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WO1990014360A1 - Inhibiteurs de la croissance de tumeurs derivees de tissus, procedes de preparation et leurs utilisations - Google Patents

Inhibiteurs de la croissance de tumeurs derivees de tissus, procedes de preparation et leurs utilisations Download PDF

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Publication number
WO1990014360A1
WO1990014360A1 PCT/US1990/002753 US9002753W WO9014360A1 WO 1990014360 A1 WO1990014360 A1 WO 1990014360A1 US 9002753 W US9002753 W US 9002753W WO 9014360 A1 WO9014360 A1 WO 9014360A1
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WIPO (PCT)
Prior art keywords
protein
tgf
activity
tumor growth
tumor
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PCT/US1990/002753
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English (en)
Inventor
Kenneth K. Iwata
John R. Stephenson
Peter Ten Dijke
Robert Franco
Leslie I. Gold
J. Gordon Foulkes
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Oncogene Science, Inc.
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Application filed by Oncogene Science, Inc. filed Critical Oncogene Science, Inc.
Priority to AU57293/90A priority Critical patent/AU668072B2/en
Publication of WO1990014360A1 publication Critical patent/WO1990014360A1/fr

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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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Bichel Nature 23JL: 449-450 (1971)
  • Injection of cell-free ascites obtained from mice bearing fully developed ascites tumors, into mice with growing ascites tumors, resulted in a pro ⁇ nounced inhibition of ascites growth.
  • Bichel, supra also observed that two surgically joined mice (para- biotic) , one mouse with an advanced tumor and the other with an early tumor, resulted in a pronounced inhibi ⁇ tion of growth of the early tumor. Based upon these observations, [Bichel, Europ. J.
  • McMahon, et al. [Proc. Natl. Acad. Sci. USA 79, 456-460 (1982)] have purified from rat liver a 26,000 dalton substance which inhibits the proliferation of nonmalignant rat liver cells, but does not inhibit the proliferation of malignant rat liver cells.
  • Other growth inhibitory substances have been identified in cultured chicken spinal cord cells [Kage, et al.. Experimental Neurology 58_: 347-360 (1970); Harrington, et al., Proc. Natl. Acad. Sci. USA 77_: 423- 427 (1980) and Steck, et al., J. Cell Biol. 8 ⁇ : 562-575 (1979)].
  • BSC-1 cells inhibited the growth of BSC-1 cells, human mammary tumor cells and normal human mammary cells.
  • Todaro [Todaro, G.J. in Epigenetic Regulation of Can ⁇ cer, Terry Fox Cancer Research Conference (University of British Columbia; Vancouver, B.C., Canada) Abs. 13 (1984) ] subsequently reported two factors with tumor cell growth inhibitory properties which were reportedly sequenced and shown to consist of 70 and 90 amino acid residues, respectively.
  • Todaro failed to report the source of the factors, their tissue type, the species the factors were derived from or the meth ⁇ od of the factor purification.
  • This invention provides a protein having tumor growth inhibitory activity comprising the 112 amino acids shown in Figure 29 beginning with alanine at position 1 and ending with serine at position 112.
  • the protein may also comprise the 412 amino acids shown in Figure 41 beginning with methionine at nucleotide position 263 and ending with serine at nucleotide position 1496.
  • this 412 amino acid sequence contains the complete precursor sequence of the protein having tumor growth inhibitory activity as well as the complete sequence of the mature protein shown in Figure 29 beginning with alanine at position 1 and ending with serine at position 112.
  • the invention provides a protein comprising the 411 amino acids shown in Figure 41 beginning with lysine at nucleotide position 266 and ending with serine at nucleotide position 1496.
  • Figure 1 shows gel filtration chromatography at 23°C. Elution pattern of gel filtration chromatography at 23*C of crude acidified, ethanol extract from human umbilical cords. Two grams of acidified, ethanol ex ⁇ tract in 150 ml of 1.0 M acetic acid was applied to a 14 x 100 cm column (Amicon; #86012) containing Bio-Gel* P10 and eluted at a flow rate of 7 ml/min. One liter fractions were collected on a SuperRac* (LKB 2211) equipped with a type C collection rack (LKB) .
