EP0378676A1 - Tumor necrosis enhancing factor and methods of preparation and use - Google Patents
Tumor necrosis enhancing factor and methods of preparation and useInfo
- Publication number
- EP0378676A1 EP0378676A1 EP89908921A EP89908921A EP0378676A1 EP 0378676 A1 EP0378676 A1 EP 0378676A1 EP 89908921 A EP89908921 A EP 89908921A EP 89908921 A EP89908921 A EP 89908921A EP 0378676 A1 EP0378676 A1 EP 0378676A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- factor
- tumor
- tumor necrosis
- tnf
- cells
- Prior art date
- Legal status (The legal status 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 status listed.)
- Ceased
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- Tumor Necrosis Factor is a cytokine (1) which has been shown to act on a variety of cellular targets including the endothelium (1) . Because endothelial cells form the luminal vascular surface, TNF-induced changes in endothelial cell properties could alter the basic blood-compatibility of the vessel wall. In this context, endothelium has been shown to provide specific receptors for TNF and a consequence of TNF interaction with these receptors includes modulation of coagulant properties (2-3). Thus, the coagulant phenotype of the endothelium is altered from the quiescent state, where mechanisms promoting the fluidity of blood predominate, to a perturbed state, where procoagulant pathways are enhanced (3) .
- mice are infused with doses of TNF in the range of 1-10 ⁇ g/animal, widespread clot formation is not observed.
- necrosis of tumors does not occur in mice bearing meth A fibrosar-'_as and clot formation throughout the tumor bed is seen (4) .
- tumor-cell derived substance could act synergistically with TNF to necrotize tumors.
- a tumor cell-derived substance might explain why certain tumors respond to TNF while others do not, even though TNF binding sites are present.
- Such a tumor necrotizing substance could act on the vessel wall as well as on other cellular elements surrounding and forming the tumor. This substance could facilitate TNF-induced hemorrhagic necrosis via several mechanisms, one of which would be by further augmenting the increase in endothelial cell procoagulant activity which substances like TNF or other onokines bring about.
- Another aspect of this investigation concerned whether tumor cell-derived substance could increase the procoagulant activity which TNF induces in the endothelium.
- TNEF tumor necrosis enhancing factor
- This invention concerns a purified tumor necrosis enhancing factor.
- the polypeptide is characterized by an apparent molecular weight between about 40,000 and about 50,000 daltons under non-reducing conditions and between about 65,000 and about 75,000 daltons under reducing conditions, and by isoelectric focussing peaks at about 6.8 and about 7.2.
- the factor is further characterized by loss of activity upon treatment wit' protease K, by an affinity for Heparin-Sepharose from which the factor may be eluted with about 0.5 M NaCl, by the ability to bind to a reverse phase FPLC-ProRPC column, and by the ability to be eluted from a reverse phase FPLC-ProRPC column to which the factor is bound in an ascending methanol gradient at about 50%, by the ability to migrate as a single band on an SDS-polyacrylamide gel, and by adsorption to Concanavalin A-Sepharose.
- This invention also concerns a purified, biologically active fragment of purified tumor necrosis enhancing factor which is characterized by an apparent molecular weight between about 10,000 and about 30,000 daltons, by the loss of activity upon treatment with trypsin or heat, by an affinity for Heparin ultrogel from which the fragment may be eluted with about 0.5 M NaCl, by the ability to bind to a Mono Q column, and by the ability to be eluted from a Mono Q column to which the fragment is bound in an ascending salt gradient at about 0.4 M NaCl.
- This invention concerns a pharmaceutical composition
- a pharmaceutical composition comprising an amount of a tumor necrosis enhancing factor effective to enhance the tumor inhibitory activity of a cytotoxic agent and a pharmaceutically acceptable carrier.
- This invention also concerns a pharmaceutical composition
- a pharmaceutical composition comprising an amount of a biologically active fragment of purified tumor necrosis enhancing factor effective to enhance the tumor inhibitory activity of a cytotoxic agent and a pharmaceutically acceptable carrier.
- Figure 1 Enhanced induction of endothelial cell tissue factor in response to TNF in the presence of tumor-conditioned medium.
- Conditioned medium from meth A sarcoma cells at the indicated dilution was incubated with endothelial cells in serum-free medium (RPMI containing lOmM HEPES, pH 7.4, 20 ⁇ g/ml transferrin, 10 ⁇ g/ml insulin, 1 ⁇ g/ml polymyxin B and 5 mg/ml human serum albumin) .
- Cultures contained either serum-free medium alone (>.,, TNF (0.1 nM) alone (TNF) , undiluted conditioned medium alone (CM) , or TNF in the presence of conditioned medium at the indicated dilution (TNF + CM) .
- Purified mouse IgG (10 ⁇ g/ml and 100 ⁇ g/ml) had no effect on effect on endothelial cell coagulant activity as measured in this assay.
