WO2000049871A9 - An anti-angiogenic kringle protein and its mutants - Google Patents

An anti-angiogenic kringle protein and its mutants

Info

Publication number
WO2000049871A9
WO2000049871A9 PCT/US2000/004798 US0004798W WO0049871A9 WO 2000049871 A9 WO2000049871 A9 WO 2000049871A9 US 0004798 W US0004798 W US 0004798W WO 0049871 A9 WO0049871 A9 WO 0049871A9
Authority
WO
WIPO (PCT)
Prior art keywords
kringle
tpa
protein
ked
cells
Prior art date
Application number
PCT/US2000/004798
Other languages
French (fr)
Other versions
WO2000049871A1 (en
Inventor
Dexian Dou
Michael Chopp
Lei Wang
Tom Mikkelsen
Original Assignee
Ford Henry Health System
Dexian Dou
Michael Chopp
Lei Wang
Tom Mikkelsen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ford Henry Health System, Dexian Dou, Michael Chopp, Lei Wang, Tom Mikkelsen filed Critical Ford Henry Health System
Priority to AU36058/00A priority Critical patent/AU3605800A/en
Publication of WO2000049871A1 publication Critical patent/WO2000049871A1/en
Publication of WO2000049871A9 publication Critical patent/WO2000049871A9/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the use of kringle proteins for the treatment of disease. More specifically, the present invention relates to the use of kringle proteins for the treatment of cancerous lesions.
  • neoangiogenesis is a critical and necessary step in the metastatic process both for tumor survival and progression.
  • the development of a network of blood vessels is a prerequisite for the local expansion of tumor colonies beyond the size (0.125 mm2) restricted by oxygen and nutrient diffusion (Liotta et al., 1991 ; Folkman et al.,1992).
  • neoangiogenesis is a novel biological target in tumors since angiogenesis involves local endothelial cell invasion, proliferation, and migration in response to many of the same cytokines frequently produced by tumors.
  • angiogenesis has been described as being regulated by angio-promoting factors (bFGF, VEGF, PDGF, TGF- ⁇ ) and angio-inhibitors (TSP-I, GDAIF).
  • Angio-inhibitors in glioma cells appear to be subject to regulation by p53 or genetic material on chromosome 10.
  • hypoxic cancer cells in the core of tumors secrete a number of growth factors such as VEGF (vascular endothelial growth factor), IL-8 (interleukin-8), HGF (hepatocyte growth factor) and bFGF (basic fibroblast growth factor) that stimulate proliferation of endothelial cells and cause rapid sprouting of new capillaries from adjacent blood vessels (White et al., 1995).
  • VEGF vascular endothelial growth factor
  • IL-8 interleukin-8
  • HGF hepatocyte growth factor
  • bFGF basic fibroblast growth factor
  • Angiostatin is a 38 kDa internal fragment of plasminogen. It has been demonstrated that human prostate carcinoma cells (PC-3. DU-145, and LN- CaP) express enzymatic activity that converts plasminogen to angiostatin (Gately et al., 1997), suggesting that generation of angiostatin is not an individual event limited to Lewis lung carcinoma.
  • Endostatin a novel angiogenesis inhibitor of the C-terminal fragment of collagen ⁇ 1 (XVIII) has also recently been identified from the conditioned media of murine hemangioendothelioma cell line EOMA (O'Reilly et al., 1994).
  • EOMA murine hemangioendothelioma cell line
  • Endostatin is a 20 kDa protein. Both wild-type and recombinant murine endostatin at a concentration of approximately 50 nM, completely inhibit the proliferation of bovine capillary endothelial cells. However, the purified human endostatin from hemofiltrate (HF) failed to inhibit the proliferation of bovine endothelial cells at the same concentration. Alignment of human circulating endostatin with murine endostatin isolated from mouse hemangioenqothelioma cell medium, indicates that the N-terminus of circulating human endostatin is 12 amino acids shorter than the mouse endostatin. There are no reports showing that human endostatin is as active as the mouse one.
  • angiostatin shows much weaker angiogenesis inhibition activity.
  • a concentration of 50 nM endostatin completely suppressed the bovine endothelial cell proliferation.
  • more than 10 times the amount of angiostatin was required. Notable homology has not been found between endostatin and angiostatin at the level of DNA or amino acid sequences.
  • the plasminogen kringle 5 alone showed inhibition of bovine endothelial cell proliferation at the same concentration as that of angiostatin which is composed of kringle 1 to 4 domains. Additional experiments indicate that individual kringles in angiostatin have varied anti-angiogenesis activities.
  • the estimated MW of "KED" is 30,001 Da, less than the 38 kDa of angiostatin.
  • tPA Human tissue plasminogen activator
  • the primary structure of kringles contains approximately 90 amino acids, with a rigidly conserved triple disulphide bond pattern.
  • the two tPA kringles show a high homology to each other sharing 51% identical amino acid residues.
  • the tPA kringles also are markedly homologous with the kringles of plasminogen. There are 28 to 35% identical residues between two tPA kringles and the five kringles in plasminogen (Fig 1).
  • Kringles are involved in basic protein-protein interactions. There are two known major biological functions of kringles, fibrinogenosis and angiogenesis. Kringles in tPA and plasminogen provide the site-specific thrombolytic reaction in the fibrinogenosis system (de Munk et al., 1989; Nesheim et al., 1990). The lysine-binding site in tPA or plasminogen kringles, which can interact with internal lysine in fibrin is responsible for fibrin binding (Horrevoets et al., 1994; Urano et al., 1991). Kringle structures of plasminogen have been demonstrated to be the key player of angiostatin.
  • Binding of a kringle to endothelial cells can be an indispensable condition for anti- angiogenic activity.
  • tPA kringle 2 binds to endothelial cells in a kringle (tPA kringle 2)-mediated manner (Mulder et al., 1997; Cheng et al., 1955), providing indirect evidence that tPA kringle 2 binds with endothelial cells.
  • tPA kringle 2 binds with endothelial cells.
  • neither the lysine-binding site nor the ability of binding with endothelial cells has been found in the tPA kringle domain.
  • tPA kringle 2 Properties of tPA kringle 2, together with high homology between tPA and plasminogen kringle suggest that the tPA kringle 2 can posses anti-angiogenesis activity, which lead the present inventors to investigate the anti-angiogenesis function of tPA kringles.
  • the tPA kringle 2 peptide is a small protein of 9.5 kDa. Therefore, the modified version with mutation of histidine to tyrosine shows strong anti-angiogenic activity which is useful for a pharmaceutical composition.
  • a pharmaceutical composition including at least one kringle protein and a pharmaceutically acceptable carrier. Also provided by the present invention is a method of treating a disease by administering an effective amount of a compound from the group consisting of kringle proteins, KED and a kringle derived from the tPA protein.
  • Figure 1 shows a schematic representation of molecular cloning and fusion of KED gene; a vector pEXdna Y containing T7 promoter and dnaY gene having been constructed for KED gene expression; KED gene generated by fusion of plasminogen kringle 5 cDNA and collagen ⁇ 1 (XVIII) fragment being shown; plasminogen kringle 5 cDNA being derived from human liver mRNA; collagen ⁇ l (XVIII) cDNA being from human kidney;
  • Figure 2 is a photograph showing the expression of the KED protein; the SDS-PAGE shows the induction of KED expression by 0.5 mM IPTG in 3 hours, shown as a Coomassie Blue-stained 10% polyacryl amide gel with 50 ug proteins on each lane;
  • Figure 3 is a photograph showing the isolation and purification of the KED protein; pure KED protein (2.5 ⁇ g) was analyzed by 10% SDS-PAGE with Coomassie Blue-staining which reveals a single KED peptide migrated at the position of 30 kDa;
  • Figures 4 A and B are graphs showing the in vitro anti-angiogenic activity of purified KED; A. Cells on each assay filter were counted; B. KED activity was analyzed in a proliferation assay using bovine endothelial cells; anti-angiogenic activity of KED is compared with endostatin and plasminogen kringle 5;
  • FIG. 5 A and B are photographs showing the inhibition of blood capillaries growth by KED., the in vivo anti-angiogenic activity of KED was tested by rat corneal assay; 25 ng bFGF intitiates an aggressive angiogenic response (A) and 25 ng bFGF +12.5 ng KED significantly suppresses (B);
  • Figure 6 shows the alignment of amino acid sequences of human tPA (K1 tPA , K2 tPA ) and plasminogen kringles (K1-5 plg ); the kringle being aligned according to their conserved cysteines (indicated by stars), positions with identical amino acids in all seven kringles are boxed;
  • Figure 7 is a photograph showing the expression of tPA kringle 2 domain which was denatured in 16% polyacrylamide gel and stained with Coomassie-blue; the tPA kringle 2 domain was purified by an affinity lysine- agarose chromatography;
  • Figure 8 is a graph showing the test of anti-angiogenic activity of tPA fragments; the proliferation assay used bovine endothelial cells wherein the Asn ⁇ -Thr 63 fragment is tPA kringle 2 domain and lle ⁇ -Thr 63 contains tPA kringle 1 and 2, also Asn 177 -Pro 527 contains tPA kringle 2 and serine protease domain;
  • Figure 9 is a photograph showing the expression of tPA kringle 2 mutants. Whole cells, equivalent to 50 g proteins, were analyzed by denatured 16% SDS-PAGE;
  • Figure 10 is a diagram showing the site-directed mutagenesis in tPA kringle 2; mutation are presented by arrows with amino acid sequence of tPA kringle 2 domain; mutations in the middle of kringle caused unstable expression; the effects of mutations on anti-angiogenic activity are compared (internal);
  • FIG 11 is a photograph showing the KED Protein; the KED gene was expressed under a T7 promoter, the recombinant E. coli strain BL21 (DE3 ) bearing the KED gene produced KED protein with an efficiency exceeding 30% of total cellular protein; the KED protein was isolated using French Press at 20.000 psi; the KED inclusion bodies were dissolved by 8M urea and refolded in a redox solution containing 1.1 mM GSH and 0.19 mM GSSG. Refolded KED protein was concentrated by ultrafiltration and was followed by Sephadex 0-25 chromatography and dialysis against PBS buffer; the purified KED protein appeared at the 30 kDa position on the SDS-PAGE;
  • Figure 12 is a bar graph showing the activity of the KED protein; KED activity was analyzed by a standard proliferation assay using Bovine endothelial cells; cells on each assay filter were counted 5 days after treatment with KED drug; the control indicates endothelial cells proliferation in the presence of bFGF; proliferation inhibition assays were performed at different concentrations of KED; proliferation (angiogenesis) of endothelial cells induced by bFGF was significantly inhibited by a concentration as low as 8 nM of KED drug;
  • Figure 13 is a bar graph showing the activity of endostatin (D); endostatin was the strongest angiogenesis inhibitor prior to the construction of the KED molecule, in identical experiments to those of Fig 12 were performed endostatin had no activity at a concentration of 12 nM;
  • Figure 14 is a graph showing the comparisons of KED with KS and D drug function; angiostatin (Plasminogen Kringle-5) showed weak anti- angiogenesis activity; Endostatin activity is stronger than angiostatin KED drug is the strongest anti-angiogenesis drug;
  • Figure 15 is a graph showing the animal treatment data, wherein the results are depicted of animals having tumors being treated with either PBS or KED;
  • Figure 16 is a graph showing the animal treatment data, wherein the results are depicted of another group of animals having tumors being treated with either PBS or KED;
  • Figure 17 is a graph showing the animal treatment data, wherein the results are depicted of another group of animals having tumors being treated with either PBS or KED;
  • Figurel ⁇ is a photograph showing the expression of tPA kringle 2
  • Figures 19 A-C are graphs and photographs showing the anti- angiogenic activity of tPA kringle 2 mutant; tPA kringle 2 H mutant inhibits
  • endothelial cell proliferation in a dose-dependent manner, endothelial migration (B) and corneal angiogenesis (C) where control bFGF pellet (C, left) is inhibited by bFGF +5 ng H mutant (C, right);
  • Figures 20 A and B are graphs showing the in vitro anti-angiogenic activity of purified KED; the activity was analyzed by proliferation assay (above) and migration (below) using bovine endothelial cells; cells on each assay filter were counted (A); anti-angiogenic activity of KED is compared with that of endostatin (B);
  • Figures 21 A-C are graphs showing the Hi-Trap Heparin-Sepharose Chromotagraphy results
  • Figures 22 A-D are graphs showing cell proliferation and migration upon treatment with KED; (A) shows the absorbance over a six day period;
  • FIG. 23 A-C are bar graphs showing comparative data between
  • KED and angiostatin shows MTT assay for KED;
  • B shows U87 cell invasion for angiostatin;
  • C shows BAC cell migration for angiostatin;
  • Figures 24 A and B are a depiction of (A) the pET system cloning sites and (B) the pET vector system circular map and polylinker.
  • the present invention provides a pharmaceutical composition for the treatment of disease including at least one kringle protein and a pharmaceutically acceptable carrier. Additionally, there is provided a pharmaceutical composition for the treatment of disease including a KED molecule or the kringle from the tPA molecule.
  • the pharmaceutical of the present invention contains therein anti-angiogenic properties, thus enabling the use of the pharmaceutical in treating cancer and other diseases in which anti-angiogenic properties will minimize or alleviate the symptoms of the disease or the disease itself.
  • the present invention includes compositions and methods for the detection and treatment of diseases and processes that are mediated by or associated with angiogenesis.
  • the composition can be isolated from body fluids including, but not limited to, serum, urine and ascites, or synthesized by chemical or biological methods (e.g. cell culture, recombinant gene expression, protein synthesis, and in vitro enzymatic catalysis of plasminogen or plasmin to yield active angiostatin).
  • Recombinant techniques include gene amplification from DNA sources using the polymerase chain reaction (PCR), and gene amplification from RNA sources using reverse transcriptase/PCR.
  • PCR polymerase chain reaction
  • the present invention inhibits the growth of blood vessels into tissues such as unvascularized or vascularized tumors.
  • the present invention also encompasses a composition comprising, a vector containing a DNA sequence encoding for the kringles of the present invention, wherein the vector is capable of expressing the kringles of the present invetion when present in a cell, a composition comprising a cell containing a vector, wherein the vector contains a DNA sequence encoding the kringles of the present invention or fragments or analogs thereof, and wherein the vector is capable of expressing the kringles of the present invention when present in the cell, and a method comprising, implanting into a human or non-human animal a cell containing a vector, wherein the vector contains a DNA sequence encoding the kringles of the present invention, and wherein the vector is capable of expressing the kringles of the present invention when present in the cell.
  • the present invention encompasses the kringles of the present invention, fragments thereof, antisera, receptor agonists or receptor antagonists that are combined with pharmaceutically acceptable carriers, and optionally sustained-release compounds or compositions, such as biodegradable polymers, to form therapeutic compositions.
  • the invention includes a composition comprising an antibody that specifically binds to the kringles of the present invention, wherein the antibody does not bind to plasminogen.
  • Immunoassays are also provided by the present invention.
  • ELISAs are the preferred immunoassays employed to assess a specimen. Both polyclonal and moloclonal antibodies can be used in the assays. The specific assay to be used can be determined by one skilled in the art.
  • the compounds of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art. Additionally, when the compound is being administered orally, there must be included a compound for preventing the degradation of the kringle.
  • the compounds of the present invention can be used in combination with other compositions and procedures for the treatment of diseases.
  • a tumor can be treated conventionally with surgery, radiation or chemotherapy combined with the kringles of the present invention and then the kringles of the present invention can be subsequently administered to the patient to extend the dormancy of micrometastases and to stabilize and inhibit the growth of any residual primary tumor.
  • the kringles of the present invention, fragments thereof, antisera, receptor agonists, receptor antagonists, or combinations thereof are combined with pharmaceutically acceptable excipients, and optionally sustained-release matrix, such as biodegradable polymers, to form therapeutic compositions.
  • a sustained- release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids.
  • the sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) polyanhydrides, poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
  • biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) polyan
  • a preferred biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and glycolic acid).
  • the angiogenesis-modulating therapeutic composition of the present invention can be a solid, liquid or aerosol and can be administered by any known route of administration.
  • solid therapeutic compositions include pills, creams, and implantable dosage units.
  • the pills can be administered orally, the therapeutic creams can be administered topically.
  • the implantable dosage unitst can be administered locally, for example at a tumor site, or which can be implanted for systemic release of the therapeutic angiogenesis- modulating composition, for example subcutaneously.
  • liquid composition examples include formulations adapted for injection subcutaneously, intravenously, intraarterially, and formulations for topical and intraocular administration.
  • aersol formulation examples include inhaler formulation for administration to the lungs.
  • the the kringles of the present invention of the present invention also can be used to generate antibodies that are specific for the inhibitor and its receptor.
  • the antibodies can be either polyclonal antibodies or monoclonal antibodies. These antibodies that specifically bind to the the kringles of the present invention its receptors can be used in diagnostic methods and kits that are well known to those of ordinary skill in the art to detect or quantify the the kringles of the present invention or it's receptors in a body fluid or tissue.
  • Results from these tests can be used to diagnose or predict the occurrence or recurrence of a cancer and other angiogenic mediated diseases.
  • the the kringles of the present invention also can be used in a diagnostic method and kit to detect and quantify antibodies capable of binding the kringles of the present invention. These kits would permit detection of circulating antibodies which indicates the spread of micrometastases in the presence of the kringles of the present invention secreted by primary tumors in situ. Patients that have such circulating anti- antibodies can be more likely to develop multiple tumors and cancers, and can be more likely to have recurrences of cancer after treatments or periods of remission.
  • the Fab fragments of these antibodies can be used as antigens to generate Fab-fragment antisera which can be used to neutralize anti the kringles of the present invention antibodies.
  • tissue plasminogen activator contains two kringle structures.
  • Angiostatin an angiogenic inhibitor, is composed of four kringles of plasminogen.
  • Computer-aided analysis revealed similarities and differences between the tPA and plasminogen kringles.
  • the tPA kringle 2 is composed of 86 amino acids (Asn ⁇ -Thr 263 in tPA molecule) and has been expressed in E. coli cells to 39.9% of the total cellular protein. The refolded peptide fragment specifically inhibited endothelial cell proliferation.
  • a series of mutations in the tPA kringle 2 domains were created by PCR-mediated site-directed mutagenesis.
  • the pharmaceutical composition of the present invention utilizes kringle proteins.
  • kringle proteins There are at least three functional sites in a single short peptide (approximate 90 amino acids) of tPA kringle 2: a lysine-binding site, an endothelial cell-binding site and a possible site for anti-angiogenesis activity.
  • tPA kringle 2 To test the functions of tPA kringle 2 the kringle domains of tPA were cloned and expressed. The tPA kringle domains have been difficult to express because very small amounts of tPA kringle 2 (0.05 mg/g cells) are produced from E.
  • tPA kringle 2 peptide was successfully expressed as high as 58.5 mg/g cells E. coli according to the methods of the present invention.
  • the recombinant tPA kringle 2 peptide inhibits the proliferation of endothelial cells.
  • a potent active site in the tPA kringle 2 structure was discovered for expressing kringle anti- angiogenic activity.
  • tPA As a thrombolytic agent, tPA has been used for treatment of stroke and myocardial infarction. It was further uncovered as part of the present invention that an internal fragment of tPA, kringle 2 possesses anti- angiogenic activity.
  • the tPA kringle 2 shares significant homology with angiostatin, an endogenous angiogenic inhibitor.
  • Angiostatin is an internal fragment of plasminogen.
  • plasminogen is not a therapeutic protein. Accordingly, in order to obtain strong anti-angiogenic tPA molecule, PCR- mediated site-directed mutagenesis has been performed.
  • tPA kringle 2 Large quantities of tPA kringle 2 can be mass produced in its modified versions in E. coli cells. Also, tPA kringle 2 can be over-expressed as high as 39.9% of total cellular proteins, more than thousand times higher than previously reported; (2) This is the first to discovery of an internal fragment (Asn ⁇ -Thr 263 , kringle 2) of tPA which possesses anti-angiogenic activity; and (3) The substitution of histidine with an aromatic residue tyrosine significantly increases anti-angiogenic activity of tPA kringle 2 domain.
  • the present invention also provides a method of treating a disease.
  • the methods includes the steps of administering an effective amount of a kringle protein, KED or tPA.
  • Applications for the present invention include but are not limited to the following diseases: atherosclerosis, arthritis, retinopathy, cancer, and other similar diseases.
  • kits containing therein all of the necessary components.
  • the kits include all of the necessary reagents for creating the anti-angiogentic activity in cells and in mammal.
  • the kits can also include the specific kringles to be used by the individual.
  • Alternative kits are made available in which the individual can create their own pharmaceutical composition based on the peptides provided by the present invention.
  • the kit provides instructions, antiserum, the proteins of the present invention, and possibly radiolabeled kringles and/or reagents for precipitation of bound antibody complexes.
  • the kit is useful for the measurement of these kringles in biological fluids and tissue extracts of animals and humans with and without tumors.
  • Another kit is used for localization of the kringles in tissues and cells.
  • This kringle immunohistochemistry kit provides instructions, kringle antiserum, and possibly blocking serum and secondary antiserum linked to a fluorescent molecule such as fluorescein isothiocyanate, or to some other reagent used to visualize the primary antiserum. Immunohistochemistry techniques are well known to those skilled in the art.
  • This kringle immunohistochemistry kit permits localization of kringle in tissue sections and cultured cells using both light and electron microscopy. It is used for both research and clinical purposes. For example, tumors are biopsied or collected and tissue sections cut with a microtome to examine sites of kringle production. Such information is useful for diagnostic and possibly therapeutic purposes in the detection and treatment of cancer.
  • Another method to visualize sites of kringle biosynthesis involves radiolabeling nucleic acids for use in in situ hybridization to probe for kringle messenger RNA. Similarly, the kringle receptor can be localized, visualized and quantitated with immunohistochemistry techniques.
  • peptides which are positives derived from the sequences of the present invention are as follows: peptide 10010281 (region 4 k2 R133-W142 k2tPA) Acetyl-RNPDGDAKPW- amide MW 1197 (10-mer) peptide 10010286 (region H2/5 H144-L151k2tPA) Acetyl-YVLKPRR-amide MW 973 (7-mer); peptide 10010284 (region K2 H144-L151k2tPA) ) Acetyl-HVLKNRR-amide MW 964 (7-mer); hK2tPA (88aa) sequence begins at N177 ⁇ T263 of tPA 1 It is important to note that the H mutant has a single point mutation H244 ⁇ Y.
  • the peptides are short 7-10 amino acid sequences which preserve the majority of the biologic activity of the parent H mutant protein, which is derived from the sequence of the second kringle domain (k2) of human tissue plasminogen activator (tPA) protein, specifically from amino acid 177 to 263.
  • PCR Polymerase chain reaction
  • ELISAs are the preferred immunoassays employed to assess a specimen.
  • ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assays. Where appropriate other immunoassays, such as radioimmunoassays (RIA) can be used as are known to those in the art. Available immunoassays are extensively described in the patent and scientific literature.
  • gene therapy refers to the transfer of genetic material (e.g DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype.
  • the genetic material of interest encodes a product (e.g. a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired.
  • the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value.
  • the genetic material of interest encodes a suicide gene.
  • in vivo gene therapy in ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to express the transfected genetic material in situ.
  • target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ, that is within the recipient.
  • the host gene if the host gene is defective, the gene is repaired in situ [Culver, 1998]. These genetically altered cells have been shown to express the transfected genetic material in situ.
  • the gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell.
  • the expression vehicle can include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR of the gene can be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore as used herein the expression vehicle may, as needed, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred and only include the specific amino acid coding region.
  • the expression vehicle can include a promotor for controlling transcription of the heterologous material and can be either a constitutive or inducible promotor to allow selective transcription. Enhancers that can be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non-translated DNA sequence which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter.
  • the expression vehicle can also include a selection gene as described herein below.
  • Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Ml (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Ml (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see United States patent 4,866,042 for vectors involving the central nervous system and also United States patents 5,464,764 and 5,487,992 for positive
  • nucleic acids by infection offers several advantages over the other listed methods. Higher efficiency can be obtained due to their infectious nature. Moreover, viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.
  • DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector AdenoTK.
  • This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences.
  • TK herpes virus thymidine kinase
  • This vector can be used to infect cells that have an adenovirus receptor which includes most cancers of epithelial origin as well as others.
  • This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vitro or ex vivo culture of cells, a tissue or a human subject.
  • Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy.
  • Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus.
  • An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation will not occur.
  • features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type.
  • recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity.
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical- type of infection in which the infectious agent spreads only through daughter progeny.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • the vector to be used in the methods of the invention will depend on desired cell type to be targeted and will be known to those skilled in the art. For example, if breast cancer is to be treated then a vector specific for such epithelial cells would be used. Likewise, if diseases or pathological conditions of the hematopoietic system are to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, would be used.
  • Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection.
  • the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles which are capable of undergoing further rounds of infection.
  • the vector's genome is also engineered to encode and express the desired recombinant gene.
  • the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection.
  • the specific type of vector will depend upon the intended application.
  • the actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.
  • the recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neuro-degenerative diseases. Following injection, the viral vectors will circulate until they recognize host cells with the appropriate target specificity for infection. An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid.
  • Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal.
  • Such non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like.
  • Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art.
  • transfection of promoter sequences, other than one normally found specifically associated with these kringles, or other sequences which would increase production of protein are also envisioned as methods of gene therapy.
  • An example of this technology is found in Transkaryotic Therapies, Inc., of Cambridge, Mass., using homologous recombination to insert a "genetic switch” that turns on an erythropoietin gene in cells. See Genetic Engineering News, Apr. 15, 1994.
  • Such "genetic switches” could be used to activate the kringles (or the receptor) in cells not normally expressing kringles (or the receptor) .
  • Gene transfer methods for gene therapy fall into three broad categories-physical (e.g., electroporation, direct gene transfer and particle bombardment), chemical (lipid-based carriers, or other non-viral vectors) and biological (virus-derived vector and receptor uptake).
  • non-viral vectors can be used which include liposomes coated with DNA.
  • liposome/DNA complexes can be directly injected intravenously into the patient. It is believed that the liposome/DNA complexes are concentrated in the liver where they deliver the DNA to macrophages and Kupffer cells. These cells are long lived and thus provide long term expression of the delivered DNA.
  • vectors or the "naked" DNA of the gene can be directly injected into the desired organ, tissue or tumor for targeted delivery of the therapeutic DNA.
  • the compound of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
  • the compound of the present invention can be administered in various ways. It should be noted that it can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants and vehicles.
  • the compounds can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and direct parenchymal infusion techniques. Implants of the compounds are also useful.
  • the patient being treated is a warm-blooded animal and, in particular, mammals including man.
  • the pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention.
  • the doses can be single doses or multiple doses over a period of several days, but single doses are preferred.
  • the doses can be single doses or multiple doses over a period of several days.
  • the treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.
  • the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, can also be used as solvent systems for compound compositions.
  • various additives which enhance the stability, sterility, and isotonicity of the compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • antibacterial and antifungal agents for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • isotonic agents for example, sugars, sodium chloride, and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds.
  • Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.
  • a pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, microspheres and programmable pumps for parenchymal infusion into organs, such as brain.
  • any compatible carrier such as various vehicle, adjuvants, additives, and diluents
  • the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, microspheres and programmable pumps for parenchymal infusion into organs, such as brain.
  • Examples of delivery systems useful in the present invention include: 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art.
  • a pharmacological formulation of the compound utilized in the present invention can be administered orally to the patient.
  • Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable.
  • Known techniques which deliver it orally or intravenously and retain the biological activity are preferred.
  • the compound of the present invention can be administered initially by intravenous injection to bring blood levels to a suitable level.
  • the patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition and as indicated above, can be used.
  • the quantity to be administered will vary for the patient being treated and will vary from about 100 ng/kg of body weight to 100 mg/kg of body weight per day.
  • these kringle proteins can be chemically coupled to isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules, chemiluminescent, bioluminescent and other compounds for a variety of applications.
  • the efficiency of the coupling reaction is determined using different techniques appropriate for the specific reaction. For example, radiolabeling of these protein with .sup.125 I is accomplished using chloramine T and Na.sup.125 I of high specific activity. The reaction is terminated with sodium metabisulfite and the mixture is desalted on disposable columns. The labeled protein is eluted from the column and fractions are collected. Aliquots are removed from each fraction and radioactivity measured in a gamma counter.
  • the unreacted Na.sup.125 I is separated from the labeled protein.
  • the protein fractions with the highest specific radioactivity are stored for subsequent use such as analysis of the ability to bind to the antisera.
  • Another application of protein conjugation is for production of polyclonal antisera.
  • the kringle proteins containing lysine residues are linked to purified bovine serum albumin using glutaraldehyde. The efficiency of the reaction is determined by measuring the incorporation of radiolabeled protein. Unreacted glutaraldehyde and protein are separated by dialysis. The conjugate is stored for subsequent use. Antiserum against these kringles, analogs thereof, protein fragments and receptor can be generated.
  • both monoclonal and polyclonal antisera are raised using established techniques known to those skilled in the art.
  • polyclonal antisera can be raised in rabbits, sheep, goats or other animals.
  • a carrier molecule such as bovine serum albumin
  • Booster injections are made at regular intervals, such as every 2 to 4 weeks. Blood samples are obtained by venipuncture, for example using the marginal ear veins after dilation, approximately 7 to 10 days after each injection. The blood samples are allowed to clot overnight at 4. degree. C.
  • the serum is removed, aliquoted, and stored at 4.degree. C. for immediate use or at -20 to - ⁇ O.degree. C. for subsequent analysis.
  • KED is also useful in the treatment of other diseases, such as but not limited to: bronchial vascular congestion, inflammatory bowel disease, adult respiratory distress syndrome, Castleman's disease, mycosis fungoides, atherosclerosis, restenosis, ischemia (limb, cerebral, myocardial), cancer, inflammation/chronic inflammatory disease, diabetes (diabetic retinopathy), ophthamology, wound healing, glomerulonephritis, Moyamoya disease, coronary artery disease, ulcer, arthritis, Kaposi sarcoma, psoriasis, peripheral arterial disease, avitreorefinopathy, Macular pucker, chronic patella tendinitis, fibrodysplasia ossifecaus progressiva, Chrohn's disease, hepatitis, neovascular glaucoma, aneurysm, rheumatoid arthritis, leiomyoma, Bartonella quintata infection
  • Human plasminogen kringle 5 is a novel angiogenesis inhibitor. It is a small protein of 10 kDa molecular weight. Angiostatin, a 38 kDa protein, contains 4 kringles of plasminogen kringle 1 through 4. A proliferation assay using bovine endothelial cells indicates that the plasminogen kringle 5 is at least as strong as anti-angiogenic effect as angiostatin. Endostatin is a newly identified angiogenic inhibitor and is the most effective inhibitor of proliferating endothelial cells so far discovered.
  • Endostatin is a 20 kDa C-terminal fragment of collagen ⁇ 1 (XVIII) sharing no homology with angiostatin and plasminogen kringle 5 domains. Hydrophobic profile analysis indicates that endostatin is highly hydrophobic and plasminogen kringle 5 is a hydrophilic protein. Refolding of endostatin protein produced from E. coli cells is not efficient ( ⁇ 1%). The cDNA of human plasminogen kringle 5 domain was fused with that of human collagen ⁇ 1 (XVIII) C-terminal fragment and this generated a novel protein with improved (approximately 100 folds) refolding efficiency and significantly increased anti-angiogenic activity.
  • the fusion protein now called "KED” has an estimated MW of 30,001 Da, has been abundantly produced in E. coli cells. Its anti-angiogenic activity has been examined in three ways: in vitro endothelial cell proliferation assay, in vivo corneal assay and in vivo tumor models.
  • the KED gene was constructed by fusion of cDNA plasminogen kringle 5 encoding Glu478 -Ala562 with a collagen ⁇ 1 (XVIII) cDNA fragment encoding Hisl154 -Lys1336.
  • the KED gene (Fig. I) is expressed in E. Coli cells.
  • AGA codon is a rare codon used in E. Coli (Wada et al., 1990).
  • the tRNA AGA is encoded by dnaY gene.
  • the dnaY gene also has been reported to be essential for DNA replication (Garcia et al., 1986). To improve productivity of the KED protein in E.
  • Coli a vector, pEXdnaY, was constructed containing a strong T7 promoter for high- speed transcription of KED mRNA and a dnaY gene supplying tRNA AGA for efficient translation of the KED product.
  • the process for KED gene fusion and cloning in pEXdnaY vector is shown in the Sequence Listing.
  • KED protein accumulated up to 47% of total cellular protein by 2 hours of induction with 0.5 mM IPTG (Fig. 2).
  • Optimal production of KED protein was performed using a fermentor.
  • the KED protein was produced as inclusion bodies in cytosol.
  • KED inclusion bodies were isolated and efficiently refolded in the presence of 0.5 M L-arginine at pH 8.6.
  • human endostatin was simultaneously expressed from pEXdnaY-hED. More than 99% of the human endostatin protein precipitated in the refolding solution as has been previously observed. No precipitation of KED fusion occurs during the refolding process. This indicates efficient productivity of KED compared to that of endostatin.
  • KED protein was obtained by HiTrap Heparin-Sepharose chromatography. Analysis of KED protein by denatured SDS-PAGE (polyacrylamide gel electrophoresis) revealed a single 30 kDa peptide that agreed with the molecular weight estimated from the deduced amino acid sequence (Fig. 3).
  • a proliferation assay using bovine endothelial cells indicated that fusion protein KED completely inhibited the endothelial cells migration at a concentration as low as 8 nM (250 ng/ml), where, human endostatin shows almost no activity at 12 nM (250 ng/ml) by the same assay (Fig. 4).
  • the IC 50 of KED observed is less than 100 ng/ml.
  • the IC 50 of human endostatin in the assay was 350 ng/ml.
  • Rodent corneal assay has been performed to test in vivo the efficacy of KED fusion protein. Different amounts, 1.25 ng, 5 ng, and 10 ng of KED were applied in the presence of 50 ng of bFGF 1.25 ng of KED fusion protein efficiently inhibited the growth of capillary blood vessels from the corneal bed (Fig. 5).
  • Gliomas were implanted in the rat brain, One mg/kg body weight of KED protein was used to treat animals for 14 days (Fig. 6).
  • the tPA kringles have been cloned and inserted into pET11a a vector for protein expression under the control of a strong T 7 phage promoter and the laclq repressor.
  • Recombinant plasmid pK1 tPA encodes r-[K1, PA ], where, r stands for recombinant and [K1 tPA ] represents amino acid sequence residues [Ile86-Asn 177] of kringle 1 domain in tPA molecule.
  • the recombinant plasmid pK2 tPA encodes r-[K2, PA ] representing amino acid residues [Asn177-Thr263] of tPA kringle 2 domain.
  • Recombinant plasmid pK12 tPA encodes a r-[K12 tPA ] protein containing kringle 1 and kringle 2 domain, an internal fragment of [Ile86-Thr263] in tPA protein sequence.
  • Recombinant proteins expressed in E. Coli were analyzed by 16% polyacrylamide gel. Whole cellular proteins of 50 ⁇ g were denatured by SDS and ⁇ -mercaptoethanol and loaded into a gel for electrophoresis. Abundant expression of 10 kDa protein has been detected from the cells carrying pK2 tPA construct (Fig. 7). 10 colonies of pK2 tPA , construct were selected and all ten expressed the same 10 kDa protein. A 20 kDa protein was found from the cells harboring pK12 tPA plasmid. All ten clones of p12 tPA expressed the 20 kDa protein.
  • the DNA deduced amino acids for r-[K2 tPA ] protein is a peptide of 88 residues including the initial methionine.
  • r-[KI2 tPA ] contains 179 residues with a MW of 19810.92 Da as expected from its DNA sequence. Expression of a 20 kDa protein from the pK12 tPA plasmid perfectly fits with that expected MW. However, even r-[K1 tPA ], sharing 92 identical amino acids with the N-terminus of r-[K12 tPA ], was not expressed.
  • tPA kringle 2 was successfully expressed, as was the tPA kringle 1 plus kringle 2. However, tPA kringle 1 domain was unable to be expressed. Expression of tPA kringle 1 was only achieved by the fusion of kringle 2 domain. The peptide containing tPA kringle 1 only, has never been expressed. Expression of tPA kringle 1 has been reported as a fusion of the plasminogen kringle domain (DeSerrano et al., 1992).
  • Endothelial proliferation assay Both recombinant proteins, r-[K2, PA ] and r-[K12 tPA ], contain tPA kringle
  • the tPA kringle 2 structure has the lysine-binding site.
  • the lysine-bind site in the tPA kringle 2 binding resins coupled with lysine ligand, can be used for affinity protein purification.
  • the stable epoxy-activated-lysine- agraose gel was used to purify the recombinant proteins r- ⁇ f ] and r-[K12 tPA ].
  • L-arginine is a competitor of the lysine ligand. Efficient binding of recombinant tPA kringles with lysine ligand occurs, when the concentration of L-arginine in the protein solution is reduced below 25 mM.
  • Elution of recombinant tPA kringles was carried out by using a competitive ligand of lysine, specifically 6-amino-n-caproic acid. There are three peaks detected in the eluted fractions from a 0-200 mM gradient. One is between 30 to 45 mM. One is 60 to 105 mM and a large peak between 125 to 150 mM of 6-amino- n-caproic acid. Samples from these fractions were subjected to SDS-PAGE analysis.
  • tPA kringle domain is a good choice for the mutation for the following reasons: (1) the tPA kringle 2 contains a strong lysine-binding site that is preferred for ease of isolation of the kringle mutants; and (2) the tPA kringle 2 has been extensively studied.
  • plasminogen kringles All five plasminogen kringles have been cloned.