  • LLB 2211 SuperRac*
  • LLB type C collection rack
  • TGI activity was determined as described in Materials and Methods. Inhibition of A549 human lung carcinoma cells is shown by triangles and mink lung (CCL 64) cells by circles. Absorbance at 280 n
  • Figure 2 shows gel filtration chromatography at 4°C. Elution pattern of gel filtration chromatography at 4'C of crude acidified, ethanol extract from human umbili- cal cords. Two grams of acidified, ethanol extract in 150 ml of 1.0 M acetic acid was applied to a 14 x 100 cm column (Amicon; #86012) containing Bio-Gel* P10 and eluted at a flow rate of 7 ml/min. One liter fractions were collected on a SuperRac* (LKB 2211) equipped with a type C collection rack (LKB) .
  • LLB 2211 SuperRac*
  • LLB type C collection rack
  • Tumor growth inhibitory activity was determined as described in Materials and Methods. Inhibition of A549 human lung*-carcinoma cells is shown by open triangles and mink lung (CCL 64) cells by open circles. .Absorbance of 280 n ( ) was detected by a Uvicord S ®
  • LLB 2138 with a full scale absorbance range of 1.0 AUFS and a single channel chart recorder (LKB 2210) with a chart speed of 1 mm/min.
  • Figure 3 shows cell growth inhibition and normal human cell stimulation by fractions from gel filtration chro ⁇ matography at 4 * C.
  • Elution pattern of gel filtration chromatography at 4 ⁇ C of crude acidified, ethanol ex ⁇ tract in 150 ml of 1.0 M acetic acid was applied to a 14 x 100 cm column (Amicon; #86012) containing Bio- Gel* P10 and eluted at a flow rate of 7 ml/min.
  • One liter fractions were collected on a SuperRac* (LKB 2211) equipped with a type C collection rack (LKB) .
  • One ml aliquots of each fraction (1 liter/fraction) were transferred to 12 x 75 mm sterile snap top tubes (Falcon 2058) .
  • Tumor growth inhibitory activity was determined as described in Materials and Methods. Inhibition of A549 human lung carcinoma cells is shown by open triangles and mink lung (CCL 64) cells by open circles. Stimulation of normal human fibroblasts is shown by open squares. Absorbance of 280 nm ( ) was detected by Uvicord S* (LKB 2138) with a full scale absorbance range of 1.0 AUFS and a single channel chart recorder (LKB 2210) with a chart speed of l mm/min.
  • Figure 4 shows reverse phase high performance liquid chromatography (HPLC) of an active fraction from gel filtration chromatography.
  • Fraction 4 derived from gel filtration chromatography on Bio-Gel* P10 of human umbilical cord acidified, ethanol extract (65.8 mg protein) was lyophilized and resuspended in 10 ml of 0.05% trifluoroacetic acid (TFA) .
  • Fraction 4 was the fir ⁇ t fraction following the major peaks of absorbance at 280 nm ( Figure 2). The sample was centrifuged on a Beckman table top centrifuge (Beckman TJ-6) at 3000 rp for 20 minutes to remove insoluble material.
  • FIG. 5 shows HPLC rechromatography of pooled TGI activity from HPLC (TGI-1) .
  • Pooled fractions of tumor growth inhibitory activity 1.5 mg
  • acetonitrile fractions 13-22
  • HPLC chroma ⁇ tography Figure 4
  • TGI-1 HPLC chroma ⁇ tography
  • the sample was load ⁇ ed onto a ⁇ BONDAPAK* C.g column (0.39 x 30 cm) (Waters #27324) .
  • the flow rate was 1 ml/min. and the effluent monitored at 206 nm ( ) with a Waters u.v. de ⁇ tector (Waters Model 481) at a sensitivity of 2.0 AUFS.
  • Elution was achieved with a linear 20 min gradient from 0-15% of increasing concentrations of 2-propanol con ⁇ taining 0.05% TFA, followed by a linear 120 min gradi ⁇ ent of 15-35% 2-propanol containing 0.05% TFA.
  • a SuperRac* (LKB 2211) was used to collect 4 ml frac ⁇ tions.
  • One ml aliquots of each fraction were trans ⁇ ferred to 12 x 75 mm polystyrene tubes (Falcon 2058) containing 50 microliters of 1.0 M acetic acid and 5C micrograms of bovine serum albumin (Sigma A-6003) and assayed for tumor growth inhibitory activity as de ⁇ scribed in Materials and Methods.