- the darkened bar corresponds to cultures incubated with tumor-conditioned medium (1/2 dilution) in the presence of TNF in which the coagulant assay was carried out in the presence of only Factor X. Results shown are the mean and SEM of triplicate determinations.
- Factor Xa generation was the same as untreated controls when tumor-conditioned medium was added directly to the Factor Vlla-X incubation mixtur in the absence of endothelial cells.
- Tumor-conditioned medium obtained as described in A above (1 ml) was applied to a Sephadex G150 column (0.9 x 55 cm), eluted with 10 mM HEPES (pH 7.4), 0.1 M NaCl and fractions (1.3 ml) were collected. Aliquots of the indicated column fractions, at a 1/4 dilution, were then incubated with endothelial cell onolayers in the presence of TNF (0.1 nM) for 7 hours at 37 'C. The tissue factor assay was carried out as described above (A) and Factor Xa formed over 7 minutes is shown (mean + SEM) .
- TNF denotes cells incubated with TNF a ⁇ -r_e (0.1 nM) and column buffer. Column fractions 1 to 9 were inactive in this assay.
- the gel filtration column was calibrated with standard proteins including ribonuclease (13,700), chymotrypsinogen A (25,000), ovalbumin (43,000) and albumin (67,000).
- This panel (left hand) demonstrates that following adsorption of TNEF activity to the resin, it was eluted with 0.5 M NaCl, with the bulk of the protein, not at higher salt concentrations where FGF and tumor cell- derived mitogens are found.
- FIG. 1 Purification of tumor necrosis enhancing factor (TNEF) by chromatography of meth A-conditioned medium on Mono S and reverse phase FPLC columns.
- TNEF tumor necrosis enhancing factor
- Meth A-conditioned medium was harvested from cultures, subjected to ammonium sulfate precipitation, dialysis, a negative adsorption step to Q-Sepharose and then chromatographed on Mono S. The column was eluted with an ascending salt gradient (dashed line) and the maximal salt concentration at the end of the gradient was 0.5 M. Protein content of the fractions is plotted versus tissue factor activity induced in endothelial cultures following a 7 hr incubation of partially purified tumor necrosis enhancing factor (TNEF) with the cells (only results of fractions with peak activity are shown) .
- TNEF tumor necrosis enhancing factor
- the factor is a polypeptide characterized by an apparent molecular weight between about 40,000 and about 50,000 daltons under non-reducing conditions and by an apparent molecular weight between about 65,000 and about 75,000 daltons under reducing conditions, preferably about 44,000 daltons on a non-reducing SDS- polyacryla ide gel, and about 70,000 daltons on a reducing SDS-polyacrylamide gel, and by isoelectric focussing peaks about 6.8 and about 7.2.
- the factor is further characterized by loss of activity upon treatment with protease K, by an affinity for Heparin- Sepharose from which the factor may be eluted with about 0.5 M NaCl, by the ability to bind to a reverse phase FPLC-ProRPC column, and by the ability to be eluted from a reverse phase FPLC-ProRPC column to which the factor is bound in an ascending methanol gradient at about 50%, by the ability to migrate as a single band on an SDS-polyacrylamide gel, and by adsorption to Concanavalin A-Sepharose.
- This invention also concerns a purified, biologically active fragment of purified tumor necrosis enhancing factor characterized by an apparent molecular weight between about 10,000 and about 30,000 daltons, preferably about 20,000 daltons, by the loss of activity upon treatment with trypsin or heat, by an affinity for Heparin Ultrogel from which the fragment may be eluted with about 0.5 M NaCl, by the ability to bind to a Mono Q column, and by the ability to be eluted from a Mono Q column to which the fragment is bound in an ascending salt gradient at about 0.4 M NaCl .
- the purified tumor necrosis enhancing factor preferably is of animal, e.g., human, rat, or mouse origin.
- Heparin Ultrogel is obtained by coupling heparin to Ultrogel A4, i.e., 4% agarose, by epichlorohydrin using a six carbon spacer arm. Heparin Ultrogel may be obtained from IBF Biotechniques Co., Savage, Maryland.
- a Mono Q* column is an ion-exchange column prepacked with Mono Q ⁇ , i.e., mono dispersed hydrophilic polymer beads with an extremely narrow particle size distribution for chromatographic resolution of proteins (pH range 2-12) .
- Mono Q* columns may be obtained from Pharmacia, Inc., Piscataway, New Jersey.
- Con A- Sepharose may be obtained by coupling Concanavalin A to Sepharose.
- Con A-Sepharose may be obtained from Pharmacia, Inc., Piscataway, New Jersey.
- Heparin- Sepharose may be obtained by coupling heparin to Sepharose.
- Heparin-Sepharose may be obtained from Pharmacia, Inc., Piscataway, New Jersey.