  • the most active plasminogen kringles are kringle 5 and 1.
  • Plasminogen kringle 4 did not show activity. Because performing the analysis of anti-angiogenic activity of tPA kringles did not include plasminogen kringles, the tPA kringle activity can not be compared with that of plasminogen kringles.
  • Sites were chosen for mutation in tPA kringle using amino acid sequences of plasminogen kringles as references, in which, plasminogen kringle 5 and 1 were selected for the positive and kringle 2, 3, 4 especially kringle 4, were selected for the negative reference.
  • positive selection or negative selection can be designed.
  • the amino acid sequence of tPA kringle 2 is aligned with the five plasminogen kringles.
  • the position(s) are identified where the amino acid residue(s) is identical between plasminogen 5 and 1 but is different from plasminogen 4 and tPA kringle 2. If a position is occupied with a similar or the same amino acid residue in tPA kringle 2 and plasminogen kringle 4, where the residue should be mutated is the top priority.
  • the amino acid residue(s) in kringle 2 is changed to an amino acid that is identical in plasminogen kringle 5 and 1.
  • the resultant mutant is expressed and tested for its effects on endothelial proliferation to see if the mutation changes (increases) its anti- angiogenic activity.
  • negative selection can be designed by changing a residue in the tPA kringle identical with both of plasminogen kringle 5 and 1 to an amino acid identical with that of plasminogen kringle 4.
  • mutations for positive selection were only performed.
  • the mutagenesis procedure utilizes a super-coiled, double-stranded and methylated DNA plasmid pK2 tPA and two synthetic oligonucleotide primers containing the desired mutation.
  • the PCR reaction employing Pfu, a proofreading DNA polymerase, efficiently produced a nicked, unmethylated daughter plasmid with a mutation which was created by the uptake of the oligonucleotide primers.
  • the methylated parent pK2 tPA plasmid was eliminated by restriction digestion using Dpn1 , which is an enzyme, cutting the methylated substrate DNA.
  • the mutated daughter plasmids were recovered by transformation into high-efficient competent E. coli cells.
  • a control PCR accompanied each set of mutation thus excluding the oligodeoxynucleotide primers.
  • Selection of transformants using the ampicillin resistance maker encoded by the vector indicated that at least a hundred fold of colonies were obtained from the PCR reaction mixture containing the mutation primers compared to that of PCR excluding the primers.
  • Dideoxynucleotide DNA sequencing of plasmids revealed that all designed mutations were successfully introduced on the tPA kringle DNA strand. Any undesired or unexpected mutation was not observed, showing that high fidelity DNA extension has been performed during the PCR process using the proof reading Pfu enzyme.
  • kringle mutants are approximately 10 kDa MW. Expression of mutants were analyzed on 16% polyacryl amide gel electrophoresis (Fig. 9), four of them were expressed. However, the Y, N and G mutants were unstable. Interestingly, the Y, N and G mutants are all located in the middle in the kringle structure, suggesting that a mutation on this region creates unstable mRNA or protein structure.
  • tPA kringle 2 mutants were solubilized and reduced with 8 M urea and 140 mM beta-mercaptoethanol. The mutants were then fractionated by Superfine Sephadex G- 75 chromatography in the resence of 8 M urea. The reduced mutant proteins appeared to be homogenous, as shown by the denatured SDS-PAGE analysis. The pure reduced mutants were refolded and pureed.
  • Kringle mutants S, K, H, W and the wild-type tPA kringle protein were tested by proliferation assay using bovine endothelial cells in the presence of bFGF. Mutations on K and W showed no effects on the anti-angiogenic activity of tPA kringle. The S mutation slightly effected the activity. The mutation on the H site generated at least 4 times higher anti-angiogenic activity compared to the parental wild-type kringle (Fig. 10), indicating that the H site is a potent active site for the expression of anti-angiogenic activity of the kringle structure.
  • Human cDNA pool was generated by reverse transcription from liver and kidney mRNA using an oligodeoxynucleotide (dT)17 primer.
  • the cDNAs were reverse transcribed in a 20 ⁇ l reaction solution containing 2.5 ⁇ g of human liver or kidney mRNA (Clontech, Palo Alto, CA), 200 ng of oligo (dT)17 primer, 5 mM DTT, 2.5 mM MgCI, 400 M of dNTP and 200 units of reverse transcriptase (BRL, Gaithersburg, MD) in 50 mM Tris buffer (pH 8.3).
  • the mixture excluding reverse transcriptase was heated at 72° C for 5 minutesto denature mRNA and was quickly chilled on ice for 10 minutesallowing primer annealing. Then the contents were incubated at 42° C for 5 min, and combined with 200 units of reverse transcriptase for an hour incubation.
  • the cDNA fragments encoding plasminogen kringle 5 domain and collagen a1 (XVIII) C-terminus were amplified by polymerase chain reaction (PCR).
  • the cDNA fragment without adaptors encoding plasminogen kringles 1 to 5, was amplified by a sense primer and a complementary primer: 5'-CTCTCAGAGTGCAAGACTGGGAATGGAAAGAAC (Leu100-Asn110), and 5'-GGCCGCACACTGAGGGACATCACAGTAGTC (Ala562-Asp553 according to the amino acid sequence of human plasminogen) (Folkman, 1996).
  • a cDNA fragment encoding collagen a 1 (XVI 11) C-terminus was amplified by a set of primers: 5'-CACAGCCACCGCGACTTCCAGCCGGTGCTC (His1154- Leu1163) for the sense 5'-end of the peptide, and 5'-
  • the PCR reaction solution (100 ⁇ l) contains 20 mM Tris (pH 8.0), 2 mM MgCI, 10 mM KCI, 0.1% Triton X-100, 10 ⁇ l first strand cDNA from the above reverse transcriptional reaction, 300 ng primers and 5 units of Pfu DNA. Twenty-five reaction cycles consisting of 45 sec at 94° C for denaturing, 45 sec at 59° C for annealing and 3 minutesat 72° C for extension were performed. PCR products were analyzed by a 1.5% agarose gel.
  • PCR reaction solution 100 ⁇ l contains 50 mM Tris (pH 8.8), 2 mM MgCI, 10 mM KCI, 10 ⁇ l of the above resulted PCR reaction mixture containing the cDNA fragments, 250 ng of each primer and 7.5 units of Pfu DNA polymerase.
  • Ten reaction PCR cycles consist of 45 sec at 94° C for denaturing, 45 sec at 37° C for annealing and 2 minutesat 68° C for DNA synthesis.
  • Vector pEXdnaY was constructed by combining of pET11a (Stratagene, La Jolla, CA) and pDC952. Five ⁇ g of pET11a plasmid (4.64 kb) was digested with 20 units of Hindlll enzyme at 37° C for 1 hour. The linearized pET11a was treated by adding 32 units of calf intestinal alkaline phosphatase (CIAP) into the restriction reaction mixture and was incubated at 30° C for 30 min, to prevent self-ligation of pET11a.
  • CIP calf intestinal alkaline phosphatase
  • the linearized and dephosphorylated pET11a DNA was then ligated with a 632 bp Hindlll-Hindlll DNA fragment containing dnaY gene (tRNA for AGG and AGA) that was isolated from pDC952 plasmid by Hindlll-digestion.
  • the ligation solution was transformed into competent E. coli TG1 cells. Recombinant plasmid pEXdnaY were isolated and identified. 3.
  • a 558 bp cDNA fragment encoding collagen a1 (XVIII) C-terminus flanked by Ndel and BamHI adaptors were cloned into the pEXdnaY vector, generating a plasmid pEXdnaY-hED. Then, a 255 bp cDNA fragment encoding plasminogen kringle 5 with Ndel adaptor at the two ends were fused in front of the collagen a1 (XVIII) C-terminal fragment, generating plasmid pECdnaY-hKED.
  • pEXdnaY DNA was double-digested with 100 units of Ndel and 150 units of BamHI enzymes, following dephosphorylation with 64 units of CIAP.
  • Pure dephosphorylated pEXdnaY vector was obtained from 0.7% agarose gel.
  • the linearized vector was ligated with Ndel-BamHI DNA fragment of collagen a1(XVIII) C-terminus created by anchored PCR and was restriction digested with Ndel and BamHI enzymes.
  • Recombinant plasmid pEXdnaY-hED was selected in TG1 cells and analyzed by double digestion of Ndel and BamHI enzymes.
  • the resulted pEXdnaY- hED plasmid (5 ⁇ g) was further linearized by 100 units of Ndel and dephosphorylated with 32 units of CIAP.
  • An Ndel-Ndel cDNA fragment encoding human plasminogen kringle 5 generated by Ndel-adaptor anchored PCR amplification was inserted into the Ndel site of pEXdnaY-hED by ligation with Ndel-linearized pEXdnaY-hED DNA.
  • the ligation solution was transformed into competent E. coli TG1 cells.
  • a recombinant plasmid pEXdnaY-hKED was identified by Ndel digestion and analysis of expression of a 30 kDa fusion protein.
  • Plasmid pEXdnaY-hKED was transformed into E. coli BL21 (DE3) cells. A single ampicillin resistance colony was picked up and inoculated in 1 ml of LB media containing 100 g/ml of ampicillin. After 12 hours incubation at 37° C, the culture was diluted 100 fold into the same medium. Incubation with shaking at 250 rpm was continued until OD reached 0.45 at 600 nm. Optimal conditions for expression of KED fusion were investigated by induction with IPTG (isopropyl-D-thiogalactopyranoside) with different concentrations from 0.1 to 10 mM and varied time from 15 minutesto 3 hours.
  • IPTG isopropyl-D-thiogalactopyranoside
  • the induced cultures were quickly chilled on ice and centrifuged. Cells were washed with ice-cold PBS (phosphate saline buffer) containing 5 mM EDTA (ethylenediaminetetraacetic acid). An aliquot of cells, equivalent to 50 ⁇ g protein was used for analysis of KED fusion expression by denatured 10% polyacrylamide gel.
  • PBS phosphate saline buffer
  • EDTA ethylenediaminetetraacetic acid
  • Optimal production of KED fusion was performed using a fermentor (Bioflo2000, New Brunswick Scientific, Edison, NJ). Over night culture (200 ml) was added into 8 I LB medium enriched with 25 g/l yeast extract. One ml of Antifoam 289 (Sigma, St. Louis, MO) was added to the medium to prevent foam-formation during fermentation. Agitation was kept 225 rpm through out the process. Airing was settled to 8 l/min until cells grown to OD 600 0.505. IPTG was added into the fermentor at the final concentration of 0.5 mM. Airing was changed to 20 l/min.
  • KED inclusion bodies were isolated by centrifugation for 20 minutesat 12,100 g at 4° C, and washed twice with ice-cold 500 ml PBS and 0.5% NP-40. Finally, the KED inclusion bodies (9.30 g wet weight, 1.60 g protein) were suspended in 50 ml of PBS.
  • the NP-40 washed KED inclusion bodies was analyzed by denatured SDS-PAGE. Contents of KED in the inclusion bodies were quantitated by scan of Comassie blue stained gel (77.2% KED).
  • To solubilize KED inclusion bodies 50 g of solid urea and 1.2 g of Tris were added into 50 ml of KED suspension. The pH was adjusted to 8.6, followed by adding of 80 ⁇ l Tween- 20, 160 ⁇ l 0.5 M EDTA and 950 ⁇ l b-mercaptoethanol. The solution was incubated at 37° C for 1 hour 30 min, and centrifuged for 20 minutesat 17,400 g at 23° C.
  • the resulted clear KED protein solution was carefully added (10 ml/min) in to 5:l of refolding solution containing 2.4 g of reduced glutathione (GSH). And that was immediately combined with another 5:l of refolding solution containing 0.5 g of oxidized glutathione (GSSG).
  • the 10:1 refolding solution used consisted of 1 kg L-arginine-HCI, 120 g Tris, 3.7 g EDTA, 10 ml of Tween-20 at pH 8.6. Refolding reaction was performed for 22h at 23° C.
  • Refolded KED solution was chilled on ice and concentrated to 650 ml by ultra-filtration using a regenerated cellulose membrane with 5 kDa weight cut-off (North Carolina SRT, Cary, NC). Precipitation of protein during ultra- filtration (16h) was excluded by centrifugation.
  • the concentrated KED protein solution was further concentrated to 32 ml in a dialysis tube (3,500 Da molecular weight cut-off) and solid polyethylene glycol (PEG-8000). The pH of concentrated KED protein solution (32 ml at pH 8.5) was changed to pH 7.4.
  • a quick passing of pi (pH 8.1) of KED was achieved by adding it drop by drop to 15 ml of 0.1 M sodium phosphate (pH 7.4) with simultaneous adding of 2N HCI to the mixture. Then, the KED protein solution was dialyzed against 5:l of 10 mM sodium phosphate (pH 7.4) containing 15 mM NaCl. Buffer was changed three times in 24 hours at 4 C. Dialyzed KED solution (145 ml) was centrifuged, and used for chromatography.
  • KED protein solution Twenty ml of dialyzed KED protein solution was diluted in 180 ml of 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. The protein solution was loaded on a Heparin-Sepharose column (HiTrap pre-packaged 5 ml column, Pharmacia, Piscataway, NJ). Columns were washed with the same buffer until the OD 280 returned to the base line. KED protein was eluted by a NaCl gradient (150-1000 mM). Pure KED protein eluted within 210-330 mM NaCl was identified by 10% polyacrylamide gel electrophoresis.
  • Acticlean Etox (Sterogene, Arcadia, CA) resins were cleaned with 1 M NaOH for 12 hours at 4° C, and packed to column. The column was washed with PBS until pH below 7.6. Pure KED protein was dialyzed against PBS, and loaded on Actilean Etox column. Each ml of Acticleas Etox gel was used for treatment 5 mg of KED protein. The KED protein was then sterilized by passing a 0.2 ⁇ m filter.
  • Bovine capillary endothelial cells (BCE cells, Folkman, 1996) were obtained for the proliferation assay (O'Reilly et al., 1997). Cells were washed with PBS and dispersed in a 0.05% trypsin solution. A cell suspension (25,000 cells/ml) was made with DMEM + 10% BCS and plated on 24-well culture plates (0.5 ml/well) and incubated at 37° C in 10% CO 2 for 24h. The medium was replaced with 0.5 ml DMEM + 5% BCS + 3 ng/ml recombinant bFGF and the test sample applied. After 72 hours , cells were dispersed in trypsin, resuspended and counted using a Coulter counter. Results were plotted as a dose-response and compared with other agents.
  • test compounds To determine the ability of the test compounds to block migration of endothelial cells toward the angiogenic factor bFGF, migration assays were performed in a modified Boyden chamber using BCE cells (Folkman, 1996). Cells were grown in DMEM supplemented with 10% FBS and supplemented at passage 15. To assess migration, the cells were starved overnight in DMEM supplemented with 0.1% BSA, harvested, suspended in DMEM/BSA, plated at 10 6 cells/ml on the lower surface of a gelatinized membrane (Nucleopore Corp., Plesanton, CA) in an inverted Boyden chamber and incubated for 1.5 hours to allow cell attachment.
  • a gelatinized membrane Nucleopore Corp., Plesanton, CA
  • DMEM with 0.1% BSA was used as a negative control and bFGF at 10 ng/ml was used as a positive control.
  • the rat corneal assay was performed as described (Folkman, 1996). Five ⁇ l Hydron pellets (Hydron Laboratories, New Brunswick, NJ) containing 10 or 20 ⁇ g/ml bFGF (25 or 50 ng) plus 5 or 25 ng test compound and 10 ⁇ g/ml sulcrafate were implanted in a corneal pocket of anesthetized rats. After 7 days, the animals were sacrificed, perfused with colloidal carbon, and the excised corneas were mounted for microscopy, photography and analysis.
  • the KED gene was constructed by fusion of cDNA plasminogen kringle 5 encoding Glu478-Ala562 with a collagen a1 (XVIII) cDNA fragment encoding His1154-Lys1336.
  • the KED gene (Fig 1) is expressed m E. coli cells.
  • AGA codon is a rare codon used in E.coli (Folkman, 1996).
  • the tRNA AGA is encoded by dnaY gene. The dnaY gene also has been reported to be essential for DNA replication (Folkman, 1996). To improve productivity of KED protein in E.
  • a vector pEXdnaY was constructed containing a strong T7 promoter for high-speed transcription of KED mRNA and a dnaY gene supplying tRNA AGA for efficient translation of KED product.
  • the process for KED gene fusion and cloning in pEXdnaY vector is shown in the Sequence Listing.
  • KED protein from pEXdnaY-hKED was induced by IPTG (isopropyl -D-thiogalactopyranoside). Abundant KED production was achieved. KED protein accumulated to 47% of total cellular protein by 2 hours induction with 0.5 mM IPTG (Fig 2). Optimal production of KED protein was performed using a fermentor. The KED protein was produced as inclusion bodies in cytosol. KED inclusion bodies were isolated and efficiently refolded in the presence of 0.5 M L-arginine at pH 8.6. As a control, human endostatin was simultaneously expressed from pEXdnaY-hED.
  • KED protein was obtained by HiTrap Heparin-Sepharose chromatography. Analysis of KED protein by denatured SDS-PAGE (polyacrylamide gel electrophoresis) revealed a single peptide of 30 kDa that agreed well with the molecular weight estimated from the deduced amino acid sequence (Fig 3).
  • Proliferation assay using bovine endothelial cells indicated that the fusion protein KED completely inhibited endothelial cell proliferation in a concentration-dependent fashion, with inhibitory activity as low as 8 nM (250 ng/ml) Human endostatin shows almost no activity at 40 nM (600 ng/ml) by the same assay (Fig 4a). These results indicate that the human fusion protein KED is a more potent inhibitor of endothelial cell proliferation than endostatin (O'Reilly et al., 1997).
  • Rodent corneal assay was performed to test the in vivo efficacy of KED fusion protein (Fig 5). 12.5 ng of KED were applied in the presence of 25 ng of bFGF. 12.5 ng of KED fusion protein (Fig. 5b) efficiently inhibited the growth of capillary blood vessel from corneal bed compared with control, containing bFGF alone (Fig. 5a).
  • the triple disulphide bond kringle structure represents a new family of angioginhibitory compounds.
  • Endostatin a highly basic protein fragment of collagen a1 (XVIII)
  • Other anti-angiogenic proteins such as platelet factor-4 (Folkman, 1996) and thrombospondin-1 (Folkman, 1996) also show high affinity with heparin. It is not yet known that how heparin affinity contributes to the anti-angiogenic function of these proteins. However, heparin affinity is a common characteristic among these proteins.
  • Proteins exhibiting heparin affinity seem to be a different class of angiogenic inhibitors compared to the kringle family.
  • Plasminogen kringle 5 is the most anti-angiogenic of the kringle structures.
  • Endostatin has the highest anti-angiogenic activity within the heparin affinity proteins. In this study, these two anti-angiogenic members were fused into a single molecule.
  • a cDNA fragment of 0.82 kb consisting of human plasminogen kringle 5 for the 5- prime end followed by the endostatin coding sequence was constructed. The cDNA has been successfully over-expressed in E. coli. The expressed product has been confirmed to be the designed fusion form, based on N- terminal sequencing and amino acid compositional analysis.
  • the kringles of the present invention can be: (i) Administered to tumor- bearing humans or animals as anti-angiogenic therapy; (ii) Monitored in human or animal serum, urine, or tissues as prognostic markers; and (iii) Used as the basis to analyze serum and urine of cancer patients for similar angiostatic molecules. It is contemplated as part of the present invention that the kringles can be isolated from a body fluid such as blood or urine of patients or the kringles can be produced by recombinant DNA methods or synthetic protein chemical methods that are well known to those of ordinary skill in the art. Protein purification methods are well known in the art and a specific example of a method for purifying the kringles, and assaying for inhibitor activity is provided in the examples below. Isolation of human endogenous kringles is accomplished using similar techniques.
  • One example of a method of producing kringles using recombinant DNA techniques entails the steps of identifying and purifying kringles as discussed above, determining the N-terminal amino acid sequence of the purified inhibitor, synthetically generating 5' and 3' DNA oligonucleotide primers for the kringle sequence, amplifying the kringle gene sequence using polymerase, inserting the amplified sequence into an appropriate vector such as an expression vector, inserting the gene containing vector into a microorganism or other expression system capable of expressing the inhibitor gene, and isolating the recombinantly produced inhibitor.
  • Appropriate vectors include viral, bacterial and eukaryotic (such as yeast) expression vectors.
  • the gene for these kringles can also be isolated from cells or tissue (such as tumor cells) that express high levels of the kringles by isolating messenger RNA from the tissue, using reverse transcriptase to generate the corresponding DNA sequence and then using the polymerase chain reaction (PCR) with the appropriate primers to amplify the DNA sequence coding for the active kringle amino acid sequence.
  • PCR polymerase chain reaction
  • Yet another method of producing kringles, or biologically active fragments thereof, is by protein synthesis. Once a biologically active fragment of a kringles is found using the assay system described more fully below, it can be sequenced, for example by automated protein sequencing methods.
  • the DNA sequence can be determined using manual or automated sequencing methods well know in the art.
  • the nucleic acid sequence provides information regarding the amino acid sequence.
  • the fragment can be synthesized by techniques well known in the art, as exemplified by "Solid Phase Protein Synthesis: A Practical Approach" E. Atherton and R. C. Sheppard, IRL Press, Oxford, England.
  • multiple fragments can be synthesized which are subsequently linked together to form larger fragments.
  • These synthetic protein fragments can also be made with amino acid substitutions at specific locations to test for agonistic and antagonistic activity in vitro and in vivo.
  • Protein fragments that possess high affinity binding to tissues can be used to isolate the receptor on affinity columns. Isolation and purification of the receptor is a fundamental step towards elucidating the mechanism of action of the kringles. Isolation of a kringle receptor and identification of the agonists and antagonists will facilitate development of drugs to modulate the activity of the receptor, the final pathway to biological activity. Isolation of the receptor enables the construction of nucleotide probes to monitor the location and synthesis of the receptor, using in situ and solution hybridization technology. Further, the gene for the receptor can be isolated, incorporated into an expression vector and transfected into cells, such as patient tumor cells to increase the ability of a cell type, tissue or tumor to bind and inhibit local angiogenesis.
  • the kringles of the present invention are effective in treating diseases or processes that are mediated by, or involve, angiogenesis.
  • the present invention includes the method of treating an angiogenesis mediated disease with an effective amount of these kringles, or a biologically active fragment thereof, or combinations of angiostatin fragments that collectively possess anti-angiogenic activity, or agonists and antagonists.
  • the angiogenesis mediated diseases include, but are not limited to, solid tumors; blood born tumors such as leukemias; tumor metastasis; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation.
  • solid tumors such as leukemias
  • tumor metastasis such as leukemias
  • benign tumors for example hemangiomas, acoustic
  • the kringles of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, Crohn's disease, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids. These kringles can be used as a birth control agent by preventing vascularization required for embryo implantation. Kringles are useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helicobacter pylori). The synthetic protein fragments of these kringles have a variety of uses.
  • the protein that binds to the receptor with high specificity and avidity is radiolabeled and employed for visualization and quantitation of binding sites using autoradiographic and membrane binding techniques.
  • This application provides important diagnostic and research tools. Knowledge of the binding properties of the receptor facilitates investigation of the transduction mechanisms linked to the receptor.
  • labeling these proteins with short lived isotopes enables visualization of receptor binding sites in vivo using positron emission tomography or other modern radiographic techniques to locate tumors with the binding sites.
  • the tPA kringle 2 carrying mutation of His244 ⁇ Tyr is fused to collagen ⁇ 1 (XVIII) C-terminus.
  • the collagen ⁇ 1 (XVIII) C-terminus flanked by Ndel and BamHI adaptors are cloned into pEXdnaY vector.
  • cDNA fragment encoding tPA kringle 2 mutant H with Ndel adaptor in both ends are fused in the front Ndel site of collagen ⁇ 1 (XVIII) C-terminus fragment, generating plasmid pEXdnaY-hHED.
  • the estimated fusion HED 1 protein MW is 30.087 kDa.
  • nude rats (Cr:NIH-rnu) harboring intracranial human glioma xenografis (U251 Mgn) are treated with daily oral doses of KED for a period of 28 days beginning 1 week following implantation. Beginning 2 weeks following implantation, 8 animals in each of the control and treatment groups and are sacrificed for histochemical and in situ detection protocols for quantitation. Subsequent groups of 8 animals in control and treatment groups are sacrificed weekly for a total of 4 sessions, a total of 6 weeks following implantation. An active drug concentration is determined in initial studies and dosage is adjusted if necessary, before proceeding.
  • the U251 MG cell line is originated by Ponten from a patient with a glioblastoma. The culture is initially mycoplasma positive and is cured by antibiotic treatment in culture (negative since 1987). U87MG is a cell line that is initiated from an anaplastic astrocytoma. All cell lines are routinely mycoplasma tested and have tested free of contamination. Cells are routinely maintained in DMEM +10% fcs in a 37°C humidiated incubator in 10% CO 2 .
  • Intracranial nude rat xenograft Intracranial nude rat xenograft:
  • nude rats are inoculated intracerebrally as follows:
  • Cells are harvested from 90% confluent culture plates by treatment with a 0.1% trypsin/EDTA for 10 minutes. Cells are rinsed off the plates with media and spun for 10 minutes at 1000 rpm. The cell pellet is resuspended in 100 ⁇ L of culture media, counted and the concentration of cells adjusted to 5 x 10 5 5 ⁇ L. Viability of the cells is tested using Trypan blue exclusion counting on a Hausser Scientific Brightline hemocytometer. Implantation:
  • Animals are anesthetized with 100 mg/kg ketamine, 1.5 mg/kg xylazine, and 0.05 ml atropine i.m.
  • the surgical zone is swabbed with betadine solution, the eyes coated with lacritube and immobilized in a small animal stereotactic device (Kopf, Cayunga, CA) After draping, a 1 cm incision is made 2 mm to the right of the midline 1 mm retro-orbitally, the skull exposed with cotton-tip applicators and a HP-4 dental drill bit is used with a micromanipulator to drill a hole 3 mm to the right of the bregma, taking care not to penetrate the dura.
  • a #270 0 ⁇ L Hamilton syringe with a #4 point, 26s gauge needle containing tumor cell suspension [500.000 viable cells] is lowered to a depth of 3.5 mm, then raised to a depth of 2.5 mm.
  • the cell suspension is injected stepwise at a rate of 0.5 ⁇ L/10 seconds until the entire volume had been injected.
  • careful note is made of any reflux from the injection site.
  • the surgical zone is flushed at least twice with sterile saline and the hole sealed with bone wax.
  • the skull is swabbed with Betadine before suturing the skin over the injection site. The animals are maintained for 1 week prior to treatment.
  • CSF is removed via suboccipital puncture for HPLC [KED] using a 20 g needle; and after a blood specimen is taken, cardiac perfusion is performed with 250 mL sterile saline followed by fixation with 250 mL of 10% buffered formalin phosphate. Finally, the brain is removed and immersed in formalin for 24 hours. It is then placed in a 200-400 g coronal rat-brain matrix (Activational Systems Inc., Warren, Ml) and cut into 1 mm blocks (same slice thickness as MRI) and subsequently imaged for histologic registration to MRI. Blocks grossly containing and adjacent to tumor are processed and paraffin embedded.
  • the plane of section is made to correspond to that of the MRI, using anatomical landmarks as closely as possible.
  • the embedded blocks are cut into serial 8 ⁇ m sections onto uncoated slides for routine hematoxylin and eosin (H&E) staining and onto poly-L-lysine coated slides for immunohistochemical and ISH.
  • H&E hematoxylin and eosin
  • In situ hybridization is carried out as described (Appendix, SD). For this 8 ⁇ m adjacent serial sections are cut and mounted on pretreated glass slides. Pretreatment of glass slides included acid-cleaning, treatment with Denhardt's medium, acetylation and dipping in dilute Elmer's glue solution (1 :100). Slides are deparaffinized in 3 changes of 100% xylene and rehydrated in graded concentrations of water in ethanol, air dried and stored at room temperature.
  • the human Alu probe Blur 2 from Dr. Carl Schmid of the
  • Random primed probes are labeled with UTP-digoxtgenin (dig) and visualized with an anti-digoxigenin polyclonal antibody conjugated to alkaline phosphatase are prepared as outlined in the Boehringer-Mannheim GeniusTM Nonradioactive DNA Labeling and Detection Kit (Cat. No. 1093 657).
  • the slides are placed in 200 mM HCI for 20 minutes followed by a brief dip in H 2 O, transferred to 2xSSC at 70°C for 30 minutes, followed by a brief dip in H 2 O and incubated for 15 minutes at 37°C in 1 mg/ml proteinase K in 20 mM Tris with 2 mM CaCI 2 , pH 7.4, followed by two 5-minute washes in H 2 O.
  • To link DNA to the nucleus they are incubated in neutral 5% paraformaldehyde for 1 hour, then washed twice in 2xSSC for 5 minutes to block non-specific binding sites.
  • the slides are then prehybridized at room temperature in the following cocktail: 5xSSC.
  • the sample is hybridized overnight at room temperature with 0.4 ng/ ⁇ l dig-labeled 300 bp Alu probe in the cocktail used for prehybridization.
  • the sample is then washed twice in 2xSSC for 5 minutes each, followed by two washes at 55°C in 2xSSC for 15 minutes each.
  • Immunological detection of the dig-labeled probe is done with dig-specific antiserum is performed as described in the GeniusTM Kit. Stained slides are digitized for quantitation.
  • KED placebo is saline
  • the primary endpoint is tumor volume, the margins measured as the infiltration border.
  • 64 nude rats are inoculated intracerebrally with tumor cells. After one week of maintenance, half the rats are randomly treated with KED and half will receive saline. Eight randomly selected animals from each treatment group is sacrificed at 1 week intervals following treatment and the volume of tumor is measured.
  • colon cancer bv KED Treatment of colon cancer bv KED: Cell lines
  • Parental colon cancer cell line LS 174T was derived from a well- differentiated Human colonic adenocarcinoma (Moose et al., 1996), and has been characterized extensively, including its ability to product mucin in vitro (Standker et al., 1997), and when grown subcutaneously in athymic nude mice (Patthy, 1985). Cells injected into the cecal wall of nude mice produce well-differentiated primary colon cancers with little metastic ability (O'Reilly et al., 1993).
  • LS LiM 6 a derivative of LS174T with high liver-metastasizing ability during cecal growth, was established by serially selecting cells which metastasized from cecum to liver as previously described (O'Reilly et al., 1993). These cells form well-differentiated mucinous carcinomas in nude mice, with large glands containing PAS-positive (Folkman, 1996) mucin comprising ⁇ 50% of the tumor area.
  • HM7 and LM12 are variants of LS174T which produce high and low amounts of mucin, respectively.
  • HM7 secretes approximately twofold more mucin in culture and contains twofold more intracellular mucin compared with LS174T, whereas LM12 produces ⁇ 30% less mucin overall compared with the parental line (Standker et al., 1997).
  • HM7 formss well differentiated tumors in nude mice, with prominent glands containing PAS-positive mucin comprising 40-50% of the tumor area.
  • LM12 xenografts contain fewer mucin-containing glands comprising ⁇ 10% of the tumor area (O'Reilly et al., 1997).
  • Tumor cells were grown to confluency in 75-cm 2 tissue culture flasks, harvested as described for cecal injection, and resuspended in serum-free DMEM at a concentration of 10 7 cells/ml.
  • Athymic nude mice were anesthetized with methodyfluorance by inhalation, prepared sterilely, and the spleen exteriorized through a flank incision.
  • the immediate fate of tumor cells after splenic injection was determined by injection of IdUR-labeled tumor cells.
  • Cells were grown as above and 0.5 ⁇ Ci/ml [ 125 l] IdUR was added to culture medium 24 hours before harvesting (Goldhaber et al., 1988). This concentration of isotope labels >95% of tumor cells without affecting viability.
  • Cells were prepared and injected into the splenic pulp as described above. Animals were sacrificed 10 minutes after injection, and livers removed. The radioactivity present in the spleen (removed after injection), liver, and remaining organs of each animal was determined by gamma-counting in a model 7000 counter (Beckman Instruments Inc.)
  • Tumor cells were grown to 50% confluency in 7-5-cm 2 tissue culture flasks as described above. The tissue culture medium was then aspirated and replaced with fresh medium containing 2 mM benzyl- ⁇ -N-acetylgalactosamine (benzyl- ⁇ -GalNAc) (Bennett et al., 1991). Cells were harvested 48 hours later and liver colonization assayed after splenic injection as above.
  • benzyl- ⁇ -GalNAc 2 mM benzyl- ⁇ -N-acetylgalactosamine
  • Growth of the recombinant strain is accomplished by making five Amp/Cm plates using the following: a. 200 ml water; b. 2 g tryptone; c. 1 g yeast extract; d. 1 g NaCl; and e. 3 g agar. Next, these compounds are mixed. The pH is then adjusted to 7.4 using 2N NaOH. The mixture is then autoclaved at 120 degree C for 15 min. Then keep it in a 50 degree C water bath for 1 hour. Next, add 0.2 ml of 100mg/ml ampicillin, the ampicillin should be dissolved in water. After this, add 0.2 ml of 35 mg/ml chloramphenicol the chloramphenicol should be dissolved in ethanol.
  • the first step requires dissolving 85 LB and 200 g of yeast extract in 10 1 of water, this equals the components of YEEM.
  • vitamin solution includes the following: 6 g/l thiamin; 3 g/l pyridoxine; 2.7 g/l DL-pantothenic acid; 0.7 g/l pyridoxine; 0.2 g/l riboflavin; 0.03 g/l biotin; 0.02 g/l folic acid.
  • metal solution add 20 ml of metal solution.
  • the metal solution includes the following: 75 g/l Na 3 Citrate; 30 g/l FeCI 3 ; 2 g/l CoCI 2 .; 2 g/l Na 2 MoO 4 ; 2 g/l CuSO 4 ; 1.5 g/l MnCI 2 ; 1.5 g/l ZnCI 2 ; 1 g/l CaCI 2 ; 0.5 g/l H 3 BO 3 . Then begin agitation at 500 rpm, pump air at 20 l/min/l culture. Add antifoam 0.5ml/hour. When 12 ⁇ OD ⁇ 15 at 600 nm, add 0.3 I of 50% glucose. When 20 ⁇ OD ⁇ 25 at 600 nm, add 0.3 I of 50% glucose.
  • membrane 0.04 micron polyethersulfone; pressure: 1 to 5 psi; temperature: 4 degree C. Concentrate to 5% of the original volume. Add equal volume of PBS (10 mM sodium phosphate buffer containing 100 mM of NaCl at pH 7.5). Concentrate to 50% volume. Repeat 10 times the previous two steps. (Caution: do not get cells by centrifugation.) The cell suspension can be frozen until uses. Membrane can be regenerated in 0.1 N NaOH for 3 hours.
  • Centrifugation is utilized for obtaining inclusion bodies. Maintain a temperature of 4 degree C. Centrifuge at 10000 x g for 30 min. Keep the pellets, because the pellets are crude product, and the pellets are called as "KED inclusion bodies". Do not measure the protein concentration of inclusion bodies. Usually, measurement of protein concentration of inclusion bodies is under estimated. Measure the wet weight of inclusion bodies. Freeze the inclusion bodies (crude product) in -80 degree C.
  • Concentration of the inclusion bodies is determined using the membrane system as follows: membrane: 0.1 micron polyethersulfone; pressure: 1 to 5 psi; and temperature: 4 degree C. Then concentrate to 10% of the original volume. Add Tris buffer (100 mM Tris at pH 8.5) to bring the volume to the original. Repeat the above steps five times. Obtain final concentration of inclusion bodies 0.1 to 0.25 wet weight / ml Tris buffer. Keep the inclusion bodies in the Tris buffer at -20 degree C as needed.
  • KED protein solution Twenty ml of dialyzed KED protein solution was diluted in 180 ml of 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. The protein solution was loaded on a Heparin-Sepharose column (HiTrap pre-packaged 5 ml column, Pharmacia, Piscataway, NJ). Columns were washed with the same buffer until the OD 280 returned to the base line. KED protein was eluted by a NaCl gradient (150-1000 mM). Pure KED protein eluted within 210-330 mM NaCl was identified by 10% polyacrylamide gel electrophoresis.
  • KED protein solution was diluted in 180 ml of 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl.
  • the protein solution was loaded on a Heparin-Sepharose column (HiTrap pre-packaged 5 ml column, Pharacia, Piscataway, NJ). Column was washed with the same buffer until the OD 280 backs to the base line.
  • KED protein was eluted by a NaCl gradient (15-1000 mM). Pure KED protein eluted within 195-345 mM NaCl was identified by 10% polyacryl amide gel electrophoresis.
  • Acticlean Etox (Sterogene, Arcadia, CA) resins were cleaned with 1 M NaOH for 12 hours at 4 °C, and packed to column. The column was washed with PBS until pH below 7.6. Pure KED protein was dialyzed against PBS, and loaded on Actilean Etox column. Each ml of Acticleas Etox gel was used for treatment 5 mg of KED protein. The KED protein was then sterilized by passing a 0.2 microm filter.
  • PMT includes the following: PBS containing 5% non-fat milk & 0.05% Tween-20 with an incubation time of 1 hour and an incubation temperature at 37 degree C. Then incubate with rabbit anti recombinant KED IgG at a concentration of 1500 to 2000 times. The incubation time should equal 1 hour with an incubation temperature of 37 degree C. Wash three times with PMT, each wash should be for 10 minutesat 37 degree C. Then incubate with rabbit anti-lgG antibodies 1 : 2000. The anti-lgG is peroxidase conjugated and incubation time is equal to 1 hour with an incubation temperature of 37 degree C. Again wash three times with PMT as before.
  • Harvesting of cells by membrane includes the following: membrane: 0.04 micron polyethersulfone at a pressure of 1 to 5 psi and a temperature of 4 degree C. Then wash cells by PBS (10 mM sodium phosphate buffer containing 100 mM of NaCl at pH 7.5). Repeat washing 3 times. Repeat 5 washing reduces endotoxin contents in the final products. Endotoxin secreted in medium from cells can be excluded by membrane system. Regenerate membrane in 0.1 N NaOH for 3 hours.
  • Stage of inclusion bodies o Method I Centrifugation occurs at a temperature of 4 degree C, at a speed of 10000 x g for 30 min. Then incubate the pellets (crude product) in liquid nitrogen for 10 min. Suspend the crude product in PBS (10 mM sodium phosphate buffer containing 100 mM of NaCl at pH 7.5). Next, adjust the concentration of inclusion bodies at 0.1 to 0.25 g wet weight / ml PBS.
  • Method III French Press Cells in a single pass.
  • Method IV The membrane system involves adding pre-cooled (4 degree C ) PBS the ten times of original volume.
  • the membrane is as follows: 0.1 micron polyethersulfone at a pressure of 1 to 5 psi with a temperature of 4 degree C. Then wash with PBS buffer.
  • Bovine capillary endothelial cells (BCE cells) were obtained for the proliferation assay, cells were washed with PBS and dispersed in a 0.05% trypsin solution. A cell suspension (25,000 cells/ml) was made with DMEM + 10% BCS and plated on 24-well culture plates (0.5 ml/well) and incubated at 37 degree C in 10% CO2 for 24hr. The medium was replaced with 0.5ml DMEM +5% BCS + 3ng/ml recombinant bFGF and the test sample applied. After 72hr, cells were dispersed in trypsin, resuspended and counted using a Coulter counter. Inhibitory activity of test compounds was expressed as a percentage inhibition of endothelial cell growth in culture compared to endothelial cells in the absence of the test compound.
  • test compounds To determine the ability of the test compounds to block migration of endothelial cells toward the angiogenic factor bFGF, migration assays were performed in a modified Boyden chamber using BCE ceils. Cells were grown in DMEM supplemented with 10% FBS and supplements at passage 15. To assess migration, the cells were starved overnight in DMEM supplemented with 0.1% BSA, harvested, suspended in DMEM/BSA, plated at 10 6 cells/ml on the lower surface of a gelatinized membrane (Nucleopore Corp., Plesanton, CA) in an inverted Boyden chamber and incubated for 1.5 hours to allow cell attachment. The chambers were then righted, test material was added to the top well and incubated for 5 hours.
  • a gelatinized membrane Nucleopore Corp., Plesanton, CA
  • Membranes were then fixed and stained and the number of cells migrating to the top of the filter in 10 high-powered fields were counted.
  • DMEM with 0.1% BSA was used as a negative control and bFGF at 10 ng/ml was used as a positive control.
  • Proliferation assay using bovine endothelial cells indicated that fusion protein KED completely inhibited endothelial cells migration at concentration as low as 8 nM (250 ng/ml). Where, human angiostatin and endostatin show almost no activity at 12 nM (250 ng/ml) by the same assay. The observed IC 50 of KED is less than 100 ng/ml. IC 50 of human endostatin in the assay was 350 ng/ml. These results indicated that the human fusion protein KED is much stronger than endostatin, the strongest anti-angiogenic agent reported to date.
  • the corneal assay was performed. 5 uL hydron pellets (Hydron Laboratories, New Brunswick, NJ) containing 50 ng bFGF or 5 ng KED protein and 10 ug/ml sulcrafate were implanted in a corneal pocket of anesthetized rats. After 7 days, the animals were sacrified, perfused with colloidal carbon, and the excised corneas were mounted for microscopy analysis.
  • the rats are anesthetized with Nembutal (29mg/kg body weight) being sure carefully clip all whiskers around the snout.
  • Nembutal 29mg/kg body weight
  • the rats are anesthetized with Nembutal (29mg/kg body weight) being sure carefully clip all whiskers around the snout.
  • Using the tip of the scalpel blade position the tip in the center of the cornea and make a 1-2mm incision into but not through the cornea.
  • iris spatula place the tip under the lip of the incision and begin to gently but firmly blunt dissect in a slightly downward direction, through the stroma to a point approximately 1- 1.5mm from the limbus. If necessary bend the iris spatula so it has a slight downward curve to it. Avoid getting too close to limbal vessels. This is the most frequent cause of non-inflammatory, false positive responses. Moisten one of Hydron pellets and using curved Dumont forceps place it near the opening of the pocket and under the scope inset it into the top of the pocket. Use your spatula to gently force the pellet down to the base of the pocket. The pellet should occupy not more than one-half the depth of the pocket.
  • the implant should occupy no more than three- fourths of the pocket. Anything larger will be extruded or the pocket will fail to seal.
  • Each 100 ml of solution contains: 600 mg of Sodium chloride; 310 mg of Sodium lactate: 4.61 ml of 60% solution; 30 mg Potassium chloride; 20 mg calcium chloride; and has a final ph 6.5 - 7.0.
  • Add sodium heparin at a final concentration of 5U/ml for perfusion. Prepare at least 2 liters of the above solution, because approximately 100-200 ml are needed for perfusion of a single 100-200 g rat.
  • the Hydron is supplied in the form of a fine powder. Make a 12% solution; (ordinarily 1 gm lots in 8 mis or 3 gm lots in 25 mis), in absolute alcohol (pure ethyl alcohol USP). To dissolve, leave at 37°C for 24 hours in a rolling apparatus (e.g., Forma-Scientific CEL-roll) or a shaker at low speed. No preservative is necessary. Store at room temperature. Incorporation of Proteins in Polymer:
  • the desired weight of lyophilized protein is added to the hydron in solution and mixed in a vortex mixer.
  • various amounts of the polymer can be pipetted into wells or troughs of the desired shape and size. Discs are good for release studies).
  • the slides Dry in a dessicator overnight.
  • the slides are placed in sterile petri dishes overnight in a laminar flow hood.