  • Inhibition of A549 human lung carcinoma cells is shown by open triangles and of mink lung (CCL 64) cells by open circles.
  • the solvent gradient is shown by large dashes ( — — — ) •
  • FIG. 6 shows reverse phase HPLC rechromatography of pooled activity from HPLC (TGI-2) .
  • Pooled fractions of tumor growth inhibitory activity 0.8 mg
  • eluting be ⁇ tween 35-39% acetonitrile fractions 25-31) by HPLC chromatography ( Figure 4)
  • TGI-2 HPLC chromatography
  • Figure 4 were lyophilized and resus- pended in 2 ml of 0.05% trifluoracetic acid (TFA).
  • TFA trifluoracetic acid
  • the sample was centrifuged on a Beckman tabletop centri ⁇ fuge (Beckman TJ-6) at 3000 rpm for 30 min to remove insoluble material.
  • Two separate injections of the supernatant were made through a Water's U6K injector equipped with a 2 ml sample loop.
  • the sample was load- ed onto a ⁇ BONDAPAK* C 18 column (0.39 x 30 cm) (Waters 27324) .
  • the flow rate was 1 ml/min and the effluent monitored at 206 nm ( ) with a Waters u.v. de ⁇ tector (Waters Model 481) at a sensitivity of 1.0 AUFS.
  • Elution was achieved with a linear 20 min gradient from 0-15% of increasing concentrations of 2-propanol con ⁇ taining 0.05% TFA, followed by a linear 120 min gradi ⁇ ent of 15-35% 2-propanol containing 0.05% TFA.
  • a Sup- erRac* (LKB 2211) was used to collect 4 ml fractions. One ml aliquots of each fraction were transferred to 12 x 75 mm polystyrene tubes (Falcon 2058) containing 50 microliters of 1.0 M acetic acid and 50 micrograms of bovine serum albumin (Sigma A-6003) and assayed for tumor growth inhibitory activity as described in Mate ⁇ rials and Methods. Inhibition of A549 human lung car- cino a cells is shown by open triangles and of mink lung (CCL 64) cells by open circles. The solvent gra ⁇ washer is shown by large dashes ( — — — ) .
  • Figure 7 shows cation exchange chromatography of human umbilical cord extracts.
  • CM-TRISACRYL* was resuspended in an equal volume of 0.1 M ammonium acetate, pH 4.0, containing 1.0 M NaCl.
  • the resin was allowed to equil ⁇ ibrate for 3 hours and degassed at 4 ⁇ C. Twenty ml of resin was packed into a 1.6 x 40 cm column (Pharmacia; #19-0362-01) and washed with 2 column volumes of 1.0 M ammonimu acetate pH 4.0, followed by 0.01 M ammonium acetate.
  • the column was washed until the effluent matched the conductivity and the pH of the equilibrat ⁇ ing buffer (0.01 M ammonium acetate pH 4.0).
  • One gram of human umbilical cord acidified, ethanol extract was resuspended in 50 ml of 1.0 M acetic acid and dialyzed against the column equilibration buffer at 4*C until the pH and the conductivity matched that of the equili ⁇ bration buffer.
  • the dialyzed acidified, ethanol ex- tract was applied to the column at a flow rate of l ml/min at 4*C and the column was washed with the equil ⁇ ibrating buffer until the absorbance ( ), A280, as monitored by a Uvicord* S (LKB 2138) with a sensi ⁇ tivity of 1.0 AUFS, was at its lowest point. This was followed by 200 ml of an ascending molarity linear gradient from 0.01 to 1.0 M ammonium acetate, pH 4.0, which was applied using a gradient mixer (Pharmacia GM- 1, #19-0495-01) .
  • Figure 8 shows rechromatography of a pooled fraction from cation exchange chromatography.
  • CM-TRISACRYL* was prepared as described in Figure 9.
  • the material from fractions containing CM III and CM IV were pooled, lyophilized, resuspended in 50 ml of 0.1 M acetic acid and dialyzed against the column equilibration buffer at 4'C until the pH and the conductivity matched that of the equilibration buffer.
  • the sample was applied to the column at a flow rate of 1 ml/min at 4*C and the column was washed with 120 ml of the equilibrating buffer.