- ProRPC is a reverse-phase column and may be obtained from Pharmacia, Inc. , Piscataway, New Jersey.
- This invention also concerns a purified DNA molecule, a cDNA molecule or an isolated genomic DNA molecule, encoding a tumor necrosis enhancing factor, and a purified DNA molecule, a cDNA molecule or an isolated genomic DNA molecule, encoding a biologically active fragment of tumor necrosis activating factor.
- DNA can be readily obtained by one skilled in the art utilizing well known methods, e.g. , the preparation of oligonucleotide probes and the use of such probes to obtain the DNA, such probes in turn being based upon the amino acid sequence of tumor necrosis enhancing factor which may be readily obtained by conventional methods such as automated DNA sequencing.
- the invention provides a pharmaceutical composition comprising an amount of a tumor necrosis enhancing factor effective to enhance the tumor inhibitory activity of a cytotoxic agent, and a pharmaceutically acceptable carrier. Also provided is a pharmaceutical composition comprising an amount of a biologically active fragment of tumor necrosis enhancing factor effective to enhance the tumor inhibitory activity of a cytotoxic agent, and a pharmaceutically acceptable carrier.
- Pharmaceutically acceptable carriers include water, and other conventional carriers, e.g., sugars, such as mannitol, in water.
- This invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising an amount of tumor necrosis enhancing factor effective to enhance the tumor inhibitory activity of a cytotoxic agent, a pharmaceutically acceptable carrier, and further comprising an amount of tumor necrosis factor effective to inhibit tumor growth.
- This invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising an amount of a biologically active fragment of tumor necrosis enhancing factor effective to enhance the tumor inhibitory activity of a cytotoxic agent, a pharmaceutically acceptable carrier, and further comprising an amount of tumor necrosis factor effective to inhibit tumor growth.
- This invention also provides a method of inhibiting the growth of tumor cells, particularly the localized inhibition of tumor cell growth within the vascular system, which comprises contacting the cells with an effective tumor inhibitory amount of a cytotoxic agent in the presence of an amount of a tumor necrosis enhancing factor effective to enhance the tumor inhibitory activity of the cytotoxic agent.
- the cytotoxic agent is a polypeptide tissue factor having tumor inhibitory activity, preferably tumor necrosis factor.
- This invention also provides a method of inhibiting the growth of tumor cells, particularly the localized inhibition of tumor cell growth within the vascular system, which comprises contacting the cells with an effective tumor inhibitory amount of a cytotoxic agent in the presence of an amount of the biologically active fragment of tumor necrosis enhancing factor effective to enhance the tumor inhibitory activity of the cytotoxic agent.
- the cytotoxic agent is a polypeptide tissue factor having tumor inhibitory activity, e.g., tumor necrosis factor.
- This invention also provides a method of inhibiting the growth of tumor cells which comprises contacting the cells with an effective amount of a pharmaceutical composition, the composition comprising an amount of a tumor necrosis enhancing factor effective to enhance the tumor inhibitory activity of a cytotoxic agent and a pharmaceutically acceptable carrier.
- This invention also provides a method of inhibiting the growth of tumor cells which comprises contacting the cells with an effective amount of a pharmaceutical composition, the composition comprising an amount of the biologically active fragment of tumor necrosi enhancing factor effective to enhance the tumo inhibitory activity of a cytotoxic agent and pharmaceutically acceptable carrier.
- This invention also provides a method of treating a subject having a tumor which comprises administering to the subject an amount of a pharmaceutical composition, the composition comprising an amount of tumor necrosis enhancing factor effective to enhance the tumor inhibitory activity of a cytotoxic agent and a pharmaceutically acceptable carrier, effective to necrotize the tumor.
- This invention also provides a method of treating a subject' having a tumor which comprises administering to the subject an amount of a pharmaceutical composition, the composition comprising an amount of the biologically active fragment of tumor necrosis enhancing factor effective to enhance the tumor inhibitory activity of a cytotoxic agent and a pharmaceutically acceptable carrier, effective to necrotize the tumor.
- This invention also provides a method of preparing a tumor necrosis enhancing factor which comprises recovering the factor from tumor cells and purifying the factor so recovered.
- This invention also provides a method of preparing a biologically active fragment of tumor necrosis enhancing factor which comprises recovering the fragment from tumor cells and purifying the fragment so recovered.
- This invention also provides a method of preparing a tumor necrosis enhancing factor which comprises expressing a purified DNA molecule encoding the tumor necrosis enhancing factor in a suitable host under conditions so that the factor is produced, recovering the factor so produced, and purifying the factor so recovered. Also provided is a method of preparing a biologically active fragment of tumor necrosis enhancing factor which comprises expressing a purified DNA molecule encoding the biologically active fragment of tumor necrosis enhancing factor in a suitable host under conditions so that the fragment is produced, recovering the fragment produced, and purifying the fragment s recovered.