  • Rehydrate by placing a drop of sterile lactated Ringer's solution on the pellet or in the trough. The hydron will swell up to a certain extent. Wait for at least 5 minutes, when most of the rehydration has occurred. The pellets can then be pulled with a tweezer or a fine spatula. Note the release starts virtually immediately on rehydration. Thus, if one needs to quantitate, zero time must be consistent.
  • the protein to be used is in solution, pipette the desired quantity of aqueous solution over the alcoholic hydron. Do not use more than 6 parts of aqueous solution to 4 parts of hydron; otherwise, the hydron will precipitate.
  • For the corneas one-to-one ratios of conditioned media and hydron are used. Mix in vortex, then will well, and put on slides or troughs as explained in Method I. Note that with this method, one ends up with less hydron, thus faster release.
  • nude mouse harboring human glioma (U251MGn) were treated with KED for a period of
  • This cancer cell line was originated by P ⁇ nten from a patient with a glioblastoma.
  • the culture was initially mycoplasma positive was cured by 5 antibiotic treatment in culture.
  • the cell line was initiated from an anaplastic astrocytoma.
  • nude mouse were inoculated with cancers as follows.
  • the cell suspension was injected stepwise at a rate of 0.5 :L/10 seconds until the entire volume had been injected. During and after the injection, careful note was made of any reflux from the injection site. Animals were maintained for 1 week prior to treatment.
  • KED Treatment This was a two-sample, placebo-controlled trial in tumor-bearing mouse of KED TREATMENT, where the placebo was saline, the drug group was suspension solution, the vehicle for administration of KED.
  • the primary endpoint is tumor volume, the margins measured as the infiltration border.
  • CB cathepsin B
  • MMP-2 Matrix metalloprotease-2
  • TIMPs endogenous tissue inhibitors of metalloproteinases
  • the plasminogen activator system also appears to be up-regulated, particularly in malignant gliomas.
  • the acquisition of cellular motility and angiogenesis are felt to be central to the process of glioma invasion.
  • Cell adhesion molecule and extracellular matrix alterations have been described in glioma model systems and, interestingly, laminin, a molecule not normally present in the brain except at the glia limitans and in the perivascular space, is upregulated in the presence of invading glioma cells, produced by surrounding normal astrocytes.
  • VEGF autocrine motility factor
  • bFGF vascular endothelial growth factor
  • Human fetal brain mRNA was used tor isolation of tPA kringle domains. Reverse transcription was carried out in 20 ⁇ l reaction solution containing 5 ⁇ g of human fetal brain mRNA (Clontech), 250 ng of oligo (dT) 17 primer, 5 mM DTT, 2.5 mM MgCI, 450 ⁇ M of dNTP and 200 units of reverse transcriptase (BRL) in 50 mM Tris buffer (pH 8.3). Briefly, reaction mixture without reverse transcriptase was heated at 72°C for 10 minutes to denature mRNA and was quickly chilled on ice for 15 minutes. After 5 minutes incubation of the contents at 42°C, an hour reaction was initiated by adding of reverse transcriptase.
  • reaction mixture without reverse transcriptase was heated at 72°C for 10 minutes to denature mRNA and was quickly chilled on ice for 15 minutes. After 5 minutes incubation of the contents at 42°C, an hour reaction was initiated by adding of reverse transcriptase.
  • the resulted cDNA from above reaction mixture were used for amplification of tPA kringles by using a proofreading thermostable Pfu DNA polymerase (Stratagene, La Jolla, CA).
  • the PCR reaction solution (100 ⁇ l) contains 20 mM Tris (pH 8.0), 2 mM MgCI, 10 mM KCI, 0.1% Triton X-100, 5 ⁇ l of cDNA, 150 ng of each primer and 7.5 units of Pfu DNA.
  • the PCR process was initiated by "hot start" (Ault et al., 1994).
  • Kringle 1 was amplified by a 5'-end primer of GGAATTC-[Ndel]- ATAGATACCAGGGCCACGTGCTACG, and a 3'-end primer of CCG- [BamHI]- TTAGTTTCCCTCAGAGCAGGCAGG.
  • Kringle 2 was amplified by a set of primers: GGAATTC-[Ndel]-AACAGTGACTGCTACTTTGGG for 5'-end and CCG-[BamHI]-TTAGGTGGAGCAGGAGGGCACATC for 3'-end.
  • a DNA fragment containing both of kringles was also amplified by a 5'-end primer of GGAATTC-[Ndel]-ATAGATACCAGGGCCACGTGCTACG, and a 3'-end primer of CCG-[BamHI]-TTAGGTGGAGCAGGAGGGCACATC.
  • GGAATTC-[Ndel]- and CCG-[BamHI]- are adaptors containing Ndel and BamHI recognition sites.
  • the PCR products containing the tPA kringle sequences were purified through the PCR Select-ll column (5 Prime-3 Prime, Boulder, CO).
  • the purified DNA fragments were digested by restriction enzymes of Ndel and BamHI at 37°C for 2 hours.
  • the digested DNA fragments with Ndel and BamHI cohesive ends were purified by phenol extraction, ethanol precipitated and used to insert into pET11a vector.
  • the pET11a vector was linearized by the same enzymes of Ndel and BamHI.
  • the linearized vector was further treated by calf intestinal alkaline phosphatase (CIAP) to prevent self-ligation of vector inter or intra molecule.
  • the dephosphorylated vector was ligated with tPA kringle fragments.
  • the ligation solution was used to transform competent E. coli TG1 cells. Recombinant plasmids were analyzed by double digestion of Ndel and BamHI enzyme. Recombinant plasmids were confirmed by DNA sequencing of dideoxy chain termination (Sanger et al., 1977).
  • Mutagenesis was carried out using recombinant plasmid pK2 tPA containing the wild type tPA kringle 2 cDNA.
  • a PCR-mediated mutation kit, Quick-Change (Stratagene, La Jolla, CA) with some modification was employed for the process. Fifty ⁇ l PCR reaction mixture was prepared on ice.
  • the mixture contains 20 mM Tris (pH 8.8), 10 mM KCI, 10 mM (NH 4 ) 2 SO 4 , 2 mM Mg SO 4 , 0.1% Triton X-100, 100 ng pure super-coiled pK2 tPA DNA, 200 ng each of sense and complementary mutation oligonucleotide primers, 200 ⁇ M of dNTP and 5 units of cloned Ptu DNA polymerase.
  • a control PCR reaction was simultaneously included in the thermal cycles by missing of both of sense and complementary mutation primers. Each PCR cycle consisted of denaturation at 95°C for 0.5 minutes, annealing at 55°C for 0.5 minutes, and extension at 68°C for 18 minutes, for a total of 12 cycles.
  • Oligonucleotide primers used for mutation PCR are listed. The mutation sites are underlined. Residue numbers are according to the amino acid sequence of tPA.
  • GEf-7 sense, 5'-GTGCCCAGGCACTGGAACTGGGCAAACATAAT-3' GEr-8, anti-sense, 5'-ATTATGTTTGCCCAGTTCCAGTGCCTGGGCAC-3'
  • E. coli BL21 (DE3) cells were co-transformed with a low-copy plasmid pDC952, a derivative of pACYC184 containing argU gene.
  • pDC952 a low-copy plasmid pDC952
  • pACYC184 a derivative of pACYC184 containing argU gene.
  • Ten colonies with ampicillin and chloramphenicol resistance for each construct were selected for gene expression analysis. Each single colony was inoculated in 2 ml of LB medium containing 100 ⁇ g/ml ampicillin and 35 ⁇ g/ml chloramphenicol.
  • Cells expressing tPA kringles were washed by ice-cold PBS solution three times in order to remove endotoxin secreted by the E. coli cells.
  • the washed cells were re-suspended in PBS solution containing 1 mM EDTA, flowed by passing through a French Press Cells (SLM Instruments, Urbana, lL) twice at 20,000 psi.
  • the inclusion bodies were collected by centrifugation of the cell lysate by 10,000 g at 4°C for 20 minutes.
  • the inclusion bodies were washed three times by ice-cold PBS containing 0.5 NP-40 detergent (Sigma, St. Louis, MO).
  • Sephadex G-75 Chromatography Super-fine Sephadex G-75 (Pharmacia) of 10 g was suspended in 100 mM Tris (pH 8.6) containing 1 mM EDTA and 8 M urea at 37°C for 24 hours. The resins were packed on several columns (250 nM x 15 nM). The columns were kept at 28-30°C for preventing the precipitation of urea. Inclusion bodies were dissolved in I00 mM Tris (pH 8.6) containing 8 M urea. 1 mM EDTA, 0.1% Tween-20 detergent, 145 mM of ⁇ -mercaptoethanol at the concentration of 10 mg/ml of proteins.
  • the reduced tPA kringles were added into refolding solution containing 100 mM Tris (pH 8.8), 1 mM EDTA, 0.1% Tween-20, 0.5 M L-arginine, 1 mM of reduced gluthionine (GSH) and 0.1 mM oxidized gluthionine (GSSG).
  • the protein concentration in the refolding solution was adjusted to 100 mg/l.
  • the refolding reaction was performed at 23 °C for 24 hours. 6. Lysine-Agarose Affinity Chromatography
  • the refolded protein solution was concentrated by using an ultra- filtration system consisting of TRIPORT Module/316L Stainless Plates with 0.1 m 2 working surface and a regenerated cellulose membrane molecular of 5 kDa weight cut-off (North Carolina SRT). The filtration was performed at pressures of 25 to 30 psi.
  • the concentrated protein solution was diluted into 100 mM Tris (pH 8.0) pre-cold at 4°C to a final concentration of 25 mM of L- arginine.
  • the diluted protein solution was then applied on a lysine-agarose column pre-equilibrated with the same 100 mMTris at pH 8.0. Approximately 1 mg protein was loaded per one ml of lysine-agarose gel.
  • the column was continued by washing using the same buffer until OD of eluant declines to zero at 280 nM.
  • the tPA kringles were eluted from the column by a strong lysine competitor of 6-amino-n-caproic acid using a gradient from zero to 200 mM. Each traction eluted from lysine column was monitored at 280 nM. Samples from each peak were analyzed by 16% SDS-PAGE
  • the pooled pure recombinant tPA kringles were dialyzed against 1000 times of PBS (v/v). Since E. coli cells secrete endotoxin, preventing contamination of tPA kringles from endotoxin was performed by using Acticlean-Etox (Sterogene, Arcadia, CA) chromatography. Acticlean-Etox resins were previously washed by 1 M NaOH for 12 hours at 4°C and packed into column. The column was washed using PBS until pH was below 7.6 before passing the recombinant tPA kringles. One ml of Acticlean-Etox gel was used for every 10 mg of tPA kringles. Finally, the recombinant kringles were sterilized by a 0.2 ⁇ m filter.
  • Protein concentration was determined by a protein assay kit (Bio-Rad
  • Protein samples were prepared by heating to 100°C for 10 minutes in SDS sample buffer. SDS-polyacryl amide gel electrophoresis (PAGE) was performed as described (Laemmli, 1970). Expression of protein was quantified by scanning and computer analysis using Alphalmage200 (Alpha Innotech Corp.).
  • BCE bovine capillary endothelial
  • the PCR products analyzed by 2% agarose gel and EtBr-staining revealed that only a single PCR product in each reaction, indicating specific amplification performed by the hot start PCR process.
  • the estimated PCR products encoding kringle 1 , kringle 2, and kringle 1 + 2 are 304 bp. 286 bp and 550 bp, respectively.
  • the sizes of each PCR product agreed well with those expected from tPA DNA sequence.
  • the tPA kringles were cloned and inserted into pET11a vector for protein expression under the control of a strong T 7 phage promoter and the laclq repressor.
  • Recombinant plasmid pK1 tPA encodes r-[K1 tPA ], where r stands for recombinant and [Kl tPA ] represents amino acid sequence residues [Ile86-Asn177] of kringle 1 domain in the tPA molecule.
  • the recombinant plasmid pK2 tPA encodes r-[K2 tPA ] representing amino acid residues [Asnl77- Thr263] of the tPA kringle 2 domain.
  • Recombinant plasmid pKI2 tPA encodes a r-[KI2 1PA ] protein containing kringle 1 and kringle 2 domain, an internal fragment of [Ile86-Thr263] in the tPA protein sequence.
  • Recombinant proteins expressed in E.coli were analyzed by 16% polyacrylamide gel. Whole cellular proteins of 50 ⁇ g were denatured by SDS and ⁇ -mercaptoethanol and loaded gel for electrophoresis. Abundant expression of a 10 kDa protein was detected from the cells carrying pK2 tPA construct. 10 colonies of pK2 tPA construct were chosen, and all ten expressed the same 10 kDa protein. A 20 kDa protein was found from the cells harboring pK12 tPA plasmid. All ten clones of pK12 tPA expressed the 20 kDa protein.
  • the DNA deduced amino acids for r-[K2 tPA ] protein is a peptide of 88 residues including the initial methionine.
  • r-[K12 tPA ] contains 179 residues with a MW of 19.810 kDa, as expected from its DNA sequence. Expression of a 20 kDa protein from the pK12, PA plasmid is congruent with that expected. However, even r-[K1 tPA ] sharing 92 identical amino acids with the N-terminus of r-[K12 tPA ], was not expressed.
  • Both recombinant proteins r-[K2 tPA ] and r-[K12 tPA ] contain tPA kringle 2 domains.
  • the tPA kringle 2 structure has the lysine-binding site. Lysine-bind site in the tPA kringle 2, binding resins coupled with lysine ligand, can be used for affinity protein purification.
  • the stable epoxy-activated lysine- agarose gel was used to purify the recombinant proteins r-[K2 tPA ] and r- [K12 tPA ].
  • L-arginine is a competitor of lysine ligand.
  • Efficient binding of recombinant tPA kringles with lysine ligand was found when the concentration of L-arginine in the protein solution is reduced below 25 mM. Elution of recombinant tPA kringles was carried out using a competitive ligand of lysine, the 6-amino-n-caproic acid. Three peaks are detected in the eluted fractions from a 0-200 mM gradient, one is between 30 to 45 mM, the second is between 60 to 105 mM, and a third large peak between 125 to 150 mM of 6- amino-n-caproic acid. Samples from these fractions were subjected to SDS- PAGE analysis.
  • Proliferation assay using bovine capillary endothelial cells indicated that both of r-[K2 tPA ] and r-[K12 tPA ] proteins are active for anti-angiogenesis.
  • the protein r-[K2 tPA ] containing tPA kringle 2 alone showed stronger activity than r-[K2 tPA ] suggesting that the tPA kringle2 is an angiogenesis inhibitor (Fig 8).
  • Fig 8 A similar result has been observed from plasminogen kringles that the plasminogen 2 alone is more active than a peptide containing plasminogen kringle 2 and 3.
  • plasminogen kringles All five plasminogen kringles have been cloned.
  • the most active plasminogen kringles are, kringles 5 and 1.
  • Plasminogen kringle 4 does nor show anti-angiogenic activity. Because analysis of anti-angiogenic activity of tPA kringles do not include plasminogen kringles, tPA kringle activity was not compared with that of plasminogen kringles.
  • Sites for mutation in tPA kringle were selected using amino acid sequences of plasminogen kringles as references, in which plasminogen kringle 5 and 1 is the positive reference and kringles 2, 3, 4 - especially kringle 4 - is the negative reference.
  • positive selection or negative selection can be designed. This is performed by aligning amino acid sequence of tPA kringle 2 with the five plasminogen kringles. Next, identify the position(s) where the amino acid residue(s) is identical between plasminogen 5 and 1 , but is different from plasminogen 4 and tPA kringle 2. If a position is occupied with a similar or the same amino acid residue in tPA kringle 2 and plasminogen kringle 4, this is where the residue should be mutated with top priority. Then, change the amino acid residue(s) in kringle 2 to an amino acid that is identical in plasminogen kringle 5 and 1.
  • negative selection can be designed by changing a residue in tPA kringle identical with both of plasminogen kringle 5 and 1 into an amino acid identical with that of plasminogen kringle 4.
  • mutations for positive selection only are performed.
  • the mutagenesis procedure utilizes a super-coiled, double-stranded and methylated DNA plasmid pK2 tPA and two synthetic oligonucleotide primers containing the desired mutation.
  • PCR employing Pfu, a proofreading DNA polymerase, efficiently produced, nicked and unmethylated, the daughter plasmid with mutation by uptake of the oligonucleotide primers.
  • the methylated parent pK2 tPA plasmid was eliminated by restriction digestion using Dpnl, which is an enzyme cutting the methylated substrate DNA.
  • the mutated daughter plasmids were recovered by transformation into high-efficiency competent E. coli cells.
  • a control PCR was accompanied with each set of mutations by excluding the oligodeoxynucleotide primers. Selection of transformants using the ampicillin resistance maker encoded by the vector, indicated that at least a hundred fold of colonies were obtained from the PCR reaction mixture containing the mutation primers compared to that of PCR excluding the primers.
  • All kringle mutants should be approximately 10 kDa. Expression of mutants was analyzed on 16% polyacryl amide gel electrophoresis (Fig 9). Four of them were expressed. However, Y, N and G mutants were unstable. Interestingly, the Y, N and G mutants are all located in the middle in the kringle structure, suggesting that mutation in this region can create unstable mRNA or protein structure.
  • tPA kringle 2 mutants were solubilized and reduced by 8 M urea and 140 mM ⁇ -mercaptoethanol, as described in Materials and Methods.
  • the reduced mutants were fractionated by Super-fine Sephadex G-75 chromatography in the presence of 8 M urea.
  • the reduced mutant proteins appeared to be homogenous by denatured SDS-PAGE analysis.
  • tPA kringle 2 Discussion tPA kringle 2, and tPA kringle 1 plus kringle 2 was expressed. tPA kringle 1 domain was unable to be expressed. Expression of tPA kringle 1 was only achieved by the fusion of kringle 2 domain, the r-[K2 tPA ] peptide. To the best of the knowledge, peptide, containing tPA kringle 1 only, has never been expressed. Expression of tPA kringle 1 has been reported as a fusion with plasminogen kringle domain (DeSerrano et al., 1992).
  • the tPA kringles have been cloned and inserted into pET11a vector for protein expression under the control of a strong T7 phage promoter and the laclq repressor.
  • Recombinant plasmid pKltPA encodes r-[K1tPA], where, r stands for recombinant and [K1tPA] represents amino acid sequence residues [Ile86-Asn177] of kringle 1 domain in tPA molecule.
  • the recombinant plasmid pK2tPA encodes r-[K2tPA] representing amino acid residues [Asn177-Thr263] of tPA kringle 2 domain.
  • Recombinant plasmid pKI2tPA encodes ar-[KI2tPA] protein containing kringle I and kringle 2 domain, an internal fragment of [Ile86-Thr263] in tPA protein sequence.
  • Recombinant proteins expressed in E. coli were analyzed by 16% polyacrylamide gel. Whole cellular proteins of 50 ⁇ g were denatured by SDS and ⁇ -mercaptoethanol and loaded gel for electrophoresis. Abundant expression of a 10 kDa protein has been detected from the cells carrying pK2tPA construct (Fig. 7). Ten colonies of pK2tPA constructs were selected and all ten expressed the same 10 kDa protein. A 20 kDa protein was found from the cells harboring pK12tPA plasmid. All ten clones of pK12tPA expressed the 20 kDa protein.
  • the DNA deduced amino acids for r-[K2tP A] protein is a peptide of 88 residues including the initial methionine.
  • Molecular weight (MW) of r-[K2tPA] estimated at 9,716.42 Da, agrees with the size of 10 kDa analyzed from the polyacrylamide gel.
  • r-[K12tPA] contains 179 residues with a MW of 19810.92 Da expected from its DNA sequence. Expression of a 20 kDa protein from the pK12tPA plasmid perfectly fits with that expected. However, even r- [K1tPA] sharing 92 identical amino acids with the N-terminus of r-[K12tPA], was not expressed.
  • the tPA kringle 2 structure has the lysine-binding site. Lysine-bind site in the tPA kringle 2, binding resins coupled with lysine ligand, can be used for affinity protein purification.
  • Lysine-agarose gel was used to purify the recombinant proteins r- [K2tPA] and r-[K12tPA].
  • the proteins were refolded in the presence of 0.5 M L-arginine as 68 described (Cao et al., 1996).
  • L-arginine is a competitor of the lysine ligand. Efficient binding of recombinant tPA kringles with lysine ligand occurs, when the concentration of L-arginine in the protein solution has been reduced below 25 mM. Elution of recombinant tPA kringles was carried out using a competitive ligand of lysine, the 6-amino-n-caproic acid.
  • r-[K2tPA] and r-[K12tPA] proteins eluted in the peak of 125-150 mM 6-amino-n-caproic acid, appeared to be homogeneous on the denaturing 16% polyacrylamide gel.
  • tPA kringles were added into the culture of bovine capillary endothelial (BCE) cells.
  • BCE bovine capillary endothelial
  • the cells were maintained in DMEM medium for five days. Cells passed through an assay filter were counted.
  • Assay results indicated that both of r-[K2tPA] and r- [K12tPA] proteins actively inhibit endothelial proliferation.
  • Protein r-[K2tPA] containing tPA kringle 2 alone showed stronger activity than r-[K2tPA], suggesting that the tPA kringle 2 can be an angiogenesis inhibitor (Fig. 8).
  • tPA kringle domain is a good choice for the mutation, since: 1) tPA kringle 2 contains a strong lysine-binding site that is preferred for ease isolation of kringle mutants; 2) tPA kringle 2 has been extensively studied and much is known about its structure and function. All five plasminogen kringles have been cloned (Cao et al., 1996).
  • the most active plasminogen kringles are kringle 5 and I. Plasminogen kringle 4 did not show activity. Because performing the analysis of anti-angiogenic activity of tPA kringles did not include plasminogen kringles, the tPA kringle activity cannot be compared with that of plasminogen kringles.
  • Sites for mutation in tPA kringle were chosen using amino acid sequences of plasminogen kringles as references, in which plasminogen kringle 5 and 1 were selected for the positive and kringle 2, 3, 4, especially kringle 4, were selected for the negative reference.
  • positive selection or negative selection can be designed.
  • the resultant mutant is expressed and its effects tested on endothelial proliferation to see if the mutation changes (increases) its anti- angiogenic activity.
  • negative selection can be designed by changing a residue in the tPA kringle identical with both of plasminogen kringle 5 and 1 to an amino acid identical with that of plasminogen kringle 4.
  • mutations for positive selection were only performed.
  • the mutagenesis procedure utilizes a super-coiled, double-stranded and methylated DNA plasmid pK2tPA and two synthetic oligonucleotide primers containing the desired mutation.
  • PCR employing Pfu, a proofreading DNA polymerase, efficiently produced, nicked and unmethylated daughter plasmid with a mutation by uptake of the oligonucleotide primers.
  • the methylated parent pK2tPA plasmid was eliminated by restriction digestion using Dpnl which is an enzyme cutting the methylated substrate DNA.
  • the mutated daughter plasmids were recovered by transformation into high- efficient competent E. coli cells.
  • a control PCR was accompanied with each set of mutation by excluding the oligodeoxynucleotide primers. Selection of transformants using the ampicillin resistance maker encoded by the vector indicated that at least a hundred fold of colonies was obtained from the PCR reaction mixture containing the mutation primers compared to that of PCR excluding the primers.
  • All kringle mutants should be approximately 10 kDa MW. Expression of mutants were analyzed on 16% polyacrylamide gel electrophoresis (Fig. 9). S (Ser186 substituted by Lys) and H (His244 substituted by Tyr) mutants were expressed. However, the Y (Tyr214 substituted by Phe) mutant was not expressed.
  • the tPA kringle 2 protein mutants were solubilized and reduced with 8M urea and 140mM ⁇ -mercaptoethanol. Proteins were fractionated by Super-fine Sephadex G-75 chromatography in the presence of 8M urea. The reduced mutant proteins appeared to be homogenous by denatured 16% SDS-PAGE analysis. The pure reduced mutants were refolded and purified by lysine-agarose chromatography.
  • Kringle mutants S, Y, H and wild-type tPA kringle 2 proteins were tested by proliferation assay using bovine endothelial cells.
  • the selected mutation sites in tPA kringle 2 domain are indicated (Fig. 10). Mutation on S showed no marked effects on activity.
  • H mutant with a single amino acid substitution of histidine by tyrosine dramatically increased inhibitory activity in the endothelial proliferation assay (Fig. 19). H mutant completely inhibits endothelial cell proliferation at 40 nM.
  • the parental wild- type tPA kringle 2 shows no activity at the same concentration.
  • the wild-type tPA kringle inhibits endothelial proliferation only approximately 45% at 200 nM.
  • the H site might be on the surface of protein:
  • Residues of Asn248- Trp253 form a lysine-binding site in tPA kringle 2 domain(Bakker et al., 1993). These residues should be exposed on the surface of protein for exhibiting lysine binding of tPA kringle 2.
  • the H site (His244) is closed to Asn248-Trp253 lysine binding site. His244 might be also exposed on the surface of kringle 2 domain for expressing anti- angiogenic activity.
  • the denatured kringle 5 can be seen as acting as a large extended peptide.
  • Different kringles with varied anti-angiogenic activity can exist in the human genome, however, there appears to be only one human endostatin. Human endostatin and mouse endostatin are highly conserved (Standker et al., 1997).
  • Anti-angiogenic peptide Design of anti-angiogenic peptide:
  • Peptides have been designed to overlap the H mutation site.
  • One is the wild-type sequence.
  • Another one is a mutant peptide with substitution of histidine by tyrosine.
  • the KED gene was constructed by fusion of cDNA plasminogen kringle 5 encoding Glu478-Ala562 with a collagen ⁇ 1 (XVIII) cDNA fragment encoding His 1154-Lys1336.
  • the KED gene is expressed in E. coli cells.
  • AGA codon is a rare codon used in E.coli (Wada et al., 1990).
  • the tRNAAGA is encoded by dna Y gene.
  • the dna Y gene also has been reported to be essential for DNA replication 72. To improve productivity of KED protein in E.
  • a vector pEXdna Y was constructed containing a strong T7 promoter for high-speed transcription of KED mRNA and a dna Y gene supplying tRNA AGA for efficient translation of the KED product.
  • the engineered KED gene encodes 271 amino acids. Expression of KED protein from pEXdnaY-hKED, was induced by IPTG (isopropyl ⁇ -D- thiogalactopyranoside). Abundant KED production was achieved. KED protein accumulated to 47% of total cellular protein by 2 hours induction with 0.5 mM IPTG (Fig. 2). Optimal production of KED protein was performed using a fermentor.
  • the KED protein was produced as inclusion bodies in cytosol. KED inclusion bodies were isolated and efficiently refolded in the presence of 0.5 M L-arginine at pH 8.6. As a control, human endostatin was simultaneously expressed from pEXdna Y-hED. More than 99% of human endostatin protein precipitated in the refolding solution as has been previously observed (O'Reilly et al., 1997). No precipitation of KED fusion occurs during the refolding.
  • KED protein was obtained by HiTrap HeparinSepharose chromatography. Analysis of KED protein by denatured SDS-PAGE (polyacrylamide gel electrophoresis) revealed a single 30 kDa peptide that agreed with the molecular weight estimated form the deduced amino acid sequence (Fig. 10). N-terminal analysis of KED protein (by Commonwealth Biotechnologies, Richmond, VA) provided an identical sequence of MEEDHMFGNGKGYRG as deduced from DNA sequence, see Sequence Listing, where the 5th residue is Cys that generally does not show up by the analysis system. Compositional analysis of KED generated similar results with that predicted, Table 1. Both of N-terminal and compositional analysis indicates that the pure protein isolated is the correct form of the KED gene product.
  • KED - fusion Proliferation assays using bovine endothelial cells indicate that fusion protein H1 completely inhibited endothelial cells migration at a concentration as low as 8 nM (250 ng/ml), where, human endostatin shown almost no activity at 12 nM (250 ng/ml) by the same assay (Fig. 20).
  • the IC 50 of human endostatin in the assay was 17.5 nM (350 ng/ml).
  • Ic50 of human plg-k5 was approximately 150 nM.
  • Ic50 for antiostatin was 135 nM.
  • migration assays are performed in a modified Boyden chamber using BCE cells (Dameron et al., 1994). Cells are grown in DMEM supplemented with 10% FBS and supplements at passage 15. To assess migration, the cells are starved overnight in DMEM supplemented with 0.1% HSA, harvested, D'YI D'Y2 :-lyi suspended in DMEM/BSA, plated at 106 cells/ml on the lower surface of a gelatinized membrane (Nucleopore Corp., Plesanton, CA) in an inverted Boyden chamber and incubated for 1.5 hours to allow cell attachment.
  • a gelatinized membrane Nucleopore Corp., Plesanton, CA
  • the chambers are then righted, and test material added to the top well incubated for 5 hours.
  • Membranes are then be fixed and stained and the number of cells migrating to the top o fthe filter in 10 high-powered fileds counted and bFGF at 10 ng/ml is used as a positive control.
  • the rat corneal assay has been perfonned to test in vivo efficacy of KED fusion protein. 12.5ng, of KED were applied in the presence of 25ng of bFGF. 12.5ng of KED fusion protein efficiently inhibited the growth of capillary blood vessels from the corneal bed (Fig. 5).
  • endostatin a highly basic protein fragment of collagen ⁇ 1 (XVIII), represents another class of angio-inhibitory compounds. Kringle-like structures are not present in the endostatin molecule. Anti-angiogenic activity of endostatin can therefore be due to a different mechanism to that of kringles. Construction of fusion protein between highly active mutated tPA kringle 2 and endostatin is described. The fusion of two completely different structures of kringle and endostatin creates a powerful anti-angiogenic molecule. Preliminary studies support this hypothesis.
  • KED a fusion between plasminogen kringle 5 and endostatin, showed significantly higher anti-angiogenic activity than either kringle 5 or endostatin. Higher activity of this chimera can reflect expression of combined activity from kringle 5 and endostatin due to simultaneously activation of different anti-angiogenic mechanisms.
  • H mutant of tPA kringle 2 that is much active than plasminogen kringle 5 which is the most active kringle reported (Nelson et al., 1995).
  • tPA kringle 2 is used as a model to identify the anti-angiogenic active site(s) of kringle structure. There are three reasons for the selection of tPA kringle 2 as a model. At first, a Preliminary Study indicated that tPA kringle 2 is an anti- angiogenic active kringle. Second, substitution of a histidine residue with tyrosine (H mutant) in tPA kringle 2 surprisingly, increases anti- angiogenic activity.
  • H mutant tyrosine
  • BEC proliferation inhibitory activity of this H mutant of tPA kringle 2 is stronger than plasminogen kringle 5 that is the most anti- angiogenic kringle reported to date.
  • kringle 2 domain as a part of tPA and rPA, has been used in clinical therapy.
  • Kringle 2 is an internal fragment of tPA and an N-terminal domain of tPA.
  • the recombinant plasmid pK2tPA containing the wild type tPA kringle 2 cDNA under aT7-driven promoter have been developed. Mutation of tPA kringle 2 is directly performed using this expression vector. No sub-cloning or other plasmid engineering is involved. A PCR-directed procedure is used which utilizes one set of complementary mutation primers overlapping the mutation-desired region. There is a recombinant plasmid pK2tPA containing the wild type tPA kringle 2 cDNA under a T7-droven promoter. Mutation of tPA kringle 2 is directly performed by using this expression vector. No sub- cloning or other plasmid engineering is involved.
  • PCR-directed procedure is used that utilizes one set of complementary mutation primers overlapping the mutation-desired region. Mutagenesis is carried out using a mutation kit, Quick-Change (Stratagene, La Jolla, CA) with some modifications. Fifty ⁇ l PCR reaction mixture are prepared on ice.
  • the mixture contains 20 mM Tris (pH 8.8), 10 mM KCI, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, 100 ng pure super-coiled pK2tPA DNA, 200 ng each of sense and complementary mutation oligonucleotide primers, 200 ⁇ M of dNTP and 5 units of cloned Pfu DNA polymerase.
  • a control PCR reaction will be simultaneously included in the thermal cycles by replacing both sense and complementary mutation primers with missense primers.
  • Kringle 2 domain contains 87 amino acids that represent residues 177 to 263 in tPA. Mutations will be generated throughout the kringle 2 sequence. Mutation sites will be specifically selected based on alignments of tPA kringle 2, plasminogen kringle 4 and 5 (Fig. 5). These two plasminogen kringles serve as references guiding selection of candidate mutation sites in tPA kringle 2. Plasminogen kringle 5 is the strongest anti- angiogenic kringle (Cao et al., 1997).
  • Kringle 4 has no anti-angiogenic activity (Urano et al., 1991). There are 24 positions, identical throughout these three kringles forming the basic kringle structure including the 3 disulphide bonds, which will not be subjected to mutation. Two groups of mutations will be performed: positive and negative selections of anti-angiogenic active site(s). A total of 17 mutants will be generated.
  • Oligonucleotide primers will be 5'-CAGTGACTGCATGTTTGGGAATGGG-3' and 5'-CCCATTCCCAAACATGCAGTCACTA-3'.
  • Oligonucleotide primers will be 5'-CCTACCGTGGCAAACACAGCCTCACC-3' and 5'-GGTGAGGCTGTGTTTGCCACGGTAGG-3'. 3. Change S193 to A. Oligonucleotide primers will be 5'-CCGTGGCACGCACGCCCTCACCGAG-3' and 5'-CTCGGTGAGGGCGTGCGTGCCACGG-3 ⁇
  • Oligonucleotide primers will be 5'-CCCGTGGAATGCCATGATCCTGATAG-3' and 5'-CTATCAGGATCATGGCATTCCACGGG-3'.
  • Oligonucleotide primers will be 5'-GCCGGAATCCTCCGGGGGATGCC-3' and 5'-GGCATCCCCCGGAGGATTCCGGC -3'.
  • Oligonucleotide primers will be 5'-GATGGGGATGCCGGGCCCTGGTGCC-3' and 5'-GGCACCAGGGCCCGGCATCCCCATC- 3'.
  • Oligonucleotide primers will be 5'-CGCAGGCTGACGTACGAGTACTGTG-3' and 5'-CACAGTACTCGTACGTCAGCCTGCG- 3 '.
  • Oligonucleotide primers will be 5'-GGCTGACGTGGGACTACTGTGATGTG-3' and
  • Oligonucleotide primers will be 5'-GTGCCCTCCTGCGCCACCTAAGGATCC-3' and 5'-GGATCCTTAGGTGGCGCAGGAGGGCAC-3'.
  • tPA kringle k2 There are 8 positions in tPA kringle k2 that are identical to those in plasminogen kringle 5, but which are not contained within plasminogen kringle 4. These 8 amino acids are F182, A223, W242, R249, L251 , D257, V258 and P259. These 8 residues will be replaced with amino acids that are identical to those in plasminogen kringle 4. These mutations are expected to produce less angiosuppressive activity in some cases. This group of mutations will serve as a negative reference for the identification of anti- angiogenic active site(s) of kringle domain proteins.
  • Oligonucleotide primers will be 5'-GACTGCTACCACGGGAATGGGTCAG-3' and 5'-CTGACCCATTCCCGTGGTAGCAGTC-3' .
  • Oligonucleotide primers will be 5'-CCCAGTGCCCAGAACCTGGGCCTGG-3' and
  • Oligonucleotide primers will be 5'-GATGCCAAGCCCACCTGCCACGTGCTG-3' and 5'-CAGCACGTGGCAGGTGGGCTTGGCATC-3 ⁇
  • Oligonucleotide primers will be 5'-GTGCTGAAGAACCCCAGGCTGACGTG-3' and ⁇ '-CACGTCAGCCTGGGGTTCTTCAGCAC -3'
  • Oligonucleotide primers will be 5'-GAACCGCAGGGTGACGTGGGAGTAC-3' and 5'-GTACTCCCACGTCACCCTGCGGTTC-3'.
  • Oligonucleotide primers will be 5'-GTGGGAGTACTGTAACGTGCCCTCC-3' and ⁇ '-GGAGGGCACGTTACAGTACTCCCAC- 3'.
  • Oligonucleotide primers will be 5'-GAGTACTGTGATCTGCCCTCCTGCTC-3' and
  • Oligonucleotide primers will be ⁇ '-GTACTGTGATGTGAAGTCCTGCTCC-S' and 5'-GGAGCAGGACTTCACATCACAGTAC-3'.
  • tPA Kringle 2 mutants Recombinant plasmids of 17 mutants will be transformed into expression strain E.coli bL2l (DE3) cells by the standard CaCI2 procedure. E..coli BL21 (DE3) cells will be co-transformed with a low-copy plasmid pDC952, a derivative of pACYC 184 containing argU gene. Ten colonies with ampicillin and chloramphenicol resistance for each construct will be selected for gene expression analysis. Each single colony will be inoculated in 2 ml of LB medium containing 100 ug/ml ampicillin and 35ug/ml chloramphenicol.
  • Anti-foam agent 289 (Sigma, St, Louis, MO) will be added to the medium at 0.005% (v/v) to prevent foam-formation during fermentation. Protein induction will be performed by 1 mM IPTG at 0.5 OD (600 nm). Cells will be collected by a filtration using 0.04 ⁇ m polyethersulfone membrane (North Carolina SRT, Cary, NC).
  • Super-fine Sephadex G- 75 (Pharmacia, Piscataway, NJ) of 10g will be equilibrated in 100mM Tris (pH 8.6) containing 1mM EDTA and 8M urea at 37°C for 24 hours .
  • the resins are packed on a columns (250nm x I5nm) at 28-30°C for preventing precipitation of urea.
  • the reduced tPA kringle 2 mutants will be added into refolding solution containing 100 mM Tris (pH 8.8), 1 mM EDTA, 0.1% Tween-20.
  • the refolded protein solution will be concentrated by using ultra- filtration system consisting ofTRIPORT Module/316L Stainless Plates with 0.1 m 2 working surface and a regenerated cellulose membrane of 5 kDa MWCO (molecular weight cut-off) (North Carolina SRT, Cary, NC).
  • the concentrated protein solution will be diluted 20 times into pre-cold(4°C)
  • Proteins eluted from lysine column will be monitored at 280nm and analyzed by 20% SDS-PAGE. E.coli cells secrete endotoxin. Prevention of endotoxin contamination will be ensured by using Acticlean Etox (Sterogene, Arcadia, CA) chromatography.
  • the pure recombinant tPA kringle 2 mutants will be dialyzed against 1000 fold of PBS (v/v) and loaded on an Acticlean Etox column previously washed with 1 M NaOH and equilibrated with PBS solution.
  • One ml of Acticlean-Etox gel will be used for every lOmg of tPA kringle mutants.
  • kringle 2 mutants will be sterilized by a 0.2 ⁇ m filter. There are total of 19 proteins, including wild-type kringle 2 and H mutants, which will be purified.
  • tPA tissue plasminogen
  • tPA kringle domain 2 which inhibit endothelial cell proliferation and/or migration, and corneal angiogenesis.
  • Polypeptide fragments of tPA kringle 2 containing functionally active anti-angiogenic site(s) exhibit anti-angiogenic activity.
  • Peptides containing the putative anti- angiogenic active site(s) are synthesized. The size for the peptides ranges from 10 to 15 amino acids.
  • Peptides as short as 10 amino acids of murine epidermal growth factor (EGF) have been shown to inhibit endothelial cell proliferation.
  • the amino acid sequence of tPAkringle 2 ( K2 tPA ; 86 residues), shown below, has certain unique chemical and enzymatic cleavage sites which are exploited to generate polypeptide fragments for functional tests.
  • the fragmentation of K2t PA into polypeptides is carried out under two different conditions. First, the intact functionally active protein is subjected to limited cleavage under mild conditions without any prior modifications to the protein structure. Thus, the chances of preserving and localizing any structural elements which could be present in the native state compact regions, usually joined by more accessible or flexible regions, is increased. Secondly, the fragments to be single linear polypeptides and therefore reduce all disulfide bridges and carboxymethylate the CysSH groups of the protein before performing the cleavage reaction. More than ten polypeptide fragments covering different regions of K2 tPA can be obtained by a combination of chemical and enzymatic cleavage methods.
  • Residues which are identical in tPA (two kringles) and plasminogen (five kringles) kringle domains are indicated by asterisks; the six cysteines involved in the three disulfide bridges of K2 tPA are in bold letters.
  • the single methionyl bond in K2 tPA is cleaved with excess cyanogen bromide (CNBr) in 0.1 ⁇ / HCI or 70% formic acid to produce two peptide fragments.
  • CNBr cyanogen bromide
  • Enzymatic fragmentation of K2 tPA at its two glutamyl bonds are performed with the GluV ⁇ form of glutamyl endopeptidase 1 , at optimal conditions to yield three peptides.
  • the reaction is stopped with Cbz-Leu-Leu-Glu-CH 2 CI and the fragments separated by hplc.
  • Prolyl oligopeptidase is used to cleave specifically the Pro-Xaa bonds in K2 tPA . Since this protease cleaves only small polypeptides, it is used in combination with CNBr and GluV ⁇ to generate smaller fragments. However, if K2 tPA is susceptible to this post-proline cleaving enzyme a 27 -residue, three 16-18 residue and two small (4-6 residue) peptide fragments spanning the K2 tPA domain can be obtained. Post-Proline cleaving enzyme; cleavage after P (Pro)
  • peptides are purified by preparative reverse phase hplc and characterized by analytical reverse phase hplc, peptide equencing and mass spectrometry. The kringle fragments are then tested in a series of in vitro (endothelial proliferation and migration) and in vivo (corneal angiogenesis) biological assays for their anti-angiogenic properties.
  • the disulfide bridges are reduced with dithiothreitol in 6.0 M guanidine solution at 50 degrees C for 30 min.
  • the free CysSH groups are then carboxymethylated with a large excess of iodoacetic acid.
  • Guanidine is removed by dialysis.
  • the reduced protein solution in guanidine is dialyzed against formic acid. When the concentration of formic acid reaches 70 %, the modified protein is ready for CNBr cleavage.
  • Analytical hplc-MS is performed by injecting into an Applied Biosystems aquapore C-8, 300e reversed phase column using a gradient over 30 minutes, from 0.1% TFA in water to 70% acetonitrile in water with 0.06% TFA. A flow rate of 30 El/minutesis used with a post column split diverting 2 El/min into the mass spectrometer. Spectra are accumulated with a scan rate of 400 amu/sec from 200 to 2000 with a nominal resolution of 1000. Preparative hplc to purify large quantities of protein fragments for biological assays are performed as described below in the peptide synthesis section.
  • Peptide synthesis Peptides are synthesized by manual solid phase peptide synthesis using 9- Fluorenylmethyloxycarbonyl (Fmoc)fetf-butyl chemistry as described previously. Fmoc-protected amino acids are activated with 2-(1H-benzotriazole-1-yl)-, 1 ,2,2-tetramethyluronium hexafluorophosphate / 1-hydroxybenzotriazole / N;N-diisopropylethylamine (1:1:2) in 1-methyl-2-pyrrolidone / dimethylformamide / dichloromethane (1 :1 :1) for all coupling steps.
  • Fmoc-protected amino acids are activated with 2-(1H-benzotriazole-1-yl)-, 1 ,2,2-tetramethyluronium hexafluorophosphate / 1-hydroxybenzotriazole / N;N-diisopropylethylamine (1
  • peptides are cleaved with reagent K (0.1 ml dimethylsulfide, 0.2 ml 1 ,2-ethanedithiol, 0.46 g phenol, 0.2 ml thioanisole, 0.2 ml water and 7 ml trifluoroacetic acid) for 2 hours at room temperature.
  • reagent K 0.1 ml dimethylsulfide, 0.2 ml 1 ,2-ethanedithiol, 0.46 g phenol, 0.2 ml thioanisole, 0.2 ml water and 7 ml trifluoroacetic acid
  • Peptides are purified by reverse-phase HPLC on a Nucleosil C 8 column (30 mn, 10 ⁇ ; 250 mm x 55 mm, Macherey and Nagel) and examined for purity by analytical reverse-phase HPLC on a Nucleosil C 8 column (3 ⁇ , 12 mn; 120 mm x 2.1 mm) as described previously (Angali et al., 1995)
  • the samples containing the pure peptides are collected and lyophilized.
  • Electrospray mass spectrometry is performed to confirm the analytical data. i.e. to confirm the amino acid sequences and the absence of modified products resulting from incomplete deprotections and side chain reactions with scavengers.
  • Table 1 Compositional analysis. Amino acid residues per KED molecule predicted from DNA sequence are compared with the results from experimental analysis.
  • NINDS National Institute of Neurological Disorders and Stroke
  • rt-PA Stroke Study Group A systems approach to immediate evaluation and management of hyperacute stroke. Experience at eight centers and implications for community practice and patient care. [Clinical Trial. Journal Article, Multicenter Study] Stroke. 28(8): 1530-40, 1997.
  • Patthy L Evolution of the proteases of blood coagulation and fibrinolysis by assembly from modules. [Review] [58 refs] [Journal Article. Review] Cell. 41(3):657-63, 1985. de Munk GA. Caspers MP. Chang GT. Pouwels PH. Enger-Valk BE. Verheijen JH. Binding of tissue-type plasminogen activator to lysine, lysine analogues, and fibrin fragments [Journal Article] Biochemistry. 28(18):7318- 25.1989.
  • EGF epidermal growth factor
  • Hu GF Limited proteolysis of angiostatin by elastase is regulated by plasminogen. J Protein Chem 1997;16:669-679.
  • Angiostatin a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994;79:315-328.
  • Liotta LA, Steeg PS, Stetler-Stevenson WG 1991: Cancer metastasis and angiogenesis; an imbalance of positive and negative regulation.
  • Sachr H. Dror a potential neurotrophic receptor gene, encodes a Drosophila homolog of the vertebrate Ror family ofTrk-related receptor tyrosine kinases. Proceedings of the National 61cademy of Sciences of the United States of America 1993;90(i5):7109-7113.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

