of the conjugate(s) with the vehicle, the resulting mixture may be a solution, suspension, emulsion or the like. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the conjugate in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease, disorder or condition treated and may be empirically determined based upon in vitro and/or in vivo data, such as the data from the mouse xenografi model for tumors or rabbit ophthalmic model. If necessary, pharmaceutically acceptable salts or other derivatives of the conjugates and complexes may be prepared.
WO 96/36362 PCT/US96/07164 The active materials can also be mixed with other active materials, that do not impair the desired action, or with materials that supplement the desired action, including viscoelastic materials, such as hyaluronic acid, which is sold under the trademark HEALON (solution of a high molecular weight (MW of about 3 millions) fraction of sodium hyaluronate; manufactured by Pharmacia, Inc. see, U.S. Patent Nos. 5,292,362, 5,282,851, 5,273,056, 5,229,127, 4,517,295 and 4,328,803),
VISCOAT
(fluorine-containing (meth)acrylates, such as, H, H, 2 H,2H-hepta_ decafluorodecylmethacrylate; see, U.S. Patent Nos. 5,278,126, 5,273,751 and 5,214,080; commercially available from Alcon Surgical, Inc.), ORCOLON (see, e.g., U.S. Patent Nos. 5,273,056; commercially available from Optical Radiation Corporation), methylcellulose, methyl hyaluronate, polyacrylamid and polymethacrylamide (see, U.S. Patent No. 5,273,751) The viscoelastic materials are present generally in amounts ranging from about 0.5 to preferably 1 to 3% by weight of the conjugate material and serve to coat and protect the treated tissues The compositions may also include a dye, such as methylene blue or other inert dye, so that the composition can be seen when injected into the eye or contacted with the surgical site during surgery.
The conjugates and complexes may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye. Such solutions, particularly those intended for ophthalmic use, may be formulated as 0.0%iuse, may be formulated as 0.01%isotonic solutions, pH about 5-7, with appropriate salts. Suitable ophthalmic solutions are known (see, U.S. Patent No. 5,116,868, which describes typical compositions of ophthalmic irrigation solutions and solutions for topical application).
Such solutions, which have a pH adjusted to about 7.4, contain, for example, 90-100 mM sodium chloride, 4-6 mM dibasic potassium phosphate, 4-6 mM dibasic sodium phosphate, 8-12 mM sodium citrate, 0.5-1.5 mM magnesium chloride, 1.5-2.5 mM calcium chloride, 15-25 mM sodium acetate, 10-20mM D.L.-sodium 1hydroxybutyrate and 5-5.5 mM glucose.
WO 96/36362 PCT/US96/07164 81 The conjugates and complexes may be prepared with carriers that protect them against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. For example, the composition may be applied during surgery using a sponge, such as a commercially available surgical sponges (see, U.S. Patent Nos. 3,956,044 and 4,045,238; available from Week, Alcon, and Mentor), that has been soaked in the composition and that releases the composition upon contact with the eye. These are particularly useful for application to the eye for ophthalmic indications following or during surgery in which only a single administration is possible. The compositions may also be applied in pellets (such as Elvax pellets(ethylene-vinyl acetate copolymer resin); about 1- 5 Ag of conjugate per 1 mg resin) that can be implanted in the eye during surgery.
Ophthalmologically effective concentrations or amounts of one or more of the conjugates and complexes are mixed with a suitable pharmaceutical carrier or vehicle. The concentrations or amounts of the conjugates and complexes that are effective requires delivery of an amount, upon administration, that prevents or substantially reduces corneal clouding, trabeculectomy closure, or pterygii recurrence.
The conjugates and complexes herein are formulated into ophthalmologically acceptable compositions and are applied to the affected area of the eye during or immediately after surgery. In particular, following excimer laser surgery, the composition is applied to the cornea; following trabeculectomy the composition is applied to the fistula; and following removal of pterygii the composition is applied to the cornea. The compositions may also be used to treat pterygii. The conjugates and complexes are applied during and immediately following surgery and may, if possible be applied post-operatively, until healing is complete. The compositions are applied as drops for topical and subconjunctival application or are injected into the eye for intraocular application. The compositions may also be absorbed to a biocompatible WO 96/36362 PCT/US96/07164 82 support, such as a cellulosic sponge or other polymer delivery device, and contacted with the affected area.
The ophthalmologic indications herein are typically be treated locally either by the application of drops to the affected tissue(s), contacting with a biocompatible sponge that has absorbed a solution of the conjugates and complexes or by injection of a composition. For the indications herein, the composition will be applied during or immediately after surgery in order to prevent closure of the trabeculectomy, prevent a proliferation of keratocytes following excimer laser surgery, or to prevent a recurrence of pterygii. The composition may also be injected into the affected tissue following surgery and applied in drops following surgery until healing is completed. For example, to administer the formulations to the eye, it can be slowly injected into the bulbar conjunctiva of the eye.
Conjugates and complexes with photocleavable linkers are among those preferred for use in the methods herein. Upon administration of such composition to the affected area of the eye, the eye is exposed to light of a wavelength, typically visible or UV that cleaves the linker, thereby releasing the cytotoxic agent.
If oral administration is desired, the conjugate should be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.
Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, 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 WO 96/36362 PCT/US96/07164 83 lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, and fruit flavoring.
When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The conjugates and complexes can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
The active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as cis-platin for treatment of tumors.
Finally, the compounds may be packaged as articles of manufacture containing packaging material, one or more conjugates and complexes or compositions as provided herein within the packaging material, and a label that indicates the indication for which the conjugate is provided.
Many methods have been developed to deliver nucleic acid into cells including retroviral vectors, electroporation, CaPO 4 precipitation and microinjection, but each of these methods has distinct disadvantages. Microinjecting nucleic acid into cells is very time consuming because each cell must be manipulated individually.
Retroviral vectors can only hold a limited length of nucleic acid and can activate oncogenes depending upon the insertion site in the target chromosome. Conditions for electroporation and CaPO 4 -mediated transfection are harsh and cause much cell death.
By comparison, receptor mediated gene delivery as described herein is a more desirable method of selectively targeting toxic genes into cells that have "more active" receptors or that overexpress the specific receptor on the cell surface.
A
receptor may be more active because it has a higher rate of internalization or higher WO 96/36362 PCT/US96/07164 84 cycling rate through the endosome to the cell surface. Advantages of this method over other gene delivery methods include increased specificity of delivery, the absence of nucleic acid length limitations, reduced toxicity, and reduced immunogenicity of the conjugate. These characteristics allow for repeated administration of the material with minimal harm to cells and may allow increased level of expression of the toxic protein.
In addition, primary cultures can also be treated using this method.
The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
EXAMPLES
EXAMPLE 1 ISOLATION OF DNA ENCODING
SAPORIN
A. Materials and methods 1. Bacterial Strains E. coli strain JA221 (lpp- hdsM+ trpE5 leuB6 lacY recAl F'[laclq lac pro+]) is publicly available from the American Type Culture Collection
(ATCC),
Rockville, MD 20852 under th l 20 Rockville, MD 20852, under the accession number ATCC 33875. (JA221 is also available from the Northern Regional Research Center (NRRL), Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604, under the accession number NRRL B-15211; see also U.S. Patent No. 4,757,013 to Inouye; and Nakamura et al., Cell 18:1109-1117, 1979). Strain INV Ia is commercially available from Invitrogen, San Diego,
CA.
2. DNA Manipulations The restriction and modification enzymes employed herein are commercially available in the U.S. Native saporin and rabbit polyclonal antiserum to saporin were obtained as previously described in Lappi et al., Biochem. Biophys. Res.
Comm. 129:934-942. Ricin A chain is commercially available from Sigma, Milwaukee, WO 96/36362 PCT/US96/07164 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. Minipreparation and maxipreparation of plasmids, preparation of competent cells, transformation, M13 manipulation, bacterial media, Western blotting, and ELISA assays were according to Sambrook et al., (Molecular Cloning:
A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
1989). The purification of DNA fragments was done using the Geneclean II kit (Bio 101) according to the manufacturer's instructions. SDS gel electrophoresis was performed on a Phastsystem (Pharmacia).
Western blotting was accomplished by transfer of the electrophoresed protein to nitrocellulose using the PhastTransfer system, as described by the manufacturer. The antiserum to SAP was used at a dilution of 1:1000. Horseradish peroxidase labeled anti-IgG was used as the second antibody (see Davis et al., Basic Methods In Molecular Biology, New York, Elsevier Science Publishing Co., pp 1-338, 1986).
B. Isolation ofDNA encoding saorin 1. Isolation of enomic DNA nd rearation of ol merase chain reaction (PCR primers Saponaria officinalis leaf genomic DNA was prepared as described in Bianchi etal., Plant Mol. Biol. 11:203-214, 1988. Primers for genomic
DNA
amplifications were synthesized in a 380B automatic DNA synthesizer. The primer corresponding to the "sense" strand of saporin CTGCAGAATTCGCATGGATCCTGCTTCAAT-3 (SEQ ID NO. 54) includes an EcoR I restriction site adapter immediately upstream of the DNA codon for amino acid of the native saporin N-terminal leader sequence. The primer CTGCAGAATTCGCCTCGTTTGACTACTTTG-3' (SEQ ID NO. 55) corresponds to the "antisense" strand of saporin and complements the coding sequence of saporin starting from the last 5 nucleotides of the DNA encoding the carboxyl end of the mature WO 96/36362 PCT/US96/07164 86 peptide. Use of this primer introduced a translation stop codon and an EcoRI restriction site after the sequence encoding mature saporin.
2. Amplification of DNA encoding sapori, Unfractionated Saponaria officinalis leaf genomic DNA (1 pl) was mixed in a final volume of 100 p1 containing 10 mM Tris-HCI (pH 50 mM KCI, 0.01% gelatin, 2 mM MgCI 2 0.2 mM dNTPs, 0.8 pg of each primer. Next, 2.5 U Taq DNA polymerase (Perkin Elmer Cetus) were added and the mixture was overlaid with pl of mineral oil (Sigma). Incubations were done in a DNA Thermal Cycler (Ericomp). One cycle included a denaturation step (94 0 C for 1 min), an annealing step 0 C for 2 min), and an elongation step (72 0 C for 3 min). After 30 cycles, a 10 tl aliquot of each reaction was run on a 1.5% agarose gel to verify the structure of the amplified product.
The amplified DNA was digested with EcoRI and subcloned into EcoRIrestricted M13mpl8 (New England Biolabs, Beverly, MA; see also Yanisch-Perron et al. (1985), "Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mpl8 and pUC19 vectors", Gene 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., Eur. J. Biochem. 183:465- 470, 1989). Nine of the M13mpl8 derivatives were sequenced and compared. Of the nine sequenced clones, five had unique sequences, set forth as SEQ ID NOs. 19-23, respectively. The clones were designated M13mpl8-G4, -G1, -G2, -G7, and -G9. Each of these clones contains all of the saporin coding sequence and 45 nucleotides of DNA encoding the native saporin N-terminal leader peptide.
Saporin DNA sequence was also cloned in the pETI la vector. Briefly, the DNA encoding SAP-6 was amplified by polymerase chain reaction (PCR) from the parental plasmid pZ1B1. The plasmid pZ1B1 contains the DNA sequence for human FGF-2 linked to SAP-6 by a two-amino-acid linker (Ala-Met). PZIB1 also includes the T7 promoter, lac operator, ribosomal binding site, and T7 terminator present in the pET- 1la vector. For SAP-6 DNA amplification, the 5' primer CATATGTGTGTCACATCAATCACATTAGAT (SEQ ID NO. 105), WO 96/36362 PCT/US96/07164 87 corresponding to the sense strand of SAP-6, incorporated a NdeI restriction enzyme site used for cloning. It also contained a Cys codon at position -1 relative to the start site of the mature protein sequence. No leader sequence was included. The 3' primer CAGGTTTGGATCCTTTACGTT (SEQ ID NO. 106) corresponding to the antisense strand of SAP-6 had a BamHI site used for cloning. The amplified DNA was gel-purified and digested with NdeI and BamHI. The digested SAP-6 DNA fragment was subcloned into the NdeI/BamHI-digested pZ1B1. This digestion removed FGF-2 and the 5' portion of SAP-6 (up to nucleotide position 650) from the parental rFGF2- SAP vector (pZ B 1) and replaced this portion with a SAP-6 molecule containing a Cys at position -1 relative to the start site of the native mature SAP-6 protein. The resultant plasmid was designated as pZ50B. pZ50B was transformed into E. coli strain NovaBlue for restriction and sequencing analysis. The appropriate clone was then transformed into E. coli strain BL21(DE3) for expression and large-scale production.
C. Mammalian codon optimization of saorin cDNA.
Mammalian expression plasmids encoding 3 -galactosidase (1-gal), pSV- 3 and pNASS-P, were obtained from Clontech (Palo Alto, CA). Plasmid pSVp expresses p-gal from the SV40 early promoter. Plasmid pNASSb is a promoterless mammalian reporter vector containing the p-gal gene.
The amino acid sequence for the plant protein saporin (SAP) was reverse translated using mammalian codons. The resulting mammalian optimized cDNA was divided into 4 fragments (designated A-D) for synthesis by PCR using overlapping oligos. To facilitate subcloning of each fragment and piecing together of the entire cDNA, restriction enzyme sites were added to the ends of each fragment, and added or removed within each fragment without changing the corresponding amino acid sequence. In addition, the 5' end of the cDNA was modified to include a Kozak sequence for optimal expression in mammalian cells. Fragments A, B, and D were each synthesized by annealing 4 oligos (2 sense, 2 antisense) with 20 base overlaps and using PCR to fill-in and amplify the fragments. The PCR products were then purified using GeneClean (BiolOl), digested with restriction enzymes recognizing the sites in the WO 96/36362 PCT/US96/07164 88 primers, and subcloned into pBluescript (Stratagene). The sequence of the inserts was verified using Sequenase Version 2.0 (United States Biochemical/Amersham).
Fragment C was synthesized in two steps: The 5' and 3' halves of the fragment were independently synthesized by PCR using 2 overlapping oligos. The products of these using 2 reactions were then purified and combined and the full-length fragment C was generated by PCR using the outermost oligos as primers. Full-length fragment C was subcloned into pBluescript for sequencing. Fragments A and B were ligated together in pBluescript at an overlapping KspI site. Fragments C and D were ligated together in pBluescript at an overlapping Pvull site. Fragments A-B and C-D were then joined in pBluescript at an overlapping AvaI site to give the full-length mammalian optimized SAP cDNA. P-gal sequences were excised from the plasmids pNASS-p and pSV-p (Clontech) by digestion with NotI and replaced with the synthetic SAP gene, which has NotI ends. Orientation of the insert was confirmed by restriction enzyme digestion.
Large scale plasmid preparations were performed using Qiagen Maxi 500 columns.
The oligos used to synthesize each SAP fragment are Al(sense):CGTATCAGGCGGCCGCCGCCATGGTGACCTCCATCACCCTGGACC TGGTGAACCCCACCGCCGGCC (SEQ ID NO.: 89)
A
2 (antisense):TTGGGGTCCTTCACGTTGTTGCGGATCTTGTCCACGAAGGAGG AGTACTGGCCGGCGGTGGGGTTCACC (SEQ ID NO.: A3(sense):AACAACGTGAAGGACCCCAACCTGAAGTACGGCGGCACCGACAT CGCCGTGATCGGCCCCCCCTC (SEQ ID NO.: 91)
A
4 (antisense):GTGCCGCGGGAGGACTGGAAGTTGATGCGCAGGAACTTCTCCT TGGAGGGGGGGCCGATCACGGC (SEQ ID NO.: 92) BI(sense):CTCCCGCGGCACCGTGTCCCTGGGCCTGAAGCGCGACAACCTGTA CGTGGTGGCCTACCTGGCCATGGACAACAC (SEQ ID NO.: 93) WO 96/36362 PCTJUS96/07164 89 B2(antisense):
GCGGTCAGCTCGGCGGAGGTGATCTCGGACTTGAGGTG
CGCGGTTCACGTTGGTGTTGTCCATGGCCAGGTA (SEQ ID NO.: 94)
B
3 (sense):GCCGAGCTGACCGCCCTGTTCCCTGAGCCCGCACG AAGGCCCTGGAGTACACCGAGGACTACCAGTC (SEQ ID NO.:
B
4 (antisense):AGCCCGAGCTCCTTGCGGGACTTGCCTGTACGG CGTTCTTCTCGATGGACTGGTAGTCCTCGGTGT (SEQ JD NO.: 96) CI (sense)
:TATAGAATTCCTCGGGCTGGGCATCGACCTGCTCGCTCT
GAGGCCGTGACAAGAGGCCCGCGTGG (SEQ ID NO.: 97)
C
2 (antisense):CGGCGGTCATCTGGATGGCGATCACGACGCTGT CTTCACCACGCGGGCCTTCTTGTTC (SEQ ID NO.: 98)
C
3 (sense):CGCCATCCAGATGACCGCCGAGGTGGCCTCCAACA GAACCTGGTGACCA&AGCTTCCCC (SEQ ID NO.: 99)
C
4 (antisense):GGCGGATCCCAGCTGACCTCGACGTACTTGCG AGTCGAACTTGTTGGGGAAGTTCTTGGTCACC (SEQ ID NO.: 100) Dl (sense):CCGGGATCCGTCAGCTGGCGCAGATCTCCCGCTTCG GACGCCAAGAACGGCG (SEQ ID NO.: 10 1)
D
2 (antisense):GCACCTTGCCGAAGCCGAAGTCGTAGCTGTACCC GTTCTTGGCGTCGCCGTAGAT (SEQ ID NO.: 102)
D
3 (sense):TTCGGCTTCGGCAAGGTGCGCCAGGTGAGCTCGTGC CTGCTGATGTACC (SEQ ID NO.: 103) WO 96/36362 PCT/US96/071 6 4
D
4 (antisense):TGAACGTGGCGGCCGCCTACTTGGGCTTGCCCAGGTACATCAG CAGGCCCAT (SEQ ID NO.: 104) D. POMPAG4 Plasmid Construction M13 mp 8-G4 was digested with EcoR I, and the resulting fragment was ligated into the EcoR I site of the vector pIN-IIIompA2 (see, see, U.S. Patent No. 4,575,013 to Inouye; and Duffaud et al., Meth. Enz. 153:492-507, 1987) using the methods described herein. The ligation was accomplished such that the DNA encoding saporin, including the N-terminal extension, was fused to the leader peptide segment of the bacterial ompA gene. The resulting plasmid pOMPAG4 contains the Ipp promoter (Nakamura et al., Cell 18:1109-1117, 1987), the E coli lac promoter operator sequence (lac 0) 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. 19. The plasmid also includes the E. coli lac repressor gene (lac I).
The M13 mpl8-G1, -G2, -G7, and -G9 clones, containing SEQ ID NOs.
20-23, respectively, are digested with EcoR I and ligated into EcoR I digested pIN- IIIompA2 as described for M13 mpl8-G4 above in this example. The resulting plasmids, labeled pOMPAGI, pOMPAG2, pOMPAG7, pOMPA9, are screened, expressed, purified, and characterized as described for the plasmid pOMPAG4.
INVla competent cells were transformed with pOMPAG4 and cultures containing the desired plasmid structure were grown further in order to obtain a large preparation of isolated pOMPAG4 plasmid using methods described herein.
E. SaDorin expression in E. coli The pOMPAG4 transformed E coli cells were grown under conditions in which the expression of the saporin-containing protein is repressed by the lac repressor until the end of the log phase of growth, at which time IPTG was added to induce expression of the saporin-encoding
DNA.
WO 96/36362 PCT/US96/07 16 4 91 To generate a large-batch culture of pOMPAG4 transformed E coli cells, an overnight culture (approximately 16 hours growth) of JA221 E coli cells transformed with the plasmid pOMPAG4 in LB broth (see, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) containing 125 mg/ml ampicillin was diluted 1:100 into a flask containing 750 ml LB broth with 125 mg/ml ampicillin. Cells were grown at logarithmic phase with shaking at 37 0 C until the optical density at 550 nm reached 0.9 measured in a spectrophotometer.
In the second step, saporin expression was induced by the addition of IPTG (Sigma) to a final concentration of 0.2 mM. Induced cultures were grown for 2 additional hours and then harvested by centrifugation (25 min., 6500 x 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 E. coli cells containing C-SAP construct in pETl la were grown in a high-cell density fed-batch fermentation with the temperature and pH controlled at 30 0
C
and 6.9, respectively. A glycerol stock (I mi) was grown in 50 ml Luria broth until the
A
600 reached 0.6 Inoculum (10 ml) was injected into a 7 -1-Applikon (Foster City CA) fermentor containing 21 complex batch medium consisting of 5 g/1 of glucose, 1.25 g/l each of yeast extract and tryptone (Difco Laboratories), 7 g/l of K 2
HPO
4 8 g/l of
KH
2
PO
4 1.66 g/l of(NH 4 2
SO
4 1 g/l ofMgSO 4 e 7H 2 0, 2 ml/i of a trace metal solution (74 g/1 of trisodium citrate, 27 g/1 of FeCI 3 6H 2 0, 2.0 g/l of CoC12, 6H,0, 2.0 g/l of Na 2 MoO 4 2H 2 0, 1.9 g/1 of CuSO 4 5H 2 0, 1.6 g/l of MnC 2 4H 2 0, 1.4 g/l of ZnC12, 4HO, 1.0 g/l of CaC1, 2H,O, 0.5 g/l of H 3
BO
3 2 ml/ of a vitamin solution (6 g/1 of thiamin HCI, 3.05 g/1 of niacin, 2.7 g/l of pantothenic acid, 0.7 g/l of pyridoxine HC1, 0.21 g/1 of riboflavin, 0.03 g/1 of biotin, 0.02 g/1 of folic acid), and 100 mg/1 of carbenicillin. The culture was grown for 12 h before initiating the continuous addition of a 40x solution of complex batch media lacking the phosphates WO 96/36362 PCT/US96/0716 4 92 and containing only 25 ml/l, each, of trace metal and vitamin solutions. The feed addition continued until the A, 60 of the culture reached 85, at which time (approximately 9 h) the culture was induced with 0.1 mM isopropyl P-D-thiogalactopyranoside. During 4 h of post-induction incubation, the culture was fed with a solution containing 100 g/1 of glucose, 100 g/1 of yeast extract, and 200 g/1 of tryptone. Finally, the cells were harvested by centrifugation 8 0 0 0xg, 10 min) and frozen at -80 0 C until further processed.
The cell pellet (-400 g wet mass) containing C-SAP was resuspended in 3 vol Buffer B (10 mM sodium phosphate pH 7.0, 5 mM EDTA, 5 mM EGTA, and 1 mM dithiothreitol). The suspension was passed through a microfluidizer three times at 124 Mpa on ice. The resultant lysate was diluted with NanoPure
H
2 0 until conductivity fell below 2.7 mS/cm. All subsequent procedures were performed at room temperature.
The diluted lysate was loaded onto an expanded bed of Streamline
SP
cation-exchange resin (300 ml) equilibrated with buffer C (20 mM sodium phosphate pH 7.0, 1 mM EDTA) at 100 ml/min upwards flow. The resin was washed with buffer C until it appeared clear. The plunger was then lowered at 2 cm/min while washing continued at 70 ml/min. Upwards flow was stopped when the plunger was approximately 8 cm away from the bed and the plunger was allowed to move to within cm of the packed bed. The resin was further washed at 70 ml/min downwards flow until A 28 0 reached baseline. Buffer C plus 0.25 M NaCI was then used to elute proteins containing C-SAP at the same flow rate.
The eluate was buffer exchanged into buffer D (50 mM sodium borate pH 8.5, 1 mM EDTA) using the Sartocon Mini crossflow filtration system with a 10000 NMolecular Massco module (Sartorius). The sample was then applied to a column of Source 15S (30 ml) equilibrated with buffer D. A 10-column-volume linear gradient of 0-0.3 M NaCI in buffer D was used to elute C-SAP at 30 ml/min.
F. Assay for cvtotoxic activity The ribosome inactivating protein activity of recombinant saporin was compared to the ribosome inactivating protein activity of native SAP in an in vitro assay measuring cell-free protein synthesis in a nuclease-treated rabbit reticulocyte WO 96/36362 PCT/US96/0716 4 93 lysate (Promega). Samples ofimmunoaffinity-purified saporin were diluted in PBS and 1l of sample was added on ice to 35 pl of rabbit reticulocyte lysate and 10 pl of a reaction mixture containing 0.5 pl of Brome Mosaic Virus RNA, 1 mM amino acid mixture minus leucine, 5 aCi of tritiated leucine and 3 1l of water. Assay tubes were incubated 1 hour in a 30 0 C water bath. The reaction was stopped by transferring the tubes to ice and adding 5 pl of the assay mixture, in triplicate, to 75 pl of 1 N sodium hydroxide, 2.5% hydrogen peroxide in the wells of a Millititer HA 9 6-well filtration plate (Millipore). When the red color had bleached from the samples, 300 pl of ice cold trichloroacetic acid (TCA) were added to each well and the plate left on ice for another 30 min. Vacuum filtration was performed with a Millipore vacuum holder.
The wells were washed three times with 300 pl of ice cold 8% TCA. After drying, the filter paper circles were punched out of the 9 6-well plate and counted by liquid scintillation techniques.
The IC 5 0 for the recombinant and native saporin were approximately pM. Therefore, recombinant saporin-containing protein has full protein synthesis inhibition activity when compared to native saporin.
EXAMPLE 2 PREPARATION OF FGF MUTEINS A. Materials and Methods 1. Reagents Restriction and modification enzymes were purchased from BRL (Gaithersburg, MD), Stratagene (La Jolla, CA) and New England Biolabs (Beverly,
MA).
Plasmid pFC80, containing the basic FGF coding sequence, was a gift of Drs. Paolo Sarmientos and Antonella Isacchi of Farmitalia Carlo Erba (Milan, Italy).
Plasmid pFC80, has been described in the PCT Application Serial No. WO 90/02800 and PCT Application Serial No. PCT/US93/05702, which are herein incorporated in WO 96/36362 PCT/US96/07164 94 their entirety by reference. The sequence of DNA encoding bFGF in pFC80 is that set forth in PCT Application Serial No. PCT/US93/05702 and in SEQ ID NO. 52.
Plasmid isolation, production of competent cells, transformation and M13 manipulations were carried out according to published procedures (Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Purification of DNA fragments was achieved using the Geneclean II kit, purchased from Bio 101 (LaJolla, CA). Sequencing of the different constructions was performed using the Sequenase kit (version 2.0) of USB (Cleveland,
OH).
2. Sodium dodecy sulhate SDS el electrohoresis and Western blottin SDS gel electrophoresis was performed on a PhastSystem utilizing gels (Pharmacia). Western blotting was accomplished by transfer of electrophoresed protein to nitrocellulose using the PhastTransfer system (Pharmacia), as described by the manufacturer. The antisera to SAP and basic FGF were used at a dilution of 1:1000.
Horseradish peroxidase labeled anti-IgG was used as the second antibody as described (Davis, Dibner et al. (1986) Basic Methods in Molecular Biology, p. 1, Elsevier Science Publishing Co., New York).
B. Prearation of the mutaenized FGF b sit-directed mutaenesis Cysteine to serine substitutions were made by oligonucleotide-directed mutagenesis using the Amersham (Arlington Heights, IL) in vitro-mutagenesis system 2.1. Oligonucleotides encoding the new amino acid were synthesized using a 380B automatic DNA synthesizer (Applied Biosystems, Foster City, CA).
1. Mutagenesis The oligonucleotide used for in vitro mutagenesis of cysteine 78 was AGGAGTGTCTGCTAACC (SEQ ID NO. 56), which spans nucleotides 225-241 of SEQ ID NO. 52). The oligonucleotide for mutagenesis of cysteine 96 was TTCTAAATCGGTTACCGATGACTG (SEQ ID NO. 57), which spans nucleotides 279-302 of SEQ ID NO. 52). The mutated replicative form DNA was transformed into WO 96/36362 PCT/US96/07164 E. coli strain JM109 and single plaques were picked and sequenced for verification of the mutation. The FGF mutated gene was then cut out of M13, ligated into the expression vector pFC80, which had the non-mutated form of the gene removed, and transformed into E. coli strain JM109. Single colonies were picked and the plasmids sequenced to verify the mutation was present. Plasmids with correct mutation were then transformed into the E. coli strain FICE 2 and single colonies from these transformations were used to obtain the mutant basic FGFs. Approximately 20 mg protein per liter of fermentation broth was obtained.