- Suitable hosts include bacteria, e.g., Escherichia coli, and mammalian cells and yeast cells. Methods for expressing DNA in such hosts are well known in the art, as are methods for recovering polypeptides produced, such as tumor necrosis enhancing factor, in such hosts and purifying factors so recovered.
- mice were injected intradermally with meth A sarcoma cells (10 cells/animal; provided by Drs. Hoffman and Old,
- mice were anestheized and subjected to whole-body beating heart perfusion fixation (90-110 mm Hg) . Incorporation of radioactivity into the mouse tissue was determined after infusion of 125I-f ⁇ br ⁇ nogen by removing a piece of tissue, weighing it and counting the sample in a gamma-counter.
- the presence of fibrin in tumor tissue was studied by excising tumor tissue, cutting it up finely with a scalpel and then extracting it with buffer containing Triton X-100 (2%) and protease inhibitors (1.5 mM PMSF, 0.3 mM leupeptin, 20 ⁇ g/ml soybean trypsin inhibitor, 500 U/ml Trasylol>.
- the extract was reacted with an equal volume of rabbit anti-mouse IgG immunobeads (Biorad, Richmond, CA) for 2 hours at 37*C to remove mouse immunoglobulin. Control experiments in which a trace of radioiodinated mouse IgG was added to rissue extra-__ indicated that greater than 99% of the IgG was absorbed by the beads.
- the extract was then made 9M in urea and 10 mg/ml in dithiothreitol, boiled for 3 minutes and added to an equal volume of sample buffer for reduced Laemmli SDS- PAGE (10) . After boiling for 3 minutes, the mixture was centrifuged (10,000 RPM; -5 min) and the supernatant was subjected to SDS-PAGE (10%) and Western blotting. In each case, the same amount of tumor tissue was processed and the total protein loaded per lane of the gel was about the same. Following Western blotting, nitrocellulose membranes were reacted with monoclonal antibody specific for human fibrin (11) followed by
- mice were maintained on drinking water supplemented with the warfarin derivative 3(o--acetonylbenzyl)-4-hyc ⁇ xycoumarin (0.7 mg/1) for three days prior to the TNF infusion (about 7 days after the meth A cells were injected) . Tumors grew to the same size in warfarin-treated and control animals. Prior to carrying out an experiment with the anticoagulated animals, a Factor X assay on the mouse plasma was performed (15) . Only animals with Factor X levels of less than 1% were used.
- TNEF Tumor Necrosis Enhancing Factor
- Endothelial cells derived from human umbilical cord veins were prepared by the method of Jaffe (16) as modified by Thornton (17) . Experiments were carried out within 24 hrs of the cells achieving confluence in 9.6 cm wells (passages 1 to 5) . Cells were characterized as endothelial based on the presence of von Willebrand Factor antigen (18) , as described previously, and thrombomodulin activity (19) . Meth A sarcoma cells, provided by Drs. Hoffman and Old (7) , were grown in RMPI 1640 containing 10% fetal calf serum.
- Conditioned medium was obtained from meth A cells by placing them in serum-free medium (RPMI containing 10 mM HEPES, pH 7.4 20 ⁇ g/ml transferrin, 10 ⁇ g/ml insulin, 1 ⁇ g/ml poly yxin B and 5 mg/ml bovine serum albumin) for 48 hrs.
- serum-free medium RPMI containing 10 mM HEPES, pH 7.4 20 ⁇ g/ml transferrin, 10 ⁇ g/ml insulin, 1 ⁇ g/ml poly yxin B and 5 mg/ml bovine serum albumin
- tissue factor activity of endothelial cell monolayers was assayed using purified human Factors Vila (8nM) (provided by Dr. R. Bach, Mc-'-tt Sina Medical School, NY, NY) and X (1.5 ⁇ M) in serum-free medium at 23'C.
- a sample (0.2 ml) of th reaction mixture was removed and assayed for Factor X activity by monitoring hydrolysis of the chromogeni substrate benz-Ile-Glu-Gly-Arg-p-nitroanilide (20) .
- monoclonal antibody which blocks human tissue facto coagulant activity was provided by Dr. R. Bach.
- TNEF tumor necrosi enhancing factor
- A In order to culture meth A sarcoma cells, a tissu explant was prepared. Adherent cells were grown t confluence in RPMI 1640 (Gibco) containing 10% feta calf serum and then split in 1:3 ratio using trypsin EDTA (Gibco) . Cells were then washed 3 times in calcium-magnesium-free Hanks' balanced salt solution and placed in serum-free medium (RPMI 1640 containin 10 mM HEPES (pH 7.4), 20 ⁇ g/ml polymyxin B and 5 mg/m bovine serum albumin) for 48 hours. Thereafter, th conditioned medium was harvested and incubated wit endothelial cell cultures as described below. B.