There is provided a pharmaceutical composition including at least one kringle protein and a pharmaceutically acceptable carrier. Also provided by the present invention is a method of treating a disease by administering an effective amount of a compound from the group consisting of kringle proteins, KED and a kringle derived from the tPA protein.

Description

AN ANTI-ANGIOGENIC KRINGLE PROTEIN AND ITS MUTANTS
BACKGROUND OF THE INVENTION
1. FIELD OF THE INVENTION
The present invention relates to the use of kringle proteins for the treatment of disease. More specifically, the present invention relates to the use of kringle proteins for the treatment of cancerous lesions.
2. DESCRIPTION OF RELATED ART
Normal physiological growth of new blood vessels in the mature organism is tightly delimited in time and restricted in localization. It occurs during wound repair, embryogenesis, corpus luteum formation, and the menstrual cycle (Folkman et al., 1996; Folkman, 1995). In contrast, nonphysiologic neovascularization is found in many disorders such as chronic inflammation, psoriasis, diabetic retinopathy, and cancer. Several avenues of experimental evidence indicate that neoangiogenesis is a critical and necessary step in the metastatic process both for tumor survival and progression. The development of a network of blood vessels is a prerequisite for the local expansion of tumor colonies beyond the size (0.125 mm2) restricted by oxygen and nutrient diffusion (Liotta et al., 1991 ; Folkman et al.,1992).
Recent data from transgenic mouse models of tumorigenesis indicate that the induction of angiogenesis appears during early stages of tumor development, suggesting that this event represents a potentially rate-limiting step (Hanahan et al., 1996). Folkman (Folkman, 1971) first hypothesized the utility of anti- angiogenic therapies, and Liotta and colleagues (Liotta et al., 1974) first demonstrated the relationship of neovascularization to metastatic progression in melanoma where tumors without signs of new vessel development rarely metastasized. Neovascular development has been proven to be a marker for invasive potential and is diagnostic of the most aggressive primary brain cancer, gliobiastoma multiforme (Mikkelsen et al., 1995). Immunohistochemical staining for Yon Willebrand's factor, factor VIII or CD34 has been used to identify tumor-related microvessels. Numeric assessment of tumor vascularity has been shown to have a negative prognostic significance in a variety of human solid tumors including breast cancer, prostate cancer, ovarian cancer, and in primary brain tumors. Thus, neoangiogenesis is a novel biological target in tumors since angiogenesis involves local endothelial cell invasion, proliferation, and migration in response to many of the same cytokines frequently produced by tumors.
The organization of proliferative neovessels in malignant tumors appears to be the result of a complex pattern of expression of a number of growth factors and their signaling pathways. In a fashion analogous to the positive-proliferative oncogenes being balanced by the growth inhibitory tumor suppressor genes, angiogenesis has been described as being regulated by angio-promoting factors (bFGF, VEGF, PDGF, TGF-β) and angio-inhibitors (TSP-I, GDAIF). Angio-inhibitors in glioma cells appear to be subject to regulation by p53 or genetic material on chromosome 10. The physiologic invasion required for neovascularization, local basement membrane remodeling capillary bud formation followed by vascular tube formation, has been shown to involve the same steps as tumor invasion (Kohn et al., 1995, Liotta et al., 1991). The balance between the martix metalloproteases (MMPs) and their inhibitors in the local environment results in local proteolysis or an inhibition of invasion. Endothelial cells have been shown to produce gelatinase A and to be sensitive to autocrine and paracrine production of tissue inhibitors of metalloproteinases (TIMPs) 1 and 2 and cathepsin B (Mikkelsen et al., 1995). Hypoxic cancer cells in the core of tumors secrete a number of growth factors such as VEGF (vascular endothelial growth factor), IL-8 (interleukin-8), HGF (hepatocyte growth factor) and bFGF (basic fibroblast growth factor) that stimulate proliferation of endothelial cells and cause rapid sprouting of new capillaries from adjacent blood vessels (White et al., 1995). The αvβ3 integrin (Hruska et al., 1995), mediates a significant parallel pathway in angiogenesis, and also has been found to be highly expressed on angiogenic blood vessels.
Recent progress in the understanding of a paradoxical clinical observation has been made, based on the phenomenon that removal of a primary tumor increases the growth rate of remote metastatic tumor (Fisher et al., 1983; Fisher et al., 1989; Simpson-Herren et al., 1976). A hypothesis has been proposed to explain this phenomenon (O'Reilly et al., 1993) which purports that a primary tumor stimulates angiogenesis in its own habitat by generating angiogenesis stimulators in excess of angiogenesis inhibitors, and that the circulating inhibitors suppress the progressive growth of latent remote metastases. Once the primary tumor is removed, the source of circulating inhibitor is eliminated and remote metastases are themselves capable of progressive uninhibited growth.
A circulating inhibitor has been identified and named angiostatin. Angiostatin is a 38 kDa internal fragment of plasminogen. It has been demonstrated that human prostate carcinoma cells (PC-3. DU-145, and LN- CaP) express enzymatic activity that converts plasminogen to angiostatin (Gately et al., 1997), suggesting that generation of angiostatin is not an individual event limited to Lewis lung carcinoma.
Increased evidence reveals that generation of angiogenesis inhibitors by primary cancers is widespread. Endostatin. a novel angiogenesis inhibitor of the C-terminal fragment of collagen α1 (XVIII) has also recently been identified from the conditioned media of murine hemangioendothelioma cell line EOMA (O'Reilly et al., 1994). A study of human blood ultrafiltrate (hemofiltrate, HF) from patients with chronic renal insufficiency found that endostatin is present in the human blood circulation (Standker et al., 1997).
Endostatin is a 20 kDa protein. Both wild-type and recombinant murine endostatin at a concentration of approximately 50 nM, completely inhibit the proliferation of bovine capillary endothelial cells. However, the purified human endostatin from hemofiltrate (HF) failed to inhibit the proliferation of bovine endothelial cells at the same concentration. Alignment of human circulating endostatin with murine endostatin isolated from mouse hemangioenqothelioma cell medium, indicates that the N-terminus of circulating human endostatin is 12 amino acids shorter than the mouse endostatin. There are no reports showing that human endostatin is as active as the mouse one.
Compared with endostatin, angiostatin shows much weaker angiogenesis inhibition activity. A concentration of 50 nM endostatin completely suppressed the bovine endothelial cell proliferation. To obtain the same effect of mouse endostatin, more than 10 times the amount of angiostatin was required. Notable homology has not been found between endostatin and angiostatin at the level of DNA or amino acid sequences. Recently, the plasminogen kringle 5 alone showed inhibition of bovine endothelial cell proliferation at the same concentration as that of angiostatin which is composed of kringle 1 to 4 domains. Additional experiments indicate that individual kringles in angiostatin have varied anti-angiogenesis activities. These data suggest that the kringle structure is essential for expression of the anti-angiogenesis activity of angiostatin. Each kringle possessing anti- angiogenesis activity can act as a "mini-angiostatin". There are no kringle-like structures present in the endostatin molecule. The anti- angiogenic mechanisms of endostatin appears completely different from angiostatin. It is important to know the anti- angiogenic activity of a single molecule containing the two complete different structures of kringle 5 and endostain. It would therefore be useful to determine whether the fusion of human plasminogen kringle 5, the mini-angiostatin, with the human endostatin, the strongest angiogenesis inhibitor so far discovered, would create a potentially potent angiogenesis inhibitor "KED". The estimated MW of "KED" is 30,001 Da, less than the 38 kDa of angiostatin.
Human tissue plasminogen activator (tPA) is a thrombolytic agent used primarily to treat myocardial infarction (Topol et al., 1988; Stump et al., 1989), ischemic stroke (National Institute, 1997; Wardlaw et al., 1997) and pulmonary embolism (Goldhaber et al., 1988; Parker et al., 1988). tPA has a modular structure, consisting of a fibronectin-like "finger", an epidermal growth factor domain, two kringle structures and a serine protease domain (Pennica et al., 1983; Patthy, 1985). The primary structure of kringles contains approximately 90 amino acids, with a rigidly conserved triple disulphide bond pattern. The two tPA kringles show a high homology to each other sharing 51% identical amino acid residues. The tPA kringles also are markedly homologous with the kringles of plasminogen. There are 28 to 35% identical residues between two tPA kringles and the five kringles in plasminogen (Fig 1).
Kringles are involved in basic protein-protein interactions. There are two known major biological functions of kringles, fibrinogenosis and angiogenesis. Kringles in tPA and plasminogen provide the site-specific thrombolytic reaction in the fibrinogenosis system (de Munk et al., 1989; Nesheim et al., 1990). The lysine-binding site in tPA or plasminogen kringles, which can interact with internal lysine in fibrin is responsible for fibrin binding (Horrevoets et al., 1994; Urano et al., 1991). Kringle structures of plasminogen have been demonstrated to be the key player of angiostatin. in the natural anti-angiogenesis reaction chain (Cao et al., 1996). However, the site(s) for expression of anti-angiogenesis activity of kringles are unknown. Also unknown is whether tPA kringles have the same anti-angiogenic activity as plasminogen kringles.
Binding of a kringle to endothelial cells can be an indispensable condition for anti- angiogenic activity. Studies of the interaction between tPA and endothelial cells showed that tPA binds to endothelial cells in a kringle (tPA kringle 2)-mediated manner (Mulder et al., 1997; Cheng et al., 1955), providing indirect evidence that tPA kringle 2 binds with endothelial cells. However, neither the lysine-binding site nor the ability of binding with endothelial cells has been found in the tPA kringle domain. Properties of tPA kringle 2, together with high homology between tPA and plasminogen kringle suggest that the tPA kringle 2 can posses anti-angiogenesis activity, which lead the present inventors to investigate the anti-angiogenesis function of tPA kringles.
It would therefore be useful to establish that distinct active sites in kringles play different roles in fibrinogenosis and angiogenesis. Additionally, it would be useful to show that kringles lacking lysine-binding site show anti- proliteration activity of endothelial cells, indicating that the lysine-binding site in kringles is not the site for anti-angiogenic function.
It would therefore be useful to clone human endostatin with the 12 amino acid at the N-terminus and to indicate that the recombinant human endostatin is as active as its mouse counterpart. It is thus set forth by the present application that the deletion of 12 amino acids from the circulating human endostatin is the cause of enzymatic degradation.
Since abnormal growth of blood vessels is associated with a number of diseases such as cancer, diabetes, arthritis, angiosarcoma and angiokeratoma, it would be useful to identify agents inhibiting growth of new blood vessels which represents a novel treatment of these diseases. The tPA kringle 2 peptide is a small protein of 9.5 kDa. Therefore, the modified version with mutation of histidine to tyrosine shows strong anti-angiogenic activity which is useful for a pharmaceutical composition.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a pharmaceutical composition including at least one kringle protein and a pharmaceutically acceptable carrier. Also provided by the present invention is a method of treating a disease by administering an effective amount of a compound from the group consisting of kringle proteins, KED and a kringle derived from the tPA protein.
DESCRIPTION OF THE DRAWINGS
Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:
Figure 1 shows a schematic representation of molecular cloning and fusion of KED gene; a vector pEXdna Y containing T7 promoter and dnaY gene having been constructed for KED gene expression; KED gene generated by fusion of plasminogen kringle 5 cDNA and collagen α1 (XVIII) fragment being shown; plasminogen kringle 5 cDNA being derived from human liver mRNA; collagen αl (XVIII) cDNA being from human kidney; Figure 2 is a photograph showing the expression of the KED protein; the SDS-PAGE shows the induction of KED expression by 0.5 mM IPTG in 3 hours, shown as a Coomassie Blue-stained 10% polyacryl amide gel with 50 ug proteins on each lane;
Figure 3 is a photograph showing the isolation and purification of the KED protein; pure KED protein (2.5 μg) was analyzed by 10% SDS-PAGE with Coomassie Blue-staining which reveals a single KED peptide migrated at the position of 30 kDa;
Figures 4 A and B are graphs showing the in vitro anti-angiogenic activity of purified KED; A. Cells on each assay filter were counted; B. KED activity was analyzed in a proliferation assay using bovine endothelial cells; anti-angiogenic activity of KED is compared with endostatin and plasminogen kringle 5;
Figure 5 A and B are photographs showing the inhibition of blood capillaries growth by KED., the in vivo anti-angiogenic activity of KED was tested by rat corneal assay; 25 ng bFGF intitiates an aggressive angiogenic response (A) and 25 ng bFGF +12.5 ng KED significantly suppresses (B);
Figure 6 shows the alignment of amino acid sequences of human tPA (K1tPA, K2tPA) and plasminogen kringles (K1-5plg); the kringle being aligned according to their conserved cysteines (indicated by stars), positions with identical amino acids in all seven kringles are boxed;
Figure 7 is a photograph showing the expression of tPA kringle 2 domain which was denatured in 16% polyacrylamide gel and stained with Coomassie-blue; the tPA kringle 2 domain was purified by an affinity lysine- agarose chromatography; Figure 8 is a graph showing the test of anti-angiogenic activity of tPA fragments; the proliferation assay used bovine endothelial cells wherein the Asn^-Thr63 fragment is tPA kringle 2 domain and lle^-Thr63 contains tPA kringle 1 and 2, also Asn177-Pro527 contains tPA kringle 2 and serine protease domain;
Figure 9 is a photograph showing the expression of tPA kringle 2 mutants; Whole cells, equivalent to 50 g proteins, were analyzed by denatured 16% SDS-PAGE;
Figure 10 is a diagram showing the site-directed mutagenesis in tPA kringle 2; mutation are presented by arrows with amino acid sequence of tPA kringle 2 domain; mutations in the middle of kringle caused unstable expression; the effects of mutations on anti-angiogenic activity are compared (internal);
Figure 11 is a photograph showing the KED Protein; the KED gene was expressed under a T7 promoter, the recombinant E. coli strain BL21 (DE3 ) bearing the KED gene produced KED protein with an efficiency exceeding 30% of total cellular protein; the KED protein was isolated using French Press at 20.000 psi; the KED inclusion bodies were dissolved by 8M urea and refolded in a redox solution containing 1.1 mM GSH and 0.19 mM GSSG. Refolded KED protein was concentrated by ultrafiltration and was followed by Sephadex 0-25 chromatography and dialysis against PBS buffer; the purified KED protein appeared at the 30 kDa position on the SDS-PAGE;
Figure 12 is a bar graph showing the activity of the KED protein; KED activity was analyzed by a standard proliferation assay using Bovine endothelial cells; cells on each assay filter were counted 5 days after treatment with KED drug; the control indicates endothelial cells proliferation in the presence of bFGF; proliferation inhibition assays were performed at different concentrations of KED; proliferation (angiogenesis) of endothelial cells induced by bFGF was significantly inhibited by a concentration as low as 8 nM of KED drug;
Figure 13 is a bar graph showing the activity of endostatin (D); endostatin was the strongest angiogenesis inhibitor prior to the construction of the KED molecule, in identical experiments to those of Fig 12 were performed endostatin had no activity at a concentration of 12 nM;
Figure 14 is a graph showing the comparisons of KED with KS and D drug function; angiostatin (Plasminogen Kringle-5) showed weak anti- angiogenesis activity; Endostatin activity is stronger than angiostatin KED drug is the strongest anti-angiogenesis drug;
Figure 15 is a graph showing the animal treatment data, wherein the results are depicted of animals having tumors being treated with either PBS or KED;
Figure 16 is a graph showing the animal treatment data, wherein the results are depicted of another group of animals having tumors being treated with either PBS or KED;
Figure 17 is a graph showing the animal treatment data, wherein the results are depicted of another group of animals having tumors being treated with either PBS or KED;
Figurelδ is a photograph showing the expression of tPA kringle 2
mutants; whole cells equivalent to 50 μg protein were analyzed by denatured
16% SDS-PAGE; M is protein, MW is a marker; the S mutant is a tPA k2 Ser186 → Lys; the Y mutant is a tPA k2 Tyr214 → Phe; the H mutant is a tPA k2
His244 → Tyr;
Figures 19 A-C are graphs and photographs showing the anti- angiogenic activity of tPA kringle 2 mutant; tPA kringle 2 H mutant inhibits
endothelial cell proliferation (A) in a dose-dependent manner, endothelial migration (B) and corneal angiogenesis (C) where control bFGF pellet (C, left) is inhibited by bFGF +5 ng H mutant (C, right);
Figures 20 A and B are graphs showing the in vitro anti-angiogenic activity of purified KED; the activity was analyzed by proliferation assay (above) and migration (below) using bovine endothelial cells; cells on each assay filter were counted (A); anti-angiogenic activity of KED is compared with that of endostatin (B);
Figures 21 A-C are graphs showing the Hi-Trap Heparin-Sepharose Chromotagraphy results;
Figures 22 A-D are graphs showing cell proliferation and migration upon treatment with KED; (A) shows the absorbance over a six day period;
(B) shows the aborbance over a four day period; (C) is a bar graph showing the BAC cell migration when treated with KED; and (D) is a comparative graph showing BAC cell proliferation when treated with angiostatin; Figures 23 A-C are bar graphs showing comparative data between
KED and angiostatin; (A) shows MTT assay for KED; (B) shows U87 cell invasion for angiostatin; (C) shows BAC cell migration for angiostatin;
Figures 24 A and B are a depiction of (A) the pET system cloning sites and (B) the pET vector system circular map and polylinker.
DETAILED DESCRIPTION OF THE INVENTION
Generally, the present invention provides a pharmaceutical composition for the treatment of disease including at least one kringle protein and a pharmaceutically acceptable carrier. Additionally, there is provided a pharmaceutical composition for the treatment of disease including a KED molecule or the kringle from the tPA molecule.
More specifically, the pharmaceutical of the present invention contains therein anti-angiogenic properties, thus enabling the use of the pharmaceutical in treating cancer and other diseases in which anti-angiogenic properties will minimize or alleviate the symptoms of the disease or the disease itself. The present invention includes compositions and methods for the detection and treatment of diseases and processes that are mediated by or associated with angiogenesis. The composition can be isolated from body fluids including, but not limited to, serum, urine and ascites, or synthesized by chemical or biological methods (e.g. cell culture, recombinant gene expression, protein synthesis, and in vitro enzymatic catalysis of plasminogen or plasmin to yield active angiostatin).
Recombinant techniques include gene amplification from DNA sources using the polymerase chain reaction (PCR), and gene amplification from RNA sources using reverse transcriptase/PCR. The present invention inhibits the growth of blood vessels into tissues such as unvascularized or vascularized tumors.
The present invention also encompasses a composition comprising, a vector containing a DNA sequence encoding for the kringles of the present invention, wherein the vector is capable of expressing the kringles of the present invetion when present in a cell, a composition comprising a cell containing a vector, wherein the vector contains a DNA sequence encoding the kringles of the present invention or fragments or analogs thereof, and wherein the vector is capable of expressing the kringles of the present invention when present in the cell, and a method comprising, implanting into a human or non-human animal a cell containing a vector, wherein the vector contains a DNA sequence encoding the kringles of the present invention, and wherein the vector is capable of expressing the kringles of the present invention when present in the cell. Still further, the present invention encompasses the kringles of the present invention, fragments thereof, antisera, receptor agonists or receptor antagonists that are combined with pharmaceutically acceptable carriers, and optionally sustained-release compounds or compositions, such as biodegradable polymers, to form therapeutic compositions. In particular, the invention includes a composition comprising an antibody that specifically binds to the kringles of the present invention, wherein the antibody does not bind to plasminogen.
Standard molecular biology techniques known in the art and not specifically described are generally followed as in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989). Additionally, standard methods in immunology known in the art and not specifically described are generally followed as in Stites et al. (eds), Basic and Clinical Immunology (&h Edition), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980).
Immunoassays are also provided by the present invention. In general, ELISAs are the preferred immunoassays employed to assess a specimen. Both polyclonal and moloclonal antibodies can be used in the assays. The specific assay to be used can be determined by one skilled in the art.
The compounds of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art. Additionally, when the compound is being administered orally, there must be included a compound for preventing the degradation of the kringle.
The compounds of the present invention can be used in combination with other compositions and procedures for the treatment of diseases. For example, a tumor can be treated conventionally with surgery, radiation or chemotherapy combined with the kringles of the present invention and then the kringles of the present invention can be subsequently administered to the patient to extend the dormancy of micrometastases and to stabilize and inhibit the growth of any residual primary tumor. Additionally, the kringles of the present invention, fragments thereof, antisera, receptor agonists, receptor antagonists, or combinations thereof, are combined with pharmaceutically acceptable excipients, and optionally sustained-release matrix, such as biodegradable polymers, to form therapeutic compositions. A sustained- release matrix, as used herein, is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. The sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) polyanhydrides, poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and glycolic acid). The angiogenesis-modulating therapeutic composition of the present invention can be a solid, liquid or aerosol and can be administered by any known route of administration. Examples of solid therapeutic compositions include pills, creams, and implantable dosage units. The pills can be administered orally, the therapeutic creams can be administered topically. The implantable dosage unitst can be administered locally, for example at a tumor site, or which can be implanted for systemic release of the therapeutic angiogenesis- modulating composition, for example subcutaneously. Examples of liquid composition include formulations adapted for injection subcutaneously, intravenously, intraarterially, and formulations for topical and intraocular administration. Examples of aersol formulation include inhaler formulation for administration to the lungs. The the kringles of the present invention of the present invention also can be used to generate antibodies that are specific for the inhibitor and its receptor. The antibodies can be either polyclonal antibodies or monoclonal antibodies. These antibodies that specifically bind to the the kringles of the present invention its receptors can be used in diagnostic methods and kits that are well known to those of ordinary skill in the art to detect or quantify the the kringles of the present invention or it's receptors in a body fluid or tissue. Results from these tests can be used to diagnose or predict the occurrence or recurrence of a cancer and other angiogenic mediated diseases. The the kringles of the present invention also can be used in a diagnostic method and kit to detect and quantify antibodies capable of binding the kringles of the present invention. These kits would permit detection of circulating antibodies which indicates the spread of micrometastases in the presence of the kringles of the present invention secreted by primary tumors in situ. Patients that have such circulating anti- antibodies can be more likely to develop multiple tumors and cancers, and can be more likely to have recurrences of cancer after treatments or periods of remission. The Fab fragments of these antibodies can be used as antigens to generate Fab-fragment antisera which can be used to neutralize anti the kringles of the present invention antibodies.
By way of background, tissue plasminogen activator (tPA) contains two kringle structures. Angiostatin, an angiogenic inhibitor, is composed of four kringles of plasminogen. Computer-aided analysis revealed similarities and differences between the tPA and plasminogen kringles. The tPA kringle 2 is composed of 86 amino acids (Asn^-Thr263 in tPA molecule) and has been expressed in E. coli cells to 39.9% of the total cellular protein. The refolded peptide fragment specifically inhibited endothelial cell proliferation. Furthermore, a series of mutations in the tPA kringle 2 domains were created by PCR-mediated site-directed mutagenesis. Among seven mutations that have been examined, a substitution of histidine with tyrosine dramatically increased its anti-angiogenic activity. The improved properties associated with this modified tPA kringle 2, such as its lower molecular weight, higher anti-angiogenic activity and the ability to produce large quantities of proteins targets these proteins for treatment of diseases related to abnormal growth of blood vessels.
The pharmaceutical composition of the present invention utilizes kringle proteins. There are at least three functional sites in a single short peptide (approximate 90 amino acids) of tPA kringle 2: a lysine-binding site, an endothelial cell-binding site and a possible site for anti-angiogenesis activity. To test the functions of tPA kringle 2 the kringle domains of tPA were cloned and expressed. The tPA kringle domains have been difficult to express because very small amounts of tPA kringle 2 (0.05 mg/g cells) are produced from E. coli and 3 ;l.9- only 0.3-0.5 mg/g cells of kringle 2 secreted from Pichia cells have been reported. However, the tPA kringle 2 peptide was successfully expressed as high as 58.5 mg/g cells E. coli according to the methods of the present invention. The recombinant tPA kringle 2 peptide inhibits the proliferation of endothelial cells. In addition, a potent active site in the tPA kringle 2 structure was discovered for expressing kringle anti- angiogenic activity.
As a thrombolytic agent, tPA has been used for treatment of stroke and myocardial infarction. It was further uncovered as part of the present invention that an internal fragment of tPA, kringle 2 possesses anti- angiogenic activity. The tPA kringle 2 shares significant homology with angiostatin, an endogenous angiogenic inhibitor. Angiostatin is an internal fragment of plasminogen. However, plasminogen is not a therapeutic protein. Accordingly, in order to obtain strong anti-angiogenic tPA molecule, PCR- mediated site-directed mutagenesis has been performed.
The benefits of using the kringles set forth in the present application include the following: (1) Large quantities of tPA kringle 2 can be mass produced in its modified versions in E. coli cells. Also, tPA kringle 2 can be over-expressed as high as 39.9% of total cellular proteins, more than thousand times higher than previously reported; (2) This is the first to discovery of an internal fragment (Asn^-Thr263, kringle 2) of tPA which possesses anti-angiogenic activity; and (3) The substitution of histidine with an aromatic residue tyrosine significantly increases anti-angiogenic activity of tPA kringle 2 domain.
The present invention also provides a method of treating a disease. The methods includes the steps of administering an effective amount of a kringle protein, KED or tPA. Applications for the present invention include but are not limited to the following diseases: atherosclerosis, arthritis, retinopathy, cancer, and other similar diseases.
The method can be used in multiple applications. In using the present method a kit is provided containing therein all of the necessary components. In order to make this procedure as easy as possible for the individual, the kits include all of the necessary reagents for creating the anti-angiogentic activity in cells and in mammal. The kits can also include the specific kringles to be used by the individual. Alternative kits are made available in which the individual can create their own pharmaceutical composition based on the peptides provided by the present invention.
The kit provides instructions, antiserum, the proteins of the present invention, and possibly radiolabeled kringles and/or reagents for precipitation of bound antibody complexes. The kit is useful for the measurement of these kringles in biological fluids and tissue extracts of animals and humans with and without tumors. Another kit is used for localization of the kringles in tissues and cells. This kringle immunohistochemistry kit provides instructions, kringle antiserum, and possibly blocking serum and secondary antiserum linked to a fluorescent molecule such as fluorescein isothiocyanate, or to some other reagent used to visualize the primary antiserum. Immunohistochemistry techniques are well known to those skilled in the art. This kringle immunohistochemistry kit permits localization of kringle in tissue sections and cultured cells using both light and electron microscopy. It is used for both research and clinical purposes. For example, tumors are biopsied or collected and tissue sections cut with a microtome to examine sites of kringle production. Such information is useful for diagnostic and possibly therapeutic purposes in the detection and treatment of cancer. Another method to visualize sites of kringle biosynthesis involves radiolabeling nucleic acids for use in in situ hybridization to probe for kringle messenger RNA. Similarly, the kringle receptor can be localized, visualized and quantitated with immunohistochemistry techniques.
Also provided by the present invention, are the peptides which are positives derived from the sequences of the present invention are as follows: peptide 10010281 (region 4 k2 R133-W142 k2tPA) Acetyl-RNPDGDAKPW- amide MW 1197 (10-mer) peptide 10010286 (region H2/5 H144-L151k2tPA) Acetyl-YVLKPRR-amide MW 973 (7-mer); peptide 10010284 (region K2 H144-L151k2tPA) ) Acetyl-HVLKNRR-amide MW 964 (7-mer); hK2tPA (88aa) sequence begins at N177→T263 of tPA 1 It is important to note that the H mutant has a single point mutation H244→Y. The peptides are short 7-10 amino acid sequences which preserve the majority of the biologic activity of the parent H mutant protein, which is derived from the sequence of the second kringle domain (k2) of human tissue plasminogen activator (tPA) protein, specifically from amino acid 177 to 263.
The above discussion provides a factual basis for the use of kringles in the treatment of disease. The methods used with and the utility of the present invention can be shown by the following non-limiting examples and accompanying figures.
EXAMPLES GENERAL METHODS:
General methods in molecular biology: Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989) and in Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988), and in Watson et al., Recombinant DNA, Scientific American Books, New York and in Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998) and methodology as set forth in United States patents 4,666,828; 4,683,202; 4,801 ,531 ; 5,192,659 and 5,272,057 and incorporated herein by reference. Polymerase chain reaction (PCR) was carried out generally as in PCR Protocols: A Guide To Methods And Applications, Academic Press, San Diego, CA (1990). In-situ (In-cell) PCR in combination with Flow Cytometry can be used for detection of cells containing specific DNA and mRNA sequences (Testoni et al, 1996, Blood 87:3822.)
General methods in immunology: Standard methods in immunology known in the art and not specifically described are generally followed as in Stites et al.(eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980).
Immunoassays
In general, ELISAs are the preferred immunoassays employed to assess a specimen. ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assays. Where appropriate other immunoassays, such as radioimmunoassays (RIA) can be used as are known to those in the art. Available immunoassays are extensively described in the patent and scientific literature. See, for example, United States patents 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901 ,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011 ,771 and 5,281 ,521 as well as Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor, New York, 1989
Recombinant Protein Purification
Marshak et al, "Strategies for Protein Purification and Characterization. A laboratory course manual." CSHL Press, 1996.
For gene therapy: By gene therapy as used herein refers to the transfer of genetic material (e.g DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype. The genetic material of interest encodes a product (e.g. a protein, polypeptide, peptide, functional RNA, antisense) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value. Alternatively, the genetic material of interest encodes a suicide gene. For a review see, in general, the text "Gene Therapy" (Advances in Pharmacology 40, Academic Press, 1997).
Two basic approaches to gene therapy have evolved: (1) ex vivo and
(2) in vivo gene therapy. In ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vitro. Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to express the transfected genetic material in situ.
\n in vivo gene therapy, target cells are not removed from the subject rather the genetic material to be transferred is introduced into the cells of the recipient organism in situ, that is within the recipient. In an alternative embodiment, if the host gene is defective, the gene is repaired in situ [Culver, 1998]. These genetically altered cells have been shown to express the transfected genetic material in situ.
The gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell. The expression vehicle can include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR of the gene can be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore as used herein the expression vehicle may, as needed, not include the 5'UTR and/or 3'UTR of the actual gene to be transferred and only include the specific amino acid coding region.
The expression vehicle can include a promotor for controlling transcription of the heterologous material and can be either a constitutive or inducible promotor to allow selective transcription. Enhancers that can be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non-translated DNA sequence which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. The expression vehicle can also include a selection gene as described herein below.
Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods can be found generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Ml (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Ml (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see United States patent 4,866,042 for vectors involving the central nervous system and also United States patents 5,464,764 and 5,487,992 for positive-negative selection methods.
Introduction of nucleic acids by infection offers several advantages over the other listed methods. Higher efficiency can be obtained due to their infectious nature. Moreover, viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events.
A specific example of DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector AdenoTK. This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences. This vector can be used to infect cells that have an adenovirus receptor which includes most cancers of epithelial origin as well as others. This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vitro or ex vivo culture of cells, a tissue or a human subject.
Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy. Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus. An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation will not occur.
Features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type.
In addition, recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical- type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. The vector to be used in the methods of the invention will depend on desired cell type to be targeted and will be known to those skilled in the art. For example, if breast cancer is to be treated then a vector specific for such epithelial cells would be used. Likewise, if diseases or pathological conditions of the hematopoietic system are to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, would be used.
Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection. In the former case, the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles which are capable of undergoing further rounds of infection. The vector's genome is also engineered to encode and express the desired recombinant gene. In the case of non-infectious viral vectors, the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection. The specific type of vector will depend upon the intended application. The actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology.
The recombinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neuro-degenerative diseases. Following injection, the viral vectors will circulate until they recognize host cells with the appropriate target specificity for infection. An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid. Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal. Such non-targeting vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like. Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art.
Alternatively, transfection of promoter sequences, other than one normally found specifically associated with these kringles, or other sequences which would increase production of protein are also envisioned as methods of gene therapy. An example of this technology is found in Transkaryotic Therapies, Inc., of Cambridge, Mass., using homologous recombination to insert a "genetic switch" that turns on an erythropoietin gene in cells. See Genetic Engineering News, Apr. 15, 1994. Such "genetic switches" could be used to activate the kringles (or the receptor) in cells not normally expressing kringles (or the receptor) . Gene transfer methods for gene therapy fall into three broad categories-physical (e.g., electroporation, direct gene transfer and particle bombardment), chemical (lipid-based carriers, or other non-viral vectors) and biological (virus-derived vector and receptor uptake). For example, non-viral vectors can be used which include liposomes coated with DNA. Such liposome/DNA complexes can be directly injected intravenously into the patient. It is believed that the liposome/DNA complexes are concentrated in the liver where they deliver the DNA to macrophages and Kupffer cells. These cells are long lived and thus provide long term expression of the delivered DNA. Additionally, vectors or the "naked" DNA of the gene can be directly injected into the desired organ, tissue or tumor for targeted delivery of the therapeutic DNA.
Delivery of gene products/therapeutics (compound):
The compound of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
In the method of the present invention, the compound of the present invention can be administered in various ways. It should be noted that it can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants and vehicles. The compounds can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and direct parenchymal infusion techniques. Implants of the compounds are also useful. The patient being treated is a warm-blooded animal and, in particular, mammals including man. The pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention.
It is noted that humans are treated generally longer than the mice or other experimental animals exemplified herein which treatment has a length proportional to the length of the disease process and drug effectiveness. The doses can be single doses or multiple doses over a period of several days, but single doses are preferred.
The doses can be single doses or multiple doses over a period of several days. The treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated.
When administering the compound of the present invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, can also be used as solvent systems for compound compositions. Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds.
Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.
A pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, microspheres and programmable pumps for parenchymal infusion into organs, such as brain. Examples of delivery systems useful in the present invention include: 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art.
A pharmacological formulation of the compound utilized in the present invention can be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable. Known techniques which deliver it orally or intravenously and retain the biological activity are preferred.
In one embodiment, the compound of the present invention can be administered initially by intravenous injection to bring blood levels to a suitable level. The patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition and as indicated above, can be used. The quantity to be administered will vary for the patient being treated and will vary from about 100 ng/kg of body weight to 100 mg/kg of body weight per day.
Additionally, these kringle proteins can be chemically coupled to isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules, chemiluminescent, bioluminescent and other compounds for a variety of applications. The efficiency of the coupling reaction is determined using different techniques appropriate for the specific reaction. For example, radiolabeling of these protein with .sup.125 I is accomplished using chloramine T and Na.sup.125 I of high specific activity. The reaction is terminated with sodium metabisulfite and the mixture is desalted on disposable columns. The labeled protein is eluted from the column and fractions are collected. Aliquots are removed from each fraction and radioactivity measured in a gamma counter. In this manner, the unreacted Na.sup.125 I is separated from the labeled protein. The protein fractions with the highest specific radioactivity are stored for subsequent use such as analysis of the ability to bind to the antisera. Another application of protein conjugation is for production of polyclonal antisera. For example, the kringle proteins containing lysine residues are linked to purified bovine serum albumin using glutaraldehyde. The efficiency of the reaction is determined by measuring the incorporation of radiolabeled protein. Unreacted glutaraldehyde and protein are separated by dialysis. The conjugate is stored for subsequent use. Antiserum against these kringles, analogs thereof, protein fragments and receptor can be generated. After protein synthesis and purification, both monoclonal and polyclonal antisera are raised using established techniques known to those skilled in the art. For example, polyclonal antisera can be raised in rabbits, sheep, goats or other animals. These kringle proteins conjugated to a carrier molecule, such as bovine serum albumin, is combined with an adjuvant mixture, emulsified and injected subcutaneously at multiple sites on the back, neck, flanks, and sometimes in the footpads. Booster injections are made at regular intervals, such as every 2 to 4 weeks. Blood samples are obtained by venipuncture, for example using the marginal ear veins after dilation, approximately 7 to 10 days after each injection. The blood samples are allowed to clot overnight at 4. degree. C. and are centrifuged at approximately 2400.times.g at 4.degree. C. for about 30 minutes. The serum is removed, aliquoted, and stored at 4.degree. C. for immediate use or at -20 to -ΘO.degree. C. for subsequent analysis.
In addition to the diseases specifically stated herein, KED is also useful in the treatment of other diseases, such as but not limited to: bronchial vascular congestion, inflammatory bowel disease, adult respiratory distress syndrome, Castleman's disease, mycosis fungoides, atherosclerosis, restenosis, ischemia (limb, cerebral, myocardial), cancer, inflammation/chronic inflammatory disease, diabetes (diabetic retinopathy), ophthamology, wound healing, glomerulonephritis, Moyamoya disease, coronary artery disease, ulcer, arthritis, Kaposi sarcoma, psoriasis, peripheral arterial disease, avitreorefinopathy, Macular pucker, chronic patella tendinitis, fibrodysplasia ossifecaus progressiva, Chrohn's disease, hepatitis, neovascular glaucoma, aneurysm, rheumatoid arthritis, leiomyoma, Bartonella quintata infection, AIDS, aortic aneurysms, renal disease, veno-occulsive disease, allergic rhinitis, and hemangioma. EXAMPLE 1
Overview of the Anti-angiogenic, KED Fusion Protein:
Human plasminogen kringle 5 is a novel angiogenesis inhibitor. It is a small protein of 10 kDa molecular weight. Angiostatin, a 38 kDa protein, contains 4 kringles of plasminogen kringle 1 through 4. A proliferation assay using bovine endothelial cells indicates that the plasminogen kringle 5 is at least as strong as anti-angiogenic effect as angiostatin. Endostatin is a newly identified angiogenic inhibitor and is the most effective inhibitor of proliferating endothelial cells so far discovered. Endostatin is a 20 kDa C-terminal fragment of collagen α1 (XVIII) sharing no homology with angiostatin and plasminogen kringle 5 domains. Hydrophobic profile analysis indicates that endostatin is highly hydrophobic and plasminogen kringle 5 is a hydrophilic protein. Refolding of endostatin protein produced from E. coli cells is not efficient (<1%). The cDNA of human plasminogen kringle 5 domain was fused with that of human collagen α1 (XVIII) C-terminal fragment and this generated a novel protein with improved (approximately 100 folds) refolding efficiency and significantly increased anti-angiogenic activity. The fusion protein, now called "KED" has an estimated MW of 30,001 Da, has been abundantly produced in E. coli cells. Its anti-angiogenic activity has been examined in three ways: in vitro endothelial cell proliferation assay, in vivo corneal assay and in vivo tumor models.
Molecular Cloning & Protein Synthesis:
The KED gene was constructed by fusion of cDNA plasminogen kringle 5 encoding Glu478 -Ala562 with a collagen α1 (XVIII) cDNA fragment encoding Hisl154 -Lys1336. The KED gene (Fig. I) is expressed in E. Coli cells. AGA codon is a rare codon used in E. Coli (Wada et al., 1990). The tRNAAGA is encoded by dnaY gene. The dnaY gene also has been reported to be essential for DNA replication (Garcia et al., 1986). To improve productivity of the KED protein in E. Coli, a vector, pEXdnaY, was constructed containing a strong T7 promoter for high- speed transcription of KED mRNA and a dnaY gene supplying tRNAAGA for efficient translation of the KED product. The process for KED gene fusion and cloning in pEXdnaY vector is shown in the Sequence Listing.
The expression of KED protein from pEXdnaY -hKED, was induced by
IPTG (isopropyl β-D-thiogalactopyranoside). Abundant KED production was achieved. KED protein accumulated up to 47% of total cellular protein by 2 hours of induction with 0.5 mM IPTG (Fig. 2). Optimal production of KED protein was performed using a fermentor. The KED protein was produced as inclusion bodies in cytosol. KED inclusion bodies were isolated and efficiently refolded in the presence of 0.5 M L-arginine at pH 8.6. As a control, human endostatin was simultaneously expressed from pEXdnaY-hED. More than 99% of the human endostatin protein precipitated in the refolding solution as has been previously observed. No precipitation of KED fusion occurs during the refolding process. This indicates efficient productivity of KED compared to that of endostatin.
Pure KED protein was obtained by HiTrap Heparin-Sepharose chromatography. Analysis of KED protein by denatured SDS-PAGE (polyacrylamide gel electrophoresis) revealed a single 30 kDa peptide that agreed with the molecular weight estimated from the deduced amino acid sequence (Fig. 3).
N-terminal analysis of KED protein (by Commonwealth Biotechnologies, Richmond, V A) provided an identical sequence of
MEEDHMFGNGKGYRG as deduced from DNA sequence (Fig. 1), where the 5th residue is Cys that generally does not show up by the analysis system. Compositional analysis of KED generated similar results with those which were predicted. Both the N-terminal and compositional analysis indicated that the pure protein isolated is the correct form of the KED gene product. Endothelial Proliferation:
A proliferation assay using bovine endothelial cells indicated that fusion protein KED completely inhibited the endothelial cells migration at a concentration as low as 8 nM (250 ng/ml), where, human endostatin shows almost no activity at 12 nM (250 ng/ml) by the same assay (Fig. 4). The IC50 of KED observed is less than 100 ng/ml. The IC50 of human endostatin in the assay was 350 ng/ml. These results indicate that the human fusion protein KED is much stronger than endostatin, the strongest anti-angiogenic agent reported to date.
Inhibition of Blood Vessel Growth in Rat Cornea:
Rodent corneal assay has been performed to test in vivo the efficacy of KED fusion protein. Different amounts, 1.25 ng, 5 ng, and 10 ng of KED were applied in the presence of 50 ng of bFGF 1.25 ng of KED fusion protein efficiently inhibited the growth of capillary blood vessels from the corneal bed (Fig. 5).
Suppression of Gliomas in Animal Model:
Gliomas were implanted in the rat brain, One mg/kg body weight of KED protein was used to treat animals for 14 days (Fig. 6).
Anti-angiogenic Mechanism of Kringle: Cloning and Expression of tPA Kringles:
The tPA kringles have been cloned and inserted into pET11a a vector for protein expression under the control of a strong T7 phage promoter and the laclq repressor. Recombinant plasmid pK1tPA encodes r-[K1,PA], where, r stands for recombinant and [K1tPA] represents amino acid sequence residues [Ile86-Asn 177] of kringle 1 domain in tPA molecule. The recombinant plasmid pK2tPA encodes r-[K2,PA] representing amino acid residues [Asn177-Thr263] of tPA kringle 2 domain. Recombinant plasmid pK12tPA encodes a r-[K12tPA] protein containing kringle 1 and kringle 2 domain, an internal fragment of [Ile86-Thr263] in tPA protein sequence.