2. Purification ofmutaenizd
FGF
Cells were grown overnight in 20 ml of LB broth containing 100 -g/ml ampicillin. The next morning the cells were pelleted and transferred to 500 ml of M9 medium with 100 ug/ml ampicillin and grown for 7 hours. The cells were pelleted and resuspended in lysis solution (10 mM TRIS, pH 7.4, 150 mM NaCI, lysozyme, 10 A g/mL, aprotinin, 10 g/mL, leupeptin, 10 lg/mL, pepstatin A, 10 pg/mL and 1 mM PMSF; 45-60 ml per 16 g of pellet) and incubated while stirring for 1 hour at room temperature. The solution was frozen and thawed three times and sonicated for minutes. The suspension was centrifuged; the supernatant saved and the pellet resuspended in another volume of lysis solution without lysozyme, centrifuged again and the supernatants pooled. Extract volumes (40 ml) were diluted to 50 ml with mM TRIS, pH 7.4 (buffer Pools were loaded onto a 5 ml Hi-Trap heparin- Sepharose column (Pharmacia, Uppsala, Sweden) equilibrated in 150 mM sodium chloride in buffer A. The column was washed with 0.6 M sodium chloride and 1 M sodium chloride in buffer A and then eluted with 2 M sodium chloride in buffer
A.
Peak fractions of the 2 M elution, as determined by optical density at 280 nm, were pooled and purity determined by gel electrophoresis. Yields were 10.5 mg of purified protein for the Cys 7 8 mutant and 10.9 mg for the Cys 9 6 mutant.
The biological activity of [C78S]FGF and [C96S]FGF was measured on adrenal capillary endothelial cells in culture. Cells were plated at 3,000 per well in a 24 well plate in 1 ml of 10% calf serum-HDMEM. Cells were allowed to attach, and samples were added in triplicate at the indicated concentration and incubated for 48 h at WO 96/36362 PCT/US96/07164 96 37'C. An equal quantity of samples was added and further incubated for 48 h. Medium was aspirated; cells were treated with trypsin (1 ml volume) to remove cells to 9 ml of Hematall diluent and counted in a Coulter Counter. The results show that the two mutants that retain virtually complete proliferative activity of native basic FGF as judged by the ability to stimulate endothelial cell proliferation in culture.
EXAMPLE 3 PREPARATION OF MONO-DERIVATIZED NUCLEIC
ACID
BINDING DOMAIN
(MYOD)
MyoD at a concentration of 4.1 mg/ml is dialyzed against 0.1 M sodium phosphate, 0.1 M sodium chloride, pH 7.5. A 1.1 molar excess (563 g in 156 p1 of anhydrous ethanol) of SPDP (Pharmacia, Uppsala, Sweden) is added and the reaction mixture immediately agitated and put on a rocker platform for 30 minutes. The solution is then dialyzed against the same buffer. An aliquot of the dialyzed solution is examined for extent of derivatization according to the Pharmacia instruction sheet. The extent of derivatization is typically 0.79 to 0.86 moles of SPDP per mole of nucleic acid binding domain.
Derivatized myoD (32.3 mg) is dialyzed in 0.1 M sodium borate, pH and applied to a Mono S 16/10 column equilibrated with 25 mM sodium chloride in dialysis buffer. A gradient of 25 mM to 125 mM sodium chloride in dialysis buffer elutes free and derivatized nucleic acid binding domain. The flow rate is 4.0 m/min, 4ml fractions are collected. Aliquots of fractions were assayed for protein concentration (BCA Protein Assay, Pierce Chemical, Chicago, IL) and for pyridylthione released by reducing agent. Individual fractions (25 to 37) are analyzed for protein concentration and pyridyl-disulfide concentration. The data indicate a separation according to the level of derivatization by SPDP. The initial eluting peak is composed of myoD that is approximately di-derivatized; the second peak is mono-derivatized and the third peak shows no derivatization. The di-derivatized material accounts for WO 96/36362 PCT/US96/07164 97 approximately 20% of the three peaks; the second accounts for approximately 48% and the third peak contains approximately 32%. Material from the second peak is pooled and gives an average ratio of pyridyl-disulfide to myoD of 0.95. Fraction 33, which showed a divergent ratio of pyridine-2-thione to protein, was excluded from the pool.
Fractions that showed a ratio of SPDP to myoD greater than 0.85 but less than 1.05 are pooled, dialyzed against 0.1 M sodium chloride, 0.1 M sodium phosphate, pH 7.5 and used for derivatization with basic FGF.
EXAMPLE 4 PREPARATION OF MODIFIED NUCLEIC ACID BINDING DOMAIN
(MYOD)
As an alternative to derivatization, myoD is modified by addition of a cysteine residue at or near the N-terminus-encoding portion of the DNA. The resulting myoD can then react with an available cysteine on an FGF or react with a linker or a linker attached to an FGF to produce conjugates that are linked via the added Cys.
Modified myoD is prepared by modifying DNA encoding the myoD (GenBank Accession No. X56677). DNA encoding Cys is inserted at position -1 or at a codon within 10 or fewer residues of the N-terminus. The resulting DNA is inserted into pET I a and pETI5b and expressed in BL21 cells (NOVAGEN, Madison,
WI).
A. Preparation of moD with an added cvsteine residue at the N- tminus Primer #1 corresponding to the sense strand of myoD, nucleotides 121- 144, incorporates a Ndel site and adds a Cys codon 5' to the start site for the mature protein 5'-CATATGTGTGAGCTACTGTCGCCACCGCTC-3' (SEQ ID NO. 58) Primer #2 is an antisense primer complementing the coding sequence of nucleic acid binding domain spanning nucleotides 1054-1077 and contains a BamHI site.
0 WO 96/36362 PCT/US96/071 6 4 98 5'-GGATCCGAGCACCTGGTATATCGGTGGGGG-3' (SEQ ID NO. 59) MyoD DNA is amplified by PCR as follows using the above primers.
A
clone containing a full-length DNA (or cDNA) for myoD (1 tl) is mixed in a final volume of 100 pl containing 10 mM Tris-HCI (pH 50 mM KCI, 0.01% gelatin, 2 mM MgC 2 0.2 mM dNTPs, 0.8 pg of each primer. Next, 2.5 U TaqI
DNA
polymerase (Boehringer Mannheim) is added and the mixture is overlaid with 30 pl of mineral oil (Sigma). Incubations are done in a DNA Thermal Cycler. Cycles include a denaturation step (94C for 1 min), an annealing step (60 0 C for 2 min), and an elongation step (72 0 C for 3 min). After 35 cycles, a 10 l aliquot of each reaction is run on a 1.5% agarose gel to verify the correct structure of the amplified product.
The amplified DNA is gel purified and digested with NdeI and BamHI and subcloned into Ndel and BamHI-digested plasmid containing FGF/myoD. This digestion and subcloning step removes the FGF-encoding DNA and 5' portion of SAP up to the BamHI site at nucleotides 555-560 (SEQ ID NO. 52) and replaces this portion with DNA encoding a myoD molecule that contains a cysteine residue at position -1 relative to the start site of the native mature SAP protein.
B. Prearatin f nucleic acid bindindomain with a csteine residue at osition 4 or 10 of the native protein These constructs are designed to introduce a cysteine residue at position 4 or 10 of the native protein by replacing the Ser residue at position 4 or the Val residue at position 10 with cysteine.
MyoD is amplified by polymerase chain reaction (PCR) from the parental plasmid encoding the FGF-nucleic acid binding domain fusion protein using primers that incorporate a TGT or TGC codon at position 4 or The PCR conditions are performed as described above, using the following cycles: denaturation step 94°C for 1 minute, annealing for 2 minutes at 60 0
C,
and extension for 2 minutes at 72 0 C for 35 cycles. The amplified DNA is gel purified, digested with NdeI and BamHI, and subcloned into NdeI and BamHI digested pET1 la.
WO 96/36362 PCT/US96/07 16 4 99 This digestion removes the FGF and 5' portion of nucleic acid binding domain (up to the newly added BamHI) from the parental FGF- myoD vector and replaces this portion with a myoD molecule containing a Cys at position 4 or 10 relative to the start site of the native protein.
The resulting plasmid is digested with NdeI/BamHI and inserted into (NOVAGEN, Madison, WI), which has a His-TagTM leader sequence (SEQ
ID
NO. 60), that has also been digested NdeI/BamHI.
DNA encoding unmodified myoD can be similarly inserted into a or pET 11A and expressed as described below for the modified SAP-encoding
DNA.
C. Ex p ression of the modified nucleic acid bindin doain-encodin
DNA
BL21(DE3) cells are transformed with the resulting plasmids and cultured as described in Example 2, except that all incubations were conducted at instead of37'C. Briefly, a single colony is grown in LB AMP0oo to and OD 60 0 of 1.0-1.5 and then induced with IPTG (final concentration 0.1 mM) for 2 h. The bacteria are spun down.
D. Purification of modifiednucleic acid bindin domai Lysis buffer (20 mM NaPO 4 pH 7.0, 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 0.5 jg/ml leupeptin, 1 gg/ml aprotinin, 0.7 tg/ml pepstatin) was added to the myoD cell paste (produced from pZ50B1 in BL21 cells, as described above) in a ratio of 1.5 ml buffer/g cells. This mixture is evenly suspended via a Polytron homogenizer and passed through a microfluidizer twice.
The resulting lysate is centrifuged at 50,000 rpm for 45 min. The supernatant is diluted with SP Buffer A (20 mM NaPO4, 1 mM EDTA, pH 7.0) so that the conductivity is below 2.5 mS/cm. The diluted lysate supernatant is then loaded onto a SP-Sepharose column, and a linear gradient of 0 to 30% SP Buffer B (1 M NaCI, mM NaPO 4 1 mM EDTA, pH 7.0) in SP Buffer A with a total of 6 column volumes is applied. Fractions containing myoD are combined and the resulting rnucleic acid binding domain had a purity of greater than 90%. A buffer exchange step is used to get the SP eluate into a buffer containing 50 mM NaBO 3 1 mM EDTA, pH 8.5 (S Buffer This sample is then applied to a Resource S column (Pharmacia, Sweden) pre- WO 96/36362 PCT/US96/07164 100 equilibrated with S Buffer A. Pure nucleic acid binding domain is eluted off the column by 10 column volumes of a linear gradient of 0 to 300 mM NaCI in SP Buffer
A.
In this preparation, ultracentrifugation is used clarify the lysate; other methods, such as filtration and using floculents also can be used. In addition, Streamline S (PHARMACIA, Sweden) may also be used for large scale preparations.
EXAMPLE PREPARATION OF CONJUGATES CONTAINING FGF MUTEINS A. Counlin ofFGF muteinto nucleic acid bindin main 1. Chemical Synthesis of C 7 8 S FGF-nucleic acid bindin domain (CCFN2) and C 9 6 S FGF-nucleic acid bindin domain (CCFN3 [C78S]FGF or [C96S]FGF (1 mg; 56 nmol) that had been dialyzed against phosphate-buffered saline is added to 2.5 mg mono-derivatized nucleic acid binding domain (a 1.5 molar excess over the basic FGF mutants) and left on a rocker platform overnight. The next morning the ultraviolet-visible wavelength spectrum is taken to determine the extent of reaction by the release of pyridylthione, which adsorbs at 343 nm with a known extinction coefficient. The ratio of pyridylthione to basic
FGF
mutant for [C78S]FGF is 1.05 and for [C96S]FGF is 0.92. The reaction mixtures are treated identically for purification in the following manner: reaction mixture is passed over a HiTrap heparin-Sepharose column (1 ml) equilibrated with 0.15 M sodium chloride in buffer A at a flow rate of 0.5 ml/min. The column is washed with 0.6 M NaCI and 1.0 M NaCI in buffer A and the product eluted with 2.0 M NaCI in buffer
A.
Fractions (0.5 ml) are analyzed by gel electrophoresis and absorbance at 280 nm. Peak tubes are pooled and dialyzed versus 10 mM sodium phosphate, pH 7.5 and applied to a Mono-S 5/5 column equilibrated with the same buffer. A 10 ml gradient between 0 and M sodium chloride in equilibration buffer is used to elute the product. Purity is determined by gel electrophoresis and peak fractions were pooled.
WO 96/36362 PCT/US96/07164 101 Under these conditions, virtually 100% of the mutant FGFs reacts with mono-derivatized myoD. Because the free surface cysteine of each mutant acts as a free sulfhydryl, it is unnecessary to reduce cysteines after purification from the bacteria.
The resulting product is purified by heparin-Sepharose (data not shown), thus establishing that heparin binding activity of the conjugate is retained.
2. Expression of the recombinant FGFC78/96S-nucleic acid bindin domain fusion proteins (FPFN4) A two-stage method is used to produce recombinant FGF[C78/96S]myoD protein (hereinafter FPFN4). Two hundred and fifty ml of LB medium containing ampicillin (100 -g/ml) are inoculated with a fresh glycerol stock of bacteria containing the plasmid. Cells are grown at 30 0 C in an incubator shaker to an OD60, of 0.7 and stored overnight at 4 0 C. The following day, cells are pelleted and resuspended in fresh LB medium (no ampicillin). The cells are divided into 5 l-liter batches and grown at 30 0 C in an incubator shaker to an OD 6 00 of 1.5. IPTG is added to a final concentration of 0.1 mM and growth is continued for about 2 to 2.5 hours at which time cells were harvested by centrifugation.
EXAMPLE 6 RECOMBINANT PRODUCTION OF FGF-NUCLEIC
ACID
BINDING DOMAIN FUSION PROTEIN A. General Descriptions 1. Bacterial Strains and Plasmids E. coli strains BL21(DE3), BL21(DE3)pLysS, HMS174(DE3) and HMS 7 4 (DE3)pLysS were purchased from NOVAGEN, Madison, WI. Plasmid described below, has been described in the WIPO International Patent Application No. WO 90/02800, except that the bFGF coding sequence in the plasmid designated pFC80 herein has the sequence set forth as SEQ ID NO. 52, nucleotides 1-465. The WO 96/36362 PCT/US96/07164 102 plasmids described herein may be prepared using pFC80 as a starting material or, alternatively, by starting with a fragment containing the clI ribosome binding site (SEQ ID NO. 61) linked to the FGF-encoding DNA (SEQ ID NO. 52).
E. coli strain JA221 (lpp- hdsM+ trpE5 leuB6 lacY recAl F'[laclq lac+ pro]) is publicly available from the American Type Culture Collection
(ATCC),
Rockville, MD 20852, under the accession number ATCC 33875. (JA221 is also available from the Northern Regional Research Center (NRRL), Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604, under the accession number NRRL B-15211; see also U.S. Patent No. 4,757,013 to Inouye; and Nakamura et al., Cell 18:1109-1117, 1979). Strain INVloa is commercially available from Invitrogen, San Diego,
CA.
B. Construction of lasmids encoding FGF/nucleic acid binding domain fusion 1. Construction of FGFM13 that contains DNA encoding the c ribosome bindin site linked to FGF A Nco I restriction site is introduced into the nucleic acid binding domain-encoding DNA by site-directed mutagenesis using the Amersham in vitromutagenesis system 2.1. The oligonucleotide employed to create the Nco I restriction site is synthesized using a 380B automatic DNA synthesizer (Applied Biosystems).
This oligonucleotide containing the Nco I site replaces the original nucleic acid binding domain-containing coding sequence.
In order to produce a bFGF coding sequence in which the stop codon was removed, the FGF-encoding DNA is subcloned into a M13 phage and subjected to site-directed mutagenesis. Plasmid pFC80 is a derivative of pDS20 (see, Duester etal., Cell 30:855-864, 1982; see also U.S. Patent Nos. 4,914,027, 5,037,744 5,100,784, and 5,187,261; see also PCT International Application No. WO 90/02800; and European Patent Application No. EP 267703 Al), which is almost the same as plasmid pKG1800 (see Bernardi et al., DNA Sequence 1:147-150, 1990; see also McKenney et al. (1981) pp. 383-415 in Gene Amplification and Analysis 2. Analysis of Nucleic Acids by Enzymatic Methods, Chirikjian et al. North Holland Publishing
F
WO 96/36362 PCT/US96/07164 103 Company, Amsterdam) except that it contains an extra 440 bp at the distal end of galK between nucleotides 2440 and 2880 in pDS20. Plasmid pKG1800 includes the 2880 bp EcoR I-Pvu II of pBR322 that contains the contains the ampicillin resistance gene and an origin of replication.
Plasmid pFC80 is prepared from pDS20 by replacing the entire galK gene with the FGF-encoding DNA of SEQ ID NO. 52, inserting the trp promoter
(SEQ
ID NO. 62) and the bacteriophage lambda cII ribosome binding site (SEQ. ID No. 61; see, Schwarz et al., Nature 272:410, 1978) upstream of and operatively linked to the FGF-encoding DNA. The Trp promoter can be obtained from plasmid pDR720 (Pharmacia PL Biochemicals) or synthesized according to SEQ ID NO. 62. Plasmid contains the 2880 bp EcoR I-BamH I fragment of plasmid pSD20, a synthetic Sal I-Nde I fragment that encodes the Trp promoter region: EcoRI
AATTCCCCTGTTGACAATTAATCATCGAACTAGTTAACTAGTACGCAGCTTGGCTGCAG
and the clI ribosome binding site (SEQ ID NO. 61)): Sail Sal Nde I
TCGACCAAGCTTGGGCATACATTCAATCAATTTTATCTAAGGAAATACTTACAT
The FGF-encoding DNA is 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 is blunt ended with Klenow's reagent and inserted into M13mpl8 that has been opened by Sma I and treated with alkaline phosphatase for blunt-end ligation. In order to remove the stop codon, an insert in the ORI minus direction is mutagenized using the Amersham kit, as described above, using the following oligonucleotide (SEQ ID NO. 63): GCTAAGAGCGCCATGGAGA, which contains one nucleotide between the FGF carboxy terminal serine codon and a Nco I WO 96/36362 PCT/US96/07 16 4 104 restriction site; it replaces the following wild type FGF encoding DNA having SEQ
ID
NO. 64: GCT AAG AGC TGA CCA TGG AGA Ala Lys Ser STOP Pro Trp Arg The resulting mutant derivative of M13mpl8, lacking a native stop codon after the carboxy terminal serine codon of bFGF, was designated FGFM13. The mutagenized region of FGFM13 contained the correct sequence (SEQ ID NO. 2. Preparation of a Dlasmid that encodes the FGF/MvoD fusion -rotein Plasmid FGFM13 is cut with NcoI and Sac I to yield a fragment containing the CII ribosome binding site linked to the bFGF coding sequence with the stop codon replaced.
An M13mpl8 derivative containing the myoD coding sequence is also cut with restriction endonucleases Nco I and Sac I, and the bFGF coding fragment from FGFM13 was inserted by ligation to DNA encoding the fusion protein bFGF- myoD into the M13mpl8 derivative to produce mpFGF- myoD, which contains the CII ribosome binding site linked to the FGF-nucleic acid binding domain fusion gene.
Plasmid mpFGF- myoD is digested with Xba I and EcoR I and the resulting fragment containing the bFGF- myoD coding sequence is isolated and ligated into plasmid pET-I la (available from NOVAGEN, Madison, WI; for a description of the plasmids see U.S. Patent No. 4,952,496; see also Studier et al., Meth. Enz. 185:60- 89, 1990; Studier et al., J. Mol. Biol. 189:113-130, 1986; Rosenberg et al., Gene 56:125-135, 1987) that has also been treated with EcoR I and Xba I.
E. coli strain BL21(DE3)pLysS (NOVAGEN, Madison WI) may be transformed with the plasmid containing the fusion gene.
Plasmid FGF/myoD may be digested with EcoR I, the ends repaired by adding nucleoside triphosphates and Klenow DNA polymerase, and then digested with Nde I to release the FGF-encoding DNA without the CII ribosome binding site. This fragment is ligated into pET 1 la, which is BamH I digested, treated to repair the ends, WO 96/36362 PCT/US96/07 16 4 105 and digested with Nde I. The resulting plasmid includes the T7 transcription terminator and the pET-1 la ribosome binding site.
Plasmid FGF/myoD may be digested with EcoR I and Nde I to release the FGF-encoding DNA without the CII ribosome binding site and ends are repaired as described above. This fragment may be ligated into pET 12a, which had been BamH
I
digested and treated to repair the ends. The resulting plasmid includes DNA encoding the OMP T secretion signal operatively linked to DNA encoding the fusion protein.
3. Pre ration of a asmid that encodes FGF2rotamine fusion rotein Protamines are small basic DNA binding proteins, approximately 6.8 kD in molecular weight with a isoelectric point of 12.175. Twenty-four of the fifty one amino acids are strongly basic. Human protamine has been shown to condense genomic DNA for packaging into the sperm head. The positive charges of the protamine react with the negative charges of the phosphate backbone of the DNA.
A FGF-protamine fusion protein that has the ability to bind to the FGF receptor and bind DNA with high affinity is constructed for expression in E. coli The sequence for the human protamine gene is obtained from GenBank (accession no.
Y00443). Four overlapping oligonucleotides 6 0mers) are generated and used to amplify the protamine gene. The amplified product is purified and ligated into the bacterial expression vector pET la (Novagen). To facilitate subcloning, a NcoI and BamHI site are incorporated into the primers. The fragment is synthesized by annealing the 4 oligos (2 sense and 2 antisense) with 20 base overlaps and using PCR to fill-in and amplify the fragments. The PCR products are digested with NcoI and BamHI, and subcloned into pBluescript SK+. The insert sequence is verified. The sequenced product is then cloned downstream and in-frame with FGF2, which has been previously cloned into the pETI la expression plasmid. The oligos used to generate fragment A are PT1:
TACATGCCATGGCCAGGTACAGATGCTGTCGCAGCCAGAGCCGGAGCAGAT
ATTACCGCC (SEQ ID NO.: WO 96/36362 PCT/US96/07 16 4 106 PT2:
GCAGCTCCGCCTCCTTCGTCTGCGACTTCTTTGTCTCTGGCGGTAATATCTGC
TCCGGCT (SEQ ID NO.: 86) PT3:
GACGAAGGAGGCGGAGCTGCCAGACACGGAGGAGAGCCATGAGGTGCTGC
CGCCCCAGGT (SEQ ID NO.: 87) PT4:
ATATATCCTAGGTTAGTGTCTTCTACATCTCGGTCTGTACCTGGGGCGGCAG
CACCTCA (SEQ ID NO.: 88) Competent bacterial cells, BL21 (DE3), are transformed with the pETI 1- FGF2-protamine construct. The cells are initially plated on LB agar plates containing 100 ig/ml ampicillin. A glycerol stock made from an individual colony added to 1 ml fresh LB broth and then to 250 ml of LB broth. The cells are grown to an OD 60 0 of 0.7 and induced with IPTG. The culture is harvested 4 hours after induction. The suspension is centrifuged; the supernatant is saved and the pellet is resuspended in lysis buffer, centrifuged again and the superatants pooled. A sample of the pellet and the supernatant are analyzed by Western analysis using antibodies to FGF2 to determine the percentage of fusion protein within each fraction. Soluble protein is purified. Briefly, the cells are pelleted and resuspended in buffer A (10 mM sodium phosphate, pH containing 10 mM EDTA, 10 mM EGTA and 50 mM NaC1) and passed through a microfluidizer (Microfluidics Corp., Newton, MA) to break open the bacteria and shear DNA. The resultant mixture is diluted and loaded onto an expanded bed Streamline
SP
cation-exchange resin. The column is washed with step gradients of increasing concentrations of NaCI. The eluted material is analyzed by Western analysis for fractions containing the fusion protein. These fractions are pooled, diluted, and loaded onto a Heparin-Sepharose affinity column. After washing, the bound proteins are eluted WO 96/36362 PCT/US96/07164 107 in a batch-wise manner in buffer containing 1 M NaCI and then in buffer containing 2 M NaC1. Peak fractions of the 2M elution, as determined by optical density at 280 nm, are pooled and the purity determined by gel electrophoresis and Western analysis. The final pool of material will be loaded onto a column of Sephacryl S-100 equilibrated with 20 mM HEPES pH 7.4, 150 mM NaCl.
Fusion protein located in the pellet is isolated, solubilized and refolded.
Briefly, each culture pellet is thawed completely and resuspended in buffer A (10 mM Tris, 1 mM EDTA, pH 8.0 0.1 mg/ml lyzozyme). The mixture is sonicated on ice, centrifuged at 16,000 X g, and the supernatant discarded. Inclusion bodies are solubilized with solubilization buffer: (6 M guanidine-HC1, 100 mM Tris, 150 mM NaC1, 50 mM EDTA, 50 mM EGTA, pH vortexed, incubated for 30 minutes at room temperature, and centrifuged at 35,000 X g for 15 minutes. The supernatant is saved and diluted 1:10 in dilution buffer (100 mM Tris, 10 mM EDTA, 1% monothioglycerol, 0.25 M L-arginine, pH The material is stirred, covered, at 4'C for 2 hours and then centrifuged at 35,000 X g for 20 minutes. The supernatant is dialyzed in against 5 liters PBS, pH 8.8, for 24 hours at 4 0 C with 3 changes of fresh PBS. The material is concentrated approximately 10-fold using size-exclusion spin columns. The soluble refolded material is then analyzed by gel electrophoresis.
Expression of the FGF-protamine fusion protein can be achieved in mammalian cells by excising the insert with restriction enzymes NdeI and BamHI and ligating into a mammalian expression vector.
C. Exression f the reombinant bFGF-nucleic acid binding domain fusion proteins A two-stage method is used to produce recombinant bFGF-myoD protein (hereinafter bFGF-nucleic acid binding domain fusion protein).
Three liters of LB broth containing ampicillin (50 ig/ml) and chloramphenicol (25 p1g/ml) are inoculated with pFS92 plasmid-containing bacterial cells (strain BL21(DE3)pLysS) from an overnight culture (1:100 dilution). Cells are grown at 37 0 C in an incubator shaker to an OD, 00 of 0.7. IPTG (Sigma Chemical, WO 96/36362 PCT/US96/07164 108 St. Louis, MO) is added to a final concentration of 0.2 mM and growth was continued for 1.5 hours at which time cells were centrifuged.
Experiments have shown that growing BL21(DE3)pLysS cells at 30 0
C
instead of37 0 C improves yields. Thus, cells are grown at 30°C to an OD6, of 1.5 prior to induction. Following induction, growth is continued for about 2 to 2.5 hours at which time the cells are harvested by centrifugation.
The pellet is resuspended in lysis solution (45-60 ml per 16 g of pellet; mM TRIS, pH 7.4, 5 mM EDTA, 10% sucrose, 150 mM NaCI, lysozyme, 100 t g/ml, aprotinin, 10 tg/ml, leupeptin, 10 tg/ml, pepstatin A, 10 g/ml and 1 mM PMSF) and incubated with stirring for 1 hour at room temperature. The solution is frozen and thawed three times and sonicated for 2.5 minutes. The suspension is centrifuged at 12,000 X g for 1 hour; the resulting first-supematant saved and the pellet is resuspended in another volume of lysis solution without lysozyme. The resuspended material is centrifuged again to produce a second-supernatant, and the two supernatants are pooled and dialyzed against borate buffered saline, pH 8.3.
D. Affinity urification of bFGF-nucleic acid bindindomain fusionotein Thirty ml of the dialyzed solution containing the bFGF-nucleic acid binding domain fusion protein from Example 5.C. is applied to HiTrap heparin-Sepharose column (Pharmacia, Uppsala, Sweden) equilibrated with 0.15 M NaCl in 10 mM TRIS, pH 7.4 (buffer The column is washed first with equilibration buffer; second with 0.6 M NaCI in buffer A; third with 1.0 M NaCI in buffer A; and finally eluted with 2 M NaCI in buffer A into 1.0 ml fractions. Samples were assayed by the ELISA method.
bFGF-nucleic acid binding domain fusion protein elutes from the heparin-Sepharose column at the same concentration (2 M NaC1) as native and recombinantly-produced bFGF, indicating that the heparin affinity is retained in the bFGF-SAP fusion protein.