  • Absorbance ( ) (280nm) was monitored by a ⁇ Uvicord S (LKB 2138) with a sensitivity of 1.0 AUFS.
  • a ⁇ Uvicord S (LKB 2138) with a sensitivity of 1.0 AUFS.
  • an addi ⁇ tional 30 ml of 1.0 M ammonium acetate, pH 4.0 was passed through the column.
  • Figure 9 shows the fractionation of TGI by cation ex ⁇ change chromatography at 4'C.
  • 1.65 mg of protein ex ⁇ tract prepared as described in the Second Series of Experiments was dialyzed extensively against 20mM ammo ⁇ nium acetate (pH 4.5) and applied to a 5 ml (1 x 6.3 cm) column of CM-TRISACRYL* previously equilibrated in 20 mM ammonium acetate (pH 4.5) and 1.65 ml fractions (12 x 100 mm polystyrene tubes) were collected.
  • Figure 10 shows the fractionation of TGI by anion ex ⁇ change chromatography at 4*C.
  • 1.65 mg of protein ex ⁇ tract prepared as described in the Second Series of Experiments was dialyzed extensively against 20 mM Tris-HCl (pH 8.0) and clarified by centrifugation at 3,000 x g for 15 minutes.
  • DEAE-TRISACRYL* was pre ⁇ pared by suspending the resin first in 20 mM Tris-HCl (pH 8.0) containing 1.0 M NaCl for 3 hours and secondly in 0.5 M Tris-HCl (pH 8.0) for 1 hour.
  • the sedimented resin was washed on a Buchner funnel with 1000 ml H 2 0 and finally resuspended in 20 mM Tris-HCl (pH 8.0), degassed and poured into a 5 ml column (l x 6.3 cm) and the resin equilibrated with 20 mM Tris, HC1 (pH 8.0).
  • the clarified sample was applied to the column and absorbance at 280 nm( ) , inhibitory activity against mink lung cells (0-0) , and the NaCl gradient ( — — — ) was determined as described in Figure 9 and under Materials and Methods.
  • the linear NaCl gra ⁇ trans in 20 mM Tris-HCl (pH 8.0) ranged from 0 to 1.0 M NaCl.
  • FIG 11 shows the fractionation of TGI by cation exchange chromatography at 4 * C.
  • CM-TRISACRYL* was prepared as described in Figure 7 with the exception that,..the final equilibration buffer was 20 mM ammonium acetate, pH 4.5.
  • Protein extract (9.9 mg) prepared as above was dialyzed extensively against 20 mM ammonium acetate (pH 4.5) and applied to a 15 ml (1.5 x 8.5 cm) column of CM-TRISACRYL* in 20 mM ammonium acetate (pH 4.5).
  • Figure 12 shows the reverse phase high performance liquid chromatography (HPLC) of active fractions from cation exchange chromatography.
  • HPLC high performance liquid chromatography
  • Fractions 59 thru 78 derived from cation exchange chromatography on CM-TRI- SACRYL* of human umbilical cord described in Figure 11 were pooled, lyophilized, and resuspended in 10 ml of 0.05% trifluoracetic acid (TFA).
  • TFA trifluoracetic acid
  • a total of twenty percent of dialyzed material containing 240 micrograms protein was injected in three separate injections through a Water's U6K injector equipped with a 2 ml sample loop. The sample was then applied onto a ⁇ BOND- APAK* C 18 column (0.39 x 30 cm) (Waters 27324).
  • the flow rate was 1 ml/min and the effluent was monitored at 206 nm ( ) with a Waters u.v. detector (Wa- ters Model 481) at a sensitivity of 0.5 AUFS.
  • Elution was achieved with a linear 5 min gradient from 0-25% of increasing concentrations of acetonitrile containing 0.05% TFA, followed by a linear 15 min gradient of 25- 45% acetonitrile containing 0.05% TFA, followed by a linear 15 min gradient of 45-80% acetonitrile contain ⁇ ing 0.05% TFA, followed by a linear 5 min gradient of 80-100% acetonitrile containing 0.05% TFA.