Recombinant proteins expressed in E. Coli were analyzed by 16% polyacrylamide gel. Whole cellular proteins of 50 μg were denatured by SDS and β-mercaptoethanol and loaded into a gel for electrophoresis. Abundant expression of 10 kDa protein has been detected from the cells carrying pK2tPA construct (Fig. 7). 10 colonies of pK2tPA, construct were selected and all ten expressed the same 10 kDa protein. A 20 kDa protein was found from the cells harboring pK12tPA plasmid. All ten clones of p12tPA expressed the 20 kDa protein. These two proteins were produced at very high levels that occupied as high as 39.9% of the total cellular protein by a density analysis of Coomassie blue-stained PAGE gel. However, no expression was observed from the cells of ten clones containing plasmid pK1tPA. No expression of kringle 1 domain was confirmed by an additional screening of 20 pK 11t.PA clones.
The DNA deduced amino acids for r-[K2tPA] protein is a peptide of 88 residues including the initial methionine. Molecular weight (MW) of r-[K2tPA] estimated at of 9,716.42 Da, agrees with the size of 10 kDA analyzed from the polyacrylamide gel. r-[KI2tPA] contains 179 residues with a MW of 19810.92 Da as expected from its DNA sequence. Expression of a 20 kDa protein from the pK12tPA plasmid perfectly fits with that expected MW. However, even r-[K1tPA], sharing 92 identical amino acids with the N-terminus of r-[K12tPA], was not expressed.
tPA kringle 2 was successfully expressed, as was the tPA kringle 1 plus kringle 2. However, tPA kringle 1 domain was unable to be expressed. Expression of tPA kringle 1 was only achieved by the fusion of kringle 2 domain. The peptide containing tPA kringle 1 only, has never been expressed. Expression of tPA kringle 1 has been reported as a fusion of the plasminogen kringle domain (DeSerrano et al., 1992).
Endothelial proliferation assay: Both recombinant proteins, r-[K2,PA] and r-[K12tPA], contain tPA kringle
2 domains. The tPA kringle 2 structure has the lysine-binding site. The lysine-bind site in the tPA kringle 2, binding resins coupled with lysine ligand, can be used for affinity protein purification. The stable epoxy-activated-lysine- agraose gel was used to purify the recombinant proteins r-^f] and r-[K12tPA]. L-arginine is a competitor of the lysine ligand. Efficient binding of recombinant tPA kringles with lysine ligand occurs, when the concentration of L-arginine in the protein solution is reduced below 25 mM. Elution of recombinant tPA kringles was carried out by using a competitive ligand of lysine, specifically 6-amino-n-caproic acid. There are three peaks detected in the eluted fractions from a 0-200 mM gradient. One is between 30 to 45 mM. One is 60 to 105 mM and a large peak between 125 to 150 mM of 6-amino- n-caproic acid. Samples from these fractions were subjected to SDS-PAGE analysis. The r-[K2tPA] and r-[K12,PA] proteins eluted in the peak of 125-150 mM 6-amino-n-caproic acid, appeared to be homogeneous on the denatured 16% polyacrylamide gel.
Different concentrations of recombinant tPA kringles were added into the culture of bovine capillary endothelial (BCE) cells. The cells were maintained in DMEM medium for five days. Cells which passed through an assay filter were counted. The assay results indicated that both the r-(K2tPA] and r-(K12lPA] proteins are active for anti-angiogenesis. Protein r-[K2tPA] containing tPA kringle 2 alone showed stronger activity than r-[K2tPA] suggesting that the tPA kringle 2 is an angiogenesis inhibitor (Fig. 8). A Potent Anti-Angiogenic Active Site in Kringle:
Obtaining a kringle possessing strong anti-angiogenesis activity benefits the development of an anti-cancer agent. Identification of active site(s) for anti-angiogenesis activity of kringles leads to an understanding of the anti-angiogenesis mechanism. A way to address this is to perform in vitro site-directed mutagenesis in kringles. The tPA kringle domain is a good choice for the mutation for the following reasons: (1) the tPA kringle 2 contains a strong lysine-binding site that is preferred for ease of isolation of the kringle mutants; and (2) the tPA kringle 2 has been extensively studied.
All five plasminogen kringles have been cloned. The sequence of anti- angiogenic activity of plasminogen kringles is kringle 5>1>3>=2>4. The most active plasminogen kringles are kringle 5 and 1. Plasminogen kringle 4 did not show activity. Because performing the analysis of anti-angiogenic activity of tPA kringles did not include plasminogen kringles, the tPA kringle activity can not be compared with that of plasminogen kringles. Sites were chosen for mutation in tPA kringle using amino acid sequences of plasminogen kringles as references, in which, plasminogen kringle 5 and 1 were selected for the positive and kringle 2, 3, 4 especially kringle 4, were selected for the negative reference.
Ideally, positive selection or negative selection can be designed. First, the amino acid sequence of tPA kringle 2 is aligned with the five plasminogen kringles. Next, the position(s) are identified where the amino acid residue(s) is identical between plasminogen 5 and 1 but is different from plasminogen 4 and tPA kringle 2. If a position is occupied with a similar or the same amino acid residue in tPA kringle 2 and plasminogen kringle 4, where the residue should be mutated is the top priority. Then the amino acid residue(s) in kringle 2 is changed to an amino acid that is identical in plasminogen kringle 5 and 1. Finally, the resultant mutant is expressed and tested for its effects on endothelial proliferation to see if the mutation changes (increases) its anti- angiogenic activity. Alternatively, negative selection can be designed by changing a residue in the tPA kringle identical with both of plasminogen kringle 5 and 1 to an amino acid identical with that of plasminogen kringle 4. Here in the present studies, mutations for positive selection were only performed.
The mutagenesis procedure utilizes a super-coiled, double-stranded and methylated DNA plasmid pK2tPA and two synthetic oligonucleotide primers containing the desired mutation. The PCR reaction employing Pfu, a proofreading DNA polymerase, efficiently produced a nicked, unmethylated daughter plasmid with a mutation which was created by the uptake of the oligonucleotide primers. The methylated parent pK2tPA plasmid was eliminated by restriction digestion using Dpn1 , which is an enzyme, cutting the methylated substrate DNA. The mutated daughter plasmids were recovered by transformation into high-efficient competent E. coli cells. A control PCR accompanied each set of mutation thus excluding the oligodeoxynucleotide primers. Selection of transformants using the ampicillin resistance maker encoded by the vector indicated that at least a hundred fold of colonies were obtained from the PCR reaction mixture containing the mutation primers compared to that of PCR excluding the primers.
Dideoxynucleotide DNA sequencing of plasmids revealed that all designed mutations were successfully introduced on the tPA kringle DNA strand. Any undesired or unexpected mutation was not observed, showing that high fidelity DNA extension has been performed during the PCR process using the proof reading Pfu enzyme.
All kringle mutants are approximately 10 kDa MW. Expression of mutants were analyzed on 16% polyacryl amide gel electrophoresis (Fig. 9), four of them were expressed. However, the Y, N and G mutants were unstable. Interestingly, the Y, N and G mutants are all located in the middle in the kringle structure, suggesting that a mutation on this region creates unstable mRNA or protein structure.
tPA kringle 2 mutants were solubilized and reduced with 8 M urea and 140 mM beta-mercaptoethanol. The mutants were then fractionated by Superfine Sephadex G- 75 chromatography in the resence of 8 M urea. The reduced mutant proteins appeared to be homogenous, as shown by the denatured SDS-PAGE analysis. The pure reduced mutants were refolded and pureed.
Kringle mutants S, K, H, W and the wild-type tPA kringle protein were tested by proliferation assay using bovine endothelial cells in the presence of bFGF. Mutations on K and W showed no effects on the anti-angiogenic activity of tPA kringle. The S mutation slightly effected the activity. The mutation on the H site generated at least 4 times higher anti-angiogenic activity compared to the parental wild-type kringle (Fig. 10), indicating that the H site is a potent active site for the expression of anti-angiogenic activity of the kringle structure.
EXAMPLE 2
Materials and methods
1. Adaptor anchored gene cloning
Human cDNA pool was generated by reverse transcription from liver and kidney mRNA using an oligodeoxynucleotide (dT)17 primer. The cDNAs were reverse transcribed in a 20 μl reaction solution containing 2.5 μg of human liver or kidney mRNA (Clontech, Palo Alto, CA), 200 ng of oligo (dT)17 primer, 5 mM DTT, 2.5 mM MgCI, 400 M of dNTP and 200 units of reverse transcriptase (BRL, Gaithersburg, MD) in 50 mM Tris buffer (pH 8.3). The mixture excluding reverse transcriptase was heated at 72° C for 5 minutesto denature mRNA and was quickly chilled on ice for 10 minutesallowing primer annealing. Then the contents were incubated at 42° C for 5 min, and combined with 200 units of reverse transcriptase for an hour incubation. The cDNA fragments encoding plasminogen kringle 5 domain and collagen a1 (XVIII) C-terminus were amplified by polymerase chain reaction (PCR).
At first, the cDNA fragment, without adaptors encoding plasminogen kringles 1 to 5, was amplified by a sense primer and a complementary primer: 5'-CTCTCAGAGTGCAAGACTGGGAATGGAAAGAAC (Leu100-Asn110), and 5'-GGCCGCACACTGAGGGACATCACAGTAGTC (Ala562-Asp553 according to the amino acid sequence of human plasminogen) (Folkman, 1996). A cDNA fragment encoding collagen a 1 (XVI 11) C-terminus was amplified by a set of primers: 5'-CACAGCCACCGCGACTTCCAGCCGGTGCTC (His1154- Leu1163) for the sense 5'-end of the peptide, and 5'-
CTACTTGGAGGCAGTCATGAAGCTGTTCTCAAT (Lys1336-lle1327) for the complementary 3'-end (Folkman, 1996). Amplification of cDNA fragments performed by using a poof-reading thermostable Pfu DNA polymerase
(Stratagene, La Jolla, CA). The PCR reaction solution (100 μl) contains 20 mM Tris (pH 8.0), 2 mM MgCI, 10 mM KCI, 0.1% Triton X-100, 10 μl first strand cDNA from the above reverse transcriptional reaction, 300 ng primers and 5 units of Pfu DNA. Twenty-five reaction cycles consisting of 45 sec at 94° C for denaturing, 45 sec at 59° C for annealing and 3 minutesat 72° C for extension were performed. PCR products were analyzed by a 1.5% agarose gel.
Addition of adaptor to the amplified cDNA fragments was performed by PCR using adaptor anchored primers. Primers for cloning of the human plasminogen kringle 5 were 5'-
GGAATTCCATATGGAAGAAGACTGTATGTTTGGG (G-[EcoRI]-[Ndel]-
[Glu478-Gly486]), and 5'-
GGAATTCCATATGGGCCGCACACTGAGGGACATC (G-[EcoRI]-[Ndel]- [Ala562-Asp556]). Collagen a1 (XVIII) C-terminus with adaptors were amplified using primers 5'- GGAATTCCATATGCACAGCCACCGCGACTTCCAG (G-[EcoRI]-[Ndel]- [His1154-lle1160]), and 5'-
CCGGGATCCCTACTTGGAGGCAGTCATGAAGCT (CCG-[BamHI]-[STOP]-[ Lys1336-Ser1330). PCR reaction solution (100 μl) contains 50 mM Tris (pH 8.8), 2 mM MgCI, 10 mM KCI, 10 μl of the above resulted PCR reaction mixture containing the cDNA fragments, 250 ng of each primer and 7.5 units of Pfu DNA polymerase. Ten reaction PCR cycles consist of 45 sec at 94° C for denaturing, 45 sec at 37° C for annealing and 2 minutesat 68° C for DNA synthesis.
A 0.57 kb DNA fragment encoding 183 amino acid residues of collagen a1 (XVIII) C-terminus with adaptors and a 0.28 kb fragment encoding 85 amino acid residues of plasminogen kringle 5, were purified from the PCR reaction mixture by spin column, and were inserted into expression vector pEXdnaY.
2. Vector pEXdnaY for expression of mammalian genes
A vector for the purpose of mammalian gene expression in E. coli was constructed. Vector pEXdnaY was constructed by combining of pET11a (Stratagene, La Jolla, CA) and pDC952. Five μg of pET11a plasmid (4.64 kb) was digested with 20 units of Hindlll enzyme at 37° C for 1 hour. The linearized pET11a was treated by adding 32 units of calf intestinal alkaline phosphatase (CIAP) into the restriction reaction mixture and was incubated at 30° C for 30 min, to prevent self-ligation of pET11a. The linearized and dephosphorylated pET11a DNA was then ligated with a 632 bp Hindlll-Hindlll DNA fragment containing dnaY gene (tRNA for AGG and AGA) that was isolated from pDC952 plasmid by Hindlll-digestion. The ligation solution was transformed into competent E. coli TG1 cells. Recombinant plasmid pEXdnaY were isolated and identified. 3. Gene fusion
A 558 bp cDNA fragment encoding collagen a1 (XVIII) C-terminus flanked by Ndel and BamHI adaptors were cloned into the pEXdnaY vector, generating a plasmid pEXdnaY-hED. Then, a 255 bp cDNA fragment encoding plasminogen kringle 5 with Ndel adaptor at the two ends were fused in front of the collagen a1 (XVIII) C-terminal fragment, generating plasmid pECdnaY-hKED. Briefly, 10 μg of pEXdnaY DNA was double-digested with 100 units of Ndel and 150 units of BamHI enzymes, following dephosphorylation with 64 units of CIAP. Pure dephosphorylated pEXdnaY vector was obtained from 0.7% agarose gel. The linearized vector was ligated with Ndel-BamHI DNA fragment of collagen a1(XVIII) C-terminus created by anchored PCR and was restriction digested with Ndel and BamHI enzymes. Recombinant plasmid pEXdnaY-hED was selected in TG1 cells and analyzed by double digestion of Ndel and BamHI enzymes. The resulted pEXdnaY- hED plasmid (5 μg) was further linearized by 100 units of Ndel and dephosphorylated with 32 units of CIAP. An Ndel-Ndel cDNA fragment encoding human plasminogen kringle 5 generated by Ndel-adaptor anchored PCR amplification was inserted into the Ndel site of pEXdnaY-hED by ligation with Ndel-linearized pEXdnaY-hED DNA. The ligation solution was transformed into competent E. coli TG1 cells. A recombinant plasmid pEXdnaY-hKED was identified by Ndel digestion and analysis of expression of a 30 kDa fusion protein.
4. Production of KED fusion Plasmid pEXdnaY-hKED was transformed into E. coli BL21 (DE3) cells. A single ampicillin resistance colony was picked up and inoculated in 1 ml of LB media containing 100 g/ml of ampicillin. After 12 hours incubation at 37° C, the culture was diluted 100 fold into the same medium. Incubation with shaking at 250 rpm was continued until OD reached 0.45 at 600 nm. Optimal conditions for expression of KED fusion were investigated by induction with IPTG (isopropyl-D-thiogalactopyranoside) with different concentrations from 0.1 to 10 mM and varied time from 15 minutesto 3 hours. The induced cultures were quickly chilled on ice and centrifuged. Cells were washed with ice-cold PBS (phosphate saline buffer) containing 5 mM EDTA (ethylenediaminetetraacetic acid). An aliquot of cells, equivalent to 50 μg protein was used for analysis of KED fusion expression by denatured 10% polyacrylamide gel.
Optimal production of KED fusion was performed using a fermentor (Bioflo2000, New Brunswick Scientific, Edison, NJ). Over night culture (200 ml) was added into 8 I LB medium enriched with 25 g/l yeast extract. One ml of Antifoam 289 (Sigma, St. Louis, MO) was added to the medium to prevent foam-formation during fermentation. Agitation was kept 225 rpm through out the process. Airing was settled to 8 l/min until cells grown to OD6000.505. IPTG was added into the fermentor at the final concentration of 0.5 mM. Airing was changed to 20 l/min. After 1 hour 55 minutesproduction (from the time IPTG added), fermentation was terminated by adding 100 ml of 0.5 M EDTA (the final OD600 was 3.1). The culture was chilled on ice and collected filtration system using 0.04 μm polyethersulfone membrane (North Carolina SRT, Cary, NC). The cells (15.2 g of wet weight) were washed with 5 I of PBS containing 1 mM EDTA, and suspended in 500 ml of the same buffer containing 0.5% of NP-40 detergent. Cells were disrupted by passing a French Press Cells at 15,000 psi. Crude KED inclusion bodies were isolated by centrifugation for 20 minutesat 12,100 g at 4° C, and washed twice with ice-cold 500 ml PBS and 0.5% NP-40. Finally, the KED inclusion bodies (9.30 g wet weight, 1.60 g protein) were suspended in 50 ml of PBS.
5. Refolding of KED fusion
The NP-40 washed KED inclusion bodies was analyzed by denatured SDS-PAGE. Contents of KED in the inclusion bodies were quantitated by scan of Comassie blue stained gel (77.2% KED). To solubilize KED inclusion bodies, 50 g of solid urea and 1.2 g of Tris were added into 50 ml of KED suspension. The pH was adjusted to 8.6, followed by adding of 80 μl Tween- 20, 160 μl 0.5 M EDTA and 950 μl b-mercaptoethanol. The solution was incubated at 37° C for 1 hour 30 min, and centrifuged for 20 minutesat 17,400 g at 23° C. The resulted clear KED protein solution was carefully added (10 ml/min) in to 5:l of refolding solution containing 2.4 g of reduced glutathione (GSH). And that was immediately combined with another 5:l of refolding solution containing 0.5 g of oxidized glutathione (GSSG). The 10:1 refolding solution used consisted of 1 kg L-arginine-HCI, 120 g Tris, 3.7 g EDTA, 10 ml of Tween-20 at pH 8.6. Refolding reaction was performed for 22h at 23° C.
Refolded KED solution was chilled on ice and concentrated to 650 ml by ultra-filtration using a regenerated cellulose membrane with 5 kDa weight cut-off (North Carolina SRT, Cary, NC). Precipitation of protein during ultra- filtration (16h) was excluded by centrifugation. The concentrated KED protein solution was further concentrated to 32 ml in a dialysis tube (3,500 Da molecular weight cut-off) and solid polyethylene glycol (PEG-8000). The pH of concentrated KED protein solution (32 ml at pH 8.5) was changed to pH 7.4. A quick passing of pi (pH 8.1) of KED was achieved by adding it drop by drop to 15 ml of 0.1 M sodium phosphate (pH 7.4) with simultaneous adding of 2N HCI to the mixture. Then, the KED protein solution was dialyzed against 5:l of 10 mM sodium phosphate (pH 7.4) containing 15 mM NaCl. Buffer was changed three times in 24 hours at 4 C. Dialyzed KED solution (145 ml) was centrifuged, and used for chromatography.
6. Heparin-Sepharose affinity chromatography
Twenty ml of dialyzed KED protein solution was diluted in 180 ml of 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. The protein solution was loaded on a Heparin-Sepharose column (HiTrap pre-packaged 5 ml column, Pharmacia, Piscataway, NJ). Columns were washed with the same buffer until the OD280 returned to the base line. KED protein was eluted by a NaCl gradient (150-1000 mM). Pure KED protein eluted within 210-330 mM NaCl was identified by 10% polyacrylamide gel electrophoresis.
7. Acticlean-Etox chromatography
Acticlean Etox (Sterogene, Arcadia, CA) resins were cleaned with 1 M NaOH for 12 hours at 4° C, and packed to column. The column was washed with PBS until pH below 7.6. Pure KED protein was dialyzed against PBS, and loaded on Actilean Etox column. Each ml of Acticleas Etox gel was used for treatment 5 mg of KED protein. The KED protein was then sterilized by passing a 0.2 μm filter.
8. Endothelial cell proliferation assay
Bovine capillary endothelial cells (BCE cells, Folkman, 1996) were obtained for the proliferation assay (O'Reilly et al., 1997). Cells were washed with PBS and dispersed in a 0.05% trypsin solution. A cell suspension (25,000 cells/ml) was made with DMEM + 10% BCS and plated on 24-well culture plates (0.5 ml/well) and incubated at 37° C in 10% CO2 for 24h. The medium was replaced with 0.5 ml DMEM + 5% BCS + 3 ng/ml recombinant bFGF and the test sample applied. After 72 hours , cells were dispersed in trypsin, resuspended and counted using a Coulter counter. Results were plotted as a dose-response and compared with other agents.
9. Endothelial cell migration assay
To determine the ability of the test compounds to block migration of endothelial cells toward the angiogenic factor bFGF, migration assays were performed in a modified Boyden chamber using BCE cells (Folkman, 1996). Cells were grown in DMEM supplemented with 10% FBS and supplemented at passage 15. To assess migration, the cells were starved overnight in DMEM supplemented with 0.1% BSA, harvested, suspended in DMEM/BSA, plated at 106 cells/ml on the lower surface of a gelatinized membrane (Nucleopore Corp., Plesanton, CA) in an inverted Boyden chamber and incubated for 1.5 hours to allow cell attachment. The chambers were then righted, test material was added to the top well and incubated for 5 hours . Membranes were then fixed and stained and the number of cells migrating to the top of the filter in 10 high-powered fields were counted. DMEM with 0.1% BSA was used as a negative control and bFGF at 10 ng/ml was used as a positive control.
10. Rat corneal angiogenesis
The rat corneal assay was performed as described (Folkman, 1996). Five μl Hydron pellets (Hydron Laboratories, New Brunswick, NJ) containing 10 or 20 μg/ml bFGF (25 or 50 ng) plus 5 or 25 ng test compound and 10 μg/ml sulcrafate were implanted in a corneal pocket of anesthetized rats. After 7 days, the animals were sacrificed, perfused with colloidal carbon, and the excised corneas were mounted for microscopy, photography and analysis.
Results
1. Molecular cloning & protein synthesis
The KED gene was constructed by fusion of cDNA plasminogen kringle 5 encoding Glu478-Ala562 with a collagen a1 (XVIII) cDNA fragment encoding His1154-Lys1336. The KED gene (Fig 1) is expressed m E. coli cells. AGA codon is a rare codon used in E.coli (Folkman, 1996). The tRNAAGA is encoded by dnaY gene. The dnaY gene also has been reported to be essential for DNA replication (Folkman, 1996). To improve productivity of KED protein in E. coli, a vector pEXdnaY was constructed containing a strong T7 promoter for high-speed transcription of KED mRNA and a dnaY gene supplying tRNAAGA for efficient translation of KED product. The process for KED gene fusion and cloning in pEXdnaY vector is shown in the Sequence Listing.
Expression of KED protein from pEXdnaY-hKED, was induced by IPTG (isopropyl -D-thiogalactopyranoside). Abundant KED production was achieved. KED protein accumulated to 47% of total cellular protein by 2 hours induction with 0.5 mM IPTG (Fig 2). Optimal production of KED protein was performed using a fermentor. The KED protein was produced as inclusion bodies in cytosol. KED inclusion bodies were isolated and efficiently refolded in the presence of 0.5 M L-arginine at pH 8.6. As a control, human endostatin was simultaneously expressed from pEXdnaY-hED. More than 99% of human endostatin protein precipitated in the refolding solution has been previously observed (O'Reilly et al., 1997). No precipitation of KED fusion occurs during the refolding process, indicating efficient productivity of KED compared to endostatin.
Pure KED protein was obtained by HiTrap Heparin-Sepharose chromatography. Analysis of KED protein by denatured SDS-PAGE (polyacrylamide gel electrophoresis) revealed a single peptide of 30 kDa that agreed well with the molecular weight estimated from the deduced amino acid sequence (Fig 3).
N-terminal analysis of KED protein (by Commonwealth Biotechnologies, Richmond, VA) resulted in the same sequence MEEDHMFGNGKGYRG as deduced from the DNA sequence (Fig 1), where the 5th residue is Cys. Compositional analysis of KED generated similar results with that predicted. Both of N-terminal and compositional analysis indicates that the pure protein isolated is the correct form of the KED gene product.
2. Endothelial cell proliferation
Proliferation assay using bovine endothelial cells indicated that the fusion protein KED completely inhibited endothelial cell proliferation in a concentration-dependent fashion, with inhibitory activity as low as 8 nM (250 ng/ml) Human endostatin shows almost no activity at 40 nM (600 ng/ml) by the same assay (Fig 4a). These results indicate that the human fusion protein KED is a more potent inhibitor of endothelial cell proliferation than endostatin (O'Reilly et al., 1997).
3. Endothelial cell migration
Migration assay using bovine endothelial cells indicated that the fusion protein KED completely inhibited endothelial cell migration at a concentration as low as 8 nM (250 ng/ml), where, human endostatin shows almost no activity at 12 nM (250 ng/ml) by the same assay (Fig 4b). The ic50 of KED is less than 100 ng/ml. IC50 of human endostatin in the assay was 350 ng/ml. These results indicate that the human fusion protein KED is a several-fold more potent inhibitor of endothelial cell migration than endostatin (O'Reilly et al., 1997).
4. Inhibition of rat corneal blood vessel growth
Rodent corneal assay was performed to test the in vivo efficacy of KED fusion protein (Fig 5). 12.5 ng of KED were applied in the presence of 25 ng of bFGF. 12.5 ng of KED fusion protein (Fig. 5b) efficiently inhibited the growth of capillary blood vessel from corneal bed compared with control, containing bFGF alone (Fig. 5a).
Discussion
Although anti-angiogenic activity varies in different kringle domain peptides, the triple disulphide bond kringle structure represents a new family of angioginhibitory compounds. Endostatin, a highly basic protein fragment of collagen a1 (XVIII), possesses high affinity with heparin. Other anti-angiogenic proteins such as platelet factor-4 (Folkman, 1996) and thrombospondin-1 (Folkman, 1996) also show high affinity with heparin. It is not yet known that how heparin affinity contributes to the anti-angiogenic function of these proteins. However, heparin affinity is a common characteristic among these proteins. Proteins exhibiting heparin affinity seem to be a different class of angiogenic inhibitors compared to the kringle family. Plasminogen kringle 5 is the most anti-angiogenic of the kringle structures. Endostatin has the highest anti-angiogenic activity within the heparin affinity proteins. In this study, these two anti-angiogenic members were fused into a single molecule. A cDNA fragment of 0.82 kb consisting of human plasminogen kringle 5 for the 5- prime end followed by the endostatin coding sequence was constructed. The cDNA has been successfully over-expressed in E. coli. The expressed product has been confirmed to be the designed fusion form, based on N- terminal sequencing and amino acid compositional analysis. A series of anti- angiogenic studies such as, endothelial proliferation and corneal assay have been performed to test the properties of this fusion protein. As expected, the fusion protein showed significantly higher anti-angiogenic activity than either kringle 5 or endostatin. Higher activity of this chimera reflects expression of combined activity from kringle 5 and endostatin. There is no structural homology between kringle 5 and endostatin. Thus, they exhibit anti- angiogenic activity by different mechanisms. Higher anti-angiogenic activity of this fusion protein is a result of simultaneous activation of different anti- angiogenic mechanisms. These results support the hypothesis that a fusion molecule of angiogenic inhibitors has, at least, additive benefit. The current study also demonstrated efficient protein production. Very inefficient production of endostatin in E. coli has been reported (O'Reilly et al., 1997). More than 99% of endostatin precipitates during refolding process. In the contrast, the fusion protein is completely soluble in the refolding solution. This improved productivity is another and unexpected advantage of gene fusion derived from the current study.
EXAMPLE 3
The kringles of the present invention can be: (i) Administered to tumor- bearing humans or animals as anti-angiogenic therapy; (ii) Monitored in human or animal serum, urine, or tissues as prognostic markers; and (iii) Used as the basis to analyze serum and urine of cancer patients for similar angiostatic molecules. It is contemplated as part of the present invention that the kringles can be isolated from a body fluid such as blood or urine of patients or the kringles can be produced by recombinant DNA methods or synthetic protein chemical methods that are well known to those of ordinary skill in the art. Protein purification methods are well known in the art and a specific example of a method for purifying the kringles, and assaying for inhibitor activity is provided in the examples below. Isolation of human endogenous kringles is accomplished using similar techniques.
One example of a method of producing kringles using recombinant DNA techniques entails the steps of identifying and purifying kringles as discussed above, determining the N-terminal amino acid sequence of the purified inhibitor, synthetically generating 5' and 3' DNA oligonucleotide primers for the kringle sequence, amplifying the kringle gene sequence using polymerase, inserting the amplified sequence into an appropriate vector such as an expression vector, inserting the gene containing vector into a microorganism or other expression system capable of expressing the inhibitor gene, and isolating the recombinantly produced inhibitor. Appropriate vectors include viral, bacterial and eukaryotic (such as yeast) expression vectors. The above techniques are more fully described in laboratory manuals such as "Molecular Cloning: A Laboratory Manual" Second Edition by Sambrook et al., Cold Spring Harbor Press, 1989. The DNA sequence of human plasminogen has been published (Browne, M. J., et al., "Expression of recombinant human plasminogen and aglycoplasminogen in HeLa cells" Fibrinolysis Vol.5 (4). 257-260, 1991) and is incorporated herein by reference. The gene for these kringles can also be isolated from cells or tissue (such as tumor cells) that express high levels of the kringles by isolating messenger RNA from the tissue, using reverse transcriptase to generate the corresponding DNA sequence and then using the polymerase chain reaction (PCR) with the appropriate primers to amplify the DNA sequence coding for the active kringle amino acid sequence. Yet another method of producing kringles, or biologically active fragments thereof, is by protein synthesis. Once a biologically active fragment of a kringles is found using the assay system described more fully below, it can be sequenced, for example by automated protein sequencing methods.
Alternatively, once the gene or DNA sequence which codes for kringles is isolated, for example by the methods described above, the DNA sequence can be determined using manual or automated sequencing methods well know in the art. The nucleic acid sequence in turn provides information regarding the amino acid sequence. Thus, if the biologically active fragment is generated by specific methods, such as tryptic digests, or if the fragment is N- terminal sequenced, the remaining amino acid sequence can be determined from the corresponding DNA sequence. Once the amino acid sequence of the protein is known, the fragment can be synthesized by techniques well known in the art, as exemplified by "Solid Phase Protein Synthesis: A Practical Approach" E. Atherton and R. C. Sheppard, IRL Press, Oxford, England. Similarly, multiple fragments can be synthesized which are subsequently linked together to form larger fragments. These synthetic protein fragments can also be made with amino acid substitutions at specific locations to test for agonistic and antagonistic activity in vitro and in vivo.
Protein fragments that possess high affinity binding to tissues can be used to isolate the receptor on affinity columns. Isolation and purification of the receptor is a fundamental step towards elucidating the mechanism of action of the kringles. Isolation of a kringle receptor and identification of the agonists and antagonists will facilitate development of drugs to modulate the activity of the receptor, the final pathway to biological activity. Isolation of the receptor enables the construction of nucleotide probes to monitor the location and synthesis of the receptor, using in situ and solution hybridization technology. Further, the gene for the receptor can be isolated, incorporated into an expression vector and transfected into cells, such as patient tumor cells to increase the ability of a cell type, tissue or tumor to bind and inhibit local angiogenesis. The kringles of the present invention are effective in treating diseases or processes that are mediated by, or involve, angiogenesis. The present invention includes the method of treating an angiogenesis mediated disease with an effective amount of these kringles, or a biologically active fragment thereof, or combinations of angiostatin fragments that collectively possess anti-angiogenic activity, or agonists and antagonists. The angiogenesis mediated diseases include, but are not limited to, solid tumors; blood born tumors such as leukemias; tumor metastasis; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and wound granulation. The kringles of the present invention are useful in the treatment of disease of excessive or abnormal stimulation of endothelial cells. These diseases include, but are not limited to, intestinal adhesions, Crohn's disease, atherosclerosis, scleroderma, and hypertrophic scars, i.e., keloids. These kringles can be used as a birth control agent by preventing vascularization required for embryo implantation. Kringles are useful in the treatment of diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa) and ulcers (Helicobacter pylori). The synthetic protein fragments of these kringles have a variety of uses. The protein that binds to the receptor with high specificity and avidity is radiolabeled and employed for visualization and quantitation of binding sites using autoradiographic and membrane binding techniques. This application provides important diagnostic and research tools. Knowledge of the binding properties of the receptor facilitates investigation of the transduction mechanisms linked to the receptor. In addition, labeling these proteins with short lived isotopes enables visualization of receptor binding sites in vivo using positron emission tomography or other modern radiographic techniques to locate tumors with the binding sites.
EXAMPLE 4 Construction of HEP Fusion Protein:
The tPA kringle 2 carrying mutation of His244 → Tyr, is fused to collagen α1 (XVIII) C-terminus. A 558 bp DNA fragment encoding 183 amino acid residues of human collagen α1 (XVIII) C-terminus with adaptors that are amplified using primers 5'- GGAATTCCATATGCACAGCCACCGCGACTTCCAG (G-[EcoRI]-[Ndel]-[His
1154 → Gin 1160]), and 5'-
CCGGGATCCCTACTTGGAGGCAGTCATGAAGCT (CCG-[BamHI]-[STOP]-
[Lys1336 → Ser1330). A 287 bp cDNA fragment encoding 87 residues of tPA kringle 2 mutant H are amplified by primers 5'- GGAATTCCATAACAGTGACTGCTACTTTGGG (G-[EcoRI]-[Ndel]-[Asn177
→ Gly 183]), and 5'-GGAATTCCATATGGGTGGAGCAGGAGGGCACATC
(G-[EcoRI]-[Ndel]-[Thr263 → Asp257]).
The collagen α1 (XVIII) C-terminus flanked by Ndel and BamHI adaptors are cloned into pEXdnaY vector. Then, cDNA fragment encoding tPA kringle 2 mutant H with Ndel adaptor in both ends are fused in the front Ndel site of collagen α1 (XVIII) C-terminus fragment, generating plasmid pEXdnaY-hHED. The estimated fusion HED 1 protein MW is 30.087 kDa.
EXAMPLE 5
To Inhibit Glioma Cell Invasion in the Rat Brain Using KED: Neuropharmacology and efficacy of KED in xenograft glioma model.
Using validated culture and implantation techniques, nude rats (Cr:NIH-rnu) harboring intracranial human glioma xenografis (U251 Mgn) are treated with daily oral doses of KED for a period of 28 days beginning 1 week following implantation. Beginning 2 weeks following implantation, 8 animals in each of the control and treatment groups and are sacrificed for histochemical and in situ detection protocols for quantitation. Subsequent groups of 8 animals in control and treatment groups are sacrificed weekly for a total of 4 sessions, a total of 6 weeks following implantation. An active drug concentration is determined in initial studies and dosage is adjusted if necessary, before proceeding. Further treatments are continued at the same dose only if adequate target KED concentrations have been achieved; otherwise a dose escalation of 25% is undertaken and the process repeated. A randomized cohort of animals implanted are then treated and survival studies undertaken. Results are fully duplicated in a repeat set of animals with a second xenografted human glioma cell line (U87MG).
Cell lines:
The U251 MG cell line is originated by Ponten from a patient with a glioblastoma. The culture is initially mycoplasma positive and is cured by antibiotic treatment in culture (negative since 1987). U87MG is a cell line that is initiated from an anaplastic astrocytoma. All cell lines are routinely mycoplasma tested and have tested free of contamination. Cells are routinely maintained in DMEM +10% fcs in a 37°C humidiated incubator in 10% CO2.
Intracranial nude rat xenograft:
Following CEAC guidelines in an approved animal use protocol, nude rats are inoculated intracerebrally as follows:
Cell culture:
Cells are harvested from 90% confluent culture plates by treatment with a 0.1% trypsin/EDTA for 10 minutes. Cells are rinsed off the plates with media and spun for 10 minutes at 1000 rpm. The cell pellet is resuspended in 100 μL of culture media, counted and the concentration of cells adjusted to 5 x 1055μL. Viability of the cells is tested using Trypan blue exclusion counting on a Hausser Scientific Brightline hemocytometer. Implantation:
Animals are anesthetized with 100 mg/kg ketamine, 1.5 mg/kg xylazine, and 0.05 ml atropine i.m. The surgical zone is swabbed with betadine solution, the eyes coated with lacritube and immobilized in a small animal stereotactic device (Kopf, Cayunga, CA) After draping, a 1 cm incision is made 2 mm to the right of the midline 1 mm retro-orbitally, the skull exposed with cotton-tip applicators and a HP-4 dental drill bit is used with a micromanipulator to drill a hole 3 mm to the right of the bregma, taking care not to penetrate the dura. A #270 0 μL Hamilton syringe with a #4 point, 26s gauge needle containing tumor cell suspension [500.000 viable cells] is lowered to a depth of 3.5 mm, then raised to a depth of 2.5 mm. The cell suspension is injected stepwise at a rate of 0.5 μL/10 seconds until the entire volume had been injected. During and after the injection, careful note is made of any reflux from the injection site. After completing the injection, there is a waiting period for 2-3 minutes before withdrawing in a stepwise manner. The surgical zone is flushed at least twice with sterile saline and the hole sealed with bone wax. Finally, the skull is swabbed with Betadine before suturing the skin over the injection site. The animals are maintained for 1 week prior to treatment.
Drug administration schedule:
Using a 22 g stainless steel feeding tube, 18.75 mg/100 μl Saline (150 mg/kg 125 gm/animal) is administered orally to treated rats daily for 28 days, beginning 1 week following tumor establishment. This dose has generated serum concentrations of 10-15 μM in other studies and a target concentration of 5-15 μM in tissue is desired. Control animals receive identical handling except for oral administration of vehicle (SALINE) alone. In the survival cohorts, treatment is given daily until death of the animal, again beginning 1 week following implantation. Tissue Collection and Processing:
At sacrifice, animals are anesthetized with ketamine/xylazine. CSF is removed via suboccipital puncture for HPLC [KED] using a 20 g needle; and after a blood specimen is taken, cardiac perfusion is performed with 250 mL sterile saline followed by fixation with 250 mL of 10% buffered formalin phosphate. Finally, the brain is removed and immersed in formalin for 24 hours. It is then placed in a 200-400 g coronal rat-brain matrix (Activational Systems Inc., Warren, Ml) and cut into 1 mm blocks (same slice thickness as MRI) and subsequently imaged for histologic registration to MRI. Blocks grossly containing and adjacent to tumor are processed and paraffin embedded. In order to achieve accurate registration with MRI slices, the plane of section is made to correspond to that of the MRI, using anatomical landmarks as closely as possible. The embedded blocks are cut into serial 8μm sections onto uncoated slides for routine hematoxylin and eosin (H&E) staining and onto poly-L-lysine coated slides for immunohistochemical and ISH.
Histologic Staining:
Conventional H&E staining is undertaken on slides from tumor and adjacent blocks. ISH and immunohistochemistry are performed on immediately adjacent sections.
Alu in situ hybridization:
In situ hybridization is carried out as described (Appendix, SD). For this 8 μm adjacent serial sections are cut and mounted on pretreated glass slides. Pretreatment of glass slides included acid-cleaning, treatment with Denhardt's medium, acetylation and dipping in dilute Elmer's glue solution (1 :100). Slides are deparaffinized in 3 changes of 100% xylene and rehydrated in graded concentrations of water in ethanol, air dried and stored at room temperature. The human Alu probe Blur 2, from Dr. Carl Schmid of the
University of California at Davis, is constructed in a pUC plasmid inserted in the Bam H1 site and represents a consensus sequence for human Alu repeat elements115. Random primed probes are labeled with UTP-digoxtgenin (dig) and visualized with an anti-digoxigenin polyclonal antibody conjugated to alkaline phosphatase are prepared as outlined in the Boehringer-Mannheim Genius™ Nonradioactive DNA Labeling and Detection Kit (Cat. No. 1093 657). Briefly, the slides are placed in 200 mM HCI for 20 minutes followed by a brief dip in H2O, transferred to 2xSSC at 70°C for 30 minutes, followed by a brief dip in H2O and incubated for 15 minutes at 37°C in 1 mg/ml proteinase K in 20 mM Tris with 2 mM CaCI2, pH 7.4, followed by two 5-minute washes in H2O. To link DNA to the nucleus, they are incubated in neutral 5% paraformaldehyde for 1 hour, then washed twice in 2xSSC for 5 minutes to block non-specific binding sites. The slides are then prehybridized at room temperature in the following cocktail: 5xSSC. 5% blocking regent (Boehringer-Manheim), 0.5% sarkosyl, 0.02% sodium dodecyl sulfate, 0.2 mg/ml sheared salmon sperm DNA and 50% formamide. The prehybridization solution is placed over the specimens and the slide placed in a humidity box for 30 minutes, the slides are rinsed twice in 2xSSC. DNA is denatured in 70% formamide-2xSSC for 5 minutes at 70°C followed by transfer to ice-cold H2O to keep it single-stranded. The section is dehydrated through 70% and 100% ethanol. After air drying, the sample is hybridized overnight at room temperature with 0.4 ng/μl dig-labeled 300 bp Alu probe in the cocktail used for prehybridization. The sample is then washed twice in 2xSSC for 5 minutes each, followed by two washes at 55°C in 2xSSC for 15 minutes each. Immunological detection of the dig-labeled probe is done with dig-specific antiserum is performed as described in the Genius™ Kit. Stained slides are digitized for quantitation.
Statistical Considerations: Inhibition of human glioma invasion in rat brain using KED. This is a two-sample, placebo-controlled trial in tumor-bearing rats of
(KED) where the placebo is saline, the vehicle for administration of KED. The primary endpoint is tumor volume, the margins measured as the infiltration border. There is a complete replication of the trial for each cell line. U251 MGn and U87MG. For each replication, 64 nude rats are inoculated intracerebrally with tumor cells. After one week of maintenance, half the rats are randomly treated with KED and half will receive saline. Eight randomly selected animals from each treatment group is sacrificed at 1 week intervals following treatment and the volume of tumor is measured.
Treatment of colon cancer bv KED: Cell lines Parental colon cancer cell line LS 174T was derived from a well- differentiated Human colonic adenocarcinoma (Moose et al., 1996), and has been characterized extensively, including its ability to product mucin in vitro (Standker et al., 1997), and when grown subcutaneously in athymic nude mice (Patthy, 1985). Cells injected into the cecal wall of nude mice produce well-differentiated primary colon cancers with little metastic ability (O'Reilly et al., 1993).
LS LiM 6, a derivative of LS174T with high liver-metastasizing ability during cecal growth, was established by serially selecting cells which metastasized from cecum to liver as previously described (O'Reilly et al., 1993). These cells form well-differentiated mucinous carcinomas in nude mice, with large glands containing PAS-positive (Folkman, 1996) mucin comprising ~50% of the tumor area. HM7 and LM12 are variants of LS174T which produce high and low amounts of mucin, respectively. Relative to the parental cell line, and were selected by a replica plating and immunoscreening method as previously described (Standker et al., 1997) HM7 secretes approximately twofold more mucin in culture and contains twofold more intracellular mucin compared with LS174T, whereas LM12 produces ~30% less mucin overall compared with the parental line (Standker et al., 1997). HM7 formss well differentiated tumors in nude mice, with prominent glands containing PAS-positive mucin comprising 40-50% of the tumor area. In contrast, LM12 xenografts contain fewer mucin-containing glands comprising <10% of the tumor area (O'Reilly et al., 1997).
Cecal injection. Confluent cultures are harvested by brief trypsinization (0.05% trypsin-
0.02% EDTA in HBSS without calcium and magnesium), washed several times with calcium and magnesium-free phosphate-buffered saline (CMF), and resuspended at a final concentration of 5x107 cells/ml in serum-free medium. The presence of single-cell suspensions was confirmed by phase- contrast microscopy and cell viability was determined by Trypan blue exclusion. Pathogen-free BALB/c NCR-NU athvmic mice (3-5 week old females obtained from the National Cancer Institute, Frederick, MD) were anesthetized. The cecum exteriorized, and 5x106 viable tumor cells in 0.1 ml injected into the cecal wall as previously described (O'Reilly et al., 1993; O'Reilly et al., 1997). The cecum was replaced in situ, and the abdomen closed with stainless steel clips. After 6 weeks animals were sacrificed by cervical dislocation and the abdominal organs and thorax examined for the presence of macroscopic "primary" cecal tumors and metastases. Macroscopic metastases were defined as those visible to the naked eye. Organs including cecum, liver, mesenteric nodes, and lungs were removed and fixed in 10% formalin for 24 hours. Representative tissues were embedded in parafin and serial 5-μm sections cut, stained with hematomylin- eosin and periodic-acid-Schiff (PAS), and examined by light microscopy to verify the presence of metastases.
Splenic injection (liver colonization assay).
The ability of the various tumor cells to colonize the liver after entry into the hepatic-portal system was tested in a modification of a splenic injection model (Pennica et al., 1983; Patthy et al., 1985), as previously described (Goldhaber et al., 1988). Tumor cells were grown to confluency in 75-cm2 tissue culture flasks, harvested as described for cecal injection, and resuspended in serum-free DMEM at a concentration of 107 cells/ml. Athymic nude mice were anesthetized with methodyfluorance by inhalation, prepared sterilely, and the spleen exteriorized through a flank incision. One million cells in 100 μl were slowly injected into the splenic pulp through a 27-gauge needle over 1 minute followed by splenectomy 1 minute later. Animals sacrificed 3-4 weeks later, the livers removed and weighed and tumor nodules were counted.
The immediate fate of tumor cells after splenic injection was determined by injection of IdUR-labeled tumor cells. Cells were grown as above and 0.5 μCi/ml [125l] IdUR was added to culture medium 24 hours before harvesting (Goldhaber et al., 1988). This concentration of isotope labels >95% of tumor cells without affecting viability. Cells were prepared and injected into the splenic pulp as described above. Animals were sacrificed 10 minutes after injection, and livers removed. The radioactivity present in the spleen (removed after injection), liver, and remaining organs of each animal was determined by gamma-counting in a model 7000 counter (Beckman Instruments Inc.)
Liver colonization after inhibition of mucin glycosylation.
Tumor cells were grown to 50% confluency in 7-5-cm2 tissue culture flasks as described above. The tissue culture medium was then aspirated and replaced with fresh medium containing 2 mM benzyl-α-N-acetylgalactosamine (benzyl-α-GalNAc) (Bennett et al., 1991). Cells were harvested 48 hours later and liver colonization assayed after splenic injection as above.
In a separate experiment, tumor cells were grown for 48 hours in medium containing 2 mM benzyl-α-GalNAc as above. The cells washed three times in CMF, and the medium will be replaced with DMEM containing 10% FBS. After an additional 48 hours the cells were harvested and liver colonization assayed after splenic-portal injection. Parallel flasks were grown for 96 hours in DMEM plus FBS or DMEM containing 2 mM benzyl-α-GalNAc (n=6 animals per group).
EXAMPLE 6 Fermentation
Growth of recombinant strain
Growth of the recombinant strain is accomplished by making five Amp/Cm plates using the following: a. 200 ml water; b. 2 g tryptone; c. 1 g yeast extract; d. 1 g NaCl; and e. 3 g agar. Next, these compounds are mixed. The pH is then adjusted to 7.4 using 2N NaOH. The mixture is then autoclaved at 120 degree C for 15 min. Then keep it in a 50 degree C water bath for 1 hour. Next, add 0.2 ml of 100mg/ml ampicillin, the ampicillin should be dissolved in water. After this, add 0.2 ml of 35 mg/ml chloramphenicol the chloramphenicol should be dissolved in ethanol. Then, put 20 ml of this in each petri dish (100 x 15 mm). Keep these at room temperature for 1 hour. Warm up 1 ml of stock solution of 3A strain at 37 degree C. After the stock solution is melted, immediately inoculate 0.05 ml of the solution on an Amp/Cm plate. Make a serial dilution. Then incubate at 37 degree C for 12- 16 hours. Pick up single colony and inoculate the single colony in 2 ml LB- Amp/Cm (tryptone 10 g/l, yeast extract 5 g/l, NaCl 5 g/l, 0.1 g/l ampicillin and 0.035 g/l chloramphenicol). Maintain the colony by shaking (250 rpm) at 37 degree C for 12-16 hours. Then transfer the above 2 ml culture to 100 ml LB- Amp/Cm. Maintain the shaking (250 rpm) at 37 degree C for 6-8 hours. Transfer above 100 ml culture to 10 I LB-Amp/Cm.
Fermentation
This is a case for 10 I fermentor. This can be linearly up scaled except of temperature, agitation and growth time for example: 100 I, x10; 300 I, x 30; 500 I, x 50; and 1000 1, x 100.
The first step requires dissolving 85 LB and 200 g of yeast extract in 10 1 of water, this equals the components of YEEM. Next, adjust pH to 7.5 using
2 N NaOH. Autoclave the entire mixture. Keep a controlled temperature at 37 degree C. Next, begin agitation at 250 rpm. Add 1 g of ampicillin, add 0.35 g of chloramphenicol, then add 1 ml antifoam. Inoculate 100 ml of 3A culture. Maintain the agitation at 250 rpm with the pH at 7.5. Keep the air at 15 l/min/l culture. Take 0.1 ml culture into 10 ml of YEEM each 0.5 hour. It is a hundred times dilution. Read the number by photometry at 600 nm., the OD number x 100 and if "n" > 5, add 0.3 I of 50% glucose. If "m" < 6, add 0.3 I of 50% glucose. Add 0.3 I of 50% sucrose. Then add 20 ml of vitamin solution, wherein the vitamin solution includes the following: 6 g/l thiamin; 3 g/l pyridoxine; 2.7 g/l DL-pantothenic acid; 0.7 g/l pyridoxine; 0.2 g/l riboflavin; 0.03 g/l biotin; 0.02 g/l folic acid. Next, add 20 ml of metal solution. The metal solution includes the following: 75 g/l Na3Citrate; 30 g/l FeCI3; 2 g/l CoCI2.; 2 g/l Na2MoO4; 2 g/l CuSO4; 1.5 g/l MnCI2; 1.5 g/l ZnCI2; 1 g/l CaCI2; 0.5 g/l H3BO3. Then begin agitation at 500 rpm, pump air at 20 l/min/l culture. Add antifoam 0.5ml/hour. When 12 < OD <15 at 600 nm, add 0.3 I of 50% glucose. When 20 < OD < 25 at 600 nm, add 0.3 I of 50% glucose.