- RPMI 1640 containin 10 mM HEPES (pH 7.4), 20 ⁇ g/ml polymyxin B and 5 mg/m bovine serum albumin
- TNF + CM Tumor Necrosis Factor
- TNF denotes cells incubated with TNF alone (0.1 nM)
- B denotes cells incubated with column buffer alone
- TNF + B H denotes cells incubated with TNF (0.1 nM) and column buffer.
- Ear ⁇ lier column fractions were inactive in this assay.
- TNEF tumor necrosis enhancing factor
- Figure 2 shows the results of heparin Ultrogel (IBF Co.) chromatography of a meth A fibrosarcoma culture supernatant (the supernatant was collected in protein- free medium) .
- the left hand panel in Figure 2A demonstrates that following adsorption of TNEF activity to the resin, it was eluted with 0.5 M NaCl, with the bulk of the protein, not at higher salt concentrations where FGF and tumor cell-derived mitogens are found.
- the salt gradient elution in the right hand panel of Figure 2B shows elution of TNF activity towards the end of the salt gradient, well separated from much of the applied protein.
- Treatment Factor Xa formed (pmole/ml/well)
- Tumor-conditioned medium obtained from meth A sarco ⁇ ma cells was subjected to no treatment (none) or the indicated procedure: heat, 100*C for 10 minutes; acid, exposure to pH 2.0 for 5 minutes; base, exposure to pH 9.0 for 5 minutes; dialysis (molecular weight cut off 3,000-4,000); or trypsin, 50 ⁇ g for 1 hour at 37 * C (trypsin was inactivated at the end of the incubation period with ' diisopropylfluorophosphate; 1 mM diisopropylfluorophosphate alone had no effect on subsequent induction of tissue factor) .
- conditioned medium was added to endo ⁇ thelial cell cultures at a 1:4 dilution in the presence of TNF (0.1 nM) for 7 hours at 37*C.
- TNF 0.1 nM
- the tissue factor assay was carried out as described above and Factor Xa formed over 7 minutes as shown. "No CM added” indicates that no conditioned medium was added (TNF alone was present at 0.1 nM) . If neither conditioned medium nor TNF was added, Factor Xa formation was less than 5 pmole/ml/well.
- the mean and SEM of triplicat determinations are shown in Table 1. trypsin and heat-sensitive polypeptide(s) , molecular weight 10,000-30,000 daltons with a low affinity for Heparin Ultrogel.
- This activity is distinct from Interleukin 1, 7 -interferon, endotoxin, tumor necrosis factor, transforming growth factor (a and ⁇ ) , Interleukin G, fibroblast growth factor, and multiple endothelial cell mitogens.
- the biologically active fragment of tumor necrosis enhancing factor is (a) trypsin and heat- sensitive polypeptide(s) fragment with a molecular weight between about 10,000-30,000 daltons and further characterized by a low affinity for Heparin Ultrogel.
- Endothelial cells derived from human umbilical cord veins were prepared by the method of Jaffe (16) as modified by Thornton et al. (17) . Experiments were carried out within 24 hr of the cells achieving confluence and cultures were characterized as described previously.
- Meth A cells provided by Drs. Hoffman and Old (Memorial Sloan- Kettering Cancer Center) (7) , were grown in RPMI 1640 containing 10% calf serum. Conditioned medium was obtained from meth A cells by placing them in serum- free medium (RPMI 1640 containing HEPES, lOmM, pH 7.4) for 12 hr at 37'C.
- TNEF tumor necrosis enhancing factor
- Tissue factor activity was determined by two-stage coagulant assay and, in a limited number of assays, vas also measured by studying formation of Factor Xa in the presence of purified Factors Vila (8 nM) and X (1.5 ⁇ M) by monitoring hydrolysis of the chromogenic substrate Benz-Ile-Glu-Gly-Arg-p-nitroanilide. Both of these assays have been previously described in this context (21) .
- Units of tissue factor activity were defined arbitrarily by assigning a value of 1U to an amount of meth A factor which induces an equivalent amount of tissue factor activity to that observed with 1 pg of purified human tissue factor reconstituted into phosphatidylserine/phosphatidylcholine vesicles (20:80) using the two-stage coagulant assay 80% phosphatidylcholine) .
- tumor necrosis enhancing factor TNEF was incubated with endothelium alone or in the presence of TNF for 7 hr at 37'C.
- Assays were carried out with whole cells obtained in suspension following scraping from the dish with a rubber policeman (cell viability was >90% based on trypan blue exclusion) or, if indicated, with intact monolayers.
- Purified recombinant TNF (10 U/ml) was provided by Dr. Lomedico of Koffman-LaRoche (Nutley, NJ) .
- Tissue factor antigen content of endothelial cultures was determined (using an ELISA assay carried out by Dr. Jim Morrissey, Scripps Clinic and Research Foundation, La Jolla, CA) .