  • a Super- Rac* (LKB 2211) was used to collect 1 ml fractions. Five hundred microliter aliquots of every other frac- tion-were transferred to 12 x 75 mm polystyrene tubes (Falcon 2058) containing 50 microliters of 1.0 M ace ⁇ tic acid and 50 micrograms of bovine serum albumin (Sigma A0281) and assayed for tumor growth inhibitory activity as described under Materials and Methods. Inhibition of A549 human lung carcinoma cells is shown by open triangles and of mink lung (CCL 64) cells by open circles. The solvent gradient is shown by large dashes ( — — — ) .
  • Figure 13 shows the hydrophobic interaction chromatog ⁇ raphy phenyl-Sepharose.
  • Phenyl-Sepharose (Pharmacia) was equilibrated with 4.0 M ammonium acetate, pH 4.5 and 15 ml of resin poured into a 1.5 x 20cm column (Pharmacia) .
  • Thirty-one mg of ether ethanol precipi- tated TGI in 36.0 ml which was equilibrated by dialysis in Spectropor* 3 (molecular weight cutoff 3,500) in 4.0 M ammonium acetate, was applied to the column at a flow rate of 1.0 ml/min.
  • Tumor growth inhibitory activity was determined for both CCL 64 mink lung cells and A549 cells as described in the initial procedure. Activity against A549 cells is not shown because the activity profiles were similar. Tumor growth inhibitory activ- ity-is plotted as percent inhibition and is illustrated by closed circles. The peak of growth inhibitory ac ⁇ tivity was eluted at 1.18 M ammonium acetate, 42% eth- ylene glycol. Protein concentration is indicated as absorbance at 280 nm and was determined using a spec- trophotometer (Baush & Lomb, Spectronic* 1001) .
  • Biologically active fractions 90-100 were pooled and dialyzed against 0.1 M acetic acid.
  • the protein con ⁇ centration of the pooled fractions was determined by absorbance at OD 280' ⁇ he recovered protein was 1.4 mg (see Table 7) .
  • the quantity of inhibitory units ap ⁇ plied was 1.56 x 10 in 30.9 mg and the amount recov ⁇ ered was 1.5 x 10 in 1.4 mg.
  • FIG 14A shows reverse phase high pressure liquid chromatography (HPLC) ( ⁇ Bondpak* C18) .
  • HPLC reverse phase high pressure liquid chromatography
  • the protein solution was sonicated for two minutes (Branson B-220 Sonicator) and particu- late matter removed by centrifugation (Beckman Model TJ6) at 3,000 rpm for 5 minutes prior to injection into a ( ⁇ BONDAPAK* C18) column (0.39 x 30 cm).
  • the protein was eluted at a flow rate of 1.0 ml per minute using a stepwise gradient.
  • the concentration of acetonitrile was initially increased to 25% in fifteen minutes and elution was continued at 25% for 10 minutes; the con ⁇ centration was then increased to 27% in two minutes and elutrion was continued at 27% for ten minutes; the con ⁇ centration increased to 28% in 2 minutes continued at 28% for 10 minutes, and finally the concentration was increased to 100% in 10 minutes.
  • the fractions were collected into siliconized glass tubes. The solvent gradient is illustrated by short dashes. .
  • .Absorbance of protein was monitored at 210 nm ( ) . Each frac ⁇ tion volume contained 1.0 ml.
  • the equipment used for RPHPLC was exactly as described in Figure 12.
  • FIG 14B is ⁇ ws the reverse phase high pressure liquid chromatography (HPLC) ( ⁇ BONDAPAK* C18) .
  • HPLC reverse phase high pressure liquid chromatography
  • TGI derived from the stro- mal component of dissected human umbilical cord tissue and obtained from pooled biologically active fractions resulting from phenyl-Sepharose chromatography were diluted in 2.0 ml of 0.05% trifluoracetic acid (TFA) and 10% acetonitrile.
  • TGI trifluoracetic acid
  • the protein was prepared and chromatographed exactly as described in Figure 14A. Ten microliters from each 1.0 ml sample were used to test for inhibitory activity. This sample represented
  • Figure 15 shows Sodium Dodecyl Sulfate Polyacrylamide Slab Gel Electrophoresis (SDS-PAGE) .
  • the lyophilized pool of biologically active protein, as marked by ar ⁇ rows, in Figure 14A from chromatography by ⁇ BONDAPAK* C18 from two identical chromatographic procedures were pooled and prepared for gel electrophoresis.