REACTIVATION Harvesting Cells
(Warning: Never use EDTA in following processes!) Isolate of cells from culture using membrane system. The membrane is as follows: membrane: 0.04 micron polyethersulfone; pressure: 1 to 5 psi; temperature: 4 degree C. Concentrate to 5% of the original volume. Add equal volume of PBS (10 mM sodium phosphate buffer containing 100 mM of NaCl at pH 7.5). Concentrate to 50% volume. Repeat 10 times the previous two steps. (Caution: do not get cells by centrifugation.) The cell suspension can be frozen until uses. Membrane can be regenerated in 0.1 N NaOH for 3 hours.
Cell Lysis
Use French Press method as set forth in previous examples. Obtain Inclusion Bodies
Centrifugation is utilized for obtaining inclusion bodies. Maintain a temperature of 4 degree C. Centrifuge at 10000 x g for 30 min. Keep the pellets, because the pellets are crude product, and the pellets are called as "KED inclusion bodies". Do not measure the protein concentration of inclusion bodies. Usually, measurement of protein concentration of inclusion bodies is under estimated. Measure the wet weight of inclusion bodies. Freeze the inclusion bodies (crude product) in -80 degree C.
Purify Inclusion Bodies
Take the inclution bodies of KED from the -80 degree C freezer. Put liquid nitrogen on the crude product. Suspend the crude product in PBS (0 mM sodium phosphate buffer containing 100 mM of NaCl at pH 7.5). Then adjust the concentration of inclusion bodies at 0.1 to 0.25 g wet weight / ml PBS. Add Tween-20 to a final concentration of 0.1% and agitate for 1 hour at 4 degree C. A single pass through the French Press Cells is all that is required. Add pre-cooled (4 degree C ) PBS the ten times of original volume.
Concentration of the inclusion bodies is determined using the membrane system as follows: membrane: 0.1 micron polyethersulfone; pressure: 1 to 5 psi; and temperature: 4 degree C. Then concentrate to 10% of the original volume. Add Tris buffer (100 mM Tris at pH 8.5) to bring the volume to the original. Repeat the above steps five times. Obtain final concentration of inclusion bodies 0.1 to 0.25 wet weight / ml Tris buffer. Keep the inclusion bodies in the Tris buffer at -20 degree C as needed.
Solublization of Inclusion Bodies
For the mild solubilization of KED protein, prepare a small fermentor 1 to 2 little. Add 0.75 I of solubilization solution. Add the components of solubilization solution (SS). Components Per little Concentration
Urea 480 g 8 M
Tween-20 1 ml 0.1%
Tris 12.1 g 0.1 M Next bring the pH to 8.5, using concentrate HCI, be careful not to use more than 0.5 I of water. Add 100 ml of crude product (inclusion bodies). Then agitate at 50 rpm. Add 1 ml of beta-mercaptoethanol. The final concentration of beta-mercaptoethanol should be 140 mM. Keep at 25 degree C for 0.5 hours. Measure the protein concentration. Control should be the SS containing 140 mM beta mercaptoethanol. Adjust the volume to 20 mg proteins / ml by SS containing 140 mM mercaptoethanol. Agitate at 50 rpm for 2.5 hours.
Refolding
Follow the refolding steps set forth in the previous examples and the composition as follows:
Components Amount/I Concentration
Tris 12.1 g/l 100 mM
L-arginine 160 g/l 0.8 M
Tween-20 1 ml 0.1%
GSH 300 mg 0.9 mM
GSSG 120 mg 0.19 mM
Zinc Chloride 1.36 mg 0.01 mM
3A 100 mg 0.003 mM
Temperature RT
Time 20 to 24 hrs pH 8.8 with HCI.
PURIFICATION
Heparin-Sepharose Chromatography
Twenty ml of dialyzed KED protein solution was diluted in 180 ml of 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. The protein solution was loaded on a Heparin-Sepharose column (HiTrap pre-packaged 5 ml column, Pharmacia, Piscataway, NJ). Columns were washed with the same buffer until the OD280 returned to the base line. KED protein was eluted by a NaCl gradient (150-1000 mM). Pure KED protein eluted within 210-330 mM NaCl was identified by 10% polyacrylamide gel electrophoresis.
The following is a typical process using "Hi-Trap Heparin-Sepharose": Twenty ml of refolded KED protein solution was diluted in 180 ml of 10 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. The protein solution was loaded on a Heparin-Sepharose column (HiTrap pre-packaged 5 ml column, Pharacia, Piscataway, NJ). Column was washed with the same buffer until the OD280 backs to the base line. KED protein was eluted by a NaCl gradient (15-1000 mM). Pure KED protein eluted within 195-345 mM NaCl was identified by 10% polyacryl amide gel electrophoresis.
Acticlean Etox Chromatography
Acticlean Etox (Sterogene, Arcadia, CA) resins were cleaned with 1 M NaOH for 12 hours at 4 °C, and packed to column. The column was washed with PBS until pH below 7.6. Pure KED protein was dialyzed against PBS, and loaded on Actilean Etox column. Each ml of Acticleas Etox gel was used for treatment 5 mg of KED protein. The KED protein was then sterilized by passing a 0.2 microm filter.
QUALITY CONTROL Polyacrylamide Gel Electrophoresis
Follow the polyacrylamide gel electrophoresis steps as set forth in the above examples.
Immunological Detection Cut two pieces of Whatman paper to cover the size of the gel with moistened transfer buffer. Place one piece of nitromembrane filter on the gel. Transfer proteins on to nitromembrane, then transfer buffer containing the following: 20% methanol; 0.025 M Tris; 0.19 M glycerol; and having pH = 8.5. Transfer volts equalling 25. The transfer time is >3 hours with a transfer temperature of 4 degree C. Alternatively the transfer volts can equal 100, with a transfer time of 1 hour and a transfer temperature RT. An incubation blot in PMT, wherein PMT includes the following: PBS containing 5% non-fat milk & 0.05% Tween-20 with an incubation time of 1 hour and an incubation temperature at 37 degree C. Then incubate with rabbit anti recombinant KED IgG at a concentration of 1500 to 2000 times. The incubation time should equal 1 hour with an incubation temperature of 37 degree C. Wash three times with PMT, each wash should be for 10 minutesat 37 degree C. Then incubate with rabbit anti-lgG antibodies 1 : 2000. The anti-lgG is peroxidase conjugated and incubation time is equal to 1 hour with an incubation temperature of 37 degree C. Again wash three times with PMT as before. Then sash with PBS. Next incubate with the following: 10 mg 4-chloro-1- naphthol; 3 ml of methanol; 17 ml of PBS; 0.02 ml H2O2 with a reaction temperature of 37 degree C and a reaction time of 5 to 30 min. Next add a stop solution of 0.05 ml 3 M NaOH.
0 Endotoxin Control
Harvesting of cells by membrane includes the following: membrane: 0.04 micron polyethersulfone at a pressure of 1 to 5 psi and a temperature of 4 degree C. Then wash cells by PBS (10 mM sodium phosphate buffer containing 100 mM of NaCl at pH 7.5). Repeat washing 3 times. Repeat 5 washing reduces endotoxin contents in the final products. Endotoxin secreted in medium from cells can be excluded by membrane system. Regenerate membrane in 0.1 N NaOH for 3 hours.
Stage of inclusion bodies o Method I: Centrifugation occurs at a temperature of 4 degree C, at a speed of 10000 x g for 30 min. Then incubate the pellets (crude product) in liquid nitrogen for 10 min. Suspend the crude product in PBS (10 mM sodium phosphate buffer containing 100 mM of NaCl at pH 7.5). Next, adjust the concentration of inclusion bodies at 0.1 to 0.25 g wet weight / ml PBS.
Method II: Extraction occurs by adding Tween-20 to a final concentration of 0.1%. Then agitating for 1 hour at a temperature of 4 degree C.
Method III: French Press Cells in a single pass.
Method IV: The membrane system involves adding pre-cooled (4 degree C ) PBS the ten times of original volume. The membrane is as follows: 0.1 micron polyethersulfone at a pressure of 1 to 5 psi with a temperature of 4 degree C. Then wash with PBS buffer.
Stage of purification
Use Acticlean Chromatography as set forth previously, because it has a high affinity with endotoxin and it reduces endotoxin contents 10 to1000- folds.
Material Qualification
Materials Comments
1. Na2HPO4 Grade I
2. (NH4)2SO4 Grade I
3. MgSO4 Grade I
4. FeCI3 Grade I
5. CoCI2 Grade I
6. Tris-HCI Grade I
7. NaMoO4 Grade I
8. CuSO4 Grade I 9. MnCI2 Grade I
10. ZnCI2 Grade I
11. CaCI2 Grade I
12. H3BO3 Grade I
13. Na3citrate Grade I
14. Thiamin 4 degree C / half year
15. Niacin 4 degree C / half year
16. Pantochenic Acid 4 degree C / half year
17. Pyridoxine 4 degree C / half year
18. Riboflavin 4 degree C / half year
19. Biotin 4 degree C / half year
20. Folic Acid 4 degree C / half year
21. Tryptone No moisture
22. Yeast Extract No moisture
23. Glucose Stable
24. LB Broth No moisture
25. Reduced Glutathione (GSH) Fresh, no moisture
26. Oxidized Glutathione (GSSG) Fresh, no moisture
27. Urea Powder, no aggregation
28. beta-Mecaptoethanol Tightly capped
29. Tris-HCI Grade I
30. HCI Keep in hood
31. NaOH No moisture
32. Na Acetate Grade I
33. Acetic Acid Stable
34. Tween-20 Stable
35. Ampicillin 4 degree C / half year
36. Chloramphenicol -20 degree C / 5 years
37. Glycerol Stable
38. Maltose Stable
39. Agarose Stable 40. Isopropyl-beta-D-Thiogalactopyranoside (IPTG)
-20 degree C / 2 years
41. NaCl Stable
42. L-Arginine Hydrochloride Stable
43. SP-Sepharose 0.1 M NaCl / 25% ethanol
44. Heprin-Sepharose 0.1 M NaCl / 25% ethanol
45. Acticlean 4 degree C
51. Antifoam Stable
52. D-Val-Leu-Lys-pNA -20 degree C
53. Glu-Plasminogen -80 degree C
54. CNBr-Fibrinogen Fragments -80 degree C
55. GLC-1000 Chromogenic LAL 4 degree C
56. Thrombin -80 degree C
57. Fibrinogen -80 degree C
PRE-CLINIC
Assav Method
Endothelial culture Bovine capillary endothelial cells (BCE cells) were obtained for the proliferation assay, cells were washed with PBS and dispersed in a 0.05% trypsin solution. A cell suspension (25,000 cells/ml) was made with DMEM + 10% BCS and plated on 24-well culture plates (0.5 ml/well) and incubated at 37 degree C in 10% CO2 for 24hr. The medium was replaced with 0.5ml DMEM +5% BCS + 3ng/ml recombinant bFGF and the test sample applied. After 72hr, cells were dispersed in trypsin, resuspended and counted using a Coulter counter. Inhibitory activity of test compounds was expressed as a percentage inhibition of endothelial cell growth in culture compared to endothelial cells in the absence of the test compound.
To determine the ability of the test compounds to block migration of endothelial cells toward the angiogenic factor bFGF, migration assays were performed in a modified Boyden chamber using BCE ceils. Cells were grown in DMEM supplemented with 10% FBS and supplements at passage 15. To assess migration, the cells were starved overnight in DMEM supplemented with 0.1% BSA, harvested, suspended in DMEM/BSA, plated at 106 cells/ml on the lower surface of a gelatinized membrane (Nucleopore Corp., Plesanton, CA) in an inverted Boyden chamber and incubated for 1.5 hours to allow cell attachment. The chambers were then righted, test material was added to the top well and incubated for 5 hours. Membranes were then fixed and stained and the number of cells migrating to the top of the filter in 10 high-powered fields were counted. DMEM with 0.1% BSA was used as a negative control and bFGF at 10 ng/ml was used as a positive control.
Assav Results
Proliferation assay using bovine endothelial cells indicated that fusion protein KED completely inhibited endothelial cells migration at concentration as low as 8 nM (250 ng/ml). Where, human angiostatin and endostatin show almost no activity at 12 nM (250 ng/ml) by the same assay. The observed IC50 of KED is less than 100 ng/ml. IC50 of human endostatin in the assay was 350 ng/ml. These results indicated that the human fusion protein KED is much stronger than endostatin, the strongest anti-angiogenic agent reported to date.
CORNEAL ASSAY
The corneal assay was performed. 5 uL hydron pellets (Hydron Laboratories, New Brunswick, NJ) containing 50 ng bFGF or 5 ng KED protein and 10 ug/ml sulcrafate were implanted in a corneal pocket of anesthetized rats. After 7 days, the animals were sacrified, perfused with colloidal carbon, and the excised corneas were mounted for microscopy analysis.
The rats are anesthetized with Nembutal (29mg/kg body weight) being sure carefully clip all whiskers around the snout. Next, position the animal under the dissecting microscope, proptose the eye and secure in place with a hemostat. Irrigate the cornea with Ringers (disclosed below) to remove any hair or debris. Make a 2mm incision into the cornea at a point just slightly off the center of the cornea using the tip of a No. 11 Bard Parker blade. Using the tip of the scalpel blade, position the tip in the center of the cornea and make a 1-2mm incision into but not through the cornea. When viewed under the dissecting scope you should just a slight separation/ effacement of the corneal surface revealing the underlying stroma. A single incision is all that is needed.
Once the corneal stroma is entered, take an iris spatula, place the tip under the lip of the incision and begin to gently but firmly blunt dissect in a slightly downward direction, through the stroma to a point approximately 1- 1.5mm from the limbus. If necessary bend the iris spatula so it has a slight downward curve to it. Avoid getting too close to limbal vessels. This is the most frequent cause of non-inflammatory, false positive responses. Moisten one of Hydron pellets and using curved Dumont forceps place it near the opening of the pocket and under the scope inset it into the top of the pocket. Use your spatula to gently force the pellet down to the base of the pocket. The pellet should occupy not more than one-half the depth of the pocket.
Anything larger will be extruded due to intraoccular pressure and will prevent the opening of the pocket from sealing spontaneously.
When the implant is in position, flush the eye with saline and proceed to the next eye. It is important to work as quickly as possible to avoid prolonged heating of the eye by the microscope lamp. It is advisable to get a cold light source if possible. Examine the corneas every other day and sacrifice animals that show any evidence of corneal exudation. Some corneal edema is expected within the first 24 to 36 hours. This should not persist after this time, however. Responses are usually fully developed within 5 to 7 days. Dissect through the stroma by rocking the spatula laterally back and forth. Continue dissecting through the stroma until you are approximately 1mm from the limbus. Irrigate the eye thoroughly with Ringers then pick up a hydrated pellet (Hydron or ethylene/vinyl acetate) with the forceps and insert into the pocket. Force it down to the base of the pocket using the spatula. Re-irrigate the eye. Ideally, the implant should occupy no more than three- fourths of the pocket. Anything larger will be extruded or the pocket will fail to seal.
When the procedure is completed, remove the hemostat and gently reposition the eye in the socket. Avoid touching the pocket. Except for some transient limbal and iris vasodiatation, the eyes will appear entirely normal after 24 hours. Examine eyes as usual for presence of developing capillary sprouts. Any corneal edema that persists for more than two days likely indicates inflammation.
Lactated Ringers
Each 100 ml of solution contains: 600 mg of Sodium chloride; 310 mg of Sodium lactate: 4.61 ml of 60% solution; 30 mg Potassium chloride; 20 mg calcium chloride; and has a final ph 6.5 - 7.0. Sodium lactate is supplied as a 60% syrup solution: FW = 112.1 g/liter, thus 6.726 g sodium lactate in a 60% solution, 4.61 ml of a 60% sodium lactate solution is 310 mg of sodium lactate. Store this at 4°C. Add sodium heparin at a final concentration of 5U/ml for perfusion. Prepare at least 2 liters of the above solution, because approximately 100-200 ml are needed for perfusion of a single 100-200 g rat.
PROCEDURES FOR PREPARATION OF HYDRON
Preparation of Hydron :The Hydron is supplied in the form of a fine powder. Make a 12% solution; (ordinarily 1 gm lots in 8 mis or 3 gm lots in 25 mis), in absolute alcohol (pure ethyl alcohol USP). To dissolve, leave at 37°C for 24 hours in a rolling apparatus (e.g., Forma-Scientific CEL-roll) or a shaker at low speed. No preservative is necessary. Store at room temperature. Incorporation of Proteins in Polymer:
Method I (for lyophilized materials):
The desired weight of lyophilized protein is added to the hydron in solution and mixed in a vortex mixer. To prepare pellets for insertion in the guinea pig cornea, aspirate 5 μl in a micropipette and drop onto a sterile glass slide. For other purposes, various amounts of the polymer can be pipetted into wells or troughs of the desired shape and size. Discs are good for release studies).
Dry in a dessicator overnight. For the corneal pellets, the slides are placed in sterile petri dishes overnight in a laminar flow hood.
Rehydrate by placing a drop of sterile lactated Ringer's solution on the pellet or in the trough. The hydron will swell up to a certain extent. Wait for at least 5 minutes, when most of the rehydration has occurred. The pellets can then be pulled with a tweezer or a fine spatula. Note the release starts virtually immediately on rehydration. Thus, if one needs to quantitate, zero time must be consistent.
Method II:
If the protein to be used is in solution, pipette the desired quantity of aqueous solution over the alcoholic hydron. Do not use more than 6 parts of aqueous solution to 4 parts of hydron; otherwise, the hydron will precipitate. For the corneas, one-to-one ratios of conditioned media and hydron are used. Mix in vortex, then will well, and put on slides or troughs as explained in Method I. Note that with this method, one ends up with less hydron, thus faster release.
Methods of Changing Release Times:
A. The more hydron, the slower the percent release. B. Triple sandwich (3-day procedure)
Pipette desired μl's of hydron (without protein) onto a glass slide or trough. Dry overnight. Add to dried pellet, a middle layer of hydron plus protein. Dry overnight. Top it off with hydron without protein. Dry overnight.
C. "Sloppy" sandwich (2-day procedure):
Pipette hydron with protein onto a glass slide, dry. Remove pellet with tweezers and dip into a puddle of hydron. Dry.
Maintain sterile and clean conditions, especially if material is to be tested in vivo. Most of the procedures are performed in a tissue culture hood, using sterile kits, which have spatulas, tweezers, etc. Hydron is much less sticky than Elvas, and can be handled more easily.
With some batches, and if a large pellet is to be dried, bubbles can form during the drying procedure, so it might be necessary to dry in a vacuum oven (equipped with millipore filter). This has not been necessary with the small pellets used in the experiments.
The mechanism of release from the polymer is unknown. Hydron is neutral, but with the small numbers of compounds tested, it appears that both negatively and positively charged proteins are released to the same extent. There are also differences in the kinetics of release, dependent on molecular weight, but these are not tremendous.
ANIMAL MODEL
Efficacy of KED in Glioma Model
Using validated culture and implantation techniques, nude mouse harboring human glioma (U251MGn) were treated with KED for a period of
14-21 days weeks beginning 1 week following implantation. Beginning 2 weeks following implantation, 8 animals in each of the control and treatment groups were undergo MR imaging and were sacrificed for histochemical and in situ detection protocols for quantitation and to allow registration of MR imaging with molecular histology. Subsequent groups of 8 animals in control and treatment groups were sacrificed weekly following neuroimaging for a total of 4 sessions, a total of 6 weeks following implantation. A randomized cohort of animals implanted had been treated and survival studies undertaken. Results have been fully duplicated in a repeat set of animals with a second human glioma cell line (U87MG). 0
Cell Lines
U251MGn:
This cancer cell line was originated by Pόnten from a patient with a glioblastoma. The culture was initially mycoplasma positive was cured by 5 antibiotic treatment in culture.
U87MG:
The cell line was initiated from an anaplastic astrocytoma.
o Culture:
All cell lines are routinely mycoplasma tested and have tested free of contamination. Cells are routinely maintained in DMEM + 10% FCS in a 37°C humidified incubator in 10% CO2.
5 Cancer Implantation
Following guidelines in an approved animal use protocol, nude mouse were inoculated with cancers as follows.
Preparation of Cancer Cells: o Cells were harvested from 90% confluent culture plates by treatment with a 0.1% trypsin/EDTA for 10 minu. Cells were rinsed off the plates with media and spun for 10 min. at 1000 rpm. The cell pellet was resuspended in 100 :L of culture media, counted and the concentration of cells adjusted to 5x105/5 :l. Viability of the cells was tested using Trypan blue exclusion counting on a Hausser Scientific Brightline hemocytometer.
Implantation:
A #2701 10 :L Hamilton syringe with a #4 point, 26s gauge needle hold tumor cell suspension [500,000 viable cells]. The cell suspension was injected stepwise at a rate of 0.5 :L/10 seconds until the entire volume had been injected. During and after the injection, careful note was made of any reflux from the injection site. Animals were maintained for 1 week prior to treatment.
KED Treatment This was a two-sample, placebo-controlled trial in tumor-bearing mouse of KED TREATMENT, where the placebo was saline, the drug group was suspension solution, the vehicle for administration of KED. The primary endpointis tumor volume, the margins measured as the infiltration border.
There were two complete replications of the trial for each cell line, U251 MGn and U87MG. For each replication, 4 nude rats were inoculated with tumor cells. After one week of maintenance, half the rats were randomly treated with KED and half were received saline.
Description of the animal model: The prognosis for patients with malignant gliomas has not significantly changed in recent years. Despite debulking surgery, radiation and cytotoxic chemotherapy, progression is the rule and median survival is still measured in weeks. These tumors have mortality ratio of 77%. Brain tumors constitute the second leading cause of cancer deaths in males and females under age 15, third for adult males and the fourth cause for females 15-34 years. In the 35-54 year age group, brain tumors remain the fourth leading cause of cancer deaths in males. Thus, brain tumors are highly fatal and often strike patients in their most productive years. Finally, the incidence of glioblastoma multiforme (GBM) is also rising, especially in older adults, the poorest prognostic group. Rapid development and assessment of novel approaches to therapy are required for this incurable malignancy.
The patterns of growth and recurrence by infiltration in these lesions confounds local approaches to therapy, such as surgery and radiation therapy. This regional infiltration during tumor progression has been most strikingly shown in the whole-mount where these tumors display a central area of necrosis, a highly cellular rim of tumor and a peripheral zone of infiltrating cells. Infiltration occurs along white matter tracts, around nerve cells, along blood vessels and beneath the pia (secondary structures of Scherer). Studies have shown that tumor cells have migrated from the primary site of malignant gliomas by the time of diagnosis in the majority of cases and are responsible for the inevitable local recurrence and tumor progression seen clinically . Recurrence of human gliomas following surgery and radiation is most commonly seen in the margin adjacent to the initial tumor but can also be remote. This infiltration zone, from which most local and regional recurrences arise, is all but invisible using conventional imaging techniques.
Gliomas in general, and more anaplastic gliomas in particular, infiltrate and spread great distances in the brain. As a result, despite aggressive surgery, radiation and adjuvant chemotherapy, few durable responses are achieved and survival continues to be measured in weeks. In histological sections, most glioblastomas contain a central area of necrosis, a highly cellular rim of tumor and a peripheral zone of infiltrating cells. Infiltration occurs along white matter tracts, around nerve cells, along blood vessels and beneath the pia (secondary structures of Scherer). Elegant studies have shown that tumor cells have migrated from the primary site of malignant gliomas by the time of diagnosis in the majority of cases and are responsible for the almost inevitable local recurrence and tumor progression seen clinically. Recurrence of human gliomas following surgery and radiation is most commonly seen in the margin adjacent to the initial tumor but can also be remote. Clearly more effective treatment approaches are required to improve local tumor control in order to prevent recurrence, progression and improve survival beyond that achievable today.
Recently, the concept of cytostatics being used to restrain tumor progression (rather than induce cyto-reduction as conventional cytotoxic chemotherapies intend) has emerged. This concept questions the current therapeutic model in cancer management, derived from microbiology, in which cancer cells are considered to be different from the host, and these differences exploited therapeutically. Continuing the analogy to infection, conventional wisdom has purported that unless cells are killed and totally eliminated, they will overwhelm the host. Research strategies, new drug development, and measures of therapeutic success have been based on this killing paradigm. A regulatory model has recently been proposed in which cancer is viewed as a dynamic maladaptive process that originates within the host, is constantly in evolution, and is potentially reversible. This model is consistent with the molecular genetic understanding of cancer processes such as clonal evolution as was demonstrated in gliomas and offers a provocative reinterpretation of long-held clinical and laboratory observations. One of the implications of such a model is that, by reimposing biological control on a cell population, functional control of a tumor can be gained and this can not require complete tumor elimination. In fact, not every cancer cell need be killed to achieve control, according to this model. Cancer is a process characterized by growth, invasion, and angiogenesis, all of which have their equivalents in normal tissues. Management of these components of the malignant phenotype constitute a novel avenue for therapeutic research. Conventional antineoplastic approaches such as surgery, radiation therapy, and cytotoxic chemotherapy play roles as debulking modalities in the regulatory model, whereas biologic strategies are intended to induce re- regulation and long-term tumor control. Anti-invasion therapy represents one of these strategies in malignant gliomas and rest on a molecular understanding of the process.
The process of glial tumor invasion has been studied in vitro for some time, but the molecular pathophysiology has not been clearly addressed. It is presumed that disordered proteolysis, adhesion, motility and angiogenesis are involved in glial tumor invasion as they appear to be in systemic cancers. The results with cysteine proteases, for example, clearly demonstrate that cathepsin B (CB) is expressed in glial tumor cells, in amounts related to the degree of malignancy. CB is seen in the infiltrating tumor cells and in the proliferative neovascular endothelium of the infiltrating margin, compatible with the hypothesis that CB is functional in the processes of tumor invasion and angiogenesis. Specific inhibitors of cathepsin B have been developed as potential anti-invasion agents in gliomas and appear active in the suppression of invasion in model systems in vitro. Many studies have also shown a role for the type IV collagenases in tumor cell invasion, angiogenesis and metastasis and also in glioma growth and invasion. Matrix metalloprotease-2 (MMP-2) in vitro has been subcellularly localized to invadopodia. Inhibition of these enzymes by endogenous tissue inhibitors of metalloproteinases (TIMPs) has shown inhibition of tumor cell invasion in experimental models. Clinical anti- invasive agents for MMP inhibition have also been developed, such as BB94 and BB-2516. Completing the list of proteases in glioma, the plasminogen activator system also appears to be up-regulated, particularly in malignant gliomas. Finally, the acquisition of cellular motility and angiogenesis are felt to be central to the process of glioma invasion. Cell adhesion molecule and extracellular matrix alterations have been described in glioma model systems and, interestingly, laminin, a molecule not normally present in the brain except at the glia limitans and in the perivascular space, is upregulated in the presence of invading glioma cells, produced by surrounding normal astrocytes. Other factors, such as the autocrine motility factor Autotaxin and Glioma-Derived Motility Factor, are expressed in malignant gliomas in vitro. Finally, VEGF, bFGF and TGF are expressed locally during the process of glioma angiogenesis, where the branching proliferation of endothelial cells is preceded by proteolysis, and endothelial migration.
EXAMPLE 7
Materials and Methods 7. Cloning of tPA Kringles
Human fetal brain mRNA was used tor isolation of tPA kringle domains. Reverse transcription was carried out in 20 μl reaction solution containing 5 μg of human fetal brain mRNA (Clontech), 250 ng of oligo (dT)17 primer, 5 mM DTT, 2.5 mM MgCI, 450 μM of dNTP and 200 units of reverse transcriptase (BRL) in 50 mM Tris buffer (pH 8.3). Briefly, reaction mixture without reverse transcriptase was heated at 72°C for 10 minutes to denature mRNA and was quickly chilled on ice for 15 minutes. After 5 minutes incubation of the contents at 42°C, an hour reaction was initiated by adding of reverse transcriptase. The resulted cDNA from above reaction mixture were used for amplification of tPA kringles by using a proofreading thermostable Pfu DNA polymerase (Stratagene, La Jolla, CA). The PCR reaction solution (100 μl) contains 20 mM Tris (pH 8.0), 2 mM MgCI, 10 mM KCI, 0.1% Triton X-100, 5 μl of cDNA, 150 ng of each primer and 7.5 units of Pfu DNA. The PCR process was initiated by "hot start" (Ault et al., 1994). Twenty-five reaction cycles consist of 45 seconds at 94°C for denaturing, 45 seconds at 59°C for annealing and 3 minutes at 68°C for DNA synthesis. Kringle 1 was amplified by a 5'-end primer of GGAATTC-[Ndel]- ATAGATACCAGGGCCACGTGCTACG, and a 3'-end primer of CCG- [BamHI]- TTAGTTTCCCTCAGAGCAGGCAGG. Kringle 2 was amplified by a set of primers: GGAATTC-[Ndel]-AACAGTGACTGCTACTTTGGG for 5'-end and CCG-[BamHI]-TTAGGTGGAGCAGGAGGGCACATC for 3'-end. A DNA fragment containing both of kringles was also amplified by a 5'-end primer of GGAATTC-[Ndel]-ATAGATACCAGGGCCACGTGCTACG, and a 3'-end primer of CCG-[BamHI]-TTAGGTGGAGCAGGAGGGCACATC. Where, GGAATTC-[Ndel]- and CCG-[BamHI]- are adaptors containing Ndel and BamHI recognition sites.
The PCR products containing the tPA kringle sequences were purified through the PCR Select-ll column (5 Prime-3 Prime, Boulder, CO). The purified DNA fragments were digested by restriction enzymes of Ndel and BamHI at 37°C for 2 hours. The digested DNA fragments with Ndel and BamHI cohesive ends were purified by phenol extraction, ethanol precipitated and used to insert into pET11a vector. The pET11a vector was linearized by the same enzymes of Ndel and BamHI. The linearized vector was further treated by calf intestinal alkaline phosphatase (CIAP) to prevent self-ligation of vector inter or intra molecule. The dephosphorylated vector was ligated with tPA kringle fragments. The ligation solution was used to transform competent E. coli TG1 cells. Recombinant plasmids were analyzed by double digestion of Ndel and BamHI enzyme. Recombinant plasmids were confirmed by DNA sequencing of dideoxy chain termination (Sanger et al., 1977).
2. PCR-Mediated Mutagenesis
Mutagenesis was carried out using recombinant plasmid pK2tPA containing the wild type tPA kringle 2 cDNA. A PCR-mediated mutation kit, Quick-Change (Stratagene, La Jolla, CA) with some modification was employed for the process. Fifty μl PCR reaction mixture was prepared on ice. The mixture contains 20 mM Tris (pH 8.8), 10 mM KCI, 10 mM (NH4)2SO4, 2 mM Mg SO4, 0.1% Triton X-100, 100 ng pure super-coiled pK2tPA DNA, 200 ng each of sense and complementary mutation oligonucleotide primers, 200 μM of dNTP and 5 units of cloned Ptu DNA polymerase. A control PCR reaction was simultaneously included in the thermal cycles by missing of both of sense and complementary mutation primers. Each PCR cycle consisted of denaturation at 95°C for 0.5 minutes, annealing at 55°C for 0.5 minutes, and extension at 68°C for 18 minutes, for a total of 12 cycles.
Oligonucleotide primers used for mutation PCR are listed. The mutation sites are underlined. Residue numbers are according to the amino acid sequence of tPA.
S mutant (Ser186 - Lys mutation): SKf-1 , sense, 5'-GCTACrrTGGGAATGGGAAAGCCTACCGTGGC-3'
SKr-2, anti-sense, 5'-GCCACGGTAGGCTTTCCCATTCCCAAAGTAGC-3'
Y mutant (Tyr214 - Phe mutation):
YFf-3, sense, 5'-CCTGATAGGCAAGGTTCACAGCACAGAACCCC-3'
YFr-4, anti-sense, 5'-GGGGTTCTGTGCTGTGAAAACCTTGCCTATCAGG-3' N mutant (Asn218 - Thr mutation):
NTf-5, sense, 5'-GTTTACACAGCACAGACCCCCAGTGCCCAGGC-3'
NTr-6, anti-sense, 5'-GCCTGGGCACTGGGGGTCTGTGCTGTGTAAAC-3'
G mutant (Gly225 - Glu mutation):
GEf-7, sense, 5'-GTGCCCAGGCACTGGAACTGGGCAAACATAAT-3' GEr-8, anti-sense, 5'-ATTATGTTTGCCCAGTTCCAGTGCCTGGGCAC-3'
K mutant (Lys240 - Gly-Gly mutation):
KGGf-9, sense, 5'-CCTGATGGGGATGCCGGTGGCCCCTGGTGCCACG-3'
KGGf-10, anti-sense, 5'-
CGTGGCACCAGGGGCCACCGGCATCCCCATCAGG-3' H mutant (His244 -Tyr mutation):
HYf-11 , sense, 5'-GCCAAGCCCTGGTGCTATGTGCTGAAGAACCGC-3'
HYr-12, anti-sense, 5'-GCGGTTCTTCAGCACATAGCACCAGGGCTTGGC-3'
W mutant (Trp253-Glu254 -Tyr-Asp mutation):
WEYDf-13, sense, δ'-CCGCAGGCTGACGT ATGATTACTGTGATGTGCCC WEYDr-14, anti-sense, 5'-
GGGCACATCACAGTAATCATACGTCAGCCTGCGG A 25 μl aliquot from the resulted reaction mixture was taken for restriction digestion with 10 units Dpnl at 37°C for 30 minutes. Two μl of digested solution mixture was used for transformation in E. coli TG1 cells. Ampicillin resistance plasmids were isolated from the TG1 cells. Confirmation of mutation was performed by dideoxynucleotide DNA sequencing.
3. Expression of tPA kringles
The purified recombinant plasmids were used to transform expression strain of E. coli BL21 (DE3) cells by the standard CaCI2 procedure. E. coli BL21 (DE3) cells were co-transformed with a low-copy plasmid pDC952, a derivative of pACYC184 containing argU gene. Ten colonies with ampicillin and chloramphenicol resistance for each construct were selected for gene expression analysis. Each single colony was inoculated in 2 ml of LB medium containing 100 μg/ml ampicillin and 35 μg/ml chloramphenicol. After 18 hours incubation at 37°C, 0.1 ml culture from each sample was diluted into pre- warmed (37°C) 10 ml LB, and continued growth was facilitated with shaking of 250 rpm. Protein expression was induced at the early log phase between 0.4 to 0.6 OD at 600 nM by adding of IPTG (isopropyl β-D-thiogalactopyranoside) to the final concentration of 0.5 nM. After 2 hours induction, the cultures were chilled on ice and centrifuged at 4°C. The resulting cell pellets were washed once by PBS (phosphate saline buffer) and stored at -20°C until use.
Large scale expression of protein was carried out using a fermentor (Bioflo2000, New Brunswick Scientific, New Brunswick, NJ). The injection of air to the fermentor was maintained at 15:1 air/1 culture/minute. The medium used in fermentor was LB enriched with 35 mg/ml yeast extract. Anti-foam agent 289 (Sigma, St. Louis, MO) was added to the medium at the concentration of 0.005% (v/v) to prevent foam-formation during fermentation. Protein induction was performed by 1 mM IPTG at 0.5 OD (600 nM). After 2 hours induction, EDTA (ethylenediaminetetraacetic acid) solution was added into fermentor at final concentration of 5mM. Cells were collected by filtration system using 0.04 μrn polyethersulfone membrane (North Carolina SRT, Cary, NC).
Cells expressing tPA kringles were washed by ice-cold PBS solution three times in order to remove endotoxin secreted by the E. coli cells. The washed cells were re-suspended in PBS solution containing 1 mM EDTA, flowed by passing through a French Press Cells (SLM Instruments, Urbana, lL) twice at 20,000 psi. The inclusion bodies were collected by centrifugation of the cell lysate by 10,000 g at 4°C for 20 minutes. The inclusion bodies were washed three times by ice-cold PBS containing 0.5 NP-40 detergent (Sigma, St. Louis, MO).
4. Sephadex G-75 Chromatography Super-fine Sephadex G-75 (Pharmacia) of 10 g was suspended in 100 mM Tris (pH 8.6) containing 1 mM EDTA and 8 M urea at 37°C for 24 hours. The resins were packed on several columns (250 nM x 15 nM). The columns were kept at 28-30°C for preventing the precipitation of urea. Inclusion bodies were dissolved in I00 mM Tris (pH 8.6) containing 8 M urea. 1 mM EDTA, 0.1% Tween-20 detergent, 145 mM of β-mercaptoethanol at the concentration of 10 mg/ml of proteins. The solution was incubated at 37°C for 1 hour and loaded (5 ml) on the above Sephadex G-75 column equilibrated by 100 mM Tris (pH 8.6). and 8 M urea. The reduced kringles were collected from the last peak from the column.
5. Refolding Kringles
The reduced tPA kringles were added into refolding solution containing 100 mM Tris (pH 8.8), 1 mM EDTA, 0.1% Tween-20, 0.5 M L-arginine, 1 mM of reduced gluthionine (GSH) and 0.1 mM oxidized gluthionine (GSSG). The protein concentration in the refolding solution was adjusted to 100 mg/l. The refolding reaction was performed at 23 °C for 24 hours. 6. Lysine-Agarose Affinity Chromatography
The refolded protein solution was concentrated by using an ultra- filtration system consisting of TRIPORT Module/316L Stainless Plates with 0.1 m2 working surface and a regenerated cellulose membrane molecular of 5 kDa weight cut-off (North Carolina SRT). The filtration was performed at pressures of 25 to 30 psi. The concentrated protein solution was diluted into 100 mM Tris (pH 8.0) pre-cold at 4°C to a final concentration of 25 mM of L- arginine. The diluted protein solution was then applied on a lysine-agarose column pre-equilibrated with the same 100 mMTris at pH 8.0. Approximately 1 mg protein was loaded per one ml of lysine-agarose gel. The column was continued by washing using the same buffer until OD of eluant declines to zero at 280 nM. The tPA kringles were eluted from the column by a strong lysine competitor of 6-amino-n-caproic acid using a gradient from zero to 200 mM. Each traction eluted from lysine column was monitored at 280 nM. Samples from each peak were analyzed by 16% SDS-PAGE
7. Acticlean-Etox Chromatography
The pooled pure recombinant tPA kringles were dialyzed against 1000 times of PBS (v/v). Since E. coli cells secrete endotoxin, preventing contamination of tPA kringles from endotoxin was performed by using Acticlean-Etox (Sterogene, Arcadia, CA) chromatography. Acticlean-Etox resins were previously washed by 1 M NaOH for 12 hours at 4°C and packed into column. The column was washed using PBS until pH was below 7.6 before passing the recombinant tPA kringles. One ml of Acticlean-Etox gel was used for every 10 mg of tPA kringles. Finally, the recombinant kringles were sterilized by a 0.2 μm filter.
8. Protein. analysis Protein concentration was determined by a protein assay kit (Bio-Rad
Laboratories). Protein samples were prepared by heating to 100°C for 10 minutes in SDS sample buffer. SDS-polyacryl amide gel electrophoresis (PAGE) was performed as described (Laemmli, 1970). Expression of protein was quantified by scanning and computer analysis using Alphalmage200 (Alpha Innotech Corp.).
9. Endothelial Proliferation Assay
Different concentrations of recombinant kringles were added into the culture of bovine capillary endothelial (BCE) cells supplied with ng/ml of bFGF. The cells were maintained in DMEM medium for five days. Cells passing through an assay filter were counted.
Results
The PCR products analyzed by 2% agarose gel and EtBr-staining revealed that only a single PCR product in each reaction, indicating specific amplification performed by the hot start PCR process. The estimated PCR products encoding kringle 1 , kringle 2, and kringle 1 + 2 are 304 bp. 286 bp and 550 bp, respectively. The sizes of each PCR product agreed well with those expected from tPA DNA sequence. All three PCR products were cloned into a T7-driven vector pET11a (5.67 kb), and generated recombinant plasmids of pK1tPA (5.95 kb), pK2tPA (5.93 kb) and pK12tPA(6.20 kb) with a selection of ampicillin resistance. DNA sequencing indicated all constructs are in frame with the ATG start codon in the right orientation. No mutants such as substitutions, insertions or deletions occur in the tPA kringles during PCR amplification and cloning.
The tPA kringles were cloned and inserted into pET11a vector for protein expression under the control of a strong T7 phage promoter and the laclq repressor. Recombinant plasmid pK1tPA encodes r-[K1tPA], where r stands for recombinant and [KltPA] represents amino acid sequence residues [Ile86-Asn177] of kringle 1 domain in the tPA molecule. The recombinant plasmid pK2tPA encodes r-[K2tPA] representing amino acid residues [Asnl77- Thr263] of the tPA kringle 2 domain. Recombinant plasmid pKI2tPA encodes a r-[KI21PA] protein containing kringle 1 and kringle 2 domain, an internal fragment of [Ile86-Thr263] in the tPA protein sequence.
Recombinant proteins expressed in E.coli, were analyzed by 16% polyacrylamide gel. Whole cellular proteins of 50 μg were denatured by SDS and β-mercaptoethanol and loaded gel for electrophoresis. Abundant expression of a 10 kDa protein was detected from the cells carrying pK2tPA construct. 10 colonies of pK2tPA construct were chosen, and all ten expressed the same 10 kDa protein. A 20 kDa protein was found from the cells harboring pK12tPA plasmid. All ten clones of pK12tPA expressed the 20 kDa protein. These two proteins were produced at very high levels and occupied as high as 39.9% of the total cellular protein as indicated by a density analysis of Coomassie blue-stained PAGE gel. However, no expression was observed from the cells of ten clones containing plasmid pK1tPA. Non expression of kringle 1 domain was confirmed by an additional screening of 20 pKltPA clones.
The DNA deduced amino acids for r-[K2tPA] protein is a peptide of 88 residues including the initial methionine. Molecular weight (MW) of r-[K2,PA] with an estimated weight 9.716 kDa, agrees with the size of 10 kDa analyzed from the polyacrylamide gel (Fig. 7 ). r-[K12tPA] contains 179 residues with a MW of 19.810 kDa, as expected from its DNA sequence. Expression of a 20 kDa protein from the pK12,PA plasmid is congruent with that expected. However, even r-[K1tPA] sharing 92 identical amino acids with the N-terminus of r-[K12tPA], was not expressed.
High-level expression of proteins in E. coli frequently results in inclusion bodies, the unfolded insoluble protein particles in the cytosol of cells. Expression of human tPA kringles accumulates as inclusion bodies, as happens with most of the mammalian proteins (Dicou, 1992). These unfolded proteins can be isolated by centrifugation. Unfolded proteins need to be solubilized, refolded and purified for testing of anti-angiogenesis activity.
Both recombinant proteins r-[K2tPA] and r-[K12tPA] contain tPA kringle 2 domains. The tPA kringle 2 structure has the lysine-binding site. Lysine-bind site in the tPA kringle 2, binding resins coupled with lysine ligand, can be used for affinity protein purification. The stable epoxy-activated lysine- agarose gel was used to purify the recombinant proteins r-[K2tPA] and r- [K12tPA]. L-arginine is a competitor of lysine ligand. Efficient binding of recombinant tPA kringles with lysine ligand was found when the concentration of L-arginine in the protein solution is reduced below 25 mM. Elution of recombinant tPA kringles was carried out using a competitive ligand of lysine, the 6-amino-n-caproic acid. Three peaks are detected in the eluted fractions from a 0-200 mM gradient, one is between 30 to 45 mM, the second is between 60 to 105 mM, and a third large peak between 125 to 150 mM of 6- amino-n-caproic acid. Samples from these fractions were subjected to SDS- PAGE analysis. The r-[K2tPA] and r-[K12tPA] proteins eluted in the peak of 125- 150 mM 6-amino-n-caproic acid, appeared homogeneous on the denatured 16% polyacryl amide gel.
Proliferation assay using bovine capillary endothelial cells indicated that both of r-[K2tPA] and r-[K12tPA] proteins are active for anti-angiogenesis. However, the protein r-[K2tPA] containing tPA kringle 2 alone showed stronger activity than r-[K2tPA] suggesting that the tPA kringle2 is an angiogenesis inhibitor (Fig 8). A similar result has been observed from plasminogen kringles that the plasminogen 2 alone is more active than a peptide containing plasminogen kringle 2 and 3.
All five plasminogen kringles have been cloned. The anti-angiogenic activity of plasminogen kringles are ranked as kringle 5>1>3>/=2>4. The most active plasminogen kringles are, kringles 5 and 1. Plasminogen kringle 4 does nor show anti-angiogenic activity. Because analysis of anti-angiogenic activity of tPA kringles do not include plasminogen kringles, tPA kringle activity was not compared with that of plasminogen kringles. Sites for mutation in tPA kringle were selected using amino acid sequences of plasminogen kringles as references, in which plasminogen kringle 5 and 1 is the positive reference and kringles 2, 3, 4 - especially kringle 4 - is the negative reference.
Ideally, positive selection or negative selection can be designed. This is performed by aligning amino acid sequence of tPA kringle 2 with the five plasminogen kringles. Next, identify the position(s) where the amino acid residue(s) is identical between plasminogen 5 and 1 , but is different from plasminogen 4 and tPA kringle 2. If a position is occupied with a similar or the same amino acid residue in tPA kringle 2 and plasminogen kringle 4, this is where the residue should be mutated with top priority. Then, change the amino acid residue(s) in kringle 2 to an amino acid that is identical in plasminogen kringle 5 and 1. Finally, express the resultant mutant and test its effects on endothelial proliferation to determine if the mutation changes (increases) anti-angiogenic activity. Alternatively, negative selection can be designed by changing a residue in tPA kringle identical with both of plasminogen kringle 5 and 1 into an amino acid identical with that of plasminogen kringle 4. Here, mutations for positive selection only are performed.
The mutagenesis procedure utilizes a super-coiled, double-stranded and methylated DNA plasmid pK2tPA and two synthetic oligonucleotide primers containing the desired mutation. PCR, employing Pfu, a proofreading DNA polymerase, efficiently produced, nicked and unmethylated, the daughter plasmid with mutation by uptake of the oligonucleotide primers. The methylated parent pK2tPA plasmid was eliminated by restriction digestion using Dpnl, which is an enzyme cutting the methylated substrate DNA. The mutated daughter plasmids were recovered by transformation into high-efficiency competent E. coli cells. A control PCR was accompanied with each set of mutations by excluding the oligodeoxynucleotide primers. Selection of transformants using the ampicillin resistance maker encoded by the vector, indicated that at least a hundred fold of colonies were obtained from the PCR reaction mixture containing the mutation primers compared to that of PCR excluding the primers.
Dideoxynucleotide DNA sequencing of plasmids revealed that all mutations designed were successfully introduced onto the tPA kringle DNA strand. Any undesired or unexpected mutation was not observed, suggesting that high fidelity DNA extension had been performed during the PCR process using the proofreading Pfu enzyme.
All kringle mutants should be approximately 10 kDa. Expression of mutants was analyzed on 16% polyacryl amide gel electrophoresis (Fig 9). Four of them were expressed. However, Y, N and G mutants were unstable. Interestingly, the Y, N and G mutants are all located in the middle in the kringle structure, suggesting that mutation in this region can create unstable mRNA or protein structure.
tPA kringle 2 mutants were solubilized and reduced by 8 M urea and 140 mM β-mercaptoethanol, as described in Materials and Methods. The reduced mutants were fractionated by Super-fine Sephadex G-75 chromatography in the presence of 8 M urea. The reduced mutant proteins appeared to be homogenous by denatured SDS-PAGE analysis.
S, K, H, W kringle mutants and the wild-type tPA kringle protein were tested by proliferation assay using bovine endothelial cells in the presence of bFGF. The primary structure of tPA kringle 2 and the mutation sites are shown in Figure 5. Mutation on K and W showed no effects on the anti- angiogenic activity of tPA kringle. S mutant slightly affected the activity. However, mutation on the H site generated at least 4 times higher anti- angiogenic activity than the parental wild-type kringle, indicating that the H site can be a potent active site for expression of anti-angiogenic activity of kringle structure.
Discussion tPA kringle 2, and tPA kringle 1 plus kringle 2 was expressed. tPA kringle 1 domain was unable to be expressed. Expression of tPA kringle 1 was only achieved by the fusion of kringle 2 domain, the r-[K2tPA] peptide. To the best of the knowledge, peptide, containing tPA kringle 1 only, has never been expressed. Expression of tPA kringle 1 has been reported as a fusion with plasminogen kringle domain (DeSerrano et al., 1992).
EXAMPLE 8
Anti-angiogenic Mechanisms of Kringles: Cloning and Expression of tPA Kringles:
The tPA kringles have been cloned and inserted into pET11a vector for protein expression under the control of a strong T7 phage promoter and the laclq repressor. Recombinant plasmid pKltPA encodes r-[K1tPA], where, r stands for recombinant and [K1tPA] represents amino acid sequence residues [Ile86-Asn177] of kringle 1 domain in tPA molecule. The recombinant plasmid pK2tPA encodes r-[K2tPA] representing amino acid residues [Asn177-Thr263] of tPA kringle 2 domain. Recombinant plasmid pKI2tPA encodes ar-[KI2tPA] protein containing kringle I and kringle 2 domain, an internal fragment of [Ile86-Thr263] in tPA protein sequence.
Recombinant proteins expressed in E. coli were analyzed by 16% polyacrylamide gel. Whole cellular proteins of 50 μg were denatured by SDS and β-mercaptoethanol and loaded gel for electrophoresis. Abundant expression of a 10 kDa protein has been detected from the cells carrying pK2tPA construct (Fig. 7). Ten colonies of pK2tPA constructs were selected and all ten expressed the same 10 kDa protein. A 20 kDa protein was found from the cells harboring pK12tPA plasmid. All ten clones of pK12tPA expressed the 20 kDa protein. These two proteins were produced at very high level that occupied as high as 39.9% of the total cellular protein by a density analysis of Coomassie blue-stained PAGE gel. However, no expression was observed from the cells of ten clones containing plasmid pKltPA. No expression of kringle 1 domain was confirmed by an additional screening of 20 pKltPA clones.
The DNA deduced amino acids for r-[K2tP A] protein is a peptide of 88 residues including the initial methionine. Molecular weight (MW) of r-[K2tPA] estimated at 9,716.42 Da, agrees with the size of 10 kDa analyzed from the polyacrylamide gel. r-[K12tPA] contains 179 residues with a MW of 19810.92 Da expected from its DNA sequence. Expression of a 20 kDa protein from the pK12tPA plasmid perfectly fits with that expected. However, even r- [K1tPA] sharing 92 identical amino acids with the N-terminus of r-[K12tPA], was not expressed.