WO 96/36362 PCT/US96/07164 109 E. Characterization of the bFGF-nucleic acid bindin, domain fusion rotein b Western blot SDS gel electrophoresis is performed on a Phastsystem utilizing acrylamide gels (Pharmacia). Western blotting is accomplished by transfer of the electrophoresed protein to nitrocellulose using the PhastTransfer system (Pharmacia) as described by the manufacturer. Antisera to bFGF is used at a dilution of 1:1000.
Horseradish peroxidase labeled anti-IgG is used as the second antibody (Davis et al., Basic Methods in Molecular Biology, New York, Elsevier Science Publishing Co., pp 1-338, 1986).
Anti-FGF antisera should bind to a protein with an approximate molecular weight of 53,000, which corresponds to the sum of the independent molecular weights of nucleic acid binding domain (35,000) and bFGF (18,000).
EXAMPLE 7 PREPARATION OF FGF-NUCLEIC ACID BINDING DOMAIN CONJUGATES THAT
CONTAIN
LINKERS ENCODING PROTEASE
SUBSTRATES
A. Snthesis ofolios en in rotease substrates Complementary single-stranded oligos in which the sense strand encodes a protease substrate, have been synthesized either using a cyclone machine (Millipore, MA) according the instructions provided by the manufacturer, or were made by Midland Certified Reagent Co. (Midland, TX) or by National Biosciences, Inc. (MN).
The following oligos have been synthesized.
1. Cathepsin B substrate linker CCATGGCCCTGGCCCTGGCCCTGGCCCTGGCCATGG SEQ ID NO: 66 2. Cathepsin D substrate linker
CCATGGGCCGATCGGGCTTCCTGGGCTTCGGCTTCCTGG
GCTTCGCCAT GG SEQ ID NO: 67 3. Trypsin substrate linker
CCATGGGCCGATCGGGCGGTGGGTGCGCTGGTAATAGAGT
WO 96/36362 PCT/US96/07164 110
CAGAAGATCAGTCGGAGCAGCCTGTCTT~IGCGGTGGTCTC
GACCTGCAGG CCATGG-3' SEQ ID NO: 68 4. Gly 4 Ser CCATGGGCGG CGGCGGCTCT GCCATGG SEQ ID NO: 47 5. (Gly 4 Ser) 2
CCATGGGCGGCGGCGGCTCTGGCGGCGGCGGCTC
TGCCATGG SEQ ID NO: 48 6. (Ser 4 GlY) 4
CCATGGCCTCGTCGTCGTCGGGCTCGTCGTCGTC
GGGCTCGTCGTCGTCGGGCTCGTCGTCGTCGGGC
GCCATGG SEQ ID NO: 49 7. (Ser 4 GlY) 2
CCATGGCCTCGTCGTCGTCGGGCTCGTCGTCGTC
GGGCGCCATGG SEQ ID NO: 8. Thrombin substrate linker CTG GTG CCG CGC GGC AGG SEQ ID NO. 69 Leu Val Pro Arg Gly Ser 9. Enterokinase substrate linker GAC GAC GAC GAG CCA SEQ ID NO. Asp Asp Asp Asp Lys Factor Xa substrate ATG GMA GGT CGT SEQ ID NO. 71 Ile Glu Gly Arg B. Prevaration of NA contuctencdn FGF-Linkerncleic ai-bndA domain The complementary oligos are annealed by heating at 95'C for 15 min., cooled to room temperature, and then incubated at 4'C for a minute to about an hour.
Following incubation, the oligos are digested with NcoI and ligated overnight at a 3:1 (insert:vector) ratio at 1 5'C to NcoI-digested plasmid which has been treated with alkaline phosphatase (Boehringer Mannheim).
Bacteria (Novablue (NOVAGEN, Madison, WI)) are transformed with the ligation mixture (I pd1) and plated on LB-amp or LB-Kan, depending upon the plasmid). Colonies are selected, clones isolated and sequenced to determine orientation of the insert. Clones with correct orientation are used to transform strain expression WO 96/36362 PCT/US96/07164 111 strain BL21(DE3) (NOVAGEN, Madison, WI). Glycerol stocks are generated from single transformed colonies. The transformed strains are cultured as described in Example 2 and fusion proteins with linkers were expressed.
The DNA and amino acid sequences of exemplary fusion proteins, containing cathepsin B substrate (FPFS9), cathepsin D substrate (FPFS5), Gly 4 Ser (FPFS7), (Gly 4 Ser) 2 (FPFS8), trypsin substrate (FPFS6), (Ser 4 Gly) 4 (FPFS12) and (Ser 4 Gly) 2 (FPFS11) linkers, respectively, are set forth in SEQ ID NOs. 72-78.
EXAMPLE 8 FGF-POLY-L-LYSINE (FGF2-K) COMPLEXED WITH
A
PLASMID ENCODING
P-GALACTOSIDASE
A. Derivatization ofpolv-L-lysine Polylysine polymer with average lengths of 13, 39, 89, 152, and 265
(K
1 3
K
39 Ks4, K 52
K
26 5 are purchased from a commercial vendor (Sigma, St. Louis, MO) and dissolved in 0.1 M NaPO4, 0.1 M NaCI, 1 mM EDTA, pH 7.5 (buffer A) at mg/ml. Approximately 30 mg ofpoly-L-lysine solution is mixed with 0.187 ml of 3 mg/ml N-succinimidyl-3(pyridyldithio)proprionate (SPDP) in anhydrous ethanol resulting in a molar ratio of SPDP/poly-L-lysine of 1.5 and incubated at room temperature for 30 minutes. The reaction mixture is then dialyzed against 4 liters of buffer A for 4 hours at room temperature.
B. Conjugation of derivatized polylysine to FGF?-3 A solution containing 28.5 mg of poly-L-lysine-SPDP is added to 12.9 mg of FGF2-3 ([C96S]-FGF2) in buffer A and incubated overnight at 4C. The molar ratio of poly-L-lysine-SPDP/FGF2-3 is approximately 1.5. Following incubation, the conjugation reaction mixture is applied to a 6 ml Resource S (Pharmacia, Uppsala, Sweden) column. A gradient of 0.15 M to 2.1 M NaCI in 20 mM NaPO4, 1 mM EDTA, pH 8.0 (Buffer B) over 24 column volumes is used for elution. The FGF2- 3 /poly-L-lysine conjugate, called FGF2-K, is eluted off the column at approximately 1.8-2 M NaCI concentration. Unreacted FGF2-3 is eluted off by 0.5-0.6 M NaC1.
WO 96/36362 PCT/US96/07164 112 The fractions containing FGF2-K are concentrated and loaded onto a gelfiltration column (Sephacryl S100) for buffer exchange into 20 mM HEPES, 0.1 M NaC1, pH 7.3. The molecular weight of FGF-K152 as determined by size exclusion HPLC is approximately 42 kD. To determine if the conjugation procedure interferes with the ability of FGF2-3 to bind heparin, the chemical conjugate FGF2-K is loaded onto a heparin column and eluted off the column at 1.8- 2.0 M NaCI. In comparison, unconjugated FGF2-3 is eluted off heparin at 1.4 1.6 M NaCI. This suggests that poly-L-lysine contributes to FGF2-3 ability to bind heparin. The ability of poly-Llysine 152 to bind heparin is not determined; poly-L-lysine 84 elutes at approximately 1.6 M NaC1. Histone HI-polylysine was purchased and cytochrome C was conjugated to polylysine as described herein.
A sample of FGF2-K is electrophoresed on SDS-PAGE under nonreducing and reducing conditions. The protein migrates at the same molecular weight as FGF. Under non-reducing conditions the conjugate does not enter the gel because of its high charge density (Figure 1, lanes 1, 2, non-reducing; lanes 3, 4, reducing).
A standard proliferation assay using aortic bovine endothelial cells is performed to determine if the conjugation procedure reduced the ability of FGF2-3 ability to stimulate mitogenesis. The results reveal that FGF2-K is equivalent to FGF2- 3 in stimulating proliferation (Figure 2).
C. FGF 2 -3-nolv-L-lvsine-nucleic acid complex formation Optimal conditions for complex formation are established. Varying quantities (0.2 to 200 pg) of P-galactosidase encoding plasmid nucleic acid pSVp or pNASS-P (lacking a promoter) are slowly mixed with 100 gg of FGF2-K in 20 mM HEPES pH7.3, 0.15 M NaC1. The reaction is incubated for 1 hour at room temperature. Nucleic acid binding to the FGF-lysine conjugate is confirmed by gel mobility shift assay using 32 P-labeled SV40--gal nucleic acid cut with HinclI restriction endonuclease. In brief, SV400-gal nucleic acid is digested with HincII restriction endonucleases; ends are labeled by T 4 PNK following dephosphorylation with calf intestinal alkaline phosphatase. To each sample of 35 ng of 32 P-labeled nucleic acid increasing amounts of FGF-polylysine conjugate is added to the mixture.
~----~11111 WO 96/36362 PCT/US96/07164 113 The protein/nucleic acid mixture is electrophoresed in an agarose gel with 1 X TAE buffer. Binding of the conjugate to the radiolabeled DNA is shown by a shift in the complex to the top of the well. (Figure As seen in Figure 3D, as little as 10 ng of
K,
4 causes a complete shift of restriction fragments indicating binding. With K,3, 100 ng of poly-L-lysine was required (Figure 3C). With
K
2 65 10 ng was required (Figure 3E).
The optimal length of poly-L-lysine and weight ratios is determined by conjugation of FGF2-3 to poly-lysine of different lengths. DNA encoding 1-galactosidase was complexed with the conjugates at 10:1, 5:1, 2:1, 1:1, and 0.5:1 (Figure 4, lanes 1-5, respectively) ratios. The ability of these FGF2-K complexes to bind DNA was determined by measuring the ability of FGF to promote the uptake of plasmid DNA into cells. FGF2-K conjugates were evaluated at various protein to DNA ratios for their ability to deliver pSVp-gal DNA into cells (Figure 4).
Briefly, the complexes were incubated for 1 hr at room temperature and then added to COS cells for 48 hrs. Cell extracts were prepared and assayed for p-gal enzyme activity. Briefly, cells are washed with 1 ml of PBS (Ca" 2 and Mg+ 2 free) and lysed. The lysate was vortexed and cell debris removed by centrifugation. The lysate was assayed for p-gal activity as recommended by the manufacturer (Promega, Madison, WI). The P-gal activity was normalized to total protein. As seen in Figure 4, lane 3, a 2:1 ratio of FGF2-K:DNA gave maximal enzyme activity.
In addition, toroid formation, which correlates with increased gene expression, was assessed by electron microscopy. A representative toroid at a protein to DNA ratio of 2:1 is shown in Figure 5, upper panel. Toroidal structures are absent, or only partially formed, at low ratios 0.5:1) (Figure 5, lower panel).
A proliferation assay is performed to determine if the condensed nucleic acid had an effect on the ability of FGF2-K to bind to cognate receptor and stimulate mitogenesis. The proliferation assay shows that only the highest dose of nucleic acid (200 pg) has a slightly inhibitory effect on proliferation as compared to FGF2-3 plus poly-L-lysine DNA (Figure 6).
WO 96/36362 PCTIUS96/07 16 4 114 A FGF2-K84-DNA at a protein:DNA ratio of 2:1 is introduced into COS cells and an endothelial cell line, ABAE, both of which express FGF receptors. The cells are subsequently assayed for P-galactosidase enzyme activity. COS and ABAE cells are grown on coverslips and incubated with the different ratios of FGF2-K:DNA for 48 hours. The cells are then fixed and stained with X-gal. Maximal P-galactosidase enzyme activity is seen when 50 pg of pSVP per 100 pg of FGF2-3-polylysine conjugate is used.
FGF2-K84-pSVp-gal at a protein to DNA ratio of 2:1 was added to various cell lines and incubated for 48 hr. Cell extracts were prepared, assayed for P-gal activity and total protein. As shown in Figure 7A, COS, B16, NIH3T3, and BHK cell lines were all able to take up complex and express p-gal.
The expression of p-gal requires FGF2 for targeting into cells. pSVp or pNASSP plasmid DNA was incubated with (Figure 7B, lanes 1, 2) or without (lanes 3, 4) FGF2-K84 for 1 hr at room temperature. Complexes were added to COS cells for 48 hr. Cell extracts were assayed for p-gal activity and normalized to total protein. Only background P-gal activity was seen unless the plasmid was complexed with FGF2/K84.
Expression of p-gal is seen to be both time and dose-dependent (Figures 7C and 7D).
Sensitivity of the receptor mediated gene delivery system is determined using the optimized FGF2-K/DNA ratio for complex formation. Increasing amounts of the FGF2-K/DNA complex is added to cells. 100 g ofFGF2-K was mixed with 50 ug of pSVP for 1 hour at room temperature. The COS and endothelial cells are incubated with increasing amounts of condensed material (0 ng, 1 ng, 10 ng, 100 ng, 1000 ng and 10,000 ng). The cells are incubated for 48 hours and then were assayed for P-galactosidase activity. In addition, cells grown on cover slips are treated with 1000 ng of FGF2-K-DNA for 48 hours, then fixed and stained using X-gal. The 3-gal enzyme assay reveals that with increasing amounts of material there is an increase in enzyme activity. (Figure 7D) Cells incubated with X-gal show blue staining throughout the cytoplasm in approximately 3% of the cells on the coverslip.
Targeting of the complexes is specific for the FGF receptor. First, as seen in Figure 8A, FGF2-K84-pSVp-gal resulted in enzyme activity (lane while only WO 96/36362 PCT/US96/07164 115 background levels of activity were seen with FGF2+K84+DNA (lane FGF2+DNA (lane K84+DNA (lane DNA (lane FGF2-K84 (lane FGF2 alone (lane 7) and K84 alone (lane The expression of 0-gal is specifically inhibited if free FGF2 is added during transfection (Figure 8B). Moreover, the addition ofheparin attenuates the expression of P-gal (Figure 8C). Moreover, histone HI and cytochrome C were ineffective in delivering pSV3-gal (Figure 8C).
Taken together, these findings support the hypothesis that the targeted DNA is introduced into receptor-bearing cells via the high affinity FGF receptor.
Because histone can bind heparin sulfate yet fails to elicit a signal, the introduction of DNA appears independent of the low affinity FGF receptor or non-specific endocytosis.
D. Effect of endosome-disuptive peptides Targeting is mediated by passage of the complex through endosomes.
Chloroquine, which was added to complexes before transfection, resulted in an 8-fold increase in P-gal activity (Figure 9A).
Based on this, the effect of endosome disruptive peptides was evaluated.
The peptide INF7, GLF EAIEGFIEN GWEGMIDGWYGC, derived from influenza virus, was synthesized. A complex between FGF2-K84 (5 jg) and pSVp-gal plasmid DNA (5 jg) was formed. At this ratio, approximately half of the negative charge of the DNA was neutralized by the conjugate. K84, poly-L-lysine, was further added to saturate binding to the remaining DNA. The INF7 peptide was added 30 minutes later.
The complex is added to COS cells and p-gal activity is assayed 48 or 72 hr later.
The amount of free polylysine necessary to neutralize the DNA and allow INF7 to complex was determined. Polylysine was added at 4, 10, or 25 pag to the FGF2-K84/pSVp-gal complex. To each of these complexes four different concentrations of INF7 were added. Maximal 1-gal expression was seen with 4 Ag of K84 and 12 jpg of INF7 (Figure 13A). When higher amounts of poly-lysine were used, more cell death resulted. The optimal amount of INF7 was determined using 4 Ag of polylysine. As seen in Figure 13B, 24 Ag of INF7 gave maximal p-gal activity. At 72 WO 96/36362 PCT/US96/071 6 4 116 hr, 48 pg of INF7 gave maximal P-gal activity (approximately 20-32 fold enhancement) (Figure 13C).
When an endosome disruptive peptide was included in the complex, expression of p-gal was increased 26-fold (Figure 9B). Concomitant with this increased level of expression was an increase in the number of cells expressing p-gal. As seen in Figure 9C, when endosome disruptive peptide (EDP) was present (right panel), of cells express 0-gal in comparison to without EDP added (left panel).
EXAMPLE 9 CYTOTOXIC ACTIVITY OF FGF/POLY-L-LYSINE BOUND TO SAP DNA PLASMID The cytotoxicity assay measures viable cells after transfection with a cytocide-encoding agent. When FGF-2 is the receptor-binding internalized ligand, COS7 cells, which express FGFR, may be used as targets, and T47D, which does not express a receptor for FGF-2 at detectable levels, may be used as negative control cells.
Cells are plated at 38,000 cells/well and 48,000 cells/well in a 1 2 -well tissue culture plate in RPMI 1640 supplemented with 5% FBS. The complex FGF2- K/pZ200M (a plasmid which expresses saporin) is incubated with COS7 or T47D cells for 48 hrs. Controls include FGF2-K alone, pZ200M alone, and FGF-2 plus poly-L-lysine plus pZ200M. Following incubation, cells are rinsed in PBS lacking Mg and Ca Trypsin at 0.1% is added for 10 min and cells are harvested and washed. Cell number from each well is determined by a Coulter particle counter (or equivalent method). A statistically significant decrease in cell number for cells incubated with FGF2-K/pZ200M compared to FGF2-K or pZ200M alone indicates sufficient cytotoxicity.
FGF
2 -polylysine-DNASAP complexes show selective cytotoxicity. To optimize the expression of the plant RIP, saporin, in mammalian cells, a synthetic saporin gene using preferred mammalian codons and introduced a "Kozak" sequence for WO 96/36362 PCT/US96/071 6 4 117 translation initiation. The synthetic gene was then cloned into SV40 promoter and promoterless expression vectors. Because the expression of SAP from SAP-encoding DNA would only be feasible if the mammalian ribosome can synthesize the protein (SAP) prior to its inactivation by the SAP synthesized, the enzymatic activity of saporin encoded by the synthetic gene was tested. SAP was cloned into a T7/SP6 promoter plasmid and sense RNA was generated using T7 RNA polymerase. The RNA was then added to a mammalian in vitro translation assay. The results from this cell-free in vitro translation assay clearly show that the saporin expressed in a mammalian system can inhibit the expression of protein mutagenesis (Figure 10). When added above to the lysate, SAP mRNA is translated into a protein that has the anticipated molecular weight of the saporin protein (lane Similarly, when luciferase mRNA is added to the lysate, a molecule consistent with the luciferase protein is detected (lane In contrast, if SAP mRNA is added to the lysate along with or 30 minutes prior to luciferase mRNA, saporin activity is detected (lanes 4 and Transfection of cells with SAP DNA demonstrates cytotoxicity. When a mammalian expression vector encoding saporin is transiently expressed in NIH 3T3 cells using CaPO4, there is a >65% decrease in cell survival (lane 3) compared to cells mock transfected (lane 1) or transfected with DNA encoding 3 -gal (lane 2) (Figure 11).
To determine whether the FGF2-K can transfer plasmid DNA encoding SAP into FGF receptor bearing cells, FGF2-K was condensed with the plasmid DNA at a ratio of 2:1 BHK 21 and NIH 3T3 cells were used as the target cells. The cells (24,000 cells/well) were incubated with either FGF2-K-DNASAP or an FGF2-K-DNAp-gal complex. After 72 hours of incubation, cell number was determined. As shown in Figure 12, there is a significant decrease in cell number when cells are incubated with the FGF2-K-DNASAP complex compared to cells incubated with the FGF2-K-DNA3-gal complex.
From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, P:\OPER\RMH\586289CLM -2 1/1/98 118various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
C
C
7. WO 96/36362 PCT/US96/07164 119 SEQUENCE LISTING GENERAL
INFORMATION:
APPLICANT: Prizm Pharmaceuticals. Inc.