  • Samples were diluted in 100 microliter sample buffer contain ⁇ ing 0.1 M Tris-HCl, pH 6.8 (Sigma), 15% glycerol (Kodak) , and 2% sodium dodecyl sulfate (SDS) .
  • the samples were boiled for two minutes to remove protein which may have adhered to the glass (siliconized) and 50 microliters transferred to 50 microliters of sample buffer containing 10% ⁇ -mercaptoethanol (BioRad*) for reduction of disulfide bonds. These samples were boiled for 2 minutes and both the unreduced and reduced samples were applied to two separate 1.5 mm wide slab gels (marked as lane 2) and electrophoresed through a 10-20% acrylamide gradient in a vertical electrophore ⁇ sis cell (BioRad, Model 155) under constant current at 30 milliamps (mA) per gel for 4.5 hours (Hoeffer power supply PS 1200 DC) .
  • Molecular weight standards both reduced by 5% / 3-mercaptoethanol and non- reduced are marked with their corresponding molecular weights. They are as follows, phosphorylase A, 96 kDa; bovine serum albumin, 68 kDa; ovalbumin, 43 kDa; car- bonic anhydrase, 30 kDa; soybean trypsin inhibitor, 21 kDa; and lysozyme 14.4 kDa.
  • the gels were stained with 0.125% Coomassie Blue R-250 (BioRad) in 5.7% ace ⁇ tic acid 47% methanol for ten minutes (to fix the pro ⁇ tein in the gel) , and destained overnight in the same solution without Coomassie Blue.
  • the gels were re- stained by a silver technique as described by Merril (BioRad silver staining kit #161-0443)
  • Lane 1 TGF- / 9) contains approximately 1,000-1,500 (50 ng) units of growth inhibitory activity
  • lane 2 contains ap ⁇ proximately 8,000-20,000 units of growth inhibitory activity.
  • Figure 16 shows reverse phase high pressure liquid chromatography (HPLC) ( ⁇ BONDAPAK* CN) of active frac ⁇ tions from the previous HPLC procedure (14B) which were combined from two separate chromatographic runs.
  • HPLC high pressure liquid chromatography
  • ⁇ BONDAPAK* CN active frac ⁇ tions from the previous HPLC procedure
  • the lyophilized material from individual tubes (siliconized glass 13 x 100 mm tubes) was suspended in 4.0 ml of 0.1% trifluoracetic acid (TFA) containing 10% propanol, sonicated for two minutes and injected onto a ⁇ BONDAPAK* CN column (0.39 x 30 cm) at 1.0 ml/minute.
  • TFA trifluoracetic acid
  • Elution of the protein was achieved by increasing the concentra ⁇ tion of 2-propanol containing 0.05% TFA from 10% to 20% in 10 minutes, the concentration was then increased from 20 to 50% in 50 minutes (0.6% per minute), and finally the concentration was increased to 100% in twenty minutes.
  • the solvent gradient is shown as short dashes.
  • Absorbance of the eluted protein was monitored at 210 nm ( ) .
  • the equipment used for RPHPLC was exactly as described in Figure 12. Each fraction volume was 1.0 ml and an aliquot of two hundred microliters was then removed from every other tube to assess biological activity (closed circles) .
  • the inhibitory activity eluted from the column between approximately 40-45% 2-propanol.
  • Figure 17 shows SDS polyacrylamide slab gel electro ⁇ phoresis and autoradiography. Lyophilized samples from specific active and inactive fractions from chromatog- raphy on a ⁇ BONDAPAK* CN column illustrated in Figure 16 were iodinated as described in the text. Samples were dissolved in both non-reducing and reducing sample buffer as described for Figure 15 and electrophoresed using a 5-20% acrylamide gradient to resolve protein bands and remove free-radioactive iodine. The gels were stained and destained until the radioactive label disappeared from the destain solution. The gels were dried using a gel dryer (Hoeffer) and subjected to autoradiography using type XAR film (Kodak) for 1 week.
  • Hoeffer gel dryer
  • Kodak type XAR film
  • Non-radioactive standards were also electrophoresed and are marked at the left of the gel.