Purification:
Both recombinant proteins, r-[K2tPA] and r-[K12tPA], contain tPA kringle 2 domains. The tPA kringle 2 structure has the lysine-binding site. Lysine-bind site in the tPA kringle 2, binding resins coupled with lysine ligand, can be used for affinity protein purification. The stable epoxy- activated
Lysine-agarose gel was used to purify the recombinant proteins r- [K2tPA] and r-[K12tPA]. The proteins were refolded in the presence of 0.5 M L-arginine as 68 described (Cao et al., 1996). L-arginine is a competitor of the lysine ligand. Efficient binding of recombinant tPA kringles with lysine ligand occurs, when the concentration of L-arginine in the protein solution has been reduced below 25 mM. Elution of recombinant tPA kringles was carried out using a competitive ligand of lysine, the 6-amino-n-caproic acid. There are three peaks detected in the eluted fractions from a 0-200 mM gradient, one is between 30 to 45 mM, one is 60 to 105 mM and a large peak between 125 to 150 mM of 6-amino-n-caproic acid. Samples from these fractions were subjected to SDS-PAGE analysis. The r-[K2tPA] and r-[K12tPA] proteins eluted in the peak of 125-150 mM 6-amino-n-caproic acid. Samples from these fractions were subjected to SDS-PAGE analysis. The r-[K2tPA] and r-[K12tPA] proteins eluted in the peak of 125-150 mM 6-amino-n-caproic acid, appeared to be homogeneous on the denaturing 16% polyacrylamide gel.
Effects on endothelial migration bv mutation:
Different concentrations of recombinant tPA kringles were added into the culture of bovine capillary endothelial (BCE) cells. The cells were maintained in DMEM medium for five days. Cells passed through an assay filter were counted. Assay results indicated that both of r-[K2tPA] and r- [K12tPA] proteins actively inhibit endothelial proliferation. Protein r-[K2tPA] containing tPA kringle 2 alone showed stronger activity than r-[K2tPA], suggesting that the tPA kringle 2 can be an angiogenesis inhibitor (Fig. 8).
A Potent Anti-angiogenic Active Site in Kringle:
Identification of active site(s) for anti-angiogenesis activity of kringles would lead to an understanding of the anti-angiogenesis mechanism, which as so far eluded investigators studying the plasminogen kringle proteins. One way to address this is to perform in vitro site-directed mutagenesis at various sites in the kringle domain. The tPA kringle domain is a good choice for the mutation, since: 1) tPA kringle 2 contains a strong lysine-binding site that is preferred for ease isolation of kringle mutants; 2) tPA kringle 2 has been extensively studied and much is known about its structure and function. All five plasminogen kringles have been cloned (Cao et al., 1996). The sequence of anti-angiogenic activity of plasminogen kringles is k5 > k1 > k3 >/= k2 > k4(Cao et al., 1996; Nelson et al., 1995). The most active plasminogen kringles are kringle 5 and I. Plasminogen kringle 4 did not show activity. Because performing the analysis of anti-angiogenic activity of tPA kringles did not include plasminogen kringles, the tPA kringle activity cannot be compared with that of plasminogen kringles. Sites for mutation in tPA kringle were chosen using amino acid sequences of plasminogen kringles as references, in which plasminogen kringle 5 and 1 were selected for the positive and kringle 2, 3, 4, especially kringle 4, were selected for the negative reference.
Ideally, positive selection or negative selection can be designed. First, align the amino acid sequence of tPA kringle 2 with the five plasminogen kringles. Next, identify the position(s) where the amino acid residue(s) is identical between plasminogen 5 and 1 , but is different from plasminogen 4 and tPA kringle 2. If a position is occupied with a similar or the same amino acid residue in tPA kringle 2, and plasminogen kringle 4, where the residue should be mutated is the top priority. Then, change the amino acid residue(s) in kringle 2 to an amino acid that is identical in plasminogen kringle 5 and 1. Finally, the resultant mutant is expressed and its effects tested on endothelial proliferation to see if the mutation changes (increases) its anti- angiogenic activity. Alternatively, negative selection can be designed by changing a residue in the tPA kringle identical with both of plasminogen kringle 5 and 1 to an amino acid identical with that of plasminogen kringle 4. Here in the present studies, mutations for positive selection were only performed.
The mutagenesis procedure utilizes a super-coiled, double-stranded and methylated DNA plasmid pK2tPA and two synthetic oligonucleotide primers containing the desired mutation. PCR employing Pfu, a proofreading DNA polymerase, efficiently produced, nicked and unmethylated daughter plasmid with a mutation by uptake of the oligonucleotide primers. The methylated parent pK2tPA plasmid was eliminated by restriction digestion using Dpnl which is an enzyme cutting the methylated substrate DNA. The mutated daughter plasmids were recovered by transformation into high- efficient competent E. coli cells. A control PCR was accompanied with each set of mutation by excluding the oligodeoxynucleotide primers. Selection of transformants using the ampicillin resistance maker encoded by the vector indicated that at least a hundred fold of colonies was obtained from the PCR reaction mixture containing the mutation primers compared to that of PCR excluding the primers.
Dideoxynucleotide DNA sequencing of plasmids revealed that all designed mutations were successfully introduced on the tPA kringle 2 cDNA. Any undesired or unexpected mutation was not observed, suggesting that high fidelity DNA extension has been performed during PCR process using the proofreading Pfu enzyme.
All kringle mutants should be approximately 10 kDa MW. Expression of mutants were analyzed on 16% polyacrylamide gel electrophoresis (Fig. 9). S (Ser186 substituted by Lys) and H (His244 substituted by Tyr) mutants were expressed. However, the Y (Tyr214 substituted by Phe) mutant was not expressed. The tPA kringle 2 protein mutants were solubilized and reduced with 8M urea and 140mM β-mercaptoethanol. Proteins were fractionated by Super-fine Sephadex G-75 chromatography in the presence of 8M urea. The reduced mutant proteins appeared to be homogenous by denatured 16% SDS-PAGE analysis. The pure reduced mutants were refolded and purified by lysine-agarose chromatography.
Effects on endothelial proliferation bv mutation:
Kringle mutants S, Y, H and wild-type tPA kringle 2 proteins were tested by proliferation assay using bovine endothelial cells. The selected mutation sites in tPA kringle 2 domain are indicated (Fig. 10). Mutation on S showed no marked effects on activity. Surprisingly, H mutant with a single amino acid substitution of histidine by tyrosine dramatically increased inhibitory activity in the endothelial proliferation assay (Fig. 19). H mutant completely inhibits endothelial cell proliferation at 40 nM. The parental wild- type tPA kringle 2 shows no activity at the same concentration. The wild-type tPA kringle inhibits endothelial proliferation only approximately 45% at 200 nM. These results indicate that the H site can be a potent active site for expression of anti-angiogenic activity of kringle structure.
Effects on corneal angiogenesis bv mutation:
As can be seen in Fig. 19 the addition of 5ng of H mutant of tPA K2 to a pellet containing 25ng bFGF is capable of significantly suppressing corneal angiogenesis.
The H site might be on the surface of protein:
Residues of Asn248- Trp253 form a lysine-binding site in tPA kringle 2 domain(Bakker et al., 1993). These residues should be exposed on the surface of protein for exhibiting lysine binding of tPA kringle 2. The H site (His244) is closed to Asn248-Trp253 lysine binding site. His244 might be also exposed on the surface of kringle 2 domain for expressing anti- angiogenic activity.
EXAMPLE 9
Design of peptide overlapping anti-angiogenic active site(s) of tPA kringle.
Peptides as short as 10 amino acids of murine epidermal growth factor (EGF) have been shown to be able to inhibit endothelial cell proliferation(Nelson et al., 1995). There are two lines of evidence which strongly support the hypothesis that synthesized peptides will inhibit endothelial cell proliferation. First, peptide fragments of fibronectin, EGF and thrombospondin have been shown to inhibit endothelial cell growth (Homandberg et al., 1985; Tolsma et al., 1993). Second, denatured human plasminogen kringle 5 even shown stronger inhibition of endothelial cell proliferation than the refolded form (Ji et al., 1998). In other words, the denatured kringle 5 can be seen as acting as a large extended peptide. Different kringles with varied anti-angiogenic activity can exist in the human genome, however, there appears to be only one human endostatin. Human endostatin and mouse endostatin are highly conserved (Standker et al., 1997).
Anti-angiogenic peptide: Design of anti-angiogenic peptide:
Peptides have been designed to overlap the H mutation site. One is the wild-type sequence. Another one is a mutant peptide with substitution of histidine by tyrosine.
Molecular Cloning & Protein Synthesis:
The KED gene was constructed by fusion of cDNA plasminogen kringle 5 encoding Glu478-Ala562 with a collagen α1 (XVIII) cDNA fragment encoding His 1154-Lys1336. The KED gene is expressed in E. coli cells. AGA codon is a rare codon used in E.coli (Wada et al., 1990). The tRNAAGA is encoded by dna Y gene. The dna Y gene also has been reported to be essential for DNA replication 72. To improve productivity of KED protein in E. coli, a vector pEXdna Y was constructed containing a strong T7 promoter for high-speed transcription of KED mRNA and a dna Y gene supplying tRNA AGA for efficient translation of the KED product. (Fig. 1) The engineered KED gene encodes 271 amino acids. Expression of KED protein from pEXdnaY-hKED, was induced by IPTG (isopropyl β-D- thiogalactopyranoside). Abundant KED production was achieved. KED protein accumulated to 47% of total cellular protein by 2 hours induction with 0.5 mM IPTG (Fig. 2). Optimal production of KED protein was performed using a fermentor. The KED protein was produced as inclusion bodies in cytosol. KED inclusion bodies were isolated and efficiently refolded in the presence of 0.5 M L-arginine at pH 8.6. As a control, human endostatin was simultaneously expressed from pEXdna Y-hED. More than 99% of human endostatin protein precipitated in the refolding solution as has been previously observed (O'Reilly et al., 1997). No precipitation of KED fusion occurs during the refolding.
Pure KED protein was obtained by HiTrap HeparinSepharose chromatography. Analysis of KED protein by denatured SDS-PAGE (polyacrylamide gel electrophoresis) revealed a single 30 kDa peptide that agreed with the molecular weight estimated form the deduced amino acid sequence (Fig. 10). N-terminal analysis of KED protein (by Commonwealth Biotechnologies, Richmond, VA) provided an identical sequence of MEEDHMFGNGKGYRG as deduced from DNA sequence, see Sequence Listing, where the 5th residue is Cys that generally does not show up by the analysis system. Compositional analysis of KED generated similar results with that predicted, Table 1. Both of N-terminal and compositional analysis indicates that the pure protein isolated is the correct form of the KED gene product.
Endothelial Cell Proliferation:
KED - fusion Proliferation assays using bovine endothelial cells indicate that fusion protein H1 completely inhibited endothelial cells migration at a concentration as low as 8 nM (250 ng/ml), where, human endostatin shown almost no activity at 12 nM (250 ng/ml) by the same assay (Fig. 20). The IC50 of human endostatin in the assay was 17.5 nM (350 ng/ml). Ic50 of human plg-k5 was approximately 150 nM. Ic50 for antiostatin was 135 nM. These results indicate that the human fusion protein KED is much stronger than endostatin, the strongest anti-angiogenic agent reported to date(O'Reilly et al., 1997). Endothelial Cell Migration: KED - fusion
To determine the ability of the test compounds to block migration of endothelial cells toward the angiogenic factor bFGF, migration assays are performed in a modified Boyden chamber using BCE cells (Dameron et al., 1994). Cells are grown in DMEM supplemented with 10% FBS and supplements at passage 15. To assess migration, the cells are starved overnight in DMEM supplemented with 0.1% HSA, harvested, D'YI D'Y2 :-lyi suspended in DMEM/BSA, plated at 106 cells/ml on the lower surface of a gelatinized membrane (Nucleopore Corp., Plesanton, CA) in an inverted Boyden chamber and incubated for 1.5 hours to allow cell attachment. The chambers are then righted, and test material added to the top well incubated for 5 hours. Membranes are then be fixed and stained and the number of cells migrating to the top o fthe filter in 10 high-powered fileds counted and bFGF at 10 ng/ml is used as a positive control.
Inhibition of Blood Vessel Growth in Rat Cornea: KED - fusion
The rat corneal assay has been perfonned to test in vivo efficacy of KED fusion protein. 12.5ng, of KED were applied in the presence of 25ng of bFGF. 12.5ng of KED fusion protein efficiently inhibited the growth of capillary blood vessels from the corneal bed (Fig. 5).
RESEARCH DESIGN AND METHODS Overall Rationale for Design of Inhibitors:
The results together with the finding that the individual plasminogen kringle possesses anti-angiogenic activity, show that the triple disulphide bond kringle structure is a new family of angio-inhibitory compounds. Understanding of anti-angiogenic mechanisms of kringle structure would benefit tumor therapy. tPA kringle 2, a well characterized protein domain, is a preferred model kringle tor investigating angio-inhibitory mechanisms.
Identification of the active site(s) for exhibiting anti-angiogenic activity of kringle would lead to understanding of anti-angiogenic mechanisms. A way to address this is to perform in vitro site-directed mutagenesis in tPA kringle 2 as proposed. Preliminary studies show that substitution of His244 with Tyr in tPA kringle 2 dramatically increases its anti-angiogenic activity, indicating the presence of a potentially active site involving the His244 residue. In order to identify anti-angiogenic active site(s), an additional 17 mutations in tPA kringle 2 structure were suggested.
Compared to kringle family, endostatin. a highly basic protein fragment of collagen α1 (XVIII), represents another class of angio-inhibitory compounds. Kringle-like structures are not present in the endostatin molecule. Anti-angiogenic activity of endostatin can therefore be due to a different mechanism to that of kringles. Construction of fusion protein between highly active mutated tPA kringle 2 and endostatin is described. The fusion of two completely different structures of kringle and endostatin creates a powerful anti-angiogenic molecule. Preliminary studies support this hypothesis. KED, a fusion between plasminogen kringle 5 and endostatin, showed significantly higher anti-angiogenic activity than either kringle 5 or endostatin. Higher activity of this chimera can reflect expression of combined activity from kringle 5 and endostatin due to simultaneously activation of different anti-angiogenic mechanisms. There is now an H mutant of tPA kringle 2 that is much active than plasminogen kringle 5 which is the most active kringle reported (Nelson et al., 1995). Together with H mutant, performing of additional mutagenesis will not only allows the study angio- inhibitory mechanism but also provides a tPA kringle 2 mutant with optimum angio-inhibitory activity for fusion with endostatin, which, in turn, result a powerful agent for efficient tumor therapy.
Identification of Anti-angiogenic Active Site(s) of Kringle: tPA kringle 2 is used as a model to identify the anti-angiogenic active site(s) of kringle structure. There are three reasons for the selection of tPA kringle 2 as a model. At first, a Preliminary Study indicated that tPA kringle 2 is an anti- angiogenic active kringle. Second, substitution of a histidine residue with tyrosine (H mutant) in tPA kringle 2 surprisingly, increases anti- angiogenic activity. BEC proliferation inhibitory activity of this H mutant of tPA kringle 2 is stronger than plasminogen kringle 5 that is the most anti- angiogenic kringle reported to date. Third, kringle 2 domain, as a part of tPA and rPA, has been used in clinical therapy. Kringle 2 is an internal fragment of tPA and an N-terminal domain of tPA.
Mutation of tPA Kringle 2:
The recombinant plasmid pK2tPA containing the wild type tPA kringle 2 cDNA under aT7-driven promoter have been developed. Mutation of tPA kringle 2 is directly performed using this expression vector. No sub-cloning or other plasmid engineering is involved. A PCR-directed procedure is used which utilizes one set of complementary mutation primers overlapping the mutation-desired region. There is a recombinant plasmid pK2tPA containing the wild type tPA kringle 2 cDNA under a T7-droven promoter. Mutation of tPA kringle 2 is directly performed by using this expression vector. No sub- cloning or other plasmid engineering is involved. PCR-directed procedure is used that utilizes one set of complementary mutation primers overlapping the mutation-desired region. Mutagenesis is carried out using a mutation kit, Quick-Change (Stratagene, La Jolla, CA) with some modifications. Fifty μl PCR reaction mixture are prepared on ice. The mixture contains 20 mM Tris (pH 8.8), 10 mM KCI, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 100 ng pure super-coiled pK2tPA DNA, 200 ng each of sense and complementary mutation oligonucleotide primers, 200 μM of dNTP and 5 units of cloned Pfu DNA polymerase. A control PCR reaction will be simultaneously included in the thermal cycles by replacing both sense and complementary mutation primers with missense primers. Each PCR cycle consists of denaturation at 95°C for 0.5 minutes, annealing at 55°C for 0.5 minutes, and extension at 68°C for 18 minutes, for a total of 12 cycles. Kringle 2 domain contains 87 amino acids that represent residues 177 to 263 in tPA. Mutations will be generated throughout the kringle 2 sequence. Mutation sites will be specifically selected based on alignments of tPA kringle 2, plasminogen kringle 4 and 5 (Fig. 5). These two plasminogen kringles serve as references guiding selection of candidate mutation sites in tPA kringle 2. Plasminogen kringle 5 is the strongest anti- angiogenic kringle (Cao et al., 1997). Kringle 4 has no anti-angiogenic activity (Urano et al., 1991). There are 24 positions, identical throughout these three kringles forming the basic kringle structure including the 3 disulphide bonds, which will not be subjected to mutation. Two groups of mutations will be performed: positive and negative selections of anti-angiogenic active site(s). A total of 17 mutants will be generated.
There are 9 positions in tPA kringle 2 occupied with the same amino acids as in plasminogen kringle 4, that will be mutated at top priority. These positions are Y181 , T191 , S193, S206, D236, K240, W253, E254 and S262. These 8 amino acids will be changed to the amino acids that are identical in plasminogen kringle 5. This group of mutations is expected to generate mutants with increased activity. Mutated position(s) with increased activity should be the candidate active site(s) for expressing anti-angiogenic activity. This group of mutations represents a positive reference for mapping anti- angiogenic active site(s).
1. Change Y181 to M. Oligonucleotide primers will be 5'-CAGTGACTGCATGTTTGGGAATGGG-3' and 5'-CCCATTCCCAAACATGCAGTCACTA-3'.
2. Change TI91 to K. Oligonucleotide primers will be 5'-CCTACCGTGGCAAACACAGCCTCACC-3' and 5'-GGTGAGGCTGTGTTTGCCACGGTAGG-3'. 3. Change S193 to A. Oligonucleotide primers will be 5'-CCGTGGCACGCACGCCCTCACCGAG-3' and 5'-CTCGGTGAGGGCGTGCGTGCCACGG-3\
4. Change S206 to A. Oligonucleotide primers will be 5'-CCCGTGGAATGCCATGATCCTGATAG-3' and 5'-CTATCAGGATCATGGCATTCCACGGG-3'.
5. Change D236 to P. Oligonucleotide primers will be 5'-GCCGGAATCCTCCGGGGGATGCC-3' and 5'-GGCATCCCCCGGAGGATTCCGGC -3'.
6. Change K240 to G. Oligonucleotide primers will be 5'-GATGGGGATGCCGGGCCCTGGTGCC-3' and 5'-GGCACCAGGGCCCGGCATCCCCATC- 3'.
7. Change W253 to Y. Oligonucleotide primers will be 5'-CGCAGGCTGACGTACGAGTACTGTG-3' and 5'-CACAGTACTCGTACGTCAGCCTGCG- 3 '.
8. Change E254 to D. Oligonucleotide primers will be 5'-GGCTGACGTGGGACTACTGTGATGTG-3' and
5'-CACATCACAGTAGTCCCACGTCAGCC- 3' .
9. Change S262 to A. Oligonucleotide primers will be 5'-GTGCCCTCCTGCGCCACCTAAGGATCC-3' and 5'-GGATCCTTAGGTGGCGCAGGAGGGCAC-3'.
There are 8 positions in tPA kringle k2 that are identical to those in plasminogen kringle 5, but which are not contained within plasminogen kringle 4. These 8 amino acids are F182, A223, W242, R249, L251 , D257, V258 and P259. These 8 residues will be replaced with amino acids that are identical to those in plasminogen kringle 4. These mutations are expected to produce less angiosuppressive activity in some cases. This group of mutations will serve as a negative reference for the identification of anti- angiogenic active site(s) of kringle domain proteins.
1. Mutate F182 to H. Oligonucleotide primers will be 5'-GACTGCTACCACGGGAATGGGTCAG-3' and 5'-CTGACCCATTCCCGTGGTAGCAGTC-3' .
2. Mutate A223 to N. Oligonucleotide primers will be 5'-CCCAGTGCCCAGAACCTGGGCCTGG-3' and
5'-CCAGGCCCAGGTTCTGGGCACTGGG-3'.
3. Mutate W242 to T. Oligonucleotide primers will be 5'-GATGCCAAGCCCACCTGCCACGTGCTG-3' and 5'-CAGCACGTGGCAGGTGGGCTTGGCATC-3\
4. Mutate R249 to P. Oligonucleotide primers will be 5'-GTGCTGAAGAACCCCAGGCTGACGTG-3' and δ'-CACGTCAGCCTGGGGTTCTTCAGCAC -3'
5. Mutate L251 to V. Oligonucleotide primers will be 5'-GAACCGCAGGGTGACGTGGGAGTAC-3' and 5'-GTACTCCCACGTCACCCTGCGGTTC-3'.
6. Mutate D257 to N. Oligonucleotide primers will be 5'-GTGGGAGTACTGTAACGTGCCCTCC-3' and δ'-GGAGGGCACGTTACAGTACTCCCAC- 3'.
7. Mutate V258 to L. Oligonucleotide primers will be 5'-GAGTACTGTGATCTGCCCTCCTGCTC-3' and
5'-GAGCAGGAGGGCAGATCACAGTACTC- 3'. 8. Mutate P259 to K. Oligonucleotide primers will be δ'-GTACTGTGATGTGAAGTCCTGCTCC-S' and 5'-GGAGCAGGACTTCACATCACAGTAC-3'.
These 17 tPA kringle2 cDNA mutants will be sequenced before protein expression.
Expression of tPA Kringle 2 mutants: Recombinant plasmids of 17 mutants will be transformed into expression strain E.coli bL2l (DE3) cells by the standard CaCI2 procedure. E..coli BL21 (DE3) cells will be co-transformed with a low-copy plasmid pDC952, a derivative of pACYC 184 containing argU gene. Ten colonies with ampicillin and chloramphenicol resistance for each construct will be selected for gene expression analysis. Each single colony will be inoculated in 2 ml of LB medium containing 100 ug/ml ampicillin and 35ug/ml chloramphenicol. After overnight incubation at 37°C, 0.1 ml culture from each sample will be diluted into pre-warmed (37°C) 10 ml LB, and continued growth will be facilitated with shaking of 250 rpm. Protein expression will be induced at the early log phase between 0.4 to 0.6 OD at 600 nm. by adding of IPTG
(isopropyl β-D-thiogalactopyranoside) to the final concentration of 0.5 nM. After 2 hours induction, the cultures will be chilled on ice and centrifuged at 4°C. The resulting cell pellets will be washed once by PBS (phosphate saline buffer) and subjected to 20% SDS-PAGE analysis. Large-scale expression of protein will be carried out using a fermentor (Bioflo2000, New Brunswick Scientific, New Brunswick. NJ). The injection of air to the fermentor will be maintained at 15 1 air/1 culture/min. The medium used is LB enriched with 35 mg/ml yeast extract. Anti-foam agent 289 (Sigma, St, Louis, MO) will be added to the medium at 0.005% (v/v) to prevent foam-formation during fermentation. Protein induction will be performed by 1 mM IPTG at 0.5 OD (600 nm). Cells will be collected by a filtration using 0.04 μm polyethersulfone membrane (North Carolina SRT, Cary, NC).
Purification of tPA Kringle 2 Mutants:
Expression of wild-type or H mutant of kringle 2 resulted in inclusion bodies in the Preliminary Study. These 17 mutants of kringle 2 can also form the inclusion bodies. These inclusion bodies will be dissolved in 100mM Tris (pH 8.6) containing 8M urea, 1 mM EDTA, 0.1 % Tween-20 detergent, 145mM of β-mercaptoethanol at the concentration of 10 mg/ml of proteins. These mutant proteins will be isolated by gel-filtration chromatography in the denatured condition, which was introduced for purification of wild-type tPA kringle 2 protein. Super-fine Sephadex G- 75 (Pharmacia, Piscataway, NJ) of 10g will be equilibrated in 100mM Tris (pH 8.6) containing 1mM EDTA and 8M urea at 37°C for 24 hours . The resins are packed on a columns (250nm x I5nm) at 28-30°C for preventing precipitation of urea. The reduced tPA kringle 2 mutants will be added into refolding solution containing 100 mM Tris (pH 8.8), 1 mM EDTA, 0.1% Tween-20. 0..5 M L-arginine, 1mM of reduced gluthionine (GSH) and 0.1 mM oxidized gluthionine (GSSG). The protein concentration in the refolding solution will be adjusted to 100 mg/l. The refolding reaction will require 24 hours at 23°C.
The refolded protein solution will be concentrated by using ultra- filtration system consisting ofTRIPORT Module/316L Stainless Plates with 0.1 m2 working surface and a regenerated cellulose membrane of 5 kDa MWCO (molecular weight cut-off) (North Carolina SRT, Cary, NC). The concentrated protein solution will be diluted 20 times into pre-cold(4°C)
100mM Tris (pH 8.0) solution. The final concentration of L-arginine should be 25mM that does not effect protein binding to the lysine ligands. The diluted protein solution will be then applied on a lysine-agarose column pre- equilibrated with the same 100 mM Tris at pH 8.0. Approximately, 1ml of lysine-agarose gel will be used for absorption of Img of kringle 2 mutants. The tPA kringle 2 mutants will be eluted from the column by using a gradient from zero to 200mM of strong lysine competitor, 6-amino-n-caproic acid. Proteins eluted from lysine column will be monitored at 280nm and analyzed by 20% SDS-PAGE. E.coli cells secrete endotoxin. Prevention of endotoxin contamination will be ensured by using Acticlean Etox (Sterogene, Arcadia, CA) chromatography. The pure recombinant tPA kringle 2 mutants will be dialyzed against 1000 fold of PBS (v/v) and loaded on an Acticlean Etox column previously washed with 1 M NaOH and equilibrated with PBS solution. One ml of Acticlean-Etox gel will be used for every lOmg of tPA kringle mutants. Finally, kringle 2 mutants will be sterilized by a 0.2 μm filter. There are total of 19 proteins, including wild-type kringle 2 and H mutants, which will be purified.
Mapping of Anti-angiogenic Active Site(s):
There are 18 mutants (including H mutant) throughout the tPA kringle 2 sequence. Anti-angiogenic activity of these mutants will be secreted in vitro by endothelial cell proliferation assay. Hot spot(s)' are identified for expression anti-angiogenic activity. Mutations occurring at the 'hot spot(s)' will affect the activity. Increased or decreased activity will be quantitated by endothelial cell proliferation assays, using different concentrations of kringle 2 mutants. Results will reveal the position of 'hot spot(s)' exhibiting enhanced or reduced anti-angiogenic activity, functionally isolating key structural sites.
To localize structural element(s) and/or animal sequence(s) in human tissue plasminogen (tPA) kringle domain 2 which inhibit endothelial cell proliferation and/or migration, and corneal angiogenesis. Polypeptide fragments of tPA kringle 2 containing functionally active anti-angiogenic site(s) exhibit anti-angiogenic activity. Peptides containing the putative anti- angiogenic active site(s) are synthesized. The size for the peptides ranges from 10 to 15 amino acids. Peptides as short as 10 amino acids of murine epidermal growth factor (EGF) have been shown to inhibit endothelial cell proliferation. The amino acid sequence of tPAkringle 2 ( K2tPA; 86 residues), shown below, has certain unique chemical and enzymatic cleavage sites which are exploited to generate polypeptide fragments for functional tests. The fragmentation of K2tPA into polypeptides is carried out under two different conditions. First, the intact functionally active protein is subjected to limited cleavage under mild conditions without any prior modifications to the protein structure. Thus, the chances of preserving and localizing any structural elements which could be present in the native state compact regions, usually joined by more accessible or flexible regions, is increased. Secondly, the fragments to be single linear polypeptides and therefore reduce all disulfide bridges and carboxymethylate the CysSH groups of the protein before performing the cleavage reaction. More than ten polypeptide fragments covering different regions of K2tPA can be obtained by a combination of chemical and enzymatic cleavage methods.
Amino Acid presentation of tPA kringle 2,( K2tPA)
NSDCYFG GSAYRGTΉSLTESGASCLPV.NSMILIGKVYTAQNPSAQALGLGKHNYCRNPDGDAKP CHVL NRRLT EYCDVPSCST
Amino Acid presentation of tPA kringle 2.( -2^ ) NSDCYFGNGSAYRGTHSLTESGASC LPWNS ILIG VYTAQNPSAQALGLGKHNYCRNPDGDAKPWCHVL NRRLTWEYC DVPSC5T
Residues which are identical in tPA (two kringles) and plasminogen (five kringles) kringle domains are indicated by asterisks; the six cysteines involved in the three disulfide bridges of K2tPA are in bold letters.
The single methionyl bond in K2tPA is cleaved with excess cyanogen bromide (CNBr) in 0.1Λ/ HCI or 70% formic acid to produce two peptide fragments. The volatile CNBr, dilute acidic medium (0.1NHCI or formic acid) and the only by-product, methyl thiocyanate, are all easily removed by lyophilization. Under appropriate conditions most proteins are cleaved in yields exceeding 80%, and often quantitatively.
Cyanogen Bromide,, cleavage after M (Met) NSDCYFG GSAYRGTHSLTESGASCLP NSM
ILIGKVYTAONPSAQALGLGKHNYCRNPOGDAKPWCHVLKNRRLTWEYCDVPSCST
Enzymatic fragmentation of K2tPA at its two glutamyl bonds are performed with the GluVδ form of glutamyl endopeptidase 1 , at optimal conditions to yield three peptides. The reaction is stopped with Cbz-Leu-Leu-Glu-CH2CI and the fragments separated by hplc.
Glutamyl endopeptidase -IStaph. aureus V-8 Protease); cleavage after E(Glu)
NSOCYFGNGSAYRGTHSLTE
SGASCLPWNS ILIG VYTAONPSAQALGLGKHNYCRNPDGDAKPWCHVLKNRRLTWE
YCDVPSCST
Prolyl oligopeptidase is used to cleave specifically the Pro-Xaa bonds in K2tPA. Since this protease cleaves only small polypeptides, it is used in combination with CNBr and GluVδ to generate smaller fragments. However, if K2tPA is susceptible to this post-proline cleaving enzyme a 27 -residue, three 16-18 residue and two small (4-6 residue) peptide fragments spanning the K2tPA domain can be obtained. Post-Proline cleaving enzyme; cleavage after P (Pro)
NSDCYFGNGSAYRGTHSLTESGASCLP NSMILIGKVYTAONP
SAQALGLGKHNYCRNP
DGDAKP CHVLKNRRLT EYCDVP
SCST
All peptides are purified by preparative reverse phase hplc and characterized by analytical reverse phase hplc, peptide equencing and mass spectrometry. The kringle fragments are then tested in a series of in vitro (endothelial proliferation and migration) and in vivo (corneal angiogenesis) biological assays for their anti-angiogenic properties.
Methods: Chemical and Enzymatic Limited Protein Digestion: The highly specific cleavage of the methionine bond in K2 is performed by adding a 10- fold molar excess of CNBr to the protein in 0.1 N HCI. Timed aliquots of the reaction mixture are taken, the volatile CNBr removed under a stream of N2 gas (in a fume hood) and the residue is analyzed by analytical hplc to determine the extent of cleavage. After the optimal conditions for the Pro-Xaa cleavage are determined, the scale of the reaction is increased, the reaction products lyophilized and purified by preparative hplc to obtain enough material for the biological assays. S. aureus V8 (ICN ImmunoBiochemicals, Lisle, IL), and post-proline protease digestions of K2tPA are carried out using 1:100 w/w protease to substrate ratio for 18 hours at 37°C in 0.1 mM phosphate buffer (V8) or NaHCO3 (post-Pro), pH 8.0. The reaction is stopped by adding 100 μM Cbz-Leu-Leu-Glu- CH2CI (V8) or Cbz-Leu-Leu-Pro-CH2CI (post-Pro) for 30 min, and the reaction mixture is lyophilized. Under the reaction conditions which require unfolding of the protein, the disulfide bridges are reduced with dithiothreitol in 6.0 M guanidine solution at 50 degrees C for 30 min. The free CysSH groups are then carboxymethylated with a large excess of iodoacetic acid. Guanidine is removed by dialysis. For CNBr cleavage of the unfolded protein, the reduced protein solution in guanidine is dialyzed against formic acid. When the concentration of formic acid reaches 70 %, the modified protein is ready for CNBr cleavage. Analytical hplc-MS is performed by injecting into an Applied Biosystems aquapore C-8, 300e reversed phase column using a gradient over 30 minutes, from 0.1% TFA in water to 70% acetonitrile in water with 0.06% TFA. A flow rate of 30 El/minutesis used with a post column split diverting 2 El/min into the mass spectrometer. Spectra are accumulated with a scan rate of 400 amu/sec from 200 to 2000 with a nominal resolution of 1000. Preparative hplc to purify large quantities of protein fragments for biological assays are performed as described below in the peptide synthesis section.
Peptide synthesis: Peptides are synthesized by manual solid phase peptide synthesis using 9- Fluorenylmethyloxycarbonyl (Fmoc)fetf-butyl chemistry as described previously. Fmoc-protected amino acids are activated with 2-(1H-benzotriazole-1-yl)-, 1 ,2,2-tetramethyluronium hexafluorophosphate / 1-hydroxybenzotriazole / N;N-diisopropylethylamine (1:1:2) in 1-methyl-2-pyrrolidone / dimethylformamide / dichloromethane (1 :1 :1) for all coupling steps. The peptides are cleaved with reagent K (0.1 ml dimethylsulfide, 0.2 ml 1 ,2-ethanedithiol, 0.46 g phenol, 0.2 ml thioanisole, 0.2 ml water and 7 ml trifluoroacetic acid) for 2 hours at room temperature. Peptides are purified by reverse-phase HPLC on a Nucleosil C8 column (30 mn, 10 μ; 250 mm x 55 mm, Macherey and Nagel) and examined for purity by analytical reverse-phase HPLC on a Nucleosil C8 column (3 μ, 12 mn; 120 mm x 2.1 mm) as described previously (Angali et al., 1995) The samples containing the pure peptides are collected and lyophilized. Electrospray mass spectrometry is performed to confirm the analytical data. i.e. to confirm the amino acid sequences and the absence of modified products resulting from incomplete deprotections and side chain reactions with scavengers.
Analytical Protein Chemistry: Mass spectrometry, protein sequencing and amino acid analysis are performed at the Protein Structure and Carbohydrate Core Facility of the University of Michigan Multipurpose Arthritis and Musculoskeletal Diseases Center (UM-MAC), where Dr. Anagli has an 0 appointment as a Faculty Participant and an Approved User of several Core Facilities (Center Grant Number NIH 5 P60 AR20557). Electrospray ionization mass spectra are generated using a Finnigan MAT mode 710 triple quadrupole mass spectrometer (Finnigan MAT, San Jose.. CA) with a 20 kV conversion dynode and a 4000 m/z mass range. The mass accuracy of the 5 data acquired is routinely better than 0.01 %. Edman degradation is performed on an Applied Biosystems 470A Sequencer and a I20A phenylthiohydantoin derivative analyzer using standard cycles supplied by the manufacturer. Protein and peptide concentrations are determined precisely by quantitative amino acid analysis using an Applied Biosystems Derivatizer o 420A with on-line PTC detection with the Applied Biosystems Analyzer I30A.
I ll Throughout this application, various publications, are referenced by author and year. Full citations for the publications are listed below. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.
Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention can be practiced otherwise than as specifically described.
Table 1 : Compositional analysis. Amino acid residues per KED molecule predicted from DNA sequence are compared with the results from experimental analysis.
Amino Acid PPrrasd-ixiccttsadd Compositional Analysis
(Count/mo . (Residues/ ol . )
Ala A 24 22.33308
Arg R 21 21.17712
Asn N a 20.22813 (AsN +• Asp)
Asp D 14 20.22813 (Asn - Asp)
Cys C 10 N/A
Gin Q 11 20.78861 (Gin + Glu)
Glu E 12 20.78861 (Gin -*- Glu)
Gly G 25 27.36785
His H 10 8.19698
He I 7 6.66840
Leu L 22 23.80118
Lys K α 7.25435
Met M 5 4.35643
Phe p 12 10.84014
Pro P 18 10.55353
Ser s 21 13.97343
Thr T 15 13.24128
Trp w 6 I / Α
Tyr V 8 7.14608
Val V 12 16.67420
Table 2
Figure imgf000115_0002
Figure imgf000115_0001
REFERENCES
Folkman J: Fighting cancer by attacking its blood supply. Scientific American 1996; 275(3): 150-154.
Folkman J, D'Amore PA: Blood vessel formation: what is its molecular basis? Cell 1996;87(7):1153-1155.
Folkman J: Angiogenesis in cancer, vascular, rheumatoid and other disease.
Nature
Medicine 1995; 1(1):27-31.
Dunn T: Oxygen and cancer. North Carolina Medical Journal 1997;58:140-143.
White FC. Carroll SM. Kamps MP: VEGF mRNA is reversibly stabilized by hypoxia and persistently stabilized in VEGF-overexpressing human tumor cell lines. Growth Factors 1995;12(4):289-301.
Hruska KA, Rolnick F, Huskey M, Alvarez U, Cheresh D: Engagement of the osteoclast integrin alpha v beta 3 by osteopontin stimulates phosphatidylinositol 3-hydroxyl kinase activity. Endocrinology 1995;136(7):2984-2992.
Fisher B, Gunduz N, Saffer EA: Influence of the interval between primary tumor removal and chemotherapy on kinetics and growth of metastases. Cancer Research;43: 1488-1492.
OIReilly M, Rosenthal R, Sage EH, Smith S, Holmgren L, Moses M, Shing Y, Folkman J: The suppression of tumor metastases by a primary tumor. Surg Forum 1993;44:474-476.
O'Reilly M, Holmgren L, Shing Y, Chen C, Rosenthal R, Moses M, Lane W, Cao Y, Sage EH, Folkman J: Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994;79:315-328.
Gately S. Twardowski P. Stack MS. Cundiff DL. Grella D. Castellino FJ. Enghild J. Kwaan HC. Lee F. Kramer RA. Volpert O. Bouck N. Soff GA: The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin. Proceedings of the National Academy of Sciences of the United States of America 1997;94(20): 10868-10872.
O'Reilly M, Boehm T, Shing Y, Fukai N, Vasios G, Lane W, Flynn E, Birkhead J, Olsen B, Folkman J: Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997;88:277-285.
Standker L, Schrader M, Kanse SM, Jurgens M, Forssmann WG, Preissner KT: Isolation and characterization of the circulating form of human endostatin. FEBS Letters 1997;420(2-3): 129-133. Ji WR. Barrientos LG. Llinas M. Gray H. Villarreal X. DeFord ME. Castellino FJ. Kramer RA. Trail PA. Selective inhibition by kringle 5 of human plasminogen on endothelial cell migration, an important process in angiogenesis: Biochemical & Biophysical Research Communications 1998;247:414-419.
Cao Y. Chen A. An SSA. Ji RW. Davidson D. Llinas M: Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. Journal of Biological Chemistry 1997;272:22924-22928.
Cao Y. Ji RW. Davidson D. Schaller J. Marti D. Sohndel S. McCance SG. O'Reilly MS. Llinas M. Folkman J: Kringle domains of human angiostatin. Characterization of the anti-proliferative activity on endothelial cells. Journal of Biological Chemistry 1996;271 :29461-29467.
Forsgren M, RAden B, Israelsson M, Larsson K, HedEn LO: Molecular cloning and characterization of a full-length cDNA clone for human plasminogen. FEBS Letters 1987;213(2):254-260.
Saarela J, Ylikarppa R, Rehn M, Purmonen S, Pihlajaniemi T: Complete primary structure of two variant forms of human type XVIII collagen and tissue-specific differences in the expression of the corresponding transcripts. Matrix Biology 1997;16:319-328.
Folkman J, Haudenschild CC, Zetter BR: Long-term culture of capillary endothelial cells. Proc Natl Acad Sci USA 1979;76:5217-5221.
Dameron KM, Volpert OV, Tainsky MA, Bouck N: Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 1994;265:1582- 1584.
Polverini PJ, Bouck NP, Rastinejad F: Assay and purification of naturally occurring inhibitor of angiogenesis. Methods Enzymol 1991 ;198:440-450.
Wada K, Aota S, Tsuchiya R, Ishibashi F, Gojibori T, Ikemura T: Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Research 1990;18:2367-2411.
Garcia G, Mar P, Mullin D, Walker J, Prather N: The E. coli dnaY gene encodes an arginine transfer RNA. Cell 1986;45:453-459.
Maione T. Gray GS. Petro AJ. Hunt AJ. Donner AL. Bauer SI. Carson HF. Sharpe RJ. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science 1990;247:77-79.
Tolsma SS. Volpert OV. Good DJ. Frazier WA. Polverini PJ. Bouck N. Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. Journal of Cell Biology 1993;97-511.
Ault GS. Ryschkewitsch CF. Stoner GL. Type-specific amplification of viral DNA using touchdown and hot start PCR. [Journal Article] Journal of Virological Methods. 46(2): 145-56, 1994
Sanger F. Air GM. Barrell BG. Brown NL. Coulson AR. Fiddes CA. Hutchison CA. Slocombe PM. Smith M. Nucliotide sequence of bacteriophage phi X174 DNA. [Journal Article] Nature. 265(5596):687-95,1977
Fisher B. Gunduz N. Coyle J. Rudock C. Saffer EA. Presence of a growth- stimulating factor in serum following primary tumor removal in mice. Cancer Research. 49: 1996 ,2001 , 1989.
Simpson-Herren L. Sanford AH. Holmquist JP. Effects of surgery on the cell kinetics on residual tumor. Cancer Treat. Rep. 60: 1749-60,1976.
Bennett WF. Paoni NF. Keyt BA. Botstein D. Jones AJ. Presta L. Wurm FM. Zoller MJ. High resolution analysis of functional determinants on human tissue- type plasminogen activator. Journal of Biological Chemistry. 266(8):5191-201 , 1991.
Moose V. Seabra M. Abedin M. Landschulz K. Marcovina S. Hobbs H. Apolipoprotein (a) kringle 4-containing fragments in human urine. J. Clin. Invest. 97:858-64,1996.
Topol EJ. Califf RM. George BS. Caracas DJ. Lee KL. Insights derived from the thrombolysis and angioplasty in myocardial infarction (TAMI) Trials. [Clinical Trial. Journal Article.
Multicenter Study] Journal of the American College of Cardiology. 12(6 Suppl A):24A-3IA. 1988.
Stump DC. Califf RM. Topol EJ. Sigmon K. Thornton D. Masek R. Anderson L. Collen D. Pharmacodynamics of thrombolysis with recombinant tissue-type plasminogen activator. Correlation with characteristics of and clinical outcomes in patients with acute myocardial infarction. The TAMI Study Group. [Journal Article] Circulation. 80(5): 1222-30, 1989.
The National Institute of Neurological Disorders and Stroke (NINDS) rt-PA Stroke Study Group. A systems approach to immediate evaluation and management of hyperacute stroke. Experience at eight centers and implications for community practice and patient care. [Clinical Trial. Journal Article, Multicenter Study] Stroke. 28(8): 1530-40, 1997.
Wardlaw JM. Warlow CP. Counsell C. Systematic review of evidence on thrombolytic therapy for acute ischaemic stroke [see comments]. [Journal Article. Meta-Analysis] Lancet. 350(9078):607-14, 1997. Goldhaber SI. Kessler CM. Heit J. Markis J. Sharma GV. Dawley D. Nagel JS. Meyerovitz M. Kim D. Vaughan DE. Randomized controlled trial of recombinant tissue plasminogen activator versus urokinase in the treatment of acute pulmonary embolism. [Clinical Trial. Journal Article. Randomized Controlled Trial] Lancet. 2(8606):293-8, 1988.
Parker JA. Markis JE. Palla A. Goldhaber SZ. Royal HD. Tumeh S. Kim D. Rustgi AK. Holman BL. Kolodny GM. Pulmonary perfusion after rt-PA therapy for acute embolism: early improvement assessed with segmental perfusion scanning. [Journal Article] Radiology. 166(2):441-5.1988.
Pennica D. Holmes WE. Kohr WJ. Harkins RN. Vehar GA. Ward CA. Bennett WF. Yelverton E. Seeburg PH. Heyneker HL. Goeddel DV. Collen D. Cloning and expression of human tissue-type plasminogen activator cDNA in E. Coli. [Journal Article] Nature. 301 (5897):114-21. 1983.
Patthy L. Evolution of the proteases of blood coagulation and fibrinolysis by assembly from modules. [Review] [58 refs] [Journal Article. Review] Cell. 41(3):657-63, 1985. de Munk GA. Caspers MP. Chang GT. Pouwels PH. Enger-Valk BE. Verheijen JH. Binding of tissue-type plasminogen activator to lysine, lysine analogues, and fibrin fragments [Journal Article] Biochemistry. 28(18):7318- 25.1989.
Nesheim M. Fredenburgh JC. Larsen GR. The dissociation constants and stoichiometries of the interactions of Lys-plasminogen and chloromethyl ketone derivatives of tissue plasminogen activator and the variant delta FE IX with intact fibrin. [Journal Article] Journal of Biological Chemistry 265(35):21541-8, 1990.
Horrevoets A. Smilde A. de Vries C. Pannekoek H. The specific roles of finger and kringle 2 domains of tissue-type plasminogen activator during in vitro fibrinolysis. Journal of Biological Chemistry 269(17): 12639-44, 1994.
Urano T. Takada Y. Takada A. Stimulation of the amidolytic activity of single chain tissue- type plasminogen activator by fibrinogen degradation product: possible fibrin binding sites on single chain tissue-type plasminogen activator molecule. Biochimica et Biophysica Acta 1077: 245-52, 1991.
Mulder M. Kohnert U. Fischer S. van Hinsbergh VW. Verheijen JH. The interaction of recombinant tissue type plasminogen activator and recombinant plasminogen activator (r-PA/BM 06.022) with human endothelial cells. [Journal Article] Blood Coagulation & Fibrinolysis. 8(2): 124-33, 1997.
Cheng XF. Brohlin M. Pohl G. Back O. Wallen P. Binding of tissue plasminogen activator to endothelial cells. Thrombosis Research. 77 (2): 149-64. 1995. Nelson SL. DeFord ME. Prorok M. Chibber BAK. Bretthauer RK. Castellino FJ. High-level secretion in Pichia pastoris and biochemical characterization of the recombinant kringle 2 domain of tissue-type plasminogen activator. Biotechnol. Appl. Biochem. 25: 63-74.1997.
Nelson J. Allen W. Scott W. Bailie J. Walker B. McFerran N. Wilson D. Murine epidermal growth factor (EGF) fragment (33-42) inhibits both EGF- and laminin-dependent endothelial cell motility and angiogenesis. Cancer Research 55: 3772-76,1995.
DeSelTano VS. Menhart N. Castellino FJ. Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator. [Journal Article] Archives of Biochemistry & Biophysics. 294(1):282-90, 1992
Hu GF. Limited proteolysis of angiostatin by elastase is regulated by plasminogen. J Protein Chem 1997;16:669-679.
Patterson BC, Sang QA. Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9). J Biol Chem 1997;272:28823-28825.
Thimme R, Spangenberg HC, Blum HE. Angiostatin. A biological inhibitor of tumor angiogenesis. Deutsche Medizinische Wochenschrift 1997;122:413-414.
Dong Z, Kumar R, Yang X, Fidler IJ. Macrophage-derived metal loelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell 1997;88:801-810.
Sim BK, O'Reilly MS, Liang H, Fortier AH, He W, Madsen JW, Lapcevich R, Nacy CA. A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer. Cancer Res 1997;57:1329-1334.
Wu Z, O'Reilly MS, Folkman J, Shing Y. Suppression of tumor growth with recombinant murine angiostatin. Biochem Biophys Res Comm 1997;236:651- 654.
Stathakis P, Fitsgerald M, Mathrhias LJ, Chesterman CN, Hogg PJ. Generation of angiostatin by reduction and proteolysis of plasmin. Catalysis by a plasmin reductase secreted by cultured cells. J Biol Chem 1997;272:20641-20645.
Bicknell R. Harris Al. mechanisms and therapeutic implications of angiogenesis. Current Opinion in Oncology 1996;8:60-65.
Jendrasschak E, Sage EH. Regulation of angiogenesis by SPARC and angiostatin: implications for tumor cell biology. Seminars in Cancer Biology 1996;7:139-146. O'Reilly MS, Holmgren L, Chen C, Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nature Med 1996;2:689-692,
Battegay EJ. Angiostatin: mechanistic insights, neovascular disease, and therapeutic prospects. J Molec Med 1996;73:333-346.
Bikfalvi A. Significance of angiogenesis in tumor progression and metastasis. European J Cancer 1995;31A:1101-1104.
O'Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS, Cao Y, Sage EH, Folkman J. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994;79:315-328.
Cao Y, Ji RW, Davidson D, Schaller J, Marti D, Sohndel S, McCance SG, O'Reilly MS, Llinas M, Folkman J. Kringle domains of human angiostatin. J Biological Chem 1996;271(46):29461 -29467
Cheng XF, Brohlin M, Pohl G, Back O, Wallen P. Binding of tissue plasminogen activator to endothelial cells. Thrombosis Research 1955;77(2):149-164.
Nilsen SL, DeFord ME, Prorok M, Chibber BAK, Bretthauer RK, Castellino FJ. High-level secretion in Pichia pastoris and biochemical characterization of the recombinant kringle 2 domain oftissue-type plasminogen activator. Biotechnol Appl Biochem 1997;25:63-74.
Sanger F, Air GM, Barrell BG, Brown NL, Coulson AR, Fiddes CA, Hutchison CA, Slocomb PM, Smith M. Nucliotide sequence of bacteriophage phi X174 DNA. Nature 1977:265(5596):687-695.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage. T4. Nature 1970;227(259):680-685.
Dicou E. Expression of recombinant human nerve growth factor in Escherichia coli. Neurochem Intl 1992;20(1): 129-134.
DeSerrano VS, Menhart N, Castellino FJ, Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator. Archives of Biochemistry & Biophysics 1992;294(1):282- 290.
Kohn EC, Liotta LA ( 1995): Molecular insights into cancer invasion: strategies for prevention and intervention. Cancer Res 55: 1856-186.
Liotta LA, Steeg PS, Stetler-Stevenson WG ( 1991): Cancer metastasis and angiogenesis; an imbalance of positive and negative regulation. Cell 64: 327- 336.
Folkman /, Shing Y (1992): Angiogenesis. 1 Biol Chem 267: 10931
Hanahan D, Folkman J (1996): Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86: 353-364.
Folkman J (1971): Tumor angiogenesis: therapeutic implications. N Engl J Med 285: 1182
Liotta LA, Kleinerman J, Saidel G (1974): Quantitative relationships of intravascular tumor cells: tumor vessels and pulmonary metastases following tumor implantation. Cancer Res 34: 997-1003.
Mikkelsen T, Yan PS, Ho KL. Sameni M, Sloane BF, Rosenblum ML (1995): Immunolocalization of cathepsin B in human glioma: implications for tumor invasion and angiogenesis. 1 Neurosurg. 83: 285-290. 10 White FC. Carroll SM. Kamps \1 P: \ .EGF mRNA is reversibly stabilized by hypoxia and persistently stabilized in VEGF-overexpressing human tumor cell lines. Growth Factors 1995;12(4):289-301.
Fisher B. Gunduz N. Coyle J. Rudock C. Saffer EA. Presence of a growth- stimulating factor in serum following primary tumor removal in mice. Cancer Research. 49: 1996-2001, 1989.
Klintworth, O.K. Corneal angiogensis: a comprehensive critical review. New York: Springer-Verlag; 1991. 56 Polverini, P.J.; Bouck, N.P.; Rastinejad, F. Assay and purification of naturally occurring inhibitor of angiogenesis. Methods Enzymol. 1991 ; 198: 440-450.
Kenyon, Barbel M; Voest, E.E.; Chen, Catherine C. Flynn, Evelyn; Folkman, J.; D' Amato, R. A model of angiogenesis in the mouse cornea. Invest Ophthlmol. Vis. Sci. 199637:1625-1632.
Wilson C. Goberdhan DC. Steller H. Dror, a potential neurotrophic receptor gene, encodes a Drosophila homolog of the vertebrate Ror family ofTrk-related receptor tyrosine kinases. Proceedings of the National 61cademy of Sciences of the United States of America 1993;90(i5):7109-7113.
Lokker NA. Presta LG. Godowski PJ. Mutational analysis and molecular modeling of the N-t~ kringle-containing domain of hepatocyte growth factor identifies amino acid side chains important for interaction with the c-Met receptor. Protein Engineering 1994;7(7):895-903. 64
DeSerrano VS. Menhart N. Castellino F J .Expression purificatio~ and characterization of the recOinbinant kringle 1 domain from tiSSVIe-type plasminogenacnvδtOr. ArCh BiOCh Biophys. 29-1 ):282-90, 1
Scott.Bailie JR.Walker B! McF erran NV. Wilson DI. Murine growth and angiogenesis. Cancer Research 1995.55 (17).3772-3776
Homandberg GA. Williams JE. Grant D. Schumacher B. Eisenstein R. Heparin- binding fragments of fibronectin are potent inhibitors of endothelial cell growth. American Journal of Pathology 1985;120(3):327-332. 68,
Anagli, J. ; Hoffinan, F.; Quadroni, M.; Voherr, T. Carafoli, E. The cahnodul in- binding domain of the inducible (macrophage) notric oxide synthase. Eu7: J: Biochem. 1995;233:701-708