(ii) TITLE OF INVENTION: COMPOSITIONS CONTAINING NUCLEIC ACIDS AND LIGANDS FOR THERAPEUTIC
TREATMENT
(iii) NUMBER OF SEQUENCES: 106 (iv) CORRESPONDENCE
ADDRESS:
ADDRESSEE: SEED and BERRY STREET: 6300 Columbia Center, 701 Fifth Avenue CITY: Seattle STATE: Washington COUNTRY:
USA
ZIP: 98104-7092 COMPUTER READABLE
FORM:
MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM:
PC-DOS/MS-DOS
SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION
DATA:
APPLICATION
NUMBER:
FILING DATE: 16-MAY-1996
CLASSIFICATION:
(viii) ATTORNEY/AGENT
INFORMATION:
NAME: Nottenburg Ph.D.. Carol REGISTRATION NUMBER: 39.317 REFERENCE/DOCKET NUMBER: 7 60100.415PC (ix) TELECOMMUNICATION
INFORMATION:
TELEPHONE: (206) 622-4900 TELEFAX: (206) 682-6031 INFORMATION FOR SEQ ID NO:1: SEQUENCE
CHARACTERISTICS:
LENGTH: 473 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 13..456 OTHER INFORMATION: /product=
"VEGF
1 ,,-encoding
DNA"
WO 96/36362 PCTIUS96/0716 4 120 (ix) FEATURE: NAME/KEY: CDS LOCATION: 13..90 OTHER INFORMATION: /product= leader-encoding sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGATCCGAAA CC ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTT Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 1 5 GCC TTG CTG Ala Leu Leu ATG GCA GAA Met Ala Glu CTC TAC CTC CAC CAT GCC Leu Tyr Leu His His Ala 20 GGA GGA GGG CAG AAT CAT Gly Gly Gly Gin Asn His 35 AAG TGG TCC CAG GCT GCA CCC Lys Trp Ser Gin Ala Ala Pro CAC GAA GTG GTG AAG TTC ATG His Glu Val Val Lys Phe Met GTC TAT CAG CGC AGC TAC TGC CAT Val Tyr Gln Arg Ser Tyr Cys His CCA ATC GAG Pro Ile Glu ACC CTG GTG GAC Thr Leu Val Asp ATC TTC CAG GAG Ile Phe Gin Glu CCT GAT GAG ATC Pro Asp Glu Ile GAG TAC Glu Tyr ATC TTC AAG CCA Ile Phe Lys Pro TGT GTG Cys Val GAG TGT Glu Cys
CCC
Pro CTG ATG Leu Met CGA TGC GGG Arg Cys Gly GTG CCC ACT GAG GAG TCC Val Pro Thr Glu Glu Ser 100 CCT CAC CAA GGC CAG CAC Pro His Gln Gly Gln His 115 GGC TGC TGC AAT GAC Gly Cys Cys Asn Asp 85 AAC ATC ACC ATG CAG Asn Ile Thr Met Gln 105 ATA GGA GAG ATG AGC Ile Gly Glu Met Ser 120 GAG GGC Glu Gly ATT ATG CGG Ile Met Arg TTC CTA CAG Phe Leu Gln AAC AAA TGT GAA Asn Lys Cys Glu TGC AGA Cys Arg 130 CCA AAG AAA GAT AGA GCA AGA CAA GAA Pro Lys Lys Asp Arg Ala Arg Gln Glu 135 lAn AAA TGT GAC AAG CCG AGG CGG TGATGAATGA
ATGAGGATCC
Lys Cys Asp Lys Pro Arg Arg 145 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 605 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: both WO 96/36362 PCT[US96/07164 (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:
CDS
LOCATION: 13..588 OTHER INFORMATION: /product= "VEGF16s-encoding
DNA"
(ix) FEATURE: NAME/KEY:
CDS
LOCATION: 13..90 OTHER INFORMATION: /product= "leader sequence-encoding
DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGATCCGAAA CC ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTT 48 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 1 5 GCC TTG CTG Ala Leu Leu ATG GCA GAA Met Ala Glu CTC TAC CTC CAC CAT Leu Tyr Leu His His 20 GCC AAG TGG TCC Ala Lys Trp Ser CAG GCT GCA CCC Gin Ala Ala Pro GGA GGA GGG CAG AAT CAT CAC GAA GTG GTG AAG TTC ATG Gly Gly Gly Gin Asn His His Glu Val Val Lys Phe Met GTC TAT CAG CGC AGC Val Tyr Gin Arg Ser TAC TGC CAT CCA ATC Tyr Cys His Pro Ile 55 GAG ACC CTG GTG GAC Glu Thr Leu Val Asp ATC TTC CAG GAG Ile Phe Gin Glu TGT GTG CCC CTG Cys Val Pro Leu GAG TGT GTG CCC Glu Cys Val Pro ATC AAA CCT CAC Ile Lys Pro His 110 CCT GAT GAG ATC Pro Asp Glu lle
GAG
Glu
TGC
Cys ATG CGA TGC GGG GGC Met Arg Cys Gly Gly 85 TAC ATC TTC AAG CCA Tyr Ile Phe Lys Pro TGC AAT GAC GAG GGC Cys Asn Asp Glu Gly ACC ATG CAG ATT ATG I Thr Met Gin Ile Met 105 192 240 288 336 384 ACT GAG GAG Thr Glu Glu :AA GGC CAG ;In Gly Gin 115 TCC AAC Ser Asn 100 CAC ATA GGA GAG ATG His Ile Gly Glu Met 120 AGC TTC CTA CAG Ser Phe Leu Gin AAC AAA TGT GAA TGC Asn Lys Cys Glu Cys 130 AGA CCA AAG AAA GAT AGA GCA AGA Arg Pro Lys Lys Asp Arg Ala Arg CAA GAA Gin Glu 140 AAT CCC TGT GGG CCT TGC TCA GAG CGG Asn Pro Cys Gly Pro Cys Ser Glu Arg 145 AGA AAG CAT TTG TTT GTA CAA Arg Lys His Leu Phe Val Gin 150 155 WO 96/36362 PCT/US96/0716 4 GAT CCG CAG Asp Pro Gin AAG GCG AGG Lys Ala Arg 175 ACG TGT Thr Cys 160 AAA TGT TCC TGC Lys Cys Ser Cys 165 AAA AAC ACA GAC TCG CGT Lys Asn Thr Asp Ser Arg 170 CAG CTT GAG TTA Gin Leu Glu Leu AAC GAA CGT Asn Glu Arg 180 ACT TGC AGA TGT GAC AAG Thr Cys Arg Cys Asp Lys 185 CCG AGG CGG TGATGAATGA
ATGAGGATCC
Pro Arg Arg 190 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 677 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:
CDS
LOCATION: 13..657 OTHER INFORMATION: /product=
"VEGF
189 -encoding
DNA"
(ix) FEATURE: NAME/KEY:
CDS
LOCATION: 13..90 OTHER INFORMATION: /product= "leader sequence-encoding
DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 GGATCCGAAA CC ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTT 48 Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 1 5 GCC TTG CTG CTC TAC CTC CAC CAT GCC AAG TGG TCC CAG GCT GCA CCC 96 Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Ser Gin Ala Ala Pro 20 GAA GGA GGA GGG Glu Gly Gly Gly CAG AAT Gin Asn CAT CAC GAA GTG GTG AAG TTC His His Glu Val Val Lys Phe GAT GTC Asp Val TAT CAG CGC Tyr Gin Arg AGC TAC Ser Tyr 50 TGC CAT CCA ATC Cys His Pro Ile GAG ACC CTG GTG Glu Thr Leu Val ATC TTC CAG GAG TAC CCT GAT GAG ATC GAG TAC ATC TTC Ile Phe Gin Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe AAG CCA TCC Lys Pro Ser TGT GTG CCC CTG ATG Cys Val Pro Leu Met CGA TGC GGG GGC Arg Cys Gly Gly 85 TGC TGC AAT GAC GAG GGC CTG Cys Cys Asn Asp Glu Gly Leu WO 96/36362 PCTfUS96/07164 GAG TGT GTG Glu Cys Val CCC ACT GAG GAG TCC Pro Thr Glu Glu Ser 100 AAC ATC ACC ATG CAG ATT ATG CGG Asn Ile Thr Met Gin Ile Met Arg 105 AAA CCT CAC CAA GGC CAG CAC Lys Pro His Gin Gly Gin His 110 115 AAC AAA TGT GAA TGC AGA CCA Asn Lys Cys Glu Cys Arg Pro 130 ATA GGA GAG ATG AGC TTC CTA CAG Ile Gly Glu Met Ser Phe Leu Gin 120 AAG AAG GAT AGA GCA AGA CAA GAA Lys Lys Asp Arg Ala Arg Gln Glu 135 140 AAA AAA TCA Lys Lys Ser AAA TCC CGG Lys Ser Arg CGG AGA AAG Arg Arg Lys I 175 TGC AAA AAC Cys Lys Asn 1 190 GTT CGA Val Arg 145 GGA AAG GGA AAG Gly Lys Gly Lys GGG CAA AAA CGA AAG CGC AAG Gly Gin Lys Arg Lys Arg Lys 150 155 CCC TGT GGG CCT TGC TCA GAG Pro Cys Gly Pro Cys Ser Glu 170 AAG TCC TGG AGC Lys Ser Trp Ser 384 432 480 528 576 624 677 CAT TTG TTT GTA CAA His Leu Phe Val Gin 180 CA GAC TCG CGT TGC hr Asp Ser Arg Cys 195 GAT CCG CAG ACG Asp Pro Gin Thr TGT AAA TGT TCC Cys Lys Cys Ser 185 AAG GCG AGG CAG CTT GAG TTA AAC Lys Ala Arg Gin Leu Glu Leu Asn 200 GAA CGT ACT TGC AGA TGT GAC AAG CCG AGG Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg 205 210 INFORMATION FOR SEQ ID NO:4: SEQUENCE
CHARACTERISTICS:
LENGTH: 728 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: both (ii) MOLECULE TYPE: cDNA TGATGAATGA
ATGAGGATCC
(ix) FEATURE: NAME/KEY:
CDS
LOCATION: 13..711 OTHER INFORMATION: (ix) FEATURE: NAME/KEY:
CDS
LOCATION: 13..90 OTHER INFORMATION: /product=
"VEGF
2 o 6 -encoding
DNA"
/product= leader sequence encoding
DNA
WO 96/36362 PCTUS96/07164 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGATCCGAAA CC ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC CTT Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu 1 5 GCC TTG CTG Ala Leu Leu CTC TAC CTC CAC CAT GCC AAG TGG TCC CAG GCT GCA Leu Tyr Leu His His Ala Lys Trp Ser Gin Ala Ala 20 GAA GGA GGA GGG Glu Gly Gly Gly CAG AAT Gin Asn CAT CAC GAA GTG GTG AAG TTC ATG His His Glu Val Val Lys Phe Met GAT GTC Asp Val TAT CAG CGC Tyr Gin Arg AGC TAC Ser Tyr 50 TGC CAT CCA ATC Cys His Pro Ile GAG ACC CTG GTG Glu Thr Leu Val ATC TTC CAG Ile Phe Gin TGT GTG CCC Cys Val Pro GAG TAC Glu Tyr CTG ATG Leu Met CCT GAT GAG ATC Pro Asp Glu Ile TAC ATC TTC AAG Tyr Ile Phe Lys CGA TGC GGG GGC TGC Arg Cys Gly Gly Cys 85 TGC AAT GAC GAG GGC CTG Cys Asn Asp Glu Gly Leu GAG TGT GTG Glu Cys Val CCC ACT GAG GAG TCC Pro Thr Glu Glu Ser 100 AAC ATC ACC ATG Asn Ile Thr Met CAG ATT ATG CGG Gin Ile Met Arg 105 ATC AAA CCT CAC Ile Lys Pro His CAA GGC CAG Gin Gly Gin 115 CAC ATA GGA GAG His Ile Gly Glu ATG AGC TTC CTA CAG Met Ser Phe Leu G1n 120 CAC AAC His Asn 125 AAA TGT GAA Lys Cys Glu AGA CCA AAG AAG GAT Arg Pro Lys Lys Asp 135 AGA GCA AGA CAA Arg Ala Arg Gin AAA AAA TCA Lys Lys Ser AAA TCC CGG Lys Ser Arg GTT CGA Val Arg 145 GGA AAG GGA AAG Gly Lys Gly Lys CAA AAA CGA AAG Gin Lys Arg Lys AAG TCC TGG AGC Lys Ser Trp Ser GTT TAC Val Tyr 165 GTT GGT GCC CGC TGC TGT Val Gly Ala Arg Cys Cys 170 CTA ATG CCC Leu Met Pro 175 TGG AGC CTC CCT GGC Trp Ser Leu Pro Gly 180 CCC CAT CCC TGT GGG CCT TGC TCA Pro His Pro Cys Gly Pro Cys Ser 185 GAG CGG Glu Arg 190 AGA AAG CAT TTG Arg Lys His Leu TTT GTA CAA Phe Val Gin 195 GAT CCG CAG ACG TGT AAA TGT Asp Pro Gin Thr Cys Lys Cys 200 TGC AAA AAC ACA GAC Cys Lys Asn Thr Asp 210 TCG CGT TGC AAG GCG AGG CAG CTT GAG TTA Ser Arg Cys Lys Ala Arg Gin Leu Glu Leu 215 220 WO 96/36362 PCT/US96/07 1 6 4 Me Le Le G1 Gl 6i G1i Lys Lys Ala Gly 145 Thr 125 AAC GAA CGT ACT TGC AGA TGT GAC AAG CCG AGG CGG TGATGAATGA Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg 225 230 235
ATGAGGATCC
INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 208 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY:
CDS
LOCATION: 1..627 OTHER INFORMATION: /note "human HBEGF pr (xi) SEQUENCE DESCRIPTION: SEQ ID et Lys Leu Leu Pro Ser Val Val Leu Lys Leu Phe Leu Ala Ala 1 5 10 eu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Arg Leu Arg Arg E 25 u Ala Ala Gly Thr Ser Asn Pro Asp Pro Pro Thr Val Ser Thr A 40 n Leu Leu Pro Leu Gly Gly Gly Arg Asp Arg Lys Val Arg Asp L 55 n Glu Ala Asp Leu Asp Leu Leu Arg Val Thr Leu Ser Ser Lys Pi 70 75 SAla Leu Ala Thr Pro Asn Lys Glu Glu His Gly Lys Arg Lys Ly 90 Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys Leu Arg Lys Ty 100 105 110 Asp Phe Cys Ile His Gly Glu Cys Lys Tyr Val Lys Glu Leu Ar 115 120 125 Pro Ser Cys Ile Cys His Pro Gly Tyr His Gly Glu Arg Cys Hi! 130 135 140 Leu Ser Leu Pro Val Glu Asn Arg Leu Tyr Thr Tyr Asp His Thr 150 155 160 Ile Leu Ala Val Val Ala Val Val Leu Ser Ser Val Cys Leu Leu 165 170 175 ecursor" Val Gly sp eu ro s /r 9 s r 0 WO 96/36362 WO 9636362 PCT/US96/07164 126 Val Ile Val Gly Leu Leu Met Phe Arg Tyr His Arg Arg Gly Gly Tyr 180 185 190 Asp Val Glu Asn Glu Glu Lys Val Lys Leu Gly Met Thr Asn Ser His 195 200 205 INFORMATION FOR SEQ ID NO:6: SEQUENCE
CHARACTERISTICS:
LENGTH: 77 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: OTHER INFORMATION: /note "human mature
HBEGF"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Arg Val Thr Leu Ser Ser Lys Pro Gin Ala Leu Ala Thr Pro Asn Lys 1 5 10 Glu Glu His Gly Lys Arg Lys Lys Lys Gly Lys Gly Leu Gly Lys Lys 25 Arg Asp Pro Cys Leu Arg Lys Tyr Lys Asp Phe Cys Ile His Gly Glu 40 Cys Lys Tyr Val Lys Glu Leu Arg Ala Pro Ser Cys Ile Cys His Pro 55 Gly Tyr His Gly Glu Arg Cys His Gly Leu Ser Leu Pro 70 INFORMATION FOR SEQ ID NO:7: SEQUENCE
CHARACTERISTICS:
LENGTH: 208 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: OTHER INFORMATION: /note "monkey HBEGF precursor" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met Lys Leu Leu Pro Ser Val Val Leu Lys Leu Leu Leu Ala Ala Val 1 5 10 Leu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Gin Leu Arg Arg Gly 25 WO 96/36362 PCT/US96/07 1 6 4 127 Leu Ala Ala Gly Thr Ser Asn Pro Asp Pro Ser Thr Gly Ser Thr Asp 40 Gin Leu Leu Arg Leu Gly Gly Gly Arg Asp Arg Lys Val Arg Asp Leu 55 Gln Glu Ala Asp Leu Asp Leu Leu Arg Val Thr Leu Ser Ser Lys Pro 70 75 Gin Ala Leu Ala Thr Pro Ser Lys Glu Glu His Gly Lys Arg Lys Lys 90 Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys Leu Arg Lys Tyr 100 105 110 Lys Asp Phe Cys Ile His Gly Glu Cys Lys Tyr Val Lys Glu Leu Arg 115 120 125 Ala Pro Ser Cys Ile Cys His Pro Gly Tyr His Gly Glu Arg Cys His 130 135 140 Gly Leu Ser Leu Pro Val Glu Asn Arg Leu Tyr Thr Tyr Asp His Thr 145 150 155 160 Thr Ile Leu Ala Val Val Ala Val Val Leu Ser Ser Val Cys Leu Leu 165 170 175 Val Ile Val Gly Leu Leu Met Phe Arg Tyr His Arg Arg Gly Gly Tyr 180 185 190 Asp Val Glu Asn Glu Glu Lys Val Lys Leu Gly Met Thr Asn Ser His 195 200 205 INFORMATION FOR SEQ ID NO:8: SEQUENCE
CHARACTERISTICS:
LENGTH: 208 amino acids TYPE: amino acid STRANDEDNESS: unknown TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (ix) FEATURE: OTHER INFORMATION: /note "rat HBEGF precursor" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Lys Leu Leu Pro Ser Val Val Leu Lys Leu Phe Leu Ala Ala Val 1 5 10 Leu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Arg Leu Arg Arg Gly 25 WO 96/36362 PCT/US96/07164 128 Leu Ala Ala Ala Thr Ser Asn Pro Asp Pro Pro Thr Gly Thr Thr Asn 40 Gin Leu Leu Pro Thr Gly Ala Asp Arg Ala Gin Glu Val Gin Asp Leu 55 Glu Gly Thr Asp Leu Asp Leu Phe Lys Val Ala Phe Ser Ser Lys Pro 70 75 G1n Ala Leu Ala Thr Pro Gly Lys Glu Lys Asn Gly Lys Lys Lys Arg 90 Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro Cys Leu Lys Lys Tyr 100 105 110 Lys Asp Tyr Cys Ile His Gly Glu Cys Arg Tyr Leu Lys Glu Leu Arg 115 120 125 lle Pro Ser Cys His Cys Leu Pro Gly Tyr His Gly Gin Arg Cys His 130 135 140 ly Leu Thr Leu Pro Val Glu Asn Pro Leu Tyr Thr Tyr Asp His Thr 145 150 155 160 Thr Val Leu Ala Val Val Ala Val Val Leu Ser Ser Val Cys Leu Leu 165 170 175 Val Ile Val Gly Leu Leu Met Phe Arg Tyr His Arg Arg Gly Gly Tyr 180 185 190 Asp Leu Glu Ser Glu Glu Lys Val Lys Leu Gly Met Ala Ser Ser His 195 200 205 INFORMATION FOR SEQ ID NO:9: SEQUENCE
CHARACTERISTICS:
LENGTH: 627 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: both (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:
CDS
LOCATION: 1..627 OTHER INFORMATION: /note "human HBEGF precursor" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ATG AG CTG CTG CCG TCG GTG GTG CTG AAG CTC TTT CTG GCT GCA GTT 48 Met Lys Leu Leu Pro Ser Val Val Leu Lys Leu Pne Leu Ala Ala Val 1 5 10 CTC TCG GCA CTG GTG ACT GGC GAG AGC CTG GAG CGG CTT CGG AGA GGG 96 Leu Ser Ala Leu Val Thr Gly Glu Ser Leu Glu Arg Leu Arg Arg Gly 25
W
WO 96/36362 PCTIUS96/0716 4 CTA GCT GCT Leu Ala Ala CAG CTG CTA Gin Leu Leu GGA ACC AGC AAC Gly Thr Ser Asn CCC CTA GGA GGC Pro Leu Gly Gly 55 CCG GAC CCT Pro Asp Pro 40 CCC ACT GTA TCC ACG GAC Pro Thr Val Ser Thr Asp GGC CGG GAC CGG AAA Gly Arg Asp Arg Lys GTC CGT GAC TTG Val Arg Asp Leu GAG GCA GAT CTG GAC Glu Ala Asp Leu Asp CTT TTG AGA GTC Leu Leu Arg Val TTA TCC TCC AAG Leu Ser Ser Lys CAA GCA CTG GCC Gin Ala Leu Ala AAA GGC AAG GGG Lys Gly Lys Gly 100 AAG GAC TTC TGC Lys Asp Phe Cys 115 ACA CCA AAC AAG Thr Pro Asn Lys GAG GAG Glu Glu CAC GGG AAA AGA His Gly Lys Arg 240 288 336 384 CTA GGG AAG AAG AGG Leu Gly Lys Lys Arg 105 GAC CCA TGT CTT Asp Pro Cys Leu CGG AAA TAC Arg Lys Tyr 110 ATC CAT GGA Ile His Gly GAA TGC AAA TAT GTG AAG GAG CTC CGG Glu Cys Lys Tyr Val Lys Glu Leu Arg 120 125 CCG GGT TAC CAT GGA GAG AGG TGT CAT Pro Gly Tyr His Gly Glu Arg Cys His 140 TCC TGC ATC TGC Ser Cys Ile Cys CTG AGC CTC CCA Leu Ser Leu Pro GAA AAT CGC TTA Glu Asn Arg Leu TAT ACC Tyr Thr 155 TAT GAC CAC ACA Tyr Asp His Thr 160 ACC ATC CTG GCC Thr Ile Leu Ala GTG GTG Val Val 165 GCT GTG GTG Ala Val Val TCA TCT GTC TGT Ser Ser Val Cys GTC ATC GTG Val Ile Val CTT CTC ATG TTT AGG Leu Leu Met Phe Arg 185 TAC CAT AGG AGA Tyr His Arg Arg GGA GGT Gly Gly 190 GAT GTG GAA AAT GAA GAG AAA GTG Asp Val Glu Asn Glu Glu Lys Val 195 200 AAG TTG GGC ATG ACT AAT TCC CAC Lys Leu Gly Met Thr Asn Ser His 205
TGA
INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 155 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WO 96/36362 PCT/US96/071 6 4 130 (ix) FEATURE: OTHER INFORMATION: /note= "FGF-1" (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5 10 Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 25 Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly 40 Thr Arg Asp Arg Ser Asp Gin His Ile Gin Leu Gin Leu Ser Ala Glu 55 Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gin Tyr Leu 70 75 Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gin Thr Pro Asn Glu 90 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110 Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gin Lys Ala 130 135 140 Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150 155 INFORMATION FOR SEQ ID NO:11: SEQUENCE
CHARACTERISTICS:
LENGTH: 155 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: OTHER INFORMATION: /note= "FGF-2" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 25 WO 96/36362 PCT/US96/0 7 1 6 4 131 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg 40 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gin Leu 55 Gln Ala Glu Glu Arg Gly Val Val Ser lie Lys Gly Val Cys Ala Asn 70 75 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 90 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys 130 135 140 Ala lle Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155 INFORMATION FOR SEQ ID NO:12: SEQUENCE
CHARACTERISTICS:
LENGTH: 239 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: OTHER INFORMATION: /note= "FGF-3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Gly Leu le Trp Leu Leu Leu Leu Ser Leu Leu Glu Pro Gly Trp 1 5 10 Pro Ala Ala Gly Pro Gly Ala Arg Leu Arg Arg Asp Ala Gly Gly Arg 25 Gly Gly Val Tyr Glu His Leu Gly Gly Ala Pro Arg Arg Arg Lys Leu 40 Tyr Cys Ala Thr Lys Tyr His Leu Gin Leu His Pro Ser Gly Arg Val 55 Asn Gly Ser Leu Glu Asn Ser Ala Tyr Ser Ile Leu Glu Ile Thr Ala 70 75 Val Glu Val Gly Ile Val Ala Ile Arg Gly Leu Phe Ser Gly Arg Tyr 90 WO 96/36362 PCT/US96/07164 132 Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr Ser 100 105 110 Ala Glu Cys Glu Phe Val Glu Arg Ile His Glu Leu Gly Tyr Asn Thr 115 120 125 Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser Ser Thr Pro Gly Ala Arg 130 135 140 Arg Gin Pro Ser Ala Glu Arg Leu Trp Tyr Val Ser Val Asn Gly Lys 145 150 155 160 Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gin Lys Ser Ser 165 170 175 Leu Phe Leu Pro Arg Val Leu Asp His Arg Asp His Glu Met Val Arg 180 185 190 Gin Leu Gln Ser Gly Leu Pro Arg Pro Pro Gly Lys Gly Val Gin Pro 195 200 205 Arg Arg Arg Arg Gin Lys Gin Ser Pro Asp Asn Leu Glu Pro Ser His 210 215 220 Val Gin Ala Ser Arg Leu Gly Ser Gin Leu Glu Ala Ser Ala His 225 230 235 INFORMATION FOR SEQ ID NO:13: SEQUENCE
CHARACTERISTICS:
LENGTH: 206 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: OTHER INFORMATION: /note= "FGF-4" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Ser Gly Pro Gly Thr Ala Ala Val Ala Leu Leu Pro Ala Val Leu 1 5 10 Leu Ala Leu Leu Ala Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala Pro 25 Thr Ala Pro Asn Gly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp Glu 40 Ser Leu Val Ala Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gin Pro 55 Lys Glu Ala Ala Val Gln Ser Gly Ala Gly Asp Tyr Leu Leu Gly Ile 70 75 WO 96/36362 PCT/US96/07 164 133 LYS Arg Leu Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe His Leu 90 Gin Ala Leu Pro Asp GiyArg IlieGly Gly Ala His Ala Asp Thr Arg 100 105 110 Asp Ser Leu Leu Glu Leu Ser Pro Val Giu Arg Gly Val Val Ser Ile 115 120 125 Phe Gly Val Ala Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys 130 135 140 Leu Tyr Gly Ser Pro Phe Phe Thr Asp Giu Cys Thr Phe Lys Glu Ile 145 150 155 160 Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Giu Ser Tyr Lys Tyr Pro Gly 165 170 175 Met Phe Ile Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg 180 185 190 Val Ser Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu 195 200 205 INFORMATION FOR SEQ ID NO:14: Ci) SEQUENCE
CHARACTERISTICS:
LENGTH: 268 amino acids TYPE: amino acid STRANOEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: OTHER INFORMATION: /note= (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Met Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu Ilie Leu 1 5 10 Ser Ala Trp Ala His Giy Giu Lys Arg Leu Ala Pro Lys Gly Gin Pro 25 Gly Pro Ala Ala Thr Asp Arg Asn Pro Ilie Gly Ser Ser Ser Arg Gin 40 Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala 55 Ala Ser Leu Gly Ser Gin Gly Ser Gly Leu Glu Gin Ser Ser Phe Gin 70 75 Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly 90 WO 96/36362 PCT/US96/07 1 6 4 134 Ile Gly Phe His Leu Gin Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser 100 105 110 His Glu Ala Asn Met Leu Ser Val Leu Glu Ile Phe Ala Val Ser Gin 115 120 125 Gly lle Val Gly Ile Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met 130 135 140 Ser Lys Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys 145 150 155 160 Lys Phe Arg Glu Arg Phe Gin Glu Asn Ser Tyr Asn Thr Tyr Ala Ser 165 170 175 Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu 180 185 190 Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro 195 200 205 Gin His Ile Ser Thr His Phe Leu Pro Arg Phe Lys Gin Ser Glu Gin 210 215 220 Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro 225 230 235 240 Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr 245 250 255 Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265 INFORMATION FOR SEQ ID SEQUENCE
CHARACTERISTICS:
LENGTH: 198 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: OTHER INFORMATION: /note= "FGF-6" (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ser Arg Gly Ala Gly Arg Leu Gin Gly Thr Leu Trp Ala Leu Val 1 5 10 Phe Leu Gly Ile Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Thr 25 Arg Ala Asn Asn Thr Leu Leu Asp Ser Arg Gly Trp Gly Thr Leu Leu 40 WO 96/36362 PCT/US96/07164 135 Ser Arg Ser Arg Ala Gly Leu Ala Gly Glu Ile Ala Gly Val Asn Trp 55 Glu Ser Gly Tyr Leu Val Gly Ile Lys Arg Gin Arg Arg Leu Tyr Cys 70 75 Asn Val Gly Ile Gly Phe His Leu Gin Val Leu Pro Asp Gly Arg Ile 90 Ser Gly Thr His Glu Glu Asn Pro Tyr Ser Leu Leu Glu Ile Ser Thr 100 105 110 Val Glu Arg Gly Val Val Ser Leu Phe Gly Val Arg Ser Ala Leu Phe 115 120 125 Val Ala Met Asn Ser Lys Gly Arg Leu Tyr Ala Thr Pro Ser Phe Gin 130 135 140 Glu Glu Cys Lys Phe Arg Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala 145 150 155 160 Tyr Glu Ser Asp Leu Tyr Gin Gly Thr Tyr Ile Ala Leu Ser Lys Tyr 165 170 175 Gly Arg Val Lys Arg Gly Ser Lys Val Ser Pro Ile Met Thr Val Thr 180 185 190 His Phe Leu Pro Arg Ile 195 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 194 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: OTHER INFORMATION: /note= "FGF-7" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met His Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr Leu Leu Tyr Arg 1 5 10 Ser Cys Phe His Ile Ile Cys Leu Val Gly Thr Ile Ser Leu Ala Cys 25 Asn Asp Met Thr Pro Glu Gin Met Ala Thr Asn Val Asn Cys Ser Ser 40 Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile 55 WO 96/36362 PCT/US96/07 1 6 4 136 Arg Val Arg Arg Leu Phe Cys Arg Thr Gin Trp Tyr Leu Arg Ile Asp 70 75 Lys Arg Gly Lys Val Lys Gly Thr Gin Glu Met Lys Asn Asn Tyr Asn 90 Ile Met Glu Ile Arg Thr Val Ala Val Gly lie Val Ala Ile Lys Gly 100 105 110 Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115 120 125 Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu 130 135 140 Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly 145 150 155 160 Gly Glu Met Phe Val Ala Leu Asn Gin Lys Gly Ile Pro Val Arg Gly 165 170 175 Lys Lys Thr Lys Lys Glu Gin Lys Thr Ala His Phe Leu Pro Met Ala 180 185 190 Ile Thr INFORMATION FOR SEQ ID NO:17: SEQUENCE
CHARACTERISTICS:
LENGTH: 215 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: OTHER INFORMATION: /note= "FGF-8" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu 1 5 10 Val Leu Cys Leu Gin Ala Gin Val Thr Val Gin Ser Ser Pro Asn Phe 25 Thr Gln His Val Arg Glu Gin Ser Leu Val Thr Asp Gin Leu Ser Arg 40 Arg Leu Ile Arg Thr Tyr G1n Leu Tyr Ser Arg Thr Ser Gly Lys His 55 Val Gin Val Leu Ala Asn Lys Arg Ile Asn Ala Met Ala Glu Asp Gly 70 75 WO 96/36362 PCT/US96/071 6 4 137 Asp Pro Phe Ala Lys Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg 90 Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile Cys Met Asn Lys 100 105 110 Lys Gly Lys Leu Ile Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys Val 115 120 125 Phe Thr Glu Ile Val Leu Glu Asn Asn Tyr Thr Ala Leu Gin Asn Ala 130 135 140 Lys Tyr Glu Gly Trp Tyr Met Ala Phe Thr Arg Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Ser Lys Thr Arg Gin His Gin Arg Glu Val His Phe Met Lys 165 170 175 Arg Leu Pro Arg Gly His His Thr Thr Glu Gin Ser Leu Arg Phe Glu 180 185 190 Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser Gin Arg 195 200 205 Thr Trp Ala Pro Glu Pro Arg 210 215 INFORMATION FOR SEQ ID NO:18: SEQUENCE
CHARACTERISTICS:
LENGTH: 208 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: OTHER INFORMATION: /note= "FGF-9" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gin Asp Ala 1 5 10 Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu 25 Leu Ser Asp His Leu Gly Gin Ser Glu Ala Gly Gly Leu Pro Arg Gly 40 Pro Ala Val Thr Asp Leu Asp His Leu Lys Gly Ile Leu Arg Arg Arg 55 Gin Leu Tyr Cys Arg Thr Gly Phe His Leu Glu lie Phe Pro Asn Gly 70 75 WO 96/36362 PCTJUS96/07164 Thr Ile Gin Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu 90 Phe Ile Ser Gly Leu Tyr 115 Ala Val Gly Leu Ser Ile Arg Gly Val Asp Ser 110 Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu 120 125 Lys Leu 130 Thr Gin Glu Cys Phe Arg Glu Gin Phe Glu Glu Asn Trp 140 Val Asp Thr Gly Arg 160 Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His 150 155 Arg Tyr Tyr Val Ala 165 Leu Asn Lys Asp Gly Thr 170 Pro Arg Glu Gly Thr 175 Arg Thr Lys Arg His Gin Lys Phe Thr 180 185 His Phe Leu Pro Arg Pro Val 190 Asp Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gin Ser 195 200 205 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 804 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..804 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..804 OTHER INFORMATION: /note= "Nucleotide sequence corresponding to the clone M13 mp18-G4 in Example I.B.2." (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 46..804 OTHER INFORMATION: /product= ""Saporin"" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48 la Trp Ile Leu Leu Gin Phe Ser Ala Trp Thr Thr Thr Asp Ala Val -10 -5 1 WO96/36362 PCTUS96/07164 139 ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA 96 Thr Ser Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser 10 TCT TTT GTG GAT AAA ATC CGA AAC AAT GTA AAG GAT CCA AAC CTG AAA 144 Ser Phe Val Asp Lys Ile Arg Asn Asn Val Lys Asp Pro Asn Leu Lys 25 TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA 192 Tyr Gly Gly Thr Asp Ile Ala Val Ile Gly Pro Pro Ser Lys Glu Lys 40 TTC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC 240 Phe Leu Arg Ile Asn Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly 55 60 CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC 288 Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn 75 ACG AAT GTT AAT CGG GCA TAT TAC TTC AAA TCA GAA ATT ACT TCC GCC 336 Thr Asn Val Asn Arg Ala Tyr Tyr Phe Lys Ser Glu Ile Thr Ser Ala 90 GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT 384 Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala 100 105 110 TTA GAA TAC ACA GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA 432 Leu Glu Tyr Thr Glu Asp Tyr Gin Ser Ile Glu Lys Asn Ala Gin Ile 115 120 125 ACA CAG GGA GAT AAA AGT AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA 480 Thr Gin Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly Ile Asp Leu 130 135 140 145 CTT TTG ACG TTC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA 528 Leu Leu Thr Phe Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys 150 155 160 AAC GAA GCT AGG TTT CTG CTT ATC GCT ATT CAA ATG ACA GCT GAG GTA 576 Asn Glu Ala Arg Phe Leu Leu Ile Ala Ile Gin Met Thr Ala Glu Val 165 170 175 GCA CGA TTT AGG TAC ATT CAA AAC TTG GTA ACT AAG AAC TTC CCC AAC 624 Ala Arg Phe Arg Tyr Ile Gin Asn Leu Val Thr Lys Asn Phe Pro Asn 180 185 190 AAG TTC GAC TCG GAT AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT 672 Lys Phe Asp Ser Asp Asn Lys Val Ile Gin Phe Glu Val Ser Trp Arg 195 200 205 AAG ATT TCT ACG GCA ATA TAC GGG GAT GCC AAA AAC GGC GTG TTT AAT 720 Lys Ile Ser Thr Ala Ile Tyr Gly Asp Ala Lys Asn Gly Val Phe Asn 210 215 220 225 WO 96/36362 PCTIUS96/07164 AAA GAT TAT GAT Lys Asp Tyr Asp CAA ATG GGA CTC Gin Met Gly Leu 245 TTC GGG TTT GGA AAA GTG Phe Gly Phe Gly Lys Val 230 235 AGG CAG GTG AAG Arg Gin Val Lys GAC TTG Asp Leu 240 CTT ATG TAT TTG Leu Met Tyr Leu GGC AAA CCA AAG Gly Lys Pro Lys 250 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 804 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:
CDS
LOCATION: 1..804 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..804 OTHER INFORMATION: /note= "Nucleotide sequence corresponding to the clone M13 mpl8-G1 in Example I.B.2." (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 46..804 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48 Ala Trp Ile Leu Leu Gin Phe Ser Ala Trp Thr Thr Thr Asp Ala Val -10 -5 1 ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT Thr Ser Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly 10 CAA TAC Gin Tyr TCT TTT GTG GAT Ser Phe Val Asp AAA ATC CGA AAC AAC GTA Lys Ile Arg Asn Asn Val 25 AAG GAT CCA AAC CTG AAA Lys Asp Pro Asn Leu Lys TAC GGT GGT Tyr Gly Gly ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA Thr Asp Ile Ala Val lle Gly Pro Pro Ser Lys Glu Lys TTC CTT AGA ATT AAT Phe Leu Arg Ile Asn TTC CAA Phe Gin AGT TCC CGA GGA ACG GTC TCA CTT GGC Ser Ser Arg Gly Thr Val Ser Leu Gly 60 WO 96/36362 PCTUS9607164 CTA AAA CGC GAT Leu Lys Arg Asp ACG AAT GTT AAT Thr Asn Val Asn GAG TTA ACC GCC Glu Leu Thr Ala 100 AAC TTG Asn Leu TAT GTG GTC GCG TAT Tyr Val Val Ala Tyr 75 CTT GCA ATG GAT AAC Leu Ala Met Asp Asn GAA ATT ACT TCC GCC Glu Ile Thr Ser Ala CGG GCA TAT TAC Arg Ala Tyr Tyr TTC AGA TCA Phe Arg Ser 90 CTT TTC CCA Leu Phe Pro GAG GCC Glu Ala 105 ACA ACT GCA AAT CAG AAA GCT Thr Thr Ala Asn Gin Lys Ala 110 TTA GAA TAC Leu Glu Tyr 115 ACA GAA GAT TAT Thr Glu Asp Tyr 120 CAG TCG ATC GAA AAG Gin Ser Ile Glu Lys 125 AAT GCC CAG ATA Asn Ala Gin Ile CAG GGA GAT AAA TCA Gin Gly Asp Lys Ser 135 AGA AAA GAA CTC Arg Lys Glu Leu CTT TTG ACG TCC Leu Leu Thr Ser TTG GGG ATC GAC Leu Gly Ile Asp GCA CGT GTG GTT Ala Arg Val Val 160 ATG GAA GCA GTG AAC Met Glu Ala Val Asn 150 AAG AAG Lys Lys 155 AAC GAA GCT Asn Glu Ala GCA CGA TTT Ala Arg Phe 180 AGG TTT Arg Phe 165 CTG CTT ATC GCT Leu Leu Ile Ala 170 ATT CAA ATG ACA GCT GAG GTA Ile Gin Met Thr Ala Glu Val 175 CGG TAC ATT CAA AAC Arg Tyr Ile Gin Asn 185 TTG GTA ACT AAG Leu Val Thr Lys AAC TTC CCC AAC Asn Phe Pro Asn 190 GAC TCG GAT AAC Asp Ser Asp Asn TCT ACG GCA ATA Ser Thr Ala Ile 215 AAG GTG Lys Val 200 ATT CAA TTT GAA GTC AGC TGG CGT Ile Gin Phe Glu Val Ser Trp Arg 205 AAG ATT Lys Ile 210 TAC GGA GAT GCC AAA Tyr Gly Asp Ala Lys 220 AAC GGC GTG TTT Asn Gly Val Phe AAA GAT TAT GAT Lys Asp Tyr Asp GGG TTT GGA AAA GTG AGG CAG GTG AAG Gly Phe Gly Lys Val Arg Gin Val Lys 235 GAC TTG Asp Leu 240 CAA ATG GGA Gin Met Gly CTT ATG TAT TTG Leu Met Tyr Leu GGC AAA CCA AAG Gly Lys Pro Lys 250 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 804 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA WO 96/36362 PCT/US96/0716 4 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..804 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..804 OTHER INFORMATION: /note= "Nucleotide sequence corresponding to the clone M13 mpl8-G2 in Example I.B.2." (ix) FEATURE: NAME/KEY: mat peptide LOCATION: 46..804 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48 Ala Trp Ile Leu Leu Gin Phe Ser Ala Trp Thr Thr Thr Asp Ala Val _in ACA TCA ATC Thr Ser Ile TCT TTT GTG Ser Phe Val ACA TTA GAT CTA GTA Thr Leu Asp Leu Val AAT CCG ACT GCG Asn Pro Thr Ala 10 GGT CAA TAC TCA Gly Gin Tyr Ser GAT AAA ATC CGA Asp Lys Ile Arg GGT ACC GAC ATA GCC Gly Thr Asp Ile Ala AAC AAC GTA AAG GAT CCA AAC CTG AAA Asn Asn Val Lys Asp Pro Asn Leu Lys 25 GTG ATA GGC CCA CCT TCT AAA GAT AAA Val Ile Gly Pro Pro Ser Lys Asp Lys AGT TCC CGA GGA ACG GTC TCA CTT GGC Ser Ser Arg Gly Thr Val Ser Leu Gly 60 CTT AGA ATT AAT Leu Arg Ile Asn TTC CAA Phe Gin 144 192 240 288 336 CTA AAA CGC Leu Lys Arg GAT AAC Asp Asn TTG TAT GTG GTC GCG Leu Tyr Val Val Ala 75 CTT GCA ATG GAT AAC Leu Ala Met Asp Asn ACG AAT GTT AAT Thr Asn Val Asn GAG TTA ACC GCC Glu Leu Thr Ala 100 CGG GCA TAT TAC Arg Ala Tyr Tyr AAA TCA GAA ATT ACT TCC GCC Lys Ser Glu Ile Thr Ser Ala CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala 105 110 TTA GAA TAC ACA GAA GAT TAT CAG TCG Leu Glu Tyr Thr Glu Asp Tyr Gin Ser 115 120 ACA CAG GGA GAT AAA AGT AGA AAA GAA Thr Gin Gly Asp Lys Ser Arg Lys Glu 130 135 ATC GAA AAG AAT GCC CAG ATA Ile Glu Lys Asn Ala Gin Ile 125 CTC GGG Leu Gly 140 TTG GGG ATC GAC TTA Leu Gly Ile Asp Leu WO 96/36362 PCT/US96/07164 CTT TTG ACG Leu Leu Thr TTC ATG Phe Met 150 GAA GCA GTG AAC AAG Glu Ala Val Asn Lys 155 AAG GCA CGT GTG Lys Ala Arg Val GTT AAA Val Lys 160 AAC GAA GCT AGG Asn Glu Ala Arg 165 GCA CGA TTT AGG Ala Arg Phe Arg 180 TTT CTG CTT ATC Phe Leu Leu Ile TAC ATT CAA AAC Tyr Ile Gln Asn 185 GCT ATT Ala Ile 170 CAA ATG ACA GCT GAG GTA Gin Met Thr Ala Glu Val 175 TTG GTA ACT AAG AAC TTC CCC AAC Leu Val Thr Lys Asn Phe Pro Asn 190 AAG TTC Lys Phe 195 TCG GAT AAC AAG Ser Asp Asn Lys 200 GTG ATT CAA TTT GAA Val Ile Gln Phe Glu 205 GGG GAT GCC AAA AAC Gly Asp Ala Lys Asn 220 ATT TCT ACG GCA Ile Ser Thr Ala GTC AGC TGG CGT Val Ser Trp Arg GGC GTG TTT AAT Gly Val Phe Asn 225 ATA TAC Ile Tyr 215 672 720 768 804 AAA GAT TAT GAT Lys Asp Tyr Asp CAA ATG GGA CTC Gln Met Gly Leu 245 GGG TTT GGA AAA Gly Phe Gly Lys AGG CAG GTG AAG Arg Gln Val Lys CTT ATG TAT TTG Leu Met Tyr Leu GGC AAA CCA AAG Gly Lys Pro Lys 250 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 804 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:
CDS
LOCATION: 1..804 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..804 OTHER INFORMATION: /note= "Nucleotide sequence corresponding to the clone M13 mpl8-G7 in Example I.B.2." (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 46..804 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: WO 96/36362 PCT/US96/07164 144 GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT GAT GCG GTC 48 Ala Trp Ile Leu Leu Gin Phe Ser Ala Trp T ThThr Thr Asp Ala Val -10 -5 1 ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT CAA TAC TCA 96 Thr Ser Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly Gin Tyr Ser 10 TCT TTT GTG GAT AAA ATC CGA AAC AAC GTA AAG GAT CCA AAC CTG AAA 144 Ser Phe Val Asp Lys Ile Arg Asn Asn Val Lys Asp Pro Asn Leu Lys 25 TAC GGT GGT ACC GAC ATA GCC GTG ATA GGC CCA CCT TCT AAA GAA AAA 192 Tyr Gly Gly Thr Asp Ile Ala Val Ile Gly Pro Pro Ser Lys Glu Lys 40 TTC CTT AGA ATT AAT TTC CAA AGT TCC CGA GGA ACG GTC TCA CTT GGC 240 Phe Leu Arg Ile Asn Phe Gin Ser Ser Arg Gly Thr Val Ser Leu Gly 55 60 CTA AAA CGC GAT AAC TTG TAT GTG GTC GCG TAT CTT GCA ATG GAT AAC 288 Leu Lys Arg Asp Asn Leu Tyr Val Val Ala Tyr Leu Ala Met Asp Asn 75 ACG AAT GTT AAT CGG GCA TAT TAC TTC AGA TCA GAA ATT ACT TCC GCC 336 Thr Asn Val Asn Arg Ala Tyr Tyr Phe Arg Ser Glu Ile Thr Ser Ala 90 GAG TTA ACC GCC CTT TTC CCA GAG GCC ACA ACT GCA AAT CAG AAA GCT 384 Glu Leu Thr Ala Leu Phe Pro Glu Ala Thr Thr Ala Asn Gin Lys Ala 100 105 110 TTA GAA TAC ACA GAA GAT TAT CAG TCG ATC GAA AAG AAT GCC CAG ATA 432 Leu Glu Tyr Thr Glu Asp Tyr Gin Ser Ile Glu Lys Asn Ala Gin lle 115 120 125 ACA CAG GGA GAT AAA TCA AGA AAA GAA CTC GGG TTG GGG ATC GAC TTA 480 Thr Gin Gly Asp Lys Ser Arg Lys Glu Leu Gly Leu Gly Ile Asp Leu 130 135 140 145 CTT TTG ACG TCC ATG GAA GCA GTG AAC AAG AAG GCA CGT GTG GTT AAA 528 Leu Leu Thr Ser Met Glu Ala Val Asn Lys Lys Ala Arg Val Val Lys 150 155 160 AAC GAA GCT AGA TTC CTT CTT ATC GCT ATT CAG ATG ACG GCT GAG GCA 576 Asn Glu Ala Arg Phe Leu Leu Ile Ala Ile Gin Met Thr Ala Glu Ala 165 170 175 GCA CGA TTT AGG TAC ATA CAA AAC TTG GTA ATC AAG AAC TTT CCC AAC 624 Ala Arg Phe Arg Tyr Ile Gin Asn Leu Val Ile Lys Asn Phe Pro Asn 180 185 190 AAG TTC AAC TCG GAA AAC AAA GTG ATT CAG TTT GAG GTT AAC TGG AAA 672 Lys Phe Asn Ser Glu Asn Lys Val Ile Gin Phe Glu Val Asn Trp Lys 195 200 205 WO 96/36362 PCTIUS96/07164 ATT TCT ACG GCA ATA TAC GGG GAT GCC Ile Ser Thr Ala Ile Tyr Gly Asp Ala 215 AAA AAC Lys Asn 220 GGC GTG TTT AAT Gly Val Phe Asn 225 AAA GAT TAT GAT Lys Asp Tyr Asp TTC GGG TTT GGA Phe Gly Phe Gly 230 AGG CAG GTG AAG Arg Gin Val Lys CAA ATG GGA Gin Met Gly CTC CTT ATG TAT TTG GGC AAA CCA AAG Leu Leu Met Tyr Leu Gly Lys Pro Lys 245 250 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 804 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..804 (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 1..804 OTHER INFORMATION: /note= "Nucleotide sequence corresponding to the clone M13 mp18-G9 in Example I.B.2." (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 46..804 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GCA TGG ATC CTG CTT CAA TTT TCA GCT TGG ACA ACA ACT Ala Trp Ile Leu Leu Gin Phe Ser Ala Trp Thr Thr Thr -10 -5 ACA TCA ATC ACA TTA GAT CTA GTA AAT CCG ACC GCG GGT Thr Ser Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly GAT GCG GTC Asp Ala Val 1 CAA TAC TCA Gin Tyr Ser TCT TTT GTG GAT AAA ATC CGA AAC Ser Phe Val Asp Lys Ile Arg Asn 25 AAC GTA AAG GAT CCA AAC CTG AAA Asn Val Lys Asp Pro Asn Leu Lys TAC GGT GGT Tyr Gly Gly ACC GAC ATA GCC GTG ATA GGC CCA Thr Asp Ile Ala Val Ile Gly Pro CCT TCT AAA GAA AAA Pro Ser Lys Glu Lys WO 96/36362 PCTIUS96/07164 TTC CTT Phe Leu CTA AAA Leu Lys AGA ATT AAT TTC Arg Ile Asn Phe CGC GAT AAC TTG Arg Asp Asn Leu CAA AGT TCC CGA GGA ACG GTC TCA CTT Gin Ser Ser Arg Gly Thr Val Ser Leu TAT GTG GTC GCG Tyr Val Val Ala 75 TAT CTT GCA ATG Tyr Leu Ala Met GAT AAC Asp Asn 240 288 336 384 ACG AAT GTT Thr Asn Val CGG GCA TAT TAC Arg Ala Tyr Tyr TTC AGA Phe Arg TCA GAA ATT ACT TCC GCC Ser Glu Ile Thr Ser Ala GAG TTA ACC GCC Glu Leu Thr Ala 100 CTT TTC CCA GAG GCC Leu Phe Pro Glu Ala 105 ACA ACT GCA AAT CAG AAA GCT Thr Thr Ala Asn Gin Lys Ala 110 TTA GAA TAC Leu Glu Tyr 115 ACA GAA GAT TAT CAG Thr Glu Asp Tyr Gin 120 TCG ATT GAA AAG Ser Ile Glu Lys 125 AAT GCC CAG ATA Asn Ala Gin Ile ACA CAA GGA GAT Thr Gin Gly Asp 130 CTT TCA ACG TCC Leu Ser Thr Ser CAA AGT Gin Ser 135 AGA AAA GAA CTC GGG Arg Lys Glu Leu Gly 140 TTG GGG ATT GAC Leu Gly Ile Asp GAA GCA GTG AAC Glu Ala Val Asn AAG GCA CGT GTG Lys Ala Arg Val GTT AAA Val Lys 160 GAC GAA GCT Asp Glu Ala TTC CTT CTT ATC Phe Leu Leu Ile GCT ATT Ala Ile 170 CAG ATG ACG GCT GAG GCA Gin Met Thr Ala Glu Ala 175 GCG CGA TTT AGG Ala Arg Phe Arg 180 AAG TTC AAC TCG Lys Phe Asn Ser I 195 TAC ATA CAA AAC TTG Tyr Ile Gin Asn Leu 185 GTA ATC AAG AAC TTT CCC AAC Val Ile Lys Asn Phe Pro Asn 190 480 528 576 624 672 720 768 AAC AAA GTG ATT CAG TTT GAG Asn Lys 200 Val Ile Gin Phe GTT AAC TGG AAA Val Asn Trp Lys ATT TCT ACG GCA ATA Ile Ser Thr Ala Ile 215 TAC GGG GAT GCC Tyr Gly Asp Ala AAA AAC GGC GTG TTT Lys Asn Gly Val Phe 220 AGG CAG GTG AAG GAC Arg Gin Val Lys Asp 240 AAA GAT TAT GAT Lys Asp Tyr Asp CAA ATG GGA CTC Gin Met Gly Leu 245 GGG TTT GGA AAA Gly Phe Gly Lys CTT ATG TAT TTG GGC AAA CCA AAG Leu Met Tyr Leu Gly Lys Pro Lys 250 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid WO 96/36362 PCTUS96/07164 147 STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..7 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Pro Lys Lys Arg Lys Val Glu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..8 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID Pro Pro Lys Lys Ala Arg Glu Val 1 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..9 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Pro Ala Ala Lys Arg Val Lys Leu Asp 1 INFORMATION FOR SEQ ID NO:27: WO 96/36362 PCT/US96/07164 148 SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Lys Arg Pro Arg Pro 1 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Lys Ile Pro Ile Lys 1 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..9 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Gly Lys Arg Lys Arg Lys Ser 1 WO 96/36362 PCT/US96/07164 149 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..9 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Lys Arg Val Ala Lys Arg Lys Leu 1 INFORMATION FOR SEQ ID NO:31: SEQUENCE
CHARACTERISTICS:
LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..9 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Ser His Trp Lys Gin Lys Arg Lys Phe 1 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY:
CDS
LOCATION: 1..8 OTHER INFORMATION: /product= nuclear translocation sequence
E
WO 96/36362 PCT/US96/07 16 4 150 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Pro Leu Leu Lys Lys Ile Lys Gin 1 INFORMATION FOR SEQ ID NO:33: SEQUENCE
CHARACTERISTICS:
LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..7 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Pro Gin Pro Lys Lys Lys Pro 1 INFORMATION FOR SEQ ID NO:34: SEQUENCE
CHARACTERISTICS:
LENGTH: 15 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY:
CDS
LOCATION: 1..15 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Pro Gly Lys Arg Lys Lys Glu Met Thr Lys Gin Lys Glu Val Pro 10 INFORMATION FOR SEQ ID SEQUENCE
CHARACTERISTICS:
LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS WO 96/36362 PCT/US96/07164 151 LOCATION: 1..12 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Arg Lys Lys Arg Arg Gin Arg Arg Arg Ala Pro 1 5 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..7 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Asn Tyr Lys Lys Pro Lys Leu 1 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..7 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: His Phe Lys Asp Pro Lys Arg 1 INFORMATION FOR SEQ ID N0:38: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide WO 96/36362 PCT/US96/07164 152 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..7 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Ala Pro Arg Arg Arg Lys Leu 1 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..6 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Ile Lys Arg Leu Arg Arg 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..6 OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID Ile Lys Arg Gin Arg Arg 1 INFORMATION FOR SEQ ID NO:41: WO96/36362 PCT/US96/07164 153 SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: OTHER INFORMATION: /product= nuclear translocation sequence (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Ile Arg Val Arg Arg 1 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Cytoplasmic Translocation Signal" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: Lys Asp Glu Leu 1 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Cytoplasmic Translocation Signal" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Arg Asp Glu Leu WO 96/36362 PCT/US96/07164 154 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: Signal" OTHER INFORMATION: /note= "Cytoplasmic Translocation Signal" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: Lys Glu Glu Leu 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 23 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Endosome-disruptive peptide INF" (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 Met Ile Asp Gly Gly Gly Cys INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Endosome-disruptive peptide INF" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 Met Ile Asp Gly Trp Tyr Gly Cys WO 96/36362 PCT/US96/07164 155 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 3..26 NAME/KEY: Gly 4 Ser with Ncol ends (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: CCATGGGCGG CGGCGGCTCT GCCATGG 27 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 3..41 NAME/KEY: (Gly 4 Ser), with Ncol ends (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: CCATGGGCGG CGGCGGCTCT GGCGGCGGCG GCTCTGCCAT GG 42 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 75 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 3..74 NAME/KEY: (Ser 4 Gly) 4 with Ncol ends (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: CCATGGCCTC GTCGTCGTCG GGCTCGTCGT CGTCGGGCTC GTCGTCGTCG GGCTCGTCGT WO 96/36362 PCT/US96/07164 156 CGTCGGGCGC CATGG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH:45 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 3..45 NAME/KEY: (Ser 4 Gly), (xi) SEQUENCE DESCRIPTION: SEQ ID CCATGGCCTC GTCGTCGTCG GGCTCGTCGT CGTCGGGCGC CATGG INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..8 OTHER INFORMATION: /product= Flexible linker (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: Ala Ala Pro Ala Ala Ala Pro Ala 1 INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 465 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..465 (ix) FEATURE: WO 96/36362 PCT/US96/07164 NAME/KEY: mat_peptide LOCATION: 1..465 OTHER INFORMATION: /product= "bFGF" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: ATG GCA GCA GGA Met Ala Ala Gly TCA ATA Ser Ile ACA ACA TTA CCC GCC TTG Thr Thr Leu Pro Ala Leu 10 CCC GAG GAT GGC Pro Glu Asp Gly GGC AGC GGC Gly Ser Gly TTC CCG CCC GGC CAC TTC Phe Pro Pro Gly His Phe GGG GGC TTC TTC CTG CGC Gly Gly Phe Phe Leu Arg 40 AAG GAC CCC AAG CGG CTG Lys Asp Pro Lys Arg Leu ATC CAC CCC GAC GGC CGA Ile His Pro Asp Gly Arg TAC TGC AAA AAC Tyr Cys Lys Asn GTT GAC Val Asp CAA GCA G1n Ala GGG GTC CGG GAG AAG Gly Val Arg Glu Lys 55 GAA GAG AGA GGA GTT Glu Glu Arg Gly Val 70 AGC GAC CCT CAC ATC AAG CTT CAA CTT Ser Asp Pro His Ile Lys Leu Gin Leu GTG TCT ATC AAA GGA GTG TGT GCT AAC Val Ser Ile Lys Gly Val Cys Ala Asn 75 CGT TAC CTG GCT Arg Tyr Leu Ala GTT ACG GAT GAG Val Thr Asp Glu 100 AAG GAA GAT GGA Lys Glu Asp Gly TTA CTG GCT TCT Leu Leu Ala Ser AAA TGT Lys Cys TGT TTC TTT TTT GAA Cys Phe Phe Phe Glu 105 CGA TTG GAA TCT Arg Leu Glu Ser AAT AAC TAC Asn Asn Tyr 110 AAT ACT TAC Asn Thr Tyr 115 CGA ACT GGG Arg Thr Gly 130 CGG TCA AGG AAA TAC Arg Ser Arg Lys Tyr 120 ACC AGT TGG TAT Thr Ser Trp Tyr GTG GCA TTG AAA Val Ala Leu Lys 125 CCT GGG CAG AAA Pro Gly Gin Lys CAG TAT AAA CTT GGA TCC Gin Tyr Lys Leu Gly Ser 135 AAA ACA GGA Lys Thr Gly 140 GCT ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 1230 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA WO 96/36362 PCT/US96/07164 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1230 (ix) FEATURE: NAME/KEY: mat peptide LOCATION: 1..465 OTHER INFORMATION: /product= "bFGF" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 472..1230 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: GCT GCT GGT TCT ATC ACT ACT CTG CCG GCT CTG CCG GAA Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu GAC GGT Asp Gly GGT TCT GGT GCT TTC Gly Ser Gly Ala Phe CCG CCC GGC CAC Pro Pro Gly His 25 TTC AAG GAC CCC Phe Lys Asp Pro AAG CGG Lys Arg TAC TGC AAA Tyr Cys Lys AAC GGG GGC TTC Asn Gly Gly Phe TTC CTG CGC ATC Phe Leu Arg lle 40 AGC GAC CCT CAC Ser Asp Pro His CAC CCC GAC GGC CGA His Pro Asp Gly Arg ATC AAG CTT CAA CTT Ile Lys Leu Gin Leu GGG GTC CGG GAG AAG Gly Val Arg Glu Lys GCA GAA GAG AGA GGA GTT GTG TCT ATC Ala Glu Glu Arg Val Val Ser Ile AAA GGA Lys Gly GTG TGT GCT AAC Val Cys Ala Asn CGT TAC CTG GCT Arg Tyr Leu Ala ATG AAG GAA GAT GGA Met Lys Glu Asp Gly TTA CTG GCT TCT AAA Leu Leu Ala Ser Lys GTT ACG GAT Val Thr Asp AAT ACT TAC Asn Thr Tyr 115 TGT TTC TTT TTT Cys Phe Phe Phe GAA CGA TTG GAA Glu Arg Leu Glu 105 ACC AGT TGG TAT Thr Ser Trp Tyr TCT AAT AAC TAC Ser Asn Asn Tyr 110 GTG GCA TTG AAA Val Ala Leu Lys 125 CGG TCA AGG AAA Arg Ser Arg Lys GGG CAG TAT AAA Gly Gin Tyr Lys GGA TCC AAA ACA GGA CCT GGG CAG AAA Gly Ser Lys Thr Gly Pro Gly Gin Lys ATA CTT TTT CTT CCA Ile Leu Phe Leu Pro 150 TCT GCT AAG AGC GCC ATG GTC Ser Ala Lys Ser Ala Met Val 155 ACA TCA Thr Ser 160 WO 96/36362 PCT/US96/07164 ATC ACA TTA GAT CTA GTA AAT CCG Ile Thr Leu Asp Leu Val Asn Pro 165 GTG GAT AAA ATC CGA AAC AAC GTA ACC GCG GGT CAA TAC TCA TCT TTT Thr Ala 170 AAG GAT Lys Asp 185 Gly Gin Tyr Ser Ser Phe 175 Val Asp Lys Ile 180 Arg Asn Asn Val CCA AAC CTG AAA TAC GGT Pro Asn Leu Lys Tyr Gly 190 GGT ACC GAC Gly Thr Asp 195 ATA GCC GTG ATA Ile Ala Val lle CCA CCT TCT AAA Pro Pro Ser Lys GAA AAA TTC CTT Glu Lys Phe Leu 205 CTT GGC CTA AAA Leu Gly Leu Lys AGA ATT Arg Ile 210 AAT TTC CAA AGT TCC CGA GGA ACG GTC Asn Phe Gin Ser Ser Arg Gly Thr Val 215 GAT AAC TTG TAT Asp Asn Leu Tyr GTC GCG TAT CTT Val Ala Tyr Leu GCA ATG GAT AAC Ala Met Asp Asn 235 ATT ACT TCC GCC Ile Thr Ser Ala ACG AAT Thr Asn 240 GTT AAT CGG Val Asn Arg GCA TAT Ala Tyr 245 TAC TTC AAA TCA Tyr Phe Lys Ser ACC GCC CTT TTC Thr Ala Leu Phe 260 CCA GAG GCC ACA Pro Glu Ala Thr GCA AAT CAG AAA Ala Asn Gin Lys GCT TTA GAA Ala Leu Glu 270 TAC ACA GAA Tyr Thr Glu 275 GGA GAT AAA Gly Asp Lys 290 GAT TAT CAG TCG Asp Tyr Gin Ser AGT AGA AAA GAA Ser Arg Lys Glu 295 ATC GAA AAG AAT GCC Ile Glu Lys Asn Ala 280 CTC GGG TTG GGG ATC Leu Gly Leu Gly Ile 300 CAG ATA ACA CAG G1n Ile Thr G1n 285 GAC TTA CTT TTG Asp Leu Leu Leu TTC ATG GAA GCA Phe Met Glu Ala AAC AAG AAG GCA Asn Lys Lys Ala GTG GTT AAA AAC Val Val Lys Asn 864 912 960 1008 1056 GCT AGG TTT CTG Ala Arg Phe Leu CTT ATC GCT Leu Ile Ala 325 ATT CAA ATG Ile Gin Met 330 ACA GCT GAG GTA GCA CGA Thr Ala Glu Val Ala Arg 335 AAC TTC CCC AAC AAG TTC Asn Phe Pro Asn Lys Phe 350 TTT AGG TAC ATT CAA AAC TTG GTA ACT AAG Phe Arg Tyr Ile Gin Asn Leu Val Thr Lys 340 345 GAC TCG GAT Asp Ser Asp 355 AAC AAG GTG ATT CAA TTT GAA GTC AGC Asn Lys Val Ile Gin Phe Glu Val Ser 360 TGG CGT AAG ATT Trp Arg Lys Ile 365 GCA ATA TAC GGG Ala Ile Tyr Gly GCC AAA AAC GGC GTG TTT AAT AAA GAT Ala Lys Asn Gly Val Phe Asn Lys Asp WO 96/36362 PCT/US96/07164 TAT GAT Tyr Asp 385 TTC GGG TTT GGA AAA GTG AGG CAG Phe Gly Phe Gly Lys Val Arg Gin 390 GTG AAG GAC TTG CAA ATG Val Lys Asp Leu Gin Met 395 400 1200 1230 GGA CTC CTT ATG Gly Leu Leu Met TAT TTG Tyr Leu 405 GGC AAA CCA AAG Gly Lys Pro Lys 410 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: miscrecomb LOCATION: 6..11 OTHER INFORMATION: /standard name= "EcoRI (ix) FEATURE: NAME/KEY: sig_peptide LOCATION: 12..30 OTHER INFORMATION: /function= "N-terminal peptide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: CTGCAGAATT CGCATGGATC CTGCTTCAAT Restriction Site" extension" /product= "Native saporin signal INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iv) ANTI-SENSE: YES (ix) FEATURE: NAME/KEY: miscrecomb LOCATION: 6..11 OTHER INFORMATION: /standard name= "EcoRI Restriction Site" (ix) FEATURE: NAME/KEY: terminator LOCATION: 23..25 OTHER INFORMATION: /note= "Anti-sense stop codon" WO 96/36362 PCT/US96/07164 161 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 26..30 OTHER INFORMATION: /note= "Anti-sense to carboxyl terminus of mature peptide" (xi) SEQUENCE DESCRIPTION: SEQ ID CTGCAGAATT CGCCTCGTTT GACTACTTTG INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: AGGAGTGTCT GCTAACC 17 INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: TTCTAAATCG GTTACCGATG ACTG 24 INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: CATATGTGTG AGCTACTGTC GCCACCGCTC 7n WO 96/36362 PCTIUS96/07164 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: GGATCCGAGC ACCTGGTATA TCGGTGGGGG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GGATCCGCCT CGTTTGACTA CTT INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: OTHER INFORMATION/product= bacteriophage lambda CII (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: GTCGACCAAG CTTGGGCATA CATTCAATCA ATTGTTATCT AAGGAAATAC TTACATATG INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: OTHER INFORMATION: /product= trp promoter ribosome binding site 59 WO 96/36362 PCT/US96/07164 163 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: AATTCCCCTG TTGACAATTA ATCATCGAAC TAGTTAACTA GTACGCAGCT TGGCTGCAG 59 INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: miscrecomb LOCATION: 11..16 OTHER INFORMATION: /standard_name= "Nco I restriction enzyme recognition site." (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..10 OTHER INFORMATION: /product= "Carboxy terminus of mature FGF protein" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: GCTAAGAGCG CCATGGAGA 19 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..12 OTHER INFORMATION: /product= "Carboxy terminus of wild type FGF" (ix) FEATURE: NAME/KEY: miscrecomb LOCATION: 13..18 OTHER INFORMATION: /standardname= "Nco I restriction enzyme recognition site" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: GCT AAG AGC TGACCATGGA GA 21 Ala Lys Ser 1 WO 96/36362 PCTIUS96/07164 164 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 102 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..96 OTHER INFORMATION: /product= "pFGFNcoI" /note= "Equals the plasmid pFC80 wih native FGF stop codon removed." (ix) FEATURE: NAME/KEY: misc recomb LOCATION: 29..34 OTHER INFORMATION: /standardname= "Nco I (xi) SEQUENCE DESCRIPTION: SEQ ID CTT TTT CTT CCA ATG TCT GCT AAG AGC GCC ATG GAG ATC CGG Leu Phe Leu Pro Met Ser Ala Lys Ser Ala Met Glu Ile Arg 1 5 10 GGT GCA GTT CTG TAC CGG TTT TCC TGT GCC GTC TTT CAG GAC Gly Ala Val Leu Tyr Arg Phe Ser Cys Ala Val Phe Gln Asp 25 restriction enzyme recognition site" CTG AAT Leu Asn TCC TGAAATCTT 102 Ser INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 3..35 NAME/KEY: Cathepsin B linker (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: CCATGGCCCT GGCCCTGGCC CTGGCCCTGG CCATGG WO 96/36362 PCT/US96/0716 4 165 INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 3..50 NAME/KEY: Cathepsin D linker (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: CCATGGGCCG ATCGGGCTTC CTGGGCTTCG GCTTCCTGGG CTTCGCCATGG 51 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 96 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 3..95 NAME/KEY: "Trypsin linker" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: CCATGGGCCG ATCGGGCGGT GGGTGCGCTG GTAATAGAGT CAGAAGATCA GTCGGAAGCA GCCTGTCTTG CGGTGGTCTC GACCTGCAGG CCATGG 96 INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..18 OTHER INFORMATION: /product= Thrombin substrate linker WO 96/36362 PCTUS96/07164 166 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: CTG GTG CCG CGC GGC AGC 18 Leu Val Pro Arg Gly Ser 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 15 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..15 OTHER INFORMATION: /product= Enterokinase substrate linker (xi) SEQUENCE DESCRIPTION: SEQ ID GAC GAC GAC GAC CCA Asp Asp Asp Asp Lys 1 INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 12 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..12 OTHER INFORMATION: /product= Factor Xa substrate (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: ATC GAA GGT CGT 12 Ile Glu Gly Arg 1 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 1260 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA WO 96/36362 PCT/US96/07164 167 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1260 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..465 OTHER INFORMATION: /product= "bFGF" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 466...501 OTHER INFORMATION: /product= "Cathepsin B linker" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 502..1260 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu GGC AGC GGC GCC Gly Ser Gly Ala TTC CCG CCC GGC CAC TTC AAG GAC CCC Phe Pro Pro Gly His Phe Lys Asp Pro AAG CGG Lys Arg TAC TGC AAA AAC GGG GGC TTC Tyr Cys Lys Asn Gly Gly Phe TTC CTG Phe Leu CGC ATC CAC CCC GAC GGC CGA Arg Ile His Pro Asp Gly Arg GGG GTC CGG GAG Gly Val Arg Glu AGC GAC CCT CAC ATC AAG CTT CAA CTT Ser Asp Pro His Ile Lys Leu Gin Leu CAA GCA Gin Ala GAA GAG AGA GGA GTT GTG TCT ATC Glu Glu Arg Gly Val Val Ser Ile AAA GGA Lys Gly 75 GTG TGT GCT AAC Val Cys Ala Asn CGT TAC CTG GCT ATG AAG GAA GAT Arg Tyr Leu Ala Met Lys Glu Asp GGA AGA Gly Arg TTA CTG GCT TCT Leu Leu Ala Ser AAA TGT Lys Cys GTT ACG GAT Val Thr Asp AAT ACT TAC Asn Thr Tyr 115 TGT TTC TTT TTT GAA CGA TTG GAA TCT Cys Phe Phe Phe Glu Arg Leu Glu Ser 105 AAT AAC TAC Asn Asn Tyr 110 CGG TCA AGG AAA TAC ACC AGT TGG Arg Ser Arg Lys Tyr Thr Ser Trp 120 TAT GTG GCA TTG AAA Tyr Val Ala Leu Lys 125 GGA CCT GGG CAG AAA Gly Pro Gly Gin Lys GGG CAG TAT AAA Gly Gin Tyr Lys CTT GGA TCC AAA ACA Leu Gly Ser Lys Thr 135 WO 96/36362 PCTI/US96/07164 ATA CTT TTT CTT Ile Leu Phe Leu CCA ATG Pro Met 150 TCT GCT AAG AGC Ser Ala Lys Ser 155 GCC ATG GCC CTG Ala Met Ala Leu CTG GCC CTG Leu Ala Leu AAT CCG ACC Asn Pro Thr AAC GTA AAG Asn Val Lys 195 ATA GGC CCA Ile Gly Pro 210 GCC CTG Ala Leu 165 GCG GGT Ala Gly 180 GCC ATG GTC ACA Ala Met Val Thr CAA TAC TCA TCT Gin Tyr Ser Ser 185 TCA ATC ACA TTA GAT CTA GTA Ser lle Thr Leu Asp Leu Val 170 175 TTT GTG GAT AAA ATC CGA AAC Phe Val Asp Lys Ile Arg Asn GAT CCA AAC CTG Asp Pro Asn Leu CCT TCT AAA GAA Pro Ser Lys Glu 215 TAC GGT GGT ACC Tyr Gly Gly Thr GAC ATA GCC GTG Asp Ile Ala Val 205 AAT TTC CAA AGT Asn Phe Gin Ser AAA TTC CTT AGA Lys Phe Leu Arg CGA GGA ACG GTC Arg Gly Thr Val CTT GGC CTA AAA Leu Gly Leu Lys GAT AAC TTG TAT GTG Asp Asn Leu Tyr Val 240 GTC GCG TAT CTT Val Ala Tyr Leu TTC AAA TCA GAA Phe Lys Ser Glu 260 ATG GAT AAC ACG AAT Met Asp Asn Thr Asn 250 GTT AAT CGG GCA Val Asn Arg Ala TAT TAC Tyr Tyr 255 ATT ACT TCC GCC Ile Thr Ser Ala GAG TTA Glu Leu 265 ACC GCC CTT TTC CCA GAG Thr Ala Leu Phe Pro Glu 270 GCC ACA ACT Ala Thr Thr 275 GCA AAT CAG AAA GCT Ala Asn Gin Lys Ala 280 TTA GAA TAC ACA Leu Glu Tyr Thr GAA GAT TAT CAG Glu Asp Tyr Gin 285 GAA AAG AAT GCC Glu Lys Asn Ala ATA ACA CAG GGA Ile Thr Gin Gly GAA CTC GGG TTG GGG Glu Leu Gly Leu Gly 305 AAC AAG AAG GCA CGT Asn Lys Lys Ala Arg 325 GAC TTA CTT TTG Asp Leu Leu Leu GAT AAA AGT AGA AAA Asp Lys Ser Arg Lys 300 TTC ATG GAA GCA GTG Phe Met Glu Ala Val 320 AGG TTT CTG CTT ATC Arg Phe Leu Leu Ile 335 GTG GTT AAA AAC Val Val Lys Asn GAA GCT Glu Ala 330 GCT ATT CAA Ala Ile G1n TTG GTA ACT Leu Val Thr 355 ACA GCT GAG GTA GCA Thr Ala Glu Val Ala 345 CGA TTT AGG TAC Arg Phe Arg Tyr ATT CAA AAC Ile Gin Asn 350 AAG AAC TTC CCC AAC AAG TTC GAC TCG Lys Asn Phe Pro Asn Lys Phe Asp Ser GAT AAC AAG GTG Asp Asn Lys Val 365 WO 96/36362 PCT/US96/07164 ATT CAA TTT Ile Gin Phe 370 GAA GTC AGC TGG Glu Val Ser Trp 375 CGT AAG ATT TCT ACG Arg Lys Ile Ser Thr 380 GCA ATA TAC GGG Ala Ile Tyr Gly GCC AAA AAC GGC Ala Lys Asn Gly TTT AAT AAA GAT TAT GAT TTC GGG TTT Phe Asn Lys Asp Tyr Asp Phe Gly Phe AAA GTG AGG CAG GTG AAG GAC TTG Lys Val Arg Gin Val Lys Asp Leu CAA ATG Gin Met 410 GGA CTC CTT ATG Gly Leu Leu Met GGC AAA CCA Gly Lys Pro INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 1275 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1275 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..465 OTHER INFORMATION: /product= "bFGF" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 466...516 OTHER INFORMATION: /product= "Cathepsin D linker" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 517..1275 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: GCA GCA GGA TCA Ala Ala Gly Ser ATA ACA ACA TTA Ile Thr Thr Leu GCC TTG CCC GAG GAT GGC Ala Leu Pro Glu Asp Gly GGC AGC GGC Gly Ser Gly TTC CCG CCC GGC Phe Pro Pro Gly TTC AAG GAC CCC Phe Lys Asp Pro AAG CGG CTG Lys Arg Leu WO 96/36362 PCT/US96/07164 TAC TGC AAA Tyr Cys Lys AAC GGG GGC TTC Asn Gly Gly Phe TTC CTG Phe Leu 40 CGC ATC CAC CCC GAC GGC CGA Arg Ile His Pro Asp Gly Arg GTT GAC Val Asp CAA GCA Gin Ala GGG GTC CGG GAG AAG Gly Val Arg Glu Lys 55 GAA GAG AGA GGA GTT Glu Glu Arg Gly Val 70 AGC GAC CCT CAC Ser Asp Pro His GTG TCT ATC AAA Val Ser Ile Lys ATC AAG CTT CAA CTT Ile Lys Leu Gin Leu GGA GTG TGT GCT Gly Val Cys Ala CGT TAC CTG Arg Tyr Leu GTT ACG GAT Val Thr Asp AAT ACT TAC Asn Thr Tyr 115 GCT ATG Ala Met AAG GAA GAT GGA AGA TTA CTG GCT Lys Glu Asp Gly Arg Leu Leu Ala 90 TCT AAA TGT Ser Lys Cys TGT TTC TTT TTT GAA Cys Phe Phe Phe Glu 105 CGA TTG GAA TCT Arg Leu Glu Ser AAT AAC Asn Asn 110 CGG TCA AGG AAA Arg Ser Arg Lys TAC ACC Tyr Thr 120 AGT TGG TAT GTG GCA TTG AAA Ser Trp Tyr Val Ala Leu Lys 125 CGA ACT Arg Thr 130 GCT ATA Ala Ile 145 GGG CAG TAT AAA CTT Gly Gin Tyr Lys Leu 135 GGA TCC AAA ACA GGA CCT GGG CAG AAA Gly Ser Lys Thr Gly Pro Gly Gin Lys CTT TTT CTT CCA ATG TCT GCT AAG Leu Phe Leu Pro Met Ser Ala Lys 150 GCC ATG GGC CGA TCG Ala Met Gly Arg Ser 160 GGC TTC CTG Gly Phe Leu ACA TTA GAT Thr Leu Asp GAT AAA ATC Asp Lys lle 195 GGC TTC Gly Phe 165 CTA GTA Leu Val 180 GGC TTC CTG GGC GLy Phe Leu GLy AAT CCG ACC GCG Asn Pro Thr Ala 185 GCC ATG GTC ACA Ala Met Val Thr CAA TAC TCA TCT Gin Tyr Ser Ser 190 TCA ATC Ser Ile 175 TTT GTG Phe Val CGA AAC AAC GTA Arg Asn Asn Val AAG GAT CCA Lys Asp Pro 200 AAC CTG AAA TAC GGT GGT Asn Leu Lys Tyr Gly Gly 205 ACC GAC Thr Asp 210 ATA GCC GTG ATA GGC Ile Ala Val Ile Gly 215 CCA CCT TCT AAA GAA Pro Pro Ser Lys Glu 220 GGA ACG GTC TCA CTT Gly Thr Val Ser Leu 235 AAA TTC CTT AGA Lys Phe Leu Arg GGC CTA AAA CGC Gly Leu Lys Arg 240 AAT TTC CAA AGT Asn Phe Gin Ser TCC CGA Ser Arg 230 GAT AAC TTG TAT Asp Asn Leu Tyr GTC GCG TAT CTT GCA Val Ala Tyr Leu Ala 250 ATG GAT AAC ACG Met Asp Asn Thr AAT GTT Asn Val 255 WO 96/36362 PCT/US96/07164 AAT CGG GCA Asn Arg Ala GCC CTT TTC Ala Leu Phe 275 TAT TAC Tyr Tyr 260 TTC AAA TCA GAA Phe Lys Ser Glu 265 ATT ACT TCC GCC Ile Thr Ser Ala AAT CAG AAA GCT Asn Gin Lys Ala 285 GAG TTA Glu Leu 270 CCA GAG GCC ACA Pro Glu Ala Thr ACT GCA Thr Ala 280 TTA GAA TAC Leu Glu Tyr ACA GAA Thr Glu 290 GAT AAA Asp Lys 305 GAT TAT CAG TCG ATC GAA AAG AAT GCC Asp Tyr Gin Ser Ile Glu Lys Asn Ala 295 CAG ATA ACA CAG GGA Gin Ile Thr Gin Gly 300 AGT AGA AAA GAA CTC Ser Arg Lys Glu Leu 310 GGG TTG GGG Gly Leu Gly ATC GAC TTA CTT TTG Ile Asp Leu Leu Leu 315 GTG GTT AAA AAC GAA Val Val Lys Asn Glu 335 TTC ATG GAA Phe Met Glu AGG TTT CTG Arg Phe Leu AGG TAC ATT Arg Tyr lle 355 GCA GTG Ala Val 325 CTT ATC Leu lle 340 AAC AAG AAG GCA Asn Lys Lys Ala GCT ATT CAA ATG ACA GCT GAG GTA Ala Ile Gin Met Thr Ala Glu Val 345 GCA CGA Ala Arg 350 CAA AAC TTG GTA Gin Asn Leu Val AAG AAC TTC CCC Lys Asn Phe Pro AAC AAG TTC GAC Asn Lys Phe Asp 365 CGT AAG ATT TCT Arg Lys Ile Ser TCG GAT Ser Asp 370 ACG GCA Thr Ala 385 AAC AAG GTG Asn Lys Val ATA TAC GGG Ile Tyr Gly
ATT
Ile TTT GAA GTC AGC Phe Glu Val Ser 1056 1104 1152 1200 1248 1275 GAT GCC AAA AAC GGC Asp Ala Lys Asn Gly 390 AAA GTG AGG CAG GTG Lys Val Arg Gin Val 410 GTG TTT AAT AAA GAT TAT Val Phe Asn Lys Asp Tyr 395 400 GAT TTC GGG TTT Asp Phe Gly Phe AAG GAC TTG CAA Lys Asp Leu Gin CTC CTT ATG Leu Leu Met TTG GGC AAA CCA Leu Gly Lys Pro INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 1251 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1251 WO 96/36362 PCT/US96/07164 172 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..465 OTHER INFORMATION: /product= "bFGF" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 466..492 OTHER INFORMATION: /product= Gly 4 Ser linker" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 493..1251 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: GCA GGA TCA Ala Gly Ser ATA ACA ACA TTA Ile Thr Thr Leu CCC GCC Pro Ala 10 TTG CCC GAG GAT Leu Pro Glu Asp GGC AGC GGC GCC Gly Ser Gly Ala TAC TGC AAA AAC Tyr Cys Lys Asn TTC CCG CCC GGC Phe Pro Pro Gly TTC AAG GAC CCC Phe Lys Asp Pro AAG CGG Lys Arg GGG GGC TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg GGG GTC CGG GAG AAG Gly Val Arg Glu Lys GCA GAA GAG AGA Ala Glu Glu Arg GGA GTT Gly Val AAG GAA Lys Glu AGC GAC CCT CAC Ser Asp Pro His GTG TCT ATC AAA Val Ser Ile Lys 75 GAT GGA AGA TTA Asp Gly Arg Leu ATC AAG CTT CAA CTT Ile Lys Leu Gin Leu GGA GTG TGT GCT AAC Gly Val Cys Ala Asn CTG GCT TCT AAA TGT Leu Ala Ser Lys Cys CGT TAC CTG GCT Arg Tyr Leu Ala GTT ACG GAT GAG TGT TTC TTT TTT GAA Val Thr Asp Glu Cys Phe Phe Phe Glu 100 105 CGA TTG GAA TCT Arg Leu Glu Ser AAT AAC Asn Asn 110 AAT ACT TAC CGG Asn Thr Tyr Arg 115 TCA AGG AAA TAC ACC AGT TGG TAT Ser Arg Lys Tyr Thr Ser Trp Tyr CGA ACT Arg Thr 130 GCT ATA Ala Ile 145 GGG CAG TAT AAA CTT Gly Gin Tyr Lys Leu 135 CTT TTT CTT CCA ATG Leu Phe Leu Pro Met 150 GGA TCC AAA ACA GGA Gly Ser Lys Thr Gly 140 TCT GCT AAG AGC GCC Ser Ala Lys Ser Ala 155 GTG GCA TTG AAA Val Ala Leu Lys 125 CCT GGG CAG AAA Pro Gly Gin Lys ATG GGC GGC GGC Met Gly Gly Gly 160 WO 96/36362 PCT/US96/07164 GGC TCT GCC ATG Gly Ser Ala Met GCG GGT CAA TAC Ala Gly Gin Tyr 180 GAT CCA AAC CTG Asp Pro Asn Leu 195 GTC ACA TCA ATC ACA Val Thr Ser Ile Thr 165 TCA TCT TTT GTG GAT Ser Ser Phe Val Asp 185 AAA TAC GGT GGT ACC Lys Tyr Gly Gly Thr 200 TTA GAT Leu Asp 170 AAA ATC Lys Ile CTA GTA AAT CCG Leu Val Asn Pro 175 CGA AAC AAC GTA Arg Asn Asn Val 190 GAC ATA GCC GTG ATA GGC CCA Asp Ile Ala Val Ile Gly Pro 205 AAA GAA AAA TTC Lys Glu Lys Phe CTT AGA Leu Arg 215 ATT AAT TTC CAA AGT TCC CGA GGA Ile Asn Phe Gin Ser Ser Arg Gly 220 GTC TCA CTT GGC CTA Val Ser Leu Gly Leu 230 AAA CGC GAT AAC Lys Arg Asp Asn AAT GTT AAT CGG Asn Val Asn Arg 250 TAT GTG GTC GCG Tyr Val Val Ala CTT GCA ATG GAT Leu Ala Met Asp AAC ACG Asn Thr 245 GCA TAT TAC TTC Ala Tyr Tyr Phe AAA TCA Lys Ser 255 GAA ATT ACT Glu Ile Thr GCA AAT CAG Ala Asn Gin 275 AAG AAT GCC Lys Asn Ala 290 GCC GAG TTA ACC Ala Glu Leu Thr CTT TTC CCA GAG GCC ACA ACT Leu Phe Pro Glu Ala Thr Thr 270 AAA GCT TTA GAA TAC Lys Ala Leu Glu Tyr 280 CAG ATA ACA CAG GGA G1n Ile Thr Gin Gly 295 ACA GAA GAT TAT Thr Glu Asp Tyr GAT AAA AGT AGA Asp Lys Ser Arg 300 CAG TCG ATC GAA Gin Ser Ile Glu 285 AAA GAA CTC GGG Lys Glu Leu Gly GTG AAC AAG AAG Val Asn Lys Lys 320 GGG ATC GAC TTA Gly Ile Asp Leu TTG ACG TTC ATG Leu Thr Phe Met GAA GCA Glu Ala 315 GCA CGT GTG GTT AAA AAC GAA GCT AGG TTT CTG CTT ATC GCT ATT CAA Ala Arg Val Val Lys Asn Glu Ala Arg Phe Leu Leu Ile Ala Ile Gin ATG ACA GCT Met Thr Ala AAG AAC TTC Lys Asn Phe 355 GAA GTC AGC Glu Val Ser 370 GTA GCA CGA TTT Val Ala Arg Phe AGG TAC Arg Tyr 345 ATT CAA AAC TTG GTA ACT Ile Gin Asn Leu Val Thr 350 CCC AAC AAG TTC GAC Pro Asn Lys Phe Asp 360 TGG CGT AAG ATT TCT Trp Arg Lys Ile Ser 375 TCG GAT AAC AAG Ser Asp Asn Lys GTG ATT CAA TTT Val Ile Gln Phe 365 1056 1104 1152 ACG GCA ATA TAC GGG GAT GCC AAA Thr Ala Ile Tyr Gly Asp Ala Lys WO 96/36362 PCT/US96/07164 AAC GGC Asn Gly 385 GTG TTT AAT AAA GAT Val Phe Asn Lys Asp 390 TAT GAT TTC GGG Tyr Asp Phe Gly 395 TTT GGA AAA GTG Phe Gly Lys Val 1200 1248 1251 CAG GTG AAG GAC Gin Val Lys Asp CAA ATG GGA CTC Gin Met Gly Leu CTT ATG TAT TTG GGC AAA Leu Met Tyr Leu Gly Lys 410 415 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1266 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1266 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..465 OTHER INFORMATION: /product= "bFGF" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 466..507 OTHER INFORMATION: /product= (Gly 4 Ser) 2 linker" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 508..1266 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu GAT GGC Asp Gly GGC AGC GGC GCC TTC CCG CCC GGC CAC TTC AAG GAC Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp TAC TGC AAA AAC Tyr Cys Lys Asn GGG GGC TTC TTC CTG CGC ATC CAC Gly Gly Phe Phe Leu Arg Ile His CCC AAG CGG CTG Pro Lys Arg Leu CCC GAC GGC CGA Pro Asp Gly Arg AAG CTT CAA CTT Lys Leu Gin Leu GGG GTC CGG GAG Gly Val Arg Glu AGC GAC CCT CAC Ser Asp Pro His WO 96/36362 PCT/US96/07164 CAA GCA GAA GAG AGA GGA GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC Ala Glu TAC CTG Glu A GCT A Arg Tyr Leu Ala GTT ACG GAT GAG Val Thr Asp Glu 100 AAT ACT TAC CGG Asn Thr Tyr Arg 115 CGA ACT GGG CAG Arg Thr Gly Gin 130 GCT ATA CTT TTT Ala Ile Leu Phe 145 GGC TCT GGC GGC Gly Ser Gly Gly CTA GTA AAT CCG Leu Val Asn Pro 180 CGA AAC AAC GTA Arg Asn Asn Val 195 GCC GTG ATA GGC Ala Val Ile Gly 210 1 1 1