  • the number of cal ⁇ culated inhibitory units applied to this gel were: from Figure 16, fraction #58 (189 units) , lane 1; #59- 65 (2,068 units), lane 2; #66-68 (46 units), lane 3; #56 (0 units), lane 4; active fraction of undissected human umbilical cord following chromatography on a ⁇ BONDAPAK* CN column as described in Figure 18 chromatogram, (408 units), lane 5; inactive fractions from same stromal/vascular preparation, lane 6; plate- let-derived TGF- ⁇ purified by Bruce Magun (256 units, approximately 0.4 ng) , lane 7.
  • Figure 18 shows reverse phase high pressure liquid chromatography (HPLC) ( ⁇ BONDAPAK* CN) .
  • Active frac- tions from a previous HPLC procedure of undissected umbilical cord (similar to Figures 14A & 14B) , which eluted at 27% acetonitrile (Pool I) from a ⁇ BONDAPAK* C18 column were pooled, lyophilized to 1.0 ml volume in a siliconized glass tube (16 x 100 mm) and diluted to a final concentration of 0.1% trifluoracetic acid (TFA) and 20% 2-propanol.
  • TPA trifluoracetic acid
  • the sample was sonicated for 2 minutes and injected onto a ⁇ BONDAPAK* CN column (0.39 x 30cm) at 1 ml per minute. Elution of the protein was achieved by increasing the concentration of 2-propanol containing 0.1% TFA from 20% to 35% in 5 minutes fol ⁇ lowed by 35% to 50% in 50 minutes (0.375% per minute), and 50% to 100% in 5 minutes.
  • the solvent gradient is shown as short dashes.
  • An aliquot of 10 microliters was removed from each 1.0 ml sample to test for biolog- ical activity (closed circles) .
  • the equipment used for RPHPLC is as described in Figure 14.
  • the active frac ⁇ tions eluted between 39 to 43% with the peak of activi ⁇ ty eluting at 40-41%.
  • the number of calculated inhibi ⁇ tory units applied to the column was 37,000. Protein concentrations could not be determined. Absorbance at 210 nm is shown by the solid line.
  • FIG 19 shows Reverse Phase High Pressure Liquid Chromatography (HPLC) ( ⁇ BONDAPAK* CN) .
  • Active fractions from a previous HPLC procedure (the same chromatograph- ic run as Figure 18 was derived) which eluted at 28-30% acetonitrile (Pool II) from a C18 resin pooled and applied to a ⁇ BONDAPAK* CN column as described in Figure 18.
  • Gradient elution and equipment are as described for Figure 18.
  • Aliquots of 100 microliters were re ⁇ moved • from every tube to test biological activity (closed circles) .
  • Biological .activity eluted from 44% to 46% with the peak of activity at 44%.
  • the number of growth inhibitory units applied to the column was 21,0 ⁇ K>. Protein concentration could not be deter ⁇ mined.
  • Figure 20 shows the Reverse Phase High Liquid Chroma ⁇ tography (HPLC) ( ⁇ BONDAPAK* CN) .
  • HPLC Reverse Phase High Liquid Chroma ⁇ tography
  • Figure 19 Figure 19
  • Pool I eluted at 40-41% and Pool II at 44%.
  • Figure 21 shows the Reverse Phase HPLC of A431 Condi ⁇ tioned Media. Lyophilized conditioned media from 4 x g
  • A431 cells (110 ml) was processed, as described in the text, for the effect of DTT on tumor growth inhibi ⁇ tory activity derived from tumor cell conditioned media.
  • Figure 22 shows the Reverse-phase HPLC of A431 Condi ⁇ tioned Media Treated with DTT.
  • Lyophilized conditioned g media from 4 x 10 A431 cells 110 ml was processed, as previously described, for the effect of DTT on tumor growth inhibitory activity in tumor cell conditioned media.
  • Lyophilized conditioned media from A431 cells was resuspended in 5 ml 4 mM HCl and centrifuged to remove insoluble material (RC5B-Sorvall* SA 600 rotor) for 15 minutes at 3,500 RPM at 4 ⁇ C. The supernatant was transferred to 1.5 ml microfuge tubes and centri ⁇ fuged in an Eppendorf* microfuge for 15 min. at 4°C.
  • Protein concentration was determined by absorbance at 280 nm. An aliquot of 0.2 ml containing 680 micrograms protein was added to 1.8 ml 0.1 M ammonium bicarbonate containing a final concentration of 65 mM DTT. The samples were incubated at room temperature for 2 h. , lyophilized, and resuspended in 2 ml of 0.05% tri ⁇ fluoroacetic acid.