- Northern blots to determine levels of tissue factor mRNA were carried out b extracting total RNA from cells using the guanidiniu thiocyanate procedure (22) , electrophoretic fractionation of the RNA on a 1.2% agarose gel (23), and transfer to nitrocellulose.
- the cDNA probe for thrombomodulin (provided by Dr. E. Sadler, Wash U. , St. Louis, MO) was labelled using random hexamer labeling (Boehringer Mannheim random primed DNA labelling kit, Indianapolis, IN) . Hybridization of the cDNA probe to normal and meth A factor-treated RNA was performed at 42'C as described previously (23).
- Tumor -.icrosis enhancing factor was studied fc * its content of lipopolysaccharide (using the Li ulus amoebocyte assay carried out by Dr. Cerami, Rockefeller University, NY, NY) and assays for Interleukin-1 activity (using the D10 assay carried out by Dr. Killian, Hoffman LaRoche, Nutley, NJ) , Interleukin-6 activity (using the B cell proliferation assay carried out by Dr. May, Rockefeller University, NY, NY) , TNF activity (using the L929 assay carried out by Mr. DiPirro, SUNY at Buffalo, Buffalo, NY) and mitogenic activity (using the 3T3 mitogenesis assay carried out by Dr. Witte, Columbia University, NY, NY) .
- Antibody to murine Interleukin la was provided by Dr. Lomedico (Hoffmann-LaRoche) and antibody to murine TNF ⁇ was purchased from Genzyme (Boston, Mass) .
- Tumor necrosis enhancing factor may be prepared as follows:
- Meth A-conditioned medium prepared as described above, was harvested from the cultures, sterile filtered (0.2 ⁇ m) , and after addition of octyl- ⁇ -glucoside (final concentration 0.1%) and protease inhibitors (benzamidine, 5 mM, PMSF, 0.2 mM) , ammonium sulfate precipitation (80% saturation) was carried out. The ammonium sulfate pellet was then dissolved in tris (20 mM; pH 7.3)/octyl- -glucoside (0.1%) and dialyzed exhaustively against the same buffer.
- the retentate was applied to a Q-Sepharose column (2.5x20 cm; Pharmacia, Piscataway, NJ) equilibrated with tris (20 mM; pH 7.3) containing octyl- ⁇ -glucoside and . protease inhibitors (as above), and step-eluted with 0.1 M NaCl in the same buffer.
- the active fractions were pooled, dialyzed versus phosphate (25 mM; pH 6.8) containing octyl-j9-glucoside (0.1%) and applied to a FPLC Mono S column (HR 10'"-D; Pharmacia) equilibrated in the same buffer containing 4M urea.
- the column was eluted with an ascending salt gradient (0 to 0.5 M NaCl) and the active fractions were pooled, the pH was adjusted to 2.2 with trifluoroacetic acid and the sample applied to an FPLC ProRPC (HR 5/10; Pharmacia).
- the column was eluted with a linear gradient of increasing methanol concentration in the presence of trifluoroacetic acid (0.1%), the active fractions were pooled, dialyzed in the presence of SDS (0.1%) and concentrated b lyophilization. Samples were then dissolved in Laemmli sample buffer and preparative SDS-PAGE (12.5%) was carried out.
- Tumor necrosis enhancing factor may also be produced from other tumor lines. This tumor- necrotizing substance may also influence other properties of the vessel wall surrounding tumors in addition to TNF-induced expression of tissue factor. Tumor necrosis enhancing factor (TNEF) may also be involved in determining the immunogenicity of meth A sarcomas and may influence the fibrinolytic potential of the tumor vasculature.
- TNF tumor necrosis factor
- 30 ⁇ g/animal or more most of the animals died with thrombi in multiple organs, especially lung and liver, consistent with previous studies indicating the severe toxicity of TNF at these concentrations (24-25) .
- a TNF concentration of 10 ⁇ g/animal resulted in less marked systemic toxicity and thrombus formation.
- 3 ⁇ g/animal most animals survived without gross lesions in the normal vasculature, but hemorrhagic changes were observed in the tumors, indicating that this lower dose of TNF was triggering hemostatic abnormalities in the vascular bed of the meth A sarcoma, without widespread thrombohemorrhagic phenomena in other tissues.