I
\rg Gly Val Val Ser Ile ATG AAG GAA GAT GGA AGA let Lys Glu Asp Gly Arg 90 [GT TTC TTT TTT GAA CGA Cys Phe Phe Phe Glu Arg 105 rCA AGG AAA TAC ACC AGT Ser Arg Lys Tyr Thr Ser 120 FAT AAA CTT GGA TCC AAA Tyr Lys Leu Gly Ser Lys 135 :TT CCA ATG TCT GCT AAG Leu Pro Met Ser Ala Lys 150 GGC GGC TCT GCC ATG GTC Gly Gly Ser Ala Met Val 165 170 \CC GCG GGT CAA TAC TCA Thr Ala Gly Gin Tyr Ser 185 AAG GAT CCA AAC CTG AAA Lys Asp Pro Asn Leu Lys 200 CCA CCT TCT AAA GAA AAA Pro Pro Ser Lys Glu Lys 215 GGA ACG GTC TCA CTT GGC Gly Thr Val Ser Leu Gly 230 TAT CTT GCA ATG GAT AAC Tyr Leu Ala Met Asp Asn 245 250 TCA GAA ATT ACT TCC GCC Ser Glu Ile Thr Ser Ala 265 ACT GCA AAT CAG AAA GCT Thr Ala Asn Gin Lys Ala 280 Lys Gly Val 75 TTA CTG GCT Leu Leu Ala TTG GAA TCT Leu Glu Ser TGG TAT GTG Trp Tyr Val 125 ACA GGA CCT Thr Gly Pro 140 AGC GCC ATG Ser Ala Met 155 ACA TCA ATC Thr Ser Ile TCT TTT GTG Ser Phe Val TAC GGT GGT Tyr Gly Gly 205 TTC CTT AGA Phe Leu Arg 220 CTA AAA CGC Cys Ala Asn TCT AAA TGT Ser Lys Cys AAT AAC TAC Asn Asn Tyr 110 GCA TTG AAA Ala Leu Lys GGG CAG AAA Gly Gin Lys GGC GGC GGC Gly Gly Gly 160 ACA TTA GAT Thr Leu Asp 175 GAT AAA ATC Asp Lys Ile 190 ACC GAC ATA Thr Asp lle ATT AAT TTC Ile Asn Phe GAT AAC TTG CAA AGT Gin Ser 225 TAT GTG Tyr Val TAT TAC Tyr Tyr CCA GAG Pro Glu
CGA
Arg
GCG
Ala
AAA
Lys 260
ACA
Thr Leu Lys Arg Asp Asn Leu ACG AAT Thr Asn GAG TTA Glu Leu TTA GAA Leu Glu GTT AAT Val Asn ACC GCC Thr Ala 270 TAC ACA Tyr Thr 285 CGG GCA Arg Ala 255 CTT TTC Leu Phe GAA GAT Glu Asp WO 96/36362 PCT/US96/07164 TAT CAG Tyr Gin 290 AGA AAA Arg Lys 305 GCA GTG Ala Val TCG ATC GAA AAG Ser Ile Glu Lys GCC CAG ATA ACA Ala Gin Ile Thr CAG GGA GAT AAA AGT Gin Gly Asp Lys Ser 300 TTG ACG TTC ATG GAA Leu Thr Phe Met Glu 320 GAA CTC GGG TTG GGG ATC GAC TTA Glu Leu Gly Leu Gly Ile Asp Leu 310 AAC AAG AAG GCA Asn Lys Lys Ala 325 CGT GTG GTT AAA Arg Val Val Lys 330 AAC GAA GCT AGG Asn Glu Ala Arg TTT CTG Phe Leu 335 CTT ATC GCT ATT Leu Ile Ala Ile 340 CAA ATG ACA GCT GAG GTA GCA CGA Gin Met Thr Ala Glu Val Ala Arg 345 CAA AAC TTG Gin Asn Leu 355 AAG GTG ATT Lys Val Ile 370 GTA ACT AAG AAC TTC Val Thr Lys Asn Phe 360 CAA TTT GAA GTC AGC Gin Phe Glu Val Ser 375 CCC AAC AAG TTC Pro Asn Lys Phe TGG CGT AAG ATT Trp Arg Lys Ile 380 TTT AGG TAC ATT Phe Arg Tyr Ile 350 GAC TCG GAT AAC Asp Ser Asp Asn 365 TCT ACG GCA ATA Ser Thr Ala Ile TAT GAT TTC GGG Tyr Asp Phe Gly 400 1008 1056 1104 GGG GAT GCC AAA AAC Gly Asp Ala Lys Asn 390 GGC GTG TTT AAT Gly Val Phe Asn GTG AAG GAC TTG Val Lys Asp Leu 410 AAA GAT Lys Asp 395 1152 1200 1248 TTT GGA AAA GTG Phe Gly Lys Val AGG CAG Arg Gin 405 CAA ATG GGA CTC CTT ATG Gin Met Gly Leu Leu Met 415 TAT TTG GGC Tyr Leu Gly AAA CCA AAG Lys Pro Lys 420 1266 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 1320 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1320 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..465 OTHER INFORMATION: /product= "bFGF" WO 96/36362 PCT/US96/07164
I
(ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 466..561 OTHER INFORMATION: /product= "Trypsin linker" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 562..1320 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro GAG GAT Glu Asp GGC AGC GGC GCC Gly Ser Gly Ala TAC TGC AAA AAC Tyr Cys Lys Asn TTC CCG CCC GGC CAC TTC AAG GAC CCC Phe Pro Pro Gly His Phe Lys Asp Pro 25 AAG CGG CTG Lys Arg Leu GGG GGC TTC TTC CTG CGC ATC CAC Gly Gly Phe Phe Leu Arg Ile His 40 GAC GGG Asp Gly GTC CGG GAG AAG AGC GAC CCT Val Arg Glu Lys Ser Asp Pro 55 CAC ATC His Ile AAA GGA Lys Gly 75 CCC GAC GGC CGA Pro Asp Gly Arg AAG CTT CAA CTT Lys Leu Gin Leu GTG TGT GCT AAC Val Cys Ala Asn CAA GCA GAA Gin Ala Glu GAG AGA GGA Glu Arg Gly 70 GTT GTG TCT ATC Val Val Ser Ile CGT TAC CTG GCT Arg Tyr Leu Ala AAG GAA GAT GGA Lys Glu Asp Gly AGA TTA CTG GCT Arg Leu Leu Ala TCT AAA Ser Lys AAT AAC Asn Asn 110 GTT ACG GAT Val Thr Asp AAT ACT TAC Asn Thr Tyr 115 CGA ACT GGG Arg Thr Gly 130 TGT TTC TTT TTT GAA CGA TTG GAA TCT Cys Phe Phe Phe Glu Arg Leu Glu Ser 105 CGG TCA AGG AAA TAC Arg Ser Arg Lys Tyr 120 CAG TAT AAA CTT GGA Gin Tyr Lys Leu Gly 135 ACC AGT TGG TAT Thr Ser Trp Tyr TCC AAA ACA GGA Ser Lys Thr Gly 140 GTG GCA TTG AAA Val Ala Leu Lys 125 CCT GGG CAG AAA Pro Gly Gin Lys ATA CTT TTT CTT CCA ATG TCT Ile Leu Phe Leu Pro Met Ser 150 GCT AAG AGC GCC ATG GGC Ala Lys Ser Ala Met Gly 155 CGA TCG Arg Ser 160 AGC AGC Ser Ser 175 GGC GGT GGG TGC Gly Gly Gly Cys GCT GGT Ala Gly 165 AAT AGA GTC AGA Asn Arg Val Arg 170 AGA TCA GTC GGA Arg Ser Val Gly WO 96/36362 PCT/US96/07164 CTG TCT TGC GGT Leu Ser Cys Gly 180 GGT CTC GAC CTG Gly Leu Asp Leu CAG GCC Gin Ala 185 ATG GTC ACA TCA ATC ACA Met Val Thr Ser Ile Thr 190 TTA GAT CTA GTA AAT CCG ACC Leu Asp Leu Val Asn Pro Thr 195 AAA ATC CGA AAC AAC GTA AAG Lys Ile Arg Asn Asn Val Lys 210 215 GAC ATA GCC GTG ATA GGC CCA Asp Ile Ala Val Ile Gly Pro 225 230 GCG GGT CAA TAC TCA Ala Gly Gin Tyr Ser 200 GAT CCA AAC CTG AAA Asp Pro Asn Leu Lys 220 CCT TCT AAA GAA AAA Pro Ser Lys Glu Lys 235 TCT TTT GTG GAT Ser Phe Val Asp 205 TAC GGT GGT ACC Tyr Gly Gly Thr TTC CTT AGA Phe Leu Arg AAT TTC CAA AGT Asn Phe Gin Ser AAC TTG TAT GTG Asn Leu Tyr Val 260 CGA GGA ACG GTC Arg Gly Thr Val TCA CTT GGC CTA Ser Leu Gly Leu 250 ATG GAT AAC ACG Met Asp Asn Thr AAA CGC Lys Arg 255 GTC GCG TAT CTT Val Ala Tyr Leu AAT GTT AAT Asn Val Asn 270 CGG GCA TAT TAC TTC AAA TCA Arg Ala Tyr Tyr Phe Lys Ser 275 CTT TTC CCA GAG GCC ACA ACT Leu Phe Pro Glu Ala Thr Thr 290 295 GAA GAT TAT CAG TCG ATC GAA Glu Asp Tyr Gin Ser Ile Glu 305 310 GAA ATT ACT TCC GCC Glu Ile Thr Ser Ala 280 GCA AAT CAG AAA GCT Ala Asn Gin Lys Ala 300 AAG AAT GCC CAG ATA Lys Asn Ala Gin Ile 315 GAG TTA ACC GCC Glu Leu Thr Ala 285 TTA GAA TAC ACA Leu Glu Tyr Thr ACA CAG GGA GAT Thr Gin Gly Asp 320 864 912 960 1008 1056 1104 AAA AGT AGA Lys Ser Arg ATG GAA GCA Met Glu Ala AAA GAA Lys Glu 325 CTC GGG TTG GGG Leu Gly Leu Gly GAC TTA CTT TTG Asp Leu Leu Leu AAC AAG AAG GCA CGT Asn Lys Lys Ala Arg 345 GCT ATT CAA ATG ACA Ala Ile Gin Met Thr 360 GTG GTT AAA AAC Val Val Lys Asn GAA GCT Glu Ala 350 TTT CTG CTT ATC Phe Leu Leu lle 355 GCT GAG GTA GCA CGA TTT AGG Ala Glu Val Ala Arg Phe Arg 365 CAA AAC TTG GTA Gin Asn Leu Val AAG AAC TTC CCC AAC AAG TTC GAC TCG Lys Asn Phe Pro Asn Lys Phe Asp Ser 380 AAC AAG GTG ATT CAA Asn Lys Val Ile Gin 390 TTT GAA GTC AGC TGG Phe Glu Val Ser Trp 395 CGT AAG ATT TCT Arg Lys Ile Ser WO 96/36362 PCT/US96/07164 GCA ATA TAC GGG Ala Ile Tyr Gly TTC GGG TTT GGA Phe Gly Phe Gly 420 CTT ATG TAT TTG Leu Met Tyr Leu 435 GCC AAA AAC GGC Ala Lys Asn Gly TTT AAT AAA GAT Phe Asn Lys Asp TAT GAT Tyr Asp 415 AAA GTG AGG CAG Lys Val Arg Gin GGC AAA CCA AAG Gly Lys Pro Lys AAG GAC TTG CAA ATG GGA CTC Lys Asp Leu Gin Met Gly Leu 430 1248 1296 1320 INFORMATION FOR SEQ ID NO:77: SEQUENCE CHARACTERISTICS: LENGTH: 1299 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1299 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..465 OTHER INFORMATION: /product= "bFGF" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 466..540 OTHER INFORMATION: /product= "(Ser,Gly),linker" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 541..1299 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: ATG GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG CCC GAG Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu GGC AGC GGC Gly Ser Gly TAC TGC AAA Tyr Cys Lys GCC TTC Ala Phe CCG CCC GGC CAC TTC AAG GAC Pro Pro Gly His Phe Lys Asp 25 CCC AAG CGG CTG Pro Lys Arg Leu AAC GGG GGC TTC Asn Gly Gly Phe CTG CGC ATC CAC CCC GAC GGC CGA Leu Arg Ile His Pro Asp Gly Arg WO 96/36362 PCT/US96/07164 GTT GAC GGG GTC Val Asp Gly Val CAA GCA GAA GAG Gin Ala Glu Glu CGT TAC CTG GCT Arg Tyr Leu Ala GTT ACG GAT GAG Val Thr Asp Glu 100 AAT ACT TAC CGG Asn Thr Tyr Arg 115 CGA ACT GGG CAG Arg Thr Gly Gin 130 GCT ATA CTT TTT Ala Ile Leu Phe 145 TCG TCG GGC TCG Ser Ser Gly Ser TCG GGC GCC ATG Ser Gly Ala Met 180 GCG GGT CAA TAC Ala Gly Gin Tyr 195 GAT CCA AAC CTG Asp Pro Asn Leu 210 CCT TCT AAA GAA Pro Ser Lys Glu 225 ACG GTC TCA CTT Thr Val Ser Leu CTT GCA ATG GAT Leu Ala Met Asp 260 CGG GAG Arg Glu AGA GGA Arg Gly 70 ATG AAG Met Lys TGT TTC AAG AGC Lys Ser 55 GTT GTG Val Val GAA GAT Glu Asp TTT TTT GAC CCT Asp Pro TCT ATC Ser Ile GGA AGA Gly Arg 90 GAA CGA
CAC
His
AAA
Lys 75
TTA
Leu
TTG
Leu Cys Phe Phe Phe Glu Arg 105
AGG
Arg
AAA
AAA TAC ACC Lys Tyr Thr 120 CTT GGA TCC Tyr Lys Leu Gly Ser 135 CTT CCA ATG TCT GCT Leu Pro Met Ser Ala 150 TCG TCG TCG GGC TCG Ser Ser Ser Gly Ser 165 GTC ACA TCA ATC ACA Val Thr Ser Ile Thr 185 TCA TCT TTT GTG GAT Ser Ser Phe Val Asp 200 AAA TAC GGT GGT ACC Lys Tyr Gly Gly Thr 215 AAA TTC CTT AGA ATT Lys Phe Leu Arg Ile 230 GGC CTA AAA CGC GAT Gly Leu Lys Arg Asp 245 AAC ACG AAT GTT AAT Asn Thr Asn Val Asn 265 AGT TGG Ser Trp AAA ACA Lys Thr AAG AGC Lys Ser 155 TCG TCG Ser Ser 170 TTA GAT Leu Asp AAA ATC Lys Ile GAC ATA Asp Ile AAT TTC Asn Phe 235 AAC TTG Asn Leu 250 CGG GCA Arg Ala
ATC
Ile
GGA
Gly
CTG
Leu
GAA
Glu
TAT
Tyr
GGA
Gly 140
GCC
Ala
TCG
Ser
CTA
Leu
CGA
Arg
GCC
Ala 220
CAA
Gin
TAT
Tyr
TAT
Tyr 180 AAG CTT CAA CTT Lys Leu Gin Leu GTG TGT GCT AAC Val Cys Ala Asn GCT TCT AAA TGT Ala Ser Lys Cys TCT AAT AAC TAC Ser Asn Asn Tyr 110 GTG GCA TTG AAA Val Ala Leu Lys 125 CCT GGG CAG AAA Pro Gly Gin Lys ATG GCC TCG TCG Met Ala Ser Ser 160 GGC TCG TCG TCG Gly Ser Ser Ser 175 GTA AAT CCG ACC Val Asn Pro Thr 190 AAC AAC GTA AAG Asn Asn Val Lys 205 GTG ATA GGC CCA Val Ile Gly Pro AGT TCC CGA GGA Ser Ser Arg Gly 240 GTG GTC GCG TAT Val Val Ala Tyr 255 TAC TTC AAA TCA Tyr Phe Lys Ser 270 WO 96/36362 PCT/US96/07164 GAA ATT ACT Glu Ile Thr 275 TCC GCC GAG TTA Ser Ala GIu Leu ACC GCC Thr Ala 280 CTT TTC CCA GAG GCC ACA ACT Leu Phe Pro Glu Ala Thr Thr 285 GCA AAT Ala Asn 290 CAG AAA GCT TTA Gin Lys Ala Leu GAA TAC Glu Tyr 295 ACA GAA GAT TAT Thr Glu Asp Tyr 300 CAG TCG ATC GAA Gin Ser Ile Glu AAT GCC CAG ATA Asn Ala Gin Ile CAG GGA GAT AAA AGT AGA AAA GAA Gin Gly Asp Lys Ser Arg Lys Glu 315 CTC GGG Leu Gly 320 TTG GGG ATC GAC Leu Gly Ile Asp CTT TTG ACG TTC Leu Leu Thr Phe GAA GCA GTG AAC Glu Ala Val Asn GCA CGT GTG GTT AAA AAC GAA GCT Ala Arg Val Val Lys Asn Glu Ala 340 TTT CTG CTT ATC Phe Leu Leu Ile GCT ATT CAA Ala Ile Gin 350 ATG ACA GCT Met Thr Ala 355 GAG GTA GCA Glu Val Ala AAG AAC Lys Asn 370 TTC CCC AAC AAG Phe Pro Asn Lys CGA TTT AGG TAC Arg Phe Arg Tyr 360 TTC GAC TCG GAT Phe Asp Ser Asp 375 ATT TCT ACG GCA Ile Ser Thr Ala ATT CAA AAC TTG GTA ACT Ile Gin Asn Leu Val Thr 365 AAC AAG GTG ATT CAA TTT Asn Lys Val Ile Gin Phe 380 ATA TAC GGG GAT GCC AAA Ile Tyr Gly Asp Ala Lys 395 400 GGG TTT GGA AAA GTG AGG Gly Phe Gly Lys Val Arg GTC AGC TGG CGT Val Ser Trp Arg 1104 1152 1200 1248 1296 AAC GGC GTG Asn Gly Val TTT AAT Phe Asn 405 AAA GAT TAT GAT Lys Asp Tyr Asp CAA ATG GGA CTC Gin Met Gly Leu 425 CAG GTG AAG GAC TTG Gin Val Lys Asp Leu 420
AAG
Lys CTT ATG TAT TTG Leu Met Tyr Leu GGC AAA Gly Lys 430 INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 1269 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1269
L
WO 96/36362 PCT/US96/07164 182 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..465 OTHER INFORMATION: /product= "bFGF" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 466..510 OTHER INFORMATION: /product= "(Ser 4 Gly), linker" (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 511..1269 OTHER INFORMATION: /product= "Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: ATG GCA GCA GGA TCA Met Ala Ala Gly Ser 1 5 ATA ACA ACA TTA CCC GCC TTG CCC GAG GAT GGC Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly GGC AGC GGC GCC Gly Ser Gly Ala TTC CCG CCC GGC CAC Phe Pro Pro Gly His 25 TTC AAG GAC CCC Phe Lys Asp Pro AAG CGG CTG Lys Arg Leu TAC TGC AAA Tyr Cys Lys AAC GGG GGC TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg GGG GTC CGG GAG AAG Gly Val Arg Glu Lys AGC GAC CCT CAC Ser Asp Pro His ATC AAG CTT CAA CTT Ile Lys Leu Gin Leu GCA GAA GAG AGA GGA GTT GTG TCT ATC Ala Glu Glu Arg Gly Val Val Ser Ile AAA GGA GTG TGT Lys Gly Val Cys 75 GCT AAC Ala Asn CGT TAC CTG Arg Tyr Leu GCT ATG Ala Met AAG GAA GAT GGA Lys Glu Asp Gly TTA CTG GCT TCT AAA Leu Leu Ala Ser Lys GTT ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu TCT AAT AAC TAC Ser Asn Asn Tyr 110 GTG GCA TTG AAA Val Ala Leu Lys 125 AAT ACT TAC Asn Thr Tyr 115 CGG TCA AGG AAA TAC Arg Ser Arg Lys Tyr 120 ACC AGT TGG TAT Thr Ser Trp Tyr GGG CAG TAT AAA CTT Gly Gin Tyr Lys Leu 135 GGA TCC AAA ACA GGA CCT GGG CAG AAA Gly Ser Lys Thr Gly Pro Gly Gin Lys 140 TCT GCT AAG AGC GCC ATG GCC TCG TCG Ser Ala Lys Ser Ala Met Ala Ser Ser 155 160 ATA CTT TTT CTT Ile Leu Phe Leu CCA ATG Pro Met 150
L
WO 96/36362 PCTIUS96/07164 TCG TCG GGC TCG TCG Ser Ser Gly Ser Ser 165 GAT CTA GTA AAT CCG Asp Leu Val Asn Pro 180 TCG TCG GGC GCC ATG Ser Ser Gly Ala Met 170 ACC GCG GGT CAA TAC Thr Ala Gly Gin Tyr 185 GTC ACA TCA ATC Val Thr Ser Ile TCA TCT TTT Ser Ser Phe GTG GAT Val Asp 190 ATC CGA AAC Ile Arg Asn 195 AAC GTA AAG GAT Asn Val Lys Asp AAC CTG AAA TAC Asn Leu Lys Tyr GGT GGT ACC GAC Gly Gly Thr Asp 205 CTT AGA ATT AAT Leu Arg Ile Asn ATA GCC Ile Ala 210 GTG ATA GGC CCA Val le Gly Pro TCT AAA GAA AAA Ser Lys Glu Lys CAA AGT TCC CGA GGA Gin Ser Ser Arg Gly 230 ACG GTC TCA CTT Thr Val Ser Leu CTA AAA CGC GAT AAC Leu Lys Arg Asp Asn 240 ACG AAT GTT AAT CGG Thr Asn Val Asn Arg 255 TTG TAT GTG GTC Leu Tyr Val Val TAT CTT GCA ATG Tyr Leu Ala Met GAT AAC Asp Asn 250 GCA TAT TAC Ala Tyr Tyr AAA TCA GAA ATT Lys Ser Glu Ile ACT TCC Thr Ser 265 GCC GAG TTA ACC GCC CTT Ala Glu Leu Thr Ala Leu 270 TTC CCA GAG GCC Phe Pro Glu Ala 275 ACT GCA AAT CAG AAA GCT TTA GAA TAC ACA GAA Thr Ala Asn 280 GAT TAT CAG Asp Tyr Gin 290 TCG ATC GAA AAG AAT Ser Ile Glu Lys Asn 295 Gin Lys Ala Leu GCC CAG ATA ACA Ala Gin Ile Thr 300 Glu Tyr Thr Glu 285 CAG GGA GAT AAA Gin Gly Asp Lys AGA AAA GAA CTC Arg Lys Glu Leu GGG TTG GGG ATC GAC Gly Leu Gly Ile Asp 310 AAG GCA CGT GTG GTT Lys Ala Arg Val Val 330 CTT TTG ACG TTC Leu Leu Thr Phe GAA GCA GTG Glu Ala Val AAC AAG Asn Lys 325 AAA AAC GAA GCT AGG Lys Asn Glu Ala Arg 335 CTG CTT ATC GCT ATT CAA ATG ACA Leu Leu Ile Ala Ile Gin Met Thr 340 GCT GAG GTA GCA CGA TTT AGG TAC Ala Glu Val Ala Arg Phe Arg Tyr 345 350 TTC CCC AAC AAG TTC GAC TCG GAT Phe Pro Asn Lys Phe Asp Ser Asp 960 1008 1056 1104 1152 ATT CAA AAC TTG GTA ACT Ile Gin Asn Leu Val Thr 355 AAG AAC Lys Asn 360 AAC AAG GTG ATT CAA TTT GAA GTC AGC TGG CGT AAG ATT TCT ACG GCA Asn Lys Val Ile Gin Phe Glu Val Ser Trp Arg Lys Ile Ser Thr Ala 370 375 380 WO 96/36362 PCT/US96/07164 TAC GGG GAT GCC Tyr Gly Asp Ala AAA AAC GGC Lys Asn Gly 390 GTG TTT AAT AAA GAT TAT GAT TTC Val Phe Asn Lys Asp Tyr Asp Phe 395 400 GGG TTT GGA AAA GTG AGG CAG GTG Gly Phe Gly Lys Val Arg Gin Val 405 AAG GAC Lys Asp 410 TTG CAA ATG GGA Leu Gin Met Gly 1248 1269 ATG TAT TTG GGC AAA CCA AAG Met Tyr Leu Gly Lys Pro Lys 420 INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 765 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..762 OTHER INFORMATION: /product= "Mammalian codon optimized saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: TG GTG ACC TCC ATC ACC CTG GAC CTG GTG AAC CCC ACC GCC GGC et Val Thr Ser Ile Thr Leu Asp Leu Val Asn Pro Thr Ala Gly 1 5 10 TAC TCC TCC TTC GTG GAC AAG ATC Tyr Ser Ser Phe Val Asp Lys Ile CGC AAC Arg Asn 25 AAC GTG AAG GAC CCC AAC Asn Val Lys Asp Pro Asn CTG AAG TAC GGC Leu Lys Tyr Gly GGC ACC GAC ATC GCC GTG ATC GGC Gly Thr Asp Ile Ala Val Ile Gly 40 CCC CCC TCC AAG Pro Pro Ser Lys GAG AAG Glu Lys CTG GGC Leu Gly TTC CTG CGC ATC Phe Leu Arg Ile CTG AAG CGC GAC Leu Lys Arg Asp 70 TTC CAG TCC TCC CGC GGC ACC GTG TCC Phe Gin Ser Ser Arg Gly Thr Val Ser AAC CTG TAC GTG Asn Leu Tyr Val GTG GCC Val Ala 75 TAC CTG GCC ATG Tyr Leu Ala Met GAC AAC ACC AAC GTG AAC CGC GCC Asp Asn Thr Asn Val Asn Arg Ala TAC TAC TTC AAG TCC GAG ATC ACC Tyr Tyr Phe Lys Ser Glu Ile Thr 90 WO 96/36362 PCTIUS96/07164 TCC GCC GAG Ser Ala Glu ACC GCC CTG TTC CCT Thr Ala Leu Phe Pro 105 GAG GCC ACC ACC Glu Ala Thr Thr GCC AAC Ala Asn 110 AAG GCC CTG GAG TAC ACC GAG GAC TAC CAG TCC ATC Lys Ala Leu Glu Tyr Thr Glu Asp Tyr Gln Ser Ile GAG AAG AAC GCC Glu Lys Asn Ala 125 GGG CTG GGC ATC Gly Leu Gly Ile CAG ATC ACC Gln Ile Thr 130 CAG GGC GAC AAG TCC Gln Gly Asp Lys Ser 135 CGC AAG GAG CTC Arg Lys Glu Leu 140
GTG
Val
GAG
Glu CTG CTG CTG ACC Leu Leu Leu Thr AAG AAC GAG GCC Lys Asn Glu Ala 165 GTG GCC CGC TTC Val Ala Arg Phe 180 ATG GAG GCC GTG AAC Met Glu Ala Val Asn 155 AAG AAG GCC CGC Lys Lys Ala Arg CGC TTC CTG CTG ATC Arg Phe Leu Leu Ile 170 CGC TAC ATC CAG AAC Arg Tyr Ile Gln Asn 185 GCC ATC CAG ATG Ala Ile Gln Met ACC GCC Thr Ala 175 CTG GTG ACC Leu Val Thr AAG AAC TTC Lys Asn Phe 190 CCC AAC AAG Pro Asn Lys 195 TTC GAC TCC GAC Phe Asp Ser Asp AAG GTG ATC CAG Lys Val Ile Gln TTC GAG GTC AGC Phe Glu Val Ser 205 TGG CGC AAG ATC TCC ACC GCC Trp Arg Lys Ile Ser Thr Ala 210 215 ATC TAC GGC GAC Ile Tyr Gly Asp GCC AAG AAC GGC GTG Ala Lys Asn Gly Val 220 TTC AAC AAG GAC Phe Asn Lys Asp 225 GAC CTG CAG ATG Asp Leu Gln Met TAC GAC TTC GGC TTC Tyr Asp Phe Gly Phe 230 GGC CTG CTG ATG TAC Gly Leu Leu Met Tyr 245 GGC AAG Gly Lys 235 GTG CGC CAG GTG Val Arg Gln Val CTG GGC AAG CCC AAG Leu Gly Lys Pro Lys 250 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1233 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 96/36362 PCT/US96/07164 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1230 OTHER INFORMATION: /product=
FGF-SAP"
coli codon optimized (xi) SEQUENCE DESCRIPTION: SEQ ID GCA GCG GGT TCT Ala Ala Gly Ser ACT ACC CTG CCG Thr Thr Leu Pro GCG CTG Ala Leu 265 CCG GAG GAC GGC Pro Glu Asp Gly 270 GGT TCT GGC GCT Gly Ser Gly Ala CCA CCG GGC CAC Pro Pro Gly His GGT TTT TTC CTG Gly Phe Phe Leu 295 AAG GAC CCG AAA CGC Lys Asp Pro Lys Arg 285 TAT TGC AAA Tyr Cys Lys AAC GGT Asn Gly 290 CGT ATC CAC CCG GAT GGC CGC Arg Ile His Pro Asp Gly Arg 300 GTC GAT GGC GTC Val Asp Gly Val 305 CGC GAA AAG TCT Arg Glu Lys Ser 310 GAT CCG CAC ATC Asp Pro His Ile AAA CTG CAA TTG Lys Leu Gin Leu 315 GTT TGC GCG AAT Val Cys Ala Asn CAA GCA Gin Ala 320 GAG GAA CGC GGT GTT Glu Glu Arg Gly Val 325 GTA AGC ATC AAG Val Ser Ile Lys TAC CTG GCG ATG Tyr Leu Ala Met GTA ACC GAT Val Thr Asp AAC ACC TAT Asn Thr Tyr GAA TGC Glu Cys 355 CGT AGC Arg Ser 370 AAA GAG GAT GGC CGC Lys Glu Asp Gly Arg 340 TTC TTC TTT GAA CGT Phe Phe Phe Glu Arg 360 CGT AAG TAC ACC TCG Arg Lys Tyr Thr Ser 375 CTG CTG GCC TCC AAG TGT Leu Leu Ala Ser Lys Cys 345 350 CTG GAG TCG AAC AAT TAT Leu Glu Ser Asn Asn Tyr 365 TGG TAC GTA GCA TTG AAA Trp Tyr Val Ala Leu Lys 380 ACG GGT CCA GGT CAG AAA Thr Gly Pro Gly Gin Lys 395
I
240 288 336 384 432 480 528 CGC ACC GGT CAG Arg Thr Gly Gin 385 TAC AAA CTG GGT TCG Tyr Lys Leu Gly Ser 390
AAG