Claims

CLAIMSWhat is claimed is:
1. A pharmaceutical composition comprising at least one kringle protein and a pharmaceutically acceptable carrier.
2. The pharmaceutical composition according to claim 1 , wherein said kringle protein is at least one from the group consisting essentially of kringle proteins 1 through 5.
3. A pharmaceutical composition comprising a molecule from the group consisting essentially of KED or a kringle derived from the tPA protein and a pharmaceutically acceptable carrier.
4. A method of treating a tumor comprising the step of administering an effective amount of at least one from the group consisting essentially of kringle proteins 1 through 5, KED and a kringle derived from the tPA protein.
PCT/US2000/004798 1999-02-24 2000-02-24 An anti-angiogenic kringle protein and its mutants WO2000049871A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU36058/00A AU3605800A (en) 1999-02-24 2000-02-24 An anti-angiogenic kringle protein and its mutants

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US12134199P 1999-02-24 1999-02-24
US60/121,341 1999-02-24
US12163399P 1999-02-25 1999-02-25
US60/121,633 1999-02-25
US16617699P 1999-11-18 1999-11-18
US60/166,176 1999-11-18

Publications (2)

Publication Number Publication Date
WO2000049871A1 WO2000049871A1 (en) 2000-08-31
WO2000049871A9 true WO2000049871A9 (en) 2002-06-20

Family

ID=27382614

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/004798 WO2000049871A1 (en) 1999-02-24 2000-02-24 An anti-angiogenic kringle protein and its mutants

Country Status (2)

Country Link
AU (1) AU3605800A (en)
WO (1) WO2000049871A1 (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0029001D0 (en) * 2000-11-28 2001-01-10 Isis Innovation Assay
EP2289542A1 (en) * 2009-08-31 2011-03-02 PAION Deutschland GmbH Treatment of neurological or neurodegenerative disorders
EP3556385A4 (en) 2016-12-15 2020-12-30 Talengen International Limited Method for mitigating heart disease
TW201822809A (en) * 2016-12-15 2018-07-01 深圳瑞健生命科學硏究院有限公司 Method for preventing and treating hyperlipidemia
TW201829448A (en) 2016-12-15 2018-08-16 大陸商深圳瑞健生命科學硏究院有限公司 Method and drug for preventing and treating obesity

Also Published As

Publication number Publication date
AU3605800A (en) 2000-09-14
WO2000049871A1 (en) 2000-08-31

Similar Documents

Publication Publication Date Title
US5801146A (en) Compound and method for inhibiting angiogenesis
US7485439B2 (en) Nucleic acids encoding plasminogen fragments and methods of use
EP0758390B1 (en) Angiostatin and method of use for inhibition of angiogenesis
EP1783215B1 (en) Angiostatin and method of use for inhibition of angiogenesis
EP0824546B1 (en) Angiostatin fragments and methods of use
US7365159B2 (en) Angiostatin protein
EP0964869B1 (en) Methods and compositions for the treatment of neoplastic diseases
US20070117180A1 (en) Enzyme producing plasma protein fragment having inhibitory activity to metastasis and growth of cancer and plasma protein fragment produced by fragmentation by said enzyme
CA2240296C (en) Endothelial cell proliferation inhibitor and method of use
JP2002510209A (en) Kringle domain 1-5 of plasminogen that can regulate angiogenesis in vivo
WO2000049871A9 (en) An anti-angiogenic kringle protein and its mutants
AU772186B2 (en) A novel anti-angiogenic agent comprising at least one kringle region and an upstream pre-activation domain of plasminogen
CA2352477A1 (en) Compositions comprising angiostatin and kringle 5 region of plasminogen and methods of use thereof
JP2002535372A (en) Plasminogen kringle 4 region fragment and methods of use
US6949511B1 (en) Methods of inhibiting angiogenesis via increasing in vivo concentrations of kringle region fragments of plasminogen
US7157556B1 (en) Deglycosylated kringle 1-3 region fragments of plasminogen and methods of use
CA2400497A1 (en) Methods and compositions for generating angiostatin
EP1867721A1 (en) Angiostatin fragments and aggregate angiostatin and methods of use
Lapcevich et al. A RecombinantHuman AngiostatinProtein Inhibits Experimental Primary and
MXPA99011041A (en) Angiostatin fragments and method of use
AU4440402A (en) Angiostatin fragments and method of use

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWE Wipo information: entry into national phase

Ref document number: 09914277

Country of ref document: US

AK Designated states

Kind code of ref document: C2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 1/25-25/25, DRAWINGS, REPLACED BY NEW PAGES 1/20-20/20; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

122 Ep: pct app. not ent. europ. phase