- Fibrin deposition in the tumor vasculature after infusion of TNF (3 ⁇ g/animal) was assessed by measuring accumulation of radioactivity in the tumor in the presence of 125I fibrinogen. About ten times more radioactivity accumulated in the tumor bed of animals co-infused with TNF than in the meth A sarcomas of saline-infused controls. In contrast, other organs showed only a minimal increase in uptake of radioactivity after TNF infusion. Heat-treatment of
- TNF which prevents its b ⁇ iing to cellular TNF receptors (8) , prevented the enhanced deposition of radioactivity in the tumor bed, indicating that TNF was the active agent. That activation of the coagulation mechanism with fibrin formation was responsible for the accumulation of radioactivity in the tumors is implied by the decreased incorporation of radioactivity in tumors from anticoagulated animals. Consistent with this hypothesis, after infusion of TNF Western blots of tumor extracts reacted strongly with a fibrin-specific monoclonal antibody. Treatment of animals with 3( ⁇ - acetonylbenzyl)-4-hydroxycoumarin considerably attenuated this band, and infusion of heat-treated TNF
- Fibrin was identified by the usual morphologic criteria (26) , and the characteristic ultrastructural periodicity of 21.09 nm.
- the scanning electron micrograph demonstrates fibrin strands apposed to the luminal endothelial cell surface, a situation never observed in control animals. Fibrin deposition was limited to the vessels in the tumor bed. At these early times, adherence of platelets and white cells on the vessel wall did not occur, and platelet thrombi were not seen in the spleen or other organs.
- TGF / 3 because of the availability of purified material and the considerable sequence homology betweeen TGF from different species (murine, porcine and human) (34-36) .
- TGF / 9 1 nor TGF0 2 at concentrations up to 500 pM
- endothelial cell procoagulant activity in the presence of TNF suggesting that the activity in meth A culture supernatants was distinct from TGF .
- the starting material was 1 liter of meth A-conditioned serum-free medium.
- TNEF tumor necrosis enhancing factor
- TNEF tumor necrosis enhancing factor
- TNEF tumor necrosis enhancing factor
- TNF tumor necrosis enhancing factor
- thrombosis is a major cause of morbidity and mortality, mechanisms involved in the pathogenesis of localized intravascular clot formation are largely uncharacterized.
- Models of intravascular clot formation derived from studies of the hemostatic plaque, rapid thrombus formation following contact of plasma factors and cellular elements of the blood with subendothelial cell components (29) , present a picture of thrombosis in which endothelial cell denudation is the critical initiating step.
- TNF an endogenously produced mediator of the host response
- TNF can selectively induce intravascular clot formation in the tumor vasculature of mice bearing meth A sarcomas in the presence of a viable endothelial monolayer and delineate another model of localized thrombosis.
- radioiodinated fibrinogen/fibrin accumulated in the tumor.
- the frank deposition of fibrin in the tumor vascular bed -indicates that activation of coagulation with clot formation was clearly involved.
- the tumor microenvironment is the result of host and tumor-derived factors which are concentrated in the tumor bed.
- the studies described here demonstrate that meth A tumor cells elaborate a polypeptide, tumor necrosis enhancing factor (TNEF) , which induces endothelial tissue factor and enhances the procoagulant response to TNF.
- Tumor necrosis enhancing facto appears to be distinct from other cytokines which have been shown to induce endothelial tissu factor activity (such as TNF and Interleukin-1) , and is a potentially important component of the tumor microenvironment contributing to alterations in vascular function observed in the tumor bed.
- TNEF tumor necrosis activating factor
- TNEF tumor necrosis enhancing factor
- TNEF tumor necrosis enhancing factor
- TNF can promote activation of the coagulation mechanism by modulating coaguxant properties of endothelial cells.
- Studies with 125I-fibrinogen showed ten-fold enhanced accumulation of radioactivity in the tumor within 2 hours after TNF infusion.
- Western blots of tumor extracts subjected to SDS-PAGE and visualized with a fibrin-specific monoclonal antibody indicated that fibrin forms in the tumor after the TNF infusion.
- a heat-stable, protease K-sensitive polypeptide with an apparent molecular weight of about 44,000 daltons on a non-reduced SDS-polyacrylamide gel and a molecular weight of about 70,000 daltons on a reduced SDS-polyacrylamide gel was purified about 5000- fold from serum-free culture supernatants of meth A cells by sequential Q-Sepharose, Mono S, reverse phase, and SDS-PAGE procedures. Based on immunologic criteria, biologic activity, and other molecular properties, tumor necrosis enhancing factor (TNEF) appears to be distinct from other cytokines and growth factors.
- TNEF tumor necrosis enhancing factor
- TNEF tumor necrosis enhancing factor
- TNF and IL-1 Cytokines with multiple overlapping biological activities. Lab. Invest 56:234.
- Tumor necrosis factor interacts with endothelial cell receptors to induce release of IL-1. J. Exp. Med. 163:1363.
- Human endothelial cells use of neparin in cloning and long-term serial cultivation. Science 222:623.