Lys GCA ATT Ala Ile 400 ATC ACG Ile Thr 415 CTG TTC CTG CCA Leu Phe Leu Pro ATG TCG GCC AAA TCG GCC Met Ser Ala Lys Ser Ala 405 410 AAC CCG ACC GCT GGT CAG Asn Pro Thr Ala Gly Gin 425 ATG GTC ACT TCT Met Val Thr Ser TAC AGC TCG TTT Tyr Ser Ser Phe 430 CTG GAT CTG Leu Asp Leu GTC GAT AAG ATT CGT AAT AAT GTG AAA GAT CCG AAT TTA AAA TAC GGT Val Asp Lys Ile Arg Asn Asn Val Lys Asp Pro Asn Leu Lys Tyr Gly 435 440 445 c WO 96/36362 PCT/US96/07164 GGC ACG GAT Gly Thr Asp ATT GCA GTG Ile Ala Val 450 ATT GGC CCG CCG Ile Gly Pro Pro 455 TCT AAG GAA AAG TTC TTG Ser Lys Glu Lys Phe Leu 460 CGT ATT AAC TTT Arg Ile Asn Phe 465 CAA AGC TCT CGC Gin Ser Ser Arg 470 GGC ACT GTG TCT Gly Thr Val Ser CTG GGC TTA AAA Leu Gly Leu Lys 475 CGC GAT Arg Asp 480 AAT TTG TAC GTT Asn Leu Tyr Val GCG TAC CTG GCG Ala Tyr Leu Ala ATG GAT AAT ACC AAT Met Asp Asn Thr Asn 490 AAC CGT GCT TAC Asn Arg Ala Tyr TTC AAA AGC GAA Phe Lys Ser Glu ATT ACC Ile Thr 505 TCT GCT GAA CTG Ser Ala Glu Leu 510 ACT GCA TTA TTC CCG GAA GCG ACT Thr Ala Leu Phe Pro Glu Ala Thr TAT ACC GAA GAT TAT Tyr Thr Glu Asp Tyr 530 CAG TCG ATT Gin Ser Ile ACT GCC Thr Ala 520 GAA AAA Glu Lys 535 GGT CTG Gly Leu AAT CAG AAA GCC CTG GAA Asn Gin Lys Ala Leu Glu 525 AAC GCG CAA ATT ACC CAG Asn Ala Gin Ile Thr Gin 540 GGT ATT GAC CTG CTG CTG Gly Ile Asp Leu Leu Leu 555 :GT GTA GTG AAA AAC GAA \rg Val Val Lys Asn Glu 816 864 912 960 GGC GAC AAA Gly Asp Lys 545 TCG CGC AAA GAG Ser Arg Lys Glu ACG TTT Thr Phe 560 GCT CGC Ala Arg 575 ATG GAG GCG GTC Met Glu Ala Val AAA AAA GCT C Lys Lys Ala A TTT CTG CTG ATC GCT ATT CAA ATG ACT Phe Leu Leu Ile Ala Ile Gin Met Thr 580 585 GCT GAA GTT GCT Ala Glu Val Ala TTC CGT TAC ATT Phe Arg Tyr Ile GAC TCC GAT AAT Asp Ser Asp Asn 610 AAC TTG GTT ACT AAG Asn Leu Val Thr Lys 600 AAC TTT CCG AAC Asn Phe Pro Asn AAA TTC Lys Phe 605 1056 1104 AAG GTT ATT CAG Lys Val Ile Gin GAA GTG AGC TGG CGC AAG ATT Glu Val Ser Trp Arg Lys Ile 620 TCG ACG GCT Ser Thr Ala 625 ATT TAT GGC GAT Ile Tyr Gly Asp AAA AAC GGC GTA Lys Asn Gly Val TTT AAC AAA GAT Phe Asn Lys Asp 635 GAT TTG CAG ATG Asp Leu Gin Met GAC TTC GGT TTT GGC AAG GTT Asp Phe Gly Phe Gly Lys Val 640 645 CGT CAG GTG Arg Gin Val 1200 GGT CTG CTG ATG TAC TTG GGC AAG CCG AAA TAA Gly Leu Leu Met Tyr Leu Gly Lys Pro Lys 655 660 WO 96/36362 PCT/US96/0716 4 INFORMATION FOR SEQ ID NO:81: SEQUENCE CHARACTERISTICS: LENGTH: 465 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..462 OTHER INFORMATION: /product= Residue 116" "FGF 2 Ile Mutation at (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: ATG GCA GCA GGA Met Ala Ala Gly GGC AGC GGC GCC Gly Ser Gly Ala 430 TAC TGC AAA AAC Tyr Cys Lys Asn 445 GAC GGG GTC CGG Asp Gly Val Arg 460 TCA ATA Ser Ile 415 ACA ACA TTA CCC GCC TTG Thr Thr Leu Pro Ala Leu 420 TTC CCG CCC GGC Phe Pro Pro Gly TTC AAG GAC Phe Lys Asp CCC GAG GAT GGC Pro Glu Asp Gly 425 CCC AAG CGG CTG Pro Lys Arg Leu 440 GAC GGC CGA GTT Asp Gly Arg Val 455 CTT CAA CTT CAA Leu Gin Leu Gin GGG GGC TTC Gly Gly Phe CTG CGC CAC CCC Leu Arg His Pro CCT CAC ATC AAG Pro His Ile Lys 470 GAG AAG Glu Lys AGC GAC Ser Asp 465 GAA GAG AGA GGA GTT GTG Glu Glu Arg Gly Val Val 480 TCT ATC AAA GGA Ser Ile Lys Gly 485 GTG TGT GCT AAC CGT Val Cys Ala Asn Arg 490 TAC CTG GCT ATG AAG Tyr Leu Ala Met Lys 495 GAA GAT GGA AGA TTA Glu Asp Gly Arg Leu 500 CTG GCT TCT AAA Leu Ala Ser Lys ACG GAT GAG Thr Asp Glu ACT TAC ATA Thr Tyr Ile 525 TTC TTT TTT GAA Phe Phe Phe Glu TTG GAA TCT AAT AAC TAC AAT Leu Glu Ser Asn Asn Tyr Asn 520 TGG TAT GTG GCA TTG AAA CGA Trp Tyr Val Ala Leu Lys Arg 535 TCA AGG AAA TAC ACC AGT Ser Arg Lys Tyr Thr Ser 530 ACT GGG CAG TAT AAA CTT GGA TCC Thr Gly Gin Tyr Lys Leu Gly Ser AAA ACA GGA CCT GGG CAG AAA GCT Lys Thr Gly Pro Gly Gin Lys Ala 550 WO 96/36362 PCTIUS96/07164 ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC TAA Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 555 560 INFORMATION FOR SEQ ID NO:82: SEQUENCE CHARACTERISTICS: LENGTH: 465 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..462 OTHER INFORMATION: /product= "FGF 2 Glu Mutation at Residue 119" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: ATG GCA GCA Met Ala Ala 155 GGA TCA ATA ACA ACA TTA CCC GCC TTG Gly Ser lle Thr Thr Leu Pro Ala Leu 160 165 GAG GAT GGC Glu Asp Gly 170 GGC AGC GGC GCC Gly Ser Gly Ala TAC TGC AAA AAC Tyr Cys Lys Asn 190 CCG CCC GGC CAC TTC AAG GAC CCC AAG Pro Pro Gly His Phe Lys Asp Pro Lys 180 CGG CTG Arg Leu 185 GGG GGC TTC TTC CTG Gly Gly Phe Phe Leu 195 CGC CAC CCC GAC Arg His Pro Asp GGC CGA Gly Arg 200 GAC GGG GTC Asp Gly Val 205 GCA GAA GAG Ala Glu Glu 220 CGG GAG AAG AGC Arg Glu Lys Ser CCT CAC ATC AAG Pro His Ile Lys CTT CAA CTT CAA Leu Gin Leu Gin 215 TGT GCT AAC CGT Cys Ala Asn Arg AGA GGA GTT GTG TCT ATC AAA GGA GTG Arg Gly Val Val Ser Ile Lys Gly Val 225 230 CTG GCT ATG AAG GAA GAT GGA AGA TTA Leu Ala Met Lys Glu Asp Gly Arg Leu 240 GCT TCT AAA TGT GTT Ala Ser Lys Cys Val 250 ACG GAT GAG Thr Asp Glu TGT TTC Cys Phe 255 TTT TTT GAA CGA TTG Phe Phe Glu Arg Leu 260 GAA TCT AAT AAC Glu Ser Asn Asn TAC AAT Tyr Asn 265 ACT TAC CGG TCA AGG GAA TAC Thr Tyr Arg Ser Arg Glu Tyr 270 ACC AGT TGG TAT GTG GCA TTG AAA CGA Thr Ser Trp Tyr Val Ala Leu Lys Arg 275 280 WO 96/36362 PCT[US96/07164 ACT GGG CAG Thr Gly Gin 285 TAT AAA CTT GGA TCC AAA Tyr Lys Leu Gly Ser Lys 290 ACA GGA CCT GGG CAG AAA GCT Thr Gly Pro Gly Gin Lys Ala 295 ATA CTT Ile Leu 300 TTT CTT CCA ATG Phe Leu Pro Met TCT GCT AAG AGC TAA Ser Ala Lys Ser 305 INFORMATION FOR SEQ ID NO:83: SEQUENCE CHARACTERISTICS: LENGTH: 465 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..462 OTHER INFORMATION: /product= Residue 120" "FGF 2 Ala Mutation at (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: ATG GCA GCA GGA TCA ATA Met Ala Ala Gly Ser Ile 155 160 ACA ACA TTA CCC Thr Thr Leu Pro TTG CCC GAG GAT Leu Pro Glu Asp GGC AGC GGC GCC Gly Ser Gly Ala CCG CCC GGC CAC TTC AAG GAC CCC AAG Pro Pro Gly His Phe Lys Asp Pro Lys 180 TAC TGC AAA Tyr Cys Lys AAC GGG Asn Gly 190 GGC TTC TTC CTG Gly Phe Phe Leu 195 CGC CAC CCC GAC Arg His Pro Asp GGC CGA Gly Arg 200 GAC GGG GTC CGG Asp Gly Val Arg 205 GAG AAG AGC GAC Glu Lys Ser Asp 210 CCT CAC ATC AAG Pro His Ile Lys CTT CAA CTT CAA Leu Gin Leu Gin 215 TGT GCT AAC CGT Cys Ala Asn Arg GCA GAA GAG Ala Glu Glu 220 AGA GGA GTT GTG TCT ATC AAA Arg Gly Val Val Ser Ile Lys 225 GGA GTG Gly Val 230 CTG GCT ATG AAG Leu Ala Met Lys GAA GAT Glu Asp 240 GGA AGA TTA CTG Gly Arg Leu Leu 245 GCT TCT AAA TGT Ala Ser Lys Cys ACG GAT GAG TGT TTC TTT TTT GAA CGA Thr Asp Glu Cys Phe Phe Phe Glu Arg GAA TCT AAT AAC Glu Ser Asn Asn TAC AAT Tyr Asn 265 WO 9 6/36362 PCT/US96/07164 ACT TAC CGG Thr Tyr Arg ACT GGG CAG Thr Gly Gin 285 TCA AGG AAA GCA Ser Arg Lys Ala 270 TAT AAA CTT GGA Tyr Lys Leu Gly ACC AGT Thr Ser 275 TGG TAT GTG GCA Trp Tyr Val Ala TTG AAA CGA Leu Lys Arg 280 TCC AAA ACA Ser Lys Thr 290 GGA CCT GGG CAG AAA GCT Gly Pro Gly Gin Lys Ala 295 ATA CTT TTT Ile Leu Phe 300 CTT CCA ATG TCT GCT AAG AGC TAA Leu Pro Met Ser Ala Lys Ser INFORMATION FOR SEQ ID NO:84: SEQUENCE CHARACTERISTICS: LENGTH: 465 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: NAME/KEY:
CDS
LOCATION: 1..462 OTHER INFORMATION: /product= Residue 123" "FGF 2 Trp Mutation at (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: GCA GCA GGA TCA ATA ACA ACA TTA CCC GCC TTG Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu 160 ic CCC GAG GAT GGC Pro Glu Asp Gly 170 GGC AGC GGC GCC Gly Ser Gly Ala TAC TGC AAA AAC Tyr Cys Lys Asn 190 CCG CCC GGC CAC Pro Pro Gly His TTC AAG Phe Lys 180 GAC CCC AAG CGG Asp Pro Lys Arg 185 GGG GGC TTC TTC Gly Gly Phe Phe CGC CAC CCC GAC Arg His Pro Asp GGC CGA Gly Arg 200 GAC GGG GTC Asp Gly Val 205 CGG GAG AAG AGC Arg Glu Lys Ser CCT CAC ATC AAG Pro His Ile Lys CTT CAA CTT CAA Leu Gin Leu Gin 215 TGT GCT AAC CGT Cys Ala Asn Arg GCA GAA GAG Ala Glu Glu 220 AGA GGA GTT Arg Gly Val GTG TCT Val Ser 225 AAA GGA GTG Lys Gly Val 230 TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT GTT Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys Val 235 240 245 250 WO 96/36362 PCTfUS96/07164 ACG GAT GAG TGT Thr Asp Glu Cys ACT TAC CGG TCA Thr Tyr Arg Ser 270 ACT GGG CAG TAT Thr Gly Gin Tyr 285 TTC TTT Phe Phe 255 TTT GAA CGA TTG Phe Glu Arg Leu 260 GAA TCT AAT Glu Ser Asn SAAC TAC AAT Asn Tyr Asn 265 TTG AAA CGA Leu Lys Arg 280 AGG AAA TAC ACC Arg Lys Tyr Thr AAA CTT GGA TCC Lys Leu Gly Ser 290 GCA TAT GTG GCA Ala Tyr Val Ala AAA ACA GGA CCT Lys Thr Gly Pro GGG CAG AAA GCT Gly Gin Lys Ala 295 ATA CTT Ile Leu 300 TTT CTT CCA ATG Phe Leu Pro Met TCT GCT AAG Ser Ala Lys 305 AGC TAA Ser INFORMATION FOR SEQ ID SEQUENCE
CHARACTERISTICS:
LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Protamine" (xi) SEQUENCE DESCRIPTION: SEQ ID TACATGCCAT GGCCAGGTAC AGATGCTGTC GCAGCCAGAG CCGGAGCAGA
TATTACCGCC
INFORMATION FOR SEQ ID NO:86: SEQUENCE
CHARACTERISTICS:
LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Protamine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: GCAGCTCCGC CTCCTTCGTC TGCGACTTCT TTGTCTCTGG CGGTAATATC
TGCTCCGGCT
WO 96/36362 PCT/US96/07164 193 INFORMATION FOR SEQ ID NO:87: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Protamine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: GACGAAGGAG GCGGAGCTGC CAGACACGGA GGAGAGCCAT GAGGTGCTGC CGCCCCAGGT INFORMATION FOR SEQ ID NO:88: SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Protamine" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: ATATATCCTA GGTTAGTGTC TTCTACATCT CGGTCTGTAC CTGGGGCGGC AGCACCTCA 59 INFORMATION FOR SEQ ID NO:89: SEQUENCE
CHARACTERISTICS:
LENGTH: 66 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: CGTATCAGGC GGCCGCCGCC ATGGTGACCT CCATCACCCT GGACCTGGTG AACCCCACCG CCGGCC 66 WO 96/36362 PCTIUS96/07164 194 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 69 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID TTGGGGTCCT TCACGTTGTT GCGGATCTTG TCCACGAAGG AGGAGTACTG GCCGGCGGTG
GGGTTCACC
INFORMATION FOR SEQ ID NO:91: SEQUENCE CHARACTERISTICS: LENGTH: 65 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: AACAACGTGA AGGACCCCAA CCTGAAGTAC GGCGGCACCG ACATCGCCGT GATCGGCCCC
CCCTC
INFORMATION FOR SEQ ID NO:92: SEQUENCE CHARACTERISTICS: LENGTH: 65 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" WO96/36362 PCT/US96/07164 195 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: GTGCCGCGGG AGGACTGGAA GTTGATGCGC AGGAACTTCT CCTTGGAGGG GGGGCCGATC ACGGC INFORMATION FOR SEQ ID NO:93: SEQUENCE CHARACTERISTICS: LENGTH: 75 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: CTCCCGCGGC ACCGTGTCCC TGGGCCTGAA GCGCGACAAC CTGTACGTGG TGGCCTACCT GGCCATGGAC AACAC INFORMATION FOR SEQ ID NO:94: SEQUENCE CHARACTERISTICS: LENGTH: 77 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: GCGGTCAGCT CGGCGGAGGT GATCTCGGAC TTGAAGTAGT AGGCGCGGTT CACGTTGGTG TTGTCCATGG CCAGGTA 77 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 96/36362 PCT/US96/071 6 4 196 (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID GCCGAGCTGA CCGCCCTGTT CCCTGAGGCC ACCACCGCCA ACCAGAAGGC CCTGGAGTAC ACCGAGGACT ACCAGTCC 78 INFORMATION FOR SEQ ID NO:96: SEQUENCE CHARACTERISTICS: LENGTH: 76 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: AGCCCGAGCT CCTTGCGGGA CTTGTCGCCC TGGGTGATCT GGGCGTTCTT CTCGATGGAC TGGTAGTCCT CGGTGT 76 INFORMATION FOR SEQ ID NO:97: SEQUENCE CHARACTERISTICS: LENGTH: 74 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: TATAGAATTC CTCGGGCTGG GCATCGACCT GCTGCTGACC TTCATGGAGG CCGTGAACAA GAAGGCCCGC GTGG 74 WO 96/36362 PCT/US96/07164 197 INFORMATION FOR SEQ ID NO:98: SEQUENCE CHARACTERISTICS: LENGTH: 68 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: CGGCGGTCAT CTGGATGGCG ATCAGCAGGA AGCGGGCCTC GTTCTTCACC ACGCGGGCCT TCTTGTTC 68 INFORMATION FOR SEQ ID NO:99: SEQUENCE
CHARACTERISTICS:
LENGTH: 70 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: CGCCATCCAG ATGACCGCCG AGGTGGCCCG CTTCCGCTAC ATCCAGAACC TGGTGACCAA GAACTTCCCC INFORMATION FOR SEQ ID NO:100: SEQUENCE
CHARACTERISTICS:
LENGTH: 76 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" WO 96/36362 PCT/US96/07164 198 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: GGCGGATCCC AGCTGACCTC GAACTGGATC ACCTTGTTGT CGGAGTCGAA CTTGTTGGGG AAGTTCTTGG TCACCA 76 INFORMATION FOR SEQ ID NO:101: SEQUENCE CHARACTERISTICS: LENGTH: 61 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: CCGGGATCCG TCAGCTGGCG CAAGATCTCC ACCGCCATCT ACGGCGACGC CAAGAACGGC G 61 INFORMATION FOR SEQ ID NO:102: SEQUENCE CHARACTERISTICS: LENGTH: 64 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: GCACCTTGCC GAAGCCGAAG TCGTAGTCCT TGTTGAACAC GCCGTTCTTG GCGTCGCCGT AGAT 64 INFORMATION FOR SEQ ID NO:103: SEQUENCE CHARACTERISTICS: LENGTH: 58 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 96/36362 PCT/US96/07164 199 (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: TTCGGCTTCG GCAAGGTGCG CCAGGTGAAG GACCTGCAGA TGGGCCTGCT GATGTACC 58 INFORMATION FOR SEQ ID NO:104: SEQUENCE CHARACTERISTICS: LENGTH: 52 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for Mammalian Codon Preferred Saporin" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: TGAACGTGGC GGCCGCCTAC TTGGGCTTGC CCAGGTACAT CAGCAGGCCC AT 52 INFORMATION FOR SEQ ID NO:105: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ix) FEATURE: OTHER INFORMATION: /note= "Primer for SAP-6" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: CATATGTGTG TCACATCAAT CACATTAGAT INFORMATION FOR SEQ ID NO:106: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 96136362 PCT/1US9610716 4 200 (ix) FEATURE: OTHER INFORMATION: /note= "Primer for SAP-6" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: CAGGTTTGGA TCCTTTACGT T 21

Claims (33)

1. A pharmaceutical composition comprising the formula: receptor-binding internalized ligand nucleic acid binding domain cytocide-encoding agent, wherein: receptor-binding internalized ligand is a polypeptide reactive with a cell surface receptor; nucleic acid binding domain binds to a nucleic acid, the domain being conjugated or fused to the receptor-binding internalized ligand; cytocide-encoding agent is a nucleic acid molecule encoding a cytocide, the agent being bound by complexation to the nucleic acid binding domain; and wherein the receptor-binding internalized ligand-nucleic acid binding domain cytocide-encoding agent binds to the cell surface receptor and internalizes the cytocide-encoding agent in cells bearing the receptor. S:
2. A pharmaceutical composition comprising the formula: receptor-binding internalized ligand nucleic acid binding domain prodrug-encoding agent, wherein: receptor-binding internalized ligand is a polypeptide reactive with a cell surface 20 receptor; nucleic acid binding domain binds to a nucleic acid, the domain being conjugated or fused to the receptor-binding internalized ligand; prodrug-encoding agent is a nucleic acid molecule encoding a prodrug, the agent being bound by complexation to the nucleic acid binding domain; and wherein the receptor-binding internalized ligand nucleic acid binding domain prodrug-encoding agent binds to the cell surface receptor and internalizes the prodrug-encoding agent in cells bearing the receptor.
3. The composition of either of claims 1 or 2 wherein the receptor-binding internalized ligand is a polypeptide reactive with an FGF receptor.
4. The composition of either of claims 1 or 2 wherein the receptor-binding internalized ligand is selected from the group consisting of any of SEQ ID NOS 10 and 12-18 P:\OPER\RMH\58628-96.CLM 10/6/99 -202- or polypeptide fragments thereof.
The composition of either of claims 1 or 2 wherein the receptor binding internalized ligand is an FGF mutein or polypeptide fragment thereof.
6. The composition of claim 1 wherein the cytocide-encoding agent encodes a protein that inhibits protein synthesis.
7. The composition of claim 6 wherein the protein is a ribosome inactivating protein.
8. The composition of claim 7 wherein the ribosome inactivating protein is saporin. 15
9. The composition of claim 7 wherein the ribosome inactivating protein is *0 t S: gelonin.
10. The composition of claim 6 wherein the protein inhibits elongation factor 2. S 20
11. The composition of claim 10 wherein the protein is diphtheria toxin.
12. The composition of claim 2 wherein the prodrug-encoding agent encodes HSV- thymidine kinase.
13. The composition of either of claims 1 or 2 wherein the receptor binding internalised ligand is a polypeptide reactive with the FGF receptor and the nucleic acid binding domain is poly-L-lysine.
14. The composition of either of claims 1 or 2 wherein the nucleic acid binding domain is selected from the group consisting of helix-tur-helix motif proteins, homeodomain proteins, zinc finger motif proteins, steroid receptor proteins, leucine zipper motif proteins, helix-loop-helix motif proteins, and P-sheet motif proteins.
A P:\OPER\RMH\5628-96.CLM 21/1/98 -203- The composition of either of claims 1 or 2 wherein the nucleic acid binding domain is selected from the group consisting of AP-1, Sp-1, rev, GCN4, cro, XcI, TFIIA, myoD, retinoic acid receptor, glucocosteroid receptor, SV40 large T antigen, and GAL 4.
16. The composition of either of claims 1 or 2 wherein the nucleic acid binding domain is selected from the group consisting of poly-L-lysine, protamine, histone, spermidine and spermine.
17. The composition of claim 1 wherein the nucleic acid binding domain 10 specifically binds a DNA molecule that encodes a ribosome inactivating protein.
S:18. The composition of claim 1 wherein the nucleic acid binding domain specifically binds the coding region of saporin DNA. 15
19. The composition of claim 1 wherein the cytocide-encoding agent further comprises a tissue-specific promoter.
20. The composition of claim 2 wherein the prodrug-encoding agent further comprises a tissue-specific promoter.
21. The composition of either of claims 19 or 20 wherein the tissue-specific promoter is selected from the group consisting of alpha-crystalline, tyrosinase, a-fetoprotein, prostate specific antigen, CEA, a-actin, VEGF receptor, erbB-2, c-myc, cyclin D, FGF receptor and gamma-crystalline promoter.
22. The composition of any one of claims 1-21, further comprising at least one linker that increases the serum stability or intracellular availability of the nucleic acid binding domain, the addition of said linker(s) resulting in the formula: receptor-binding internalized ligand-(L)q-nucleic acid binding domain-cytocide encoding agent or the formula: receptor-binding internalized ligand-(L)q-nucleic acid binding domain-prodrug encoding agent wherein: L is at least one linker; and P:\OPER\RMH\5628-%.CLM 211/98 -204- q is 1 or more, such that the conjugate retains the ability to bind to a cell surface receptor and internalize the cytocide-encoding agent or prodrug-encoding agent, and wherein the cytocide-encoding agent or prodrug-encoding agent is bound to the nucleic acid binding domain.
23. The composition of claim 22 wherein the linker increases the flexibility of the conjugate.
24. The composition of claim 23 wherein the linker is selected from the group 10 consisting of (GlymSerp)n, (SermGlyp)n and (AlaAlaProAla)n in which n is 1 to 6, m is 1 to 6 and p is 1 to 4.
25. The composition of claim 24 wherein m is 4, p is 1 and n is 2 to 4. 15
26. A method for the treatment or prophylaxis of excessive cell proliferation in the eye which comprises administering to a patient a therapeutically effective amount of a composition according to any one of claims 1 to 25, wherein the cells are epithelial cells, endothelial cells, fibroblast cells or keratocytes. S* 20
27. A method according to claim 26, wherein the composition is administered by contacting the eye with the composition.
28. A method for the treatment or prophylaxis of cancer which comprises administering to a patient a therapeutically effective amount of a composition according to any one of claims 1 to
29. A method according to claim 28, wherein the composition is administered by contacting cancer cells to be treated with the composition.
30. A method for the treatment or prophylaxis of smooth muscle cell hyperplasia which comprises administering to a patient a therapeutically effective amount of a composition according to any one of claims 1 to P:\OPER\RMH\58628-96.CLM 10/6/99 -205-
31. A method according to claim 30, wherein the composition is administered by contacting the smooth muscles cells with the composition.
32. A method for the manufacture of a medicament including the step of bringing a composition according to any one of claims 1 to 25 into a form suitable for administration.
33. A pharmaceutical composition having the formula: receptor-binding internalized ligand nucleic acid binding domain targeted agent, wherein: receptor-binding internalized ligand is a polypeptide reactive with a cell surface receptor; nucleic acid binding domain binds to a nucleic acid, the domain being chemically conjugated or fused to the receptor-binding internalized ligand; targeted agent is a therapeutic nucleic acid molecule, the agent being bound by 15 complexation to the nucleic acid binding domain; and wherein the receptor-binding internalized ligand nucleic acid binding domain targeted agent binds to the cell surface receptor and internalizes the therapeutic nucleic acid in cells bearing the receptor. 0* DATED this 10th day of June, 1999 Prizm Pharmaceuticals, Inc. byDAVIES COLLISON CAVE Patent Attorneys for the Applicants
AU58628/96A 1995-05-16 1996-05-16 Compositions containing nucleic acids and ligands for therapeutic treatment Ceased AU710309B2 (en)

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