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Des facteurs qui agissent en synergie avec un facteur de nécrose tumorale (TNF), pour produire l'expression d'un facteur de croissance tissulaire (du type thromboplastine ou facteur III) dans des cellules endothéliales, ont pu être isolées. Les deux facteurs protéiques sont obtenus à partir de cellules de sarcome de type meth A de la souris. Le premier facteur se caractérise par un poids moléculaire de 40 à 50 KDA dans des conditions non réductrices et de 65 à 75 KDA dans des conditions réductrices et par des points isoélectriques compris entre 6,8 et 7,2. Ledit facteur se caractérise en outre: par sa perte d'activité lorsqu'il est traité avec la protéase K, par sa capacité d'élution à partir d'héparine-sépharose avec 0,5 M de NaCl, par sa capacité de liaison à une colonne de FPLC-ProRPC en phase inverse, par sa capacité d'élution à partir de la colonne de FPLC-ProRPC en phase inverse par gradient de méthanol ascendant à environ 50 %, par sa capacité de migration sous la forme d'une bande unitaire sur un gel de SDS-polyacrylamide et par sa capacité d'adsorption sur de la concanavaline A-Sépharose. Le second facteur se caractérise par un poids moléculaire de 10 à 30 KDa par filtrage par gel, par sa perte d'activité lorsqu'il est traité avec de la trypsine, par sa thermosensibilité, par sa capacité d'élution à partir d'Ultrogel d'héparine avec 0,5 M de NaCl, par sa capacité de liaison sur une colonne mono Q et par sa séparation par élution par gradient salin ascendant avec 0,4 M de NaCl.Factors which act in synergy with a tumor necrosis factor (TNF), to produce the expression of a tissue growth factor (of the thromboplastin or factor III type) in endothelial cells, have been isolated. Both protein factors are obtained from mouse meth A sarcoma cells. The first factor is characterized by a molecular weight of 40 to 50 KDA under non-reducing conditions and from 65 to 75 KDA under reducing conditions and by isoelectric points between 6.8 and 7.2. Said factor is further characterized: by its loss of activity when treated with protease K, by its capacity for elution from heparin-sepharose with 0.5 M NaCl, by its capacity for binding to a column of FPLC-ProRPC in reverse phase, by its capacity of elution from the column of FPLC-ProRPC in reverse phase by methanol gradient rising to about 50%, by its capacity of migration in the form of a band unit on an SDS-polyacrylamide gel and by its adsorption capacity on concanavalin A-Sepharose. The second factor is characterized by a molecular weight of 10 to 30 KDa by gel filtration, by its loss of activity when it is treated with trypsin, by its thermosensitivity, by its capacity of elution from Ultrogel. heparin with 0.5 M NaCl, by its binding capacity on a mono Q column and by its separation by elution using an ascending salt gradient with 0.4 M NaCl.
Description
Claims
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US21965088A | 1988-07-15 | 1988-07-15 | |
US219650 | 1988-07-15 |
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EP0378676A1 true EP0378676A1 (en) | 1990-07-25 |
EP0378676A4 EP0378676A4 (en) | 1991-01-16 |
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EP19890908921 Ceased EP0378676A4 (en) | 1988-07-15 | 1989-07-14 | Tumor necrosis enhancing factor and methods of preparation and use |
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EP (1) | EP0378676A4 (en) |
JP (1) | JPH03501620A (en) |
AU (1) | AU630106B2 (en) |
WO (1) | WO1990000400A1 (en) |
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US5830448A (en) * | 1994-06-16 | 1998-11-03 | Genentech, Inc. | Compositions and methods for the treatment of tumors |
JP6286689B2 (en) * | 2012-02-10 | 2018-03-14 | 株式会社ジャパニック | Cosmetic or skin regeneration promoter using non-human stem cell culture supernatant as raw material, and protein iontophoresis method |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
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FR2513124B1 (en) * | 1981-07-21 | 1989-11-17 | Hayashibara Biochem Lab | PRODUCTION AND APPLICATIONS OF THE TARGET CELL LYSE FACTOR |
US4481137A (en) * | 1982-02-26 | 1984-11-06 | Mochida Pharmaceutical Co., Ltd. | Glycoproteins and processes for their production |
US4456550A (en) * | 1982-11-22 | 1984-06-26 | President And Fellows Of Harvard College | Vascular permeability factor |
-
1989
- 1989-07-14 JP JP1508421A patent/JPH03501620A/en active Pending
- 1989-07-14 WO PCT/US1989/003108 patent/WO1990000400A1/en not_active Application Discontinuation
- 1989-07-14 EP EP19890908921 patent/EP0378676A4/en not_active Ceased
- 1989-07-14 AU AU40348/89A patent/AU630106B2/en not_active Ceased
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See references of WO9000400A1 * |
Also Published As
Publication number | Publication date |
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WO1990000400A1 (en) | 1990-01-25 |
EP0378676A4 (en) | 1991-01-16 |
AU630106B2 (en) | 1992-10-22 |
JPH03501620A (en) | 1991-04-11 |
AU4034889A (en) | 1990-02-05 |
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