  • the material (1.0 ml) was injected onto a reverse phase semipreparative ⁇ BONDAPAK* C18 column, and 2.0 ml fractions were collected at the start of a linear gradient and assayed for growth in ⁇ hibitory activity against mink lung cell line (CCL 64) ( —o—0— ) and human tumor cell line (A549) ( —D— D— ) as previously described. Absorbance at 206 nm is indicated by the solid line.
  • FIG 23 in a schematic representation of trpE: :TGF- ⁇ l plasmid constructs using pATH 11 and pKS-1 expres- sion vectors for the production of TGF-,51 polypeptide in bacteria.
  • Figure 24 shows a Southern blot analysis of human tumor DNAs hybridized with a Pvu II-Pvu II TGF- ⁇ l cDNA probe.
  • SCC - is standard saline-citrate buffer, which consists of : 0.15M sodium chloride and 0.15M sodium citrate. (pH 7.0) .
  • Figure 25 shows a restriction map of the phage sub- clone that hybridized to the TGF- ⁇ l cDNA probe at high stringency wash.
  • the clone corresponds to TGF- ⁇ l genomic locus.
  • the Sal I-Sal I fragment of the phage clone was subcloned into pUC. Abreviations for re ⁇ striction enzyme sites are: S-Sal I; K-Kpnl; E-Eco RI; H-Hind III; B-Bam HI; Bg-Bgl II.
  • Figure 26 shows a restriction map of the phage sub- clone that hybridized to the TGF- ⁇ l cDNA probe only under conditions of low stringency.
  • Figure 27 shows a comparison of the nucleotide sequence and the predicted amino acid sequence of TGF-ySl and the related gene encoding the protein with tumor inhibitory activity. Indentical amino acids are boxed. (A) cor ⁇ responds to the gene encoding the protein having tumor growth inhibitory activity.
  • Figure 28 shows a restriction map of the Bam HI frag ⁇ ment of the related gene encoding the protein having tumor growth inhibitory activity subcloned into pUC.
  • the position of the repeat free fragment (BamHI-TaqI) is indicated with a bar.
  • Figure 29 shows a partial nucleotide sequence of the 1.7 kb cDNA encoding the protein having tumor growth inhibitory activity and its corresponding amino acid sequence.
  • Figure 30 shows the restriction map of the 1.7 kb Eco RI subclone of the TGF- ⁇ l related gene encoding the protein having tumor growth inhibitory activity.
  • Figure 31 shows a nucleotide and predicted amino acid sequence comparison of the gene encoding the protein having tumor growth inhibitory activity with TGF- ⁇ l and TGF- ⁇ 2.
  • (A) corresponds to the gene encoding the pro ⁇ tein having tumor growth inhibitory activity.
  • Figure 32 shows a Northern blot analysis of A673, A549, and A498 cell lines using an Eco RI-Bgl II 1.7 kb cDNA fragment of the gene encoding the protein having tumor growth inhibitory activity as a probe.
  • Figure 33 shows a Northern blot analysis of A673, A549, and A498 cell lines using a Pvu II-Taq I probe from genomic sequences of the gene related to TGF- ⁇ l and encoding the protein having tumor inhibitory activity.
  • Figure 34 shows a Northern blot analysis of A673, A549, and A498 cell lines using a Pst I-Bal I TGF- / 31 probe.
  • Figure 35 shows a Northern blot analysis of A673, A549 and A498 cell lines using TGF- ⁇ l cDNA containing the complete coding sequence of TGF- ⁇ l precursor as a probe.
  • Figure 36 shows a Northern blot analysis of mRNA from umbilical cord and A673 cell line using an Eco RI - Bgl II cDNA fragment of the gene encoding the protein hav ⁇ ing tumor growth inhibitory activity as a probe.
  • Figure 37 shows the production of trpE: :protein having tumor growth inhibitory activity fusion protein of three lysates by SDS polyacrylamide gel electrophore ⁇ sis.
  • (A) corresponds to the gene encoding the protein having tumor growth inhibitory activity.
  • Figure 38 shows a Western blot analysis of an antibody recognizing a fusion protein of the protein having tumor growth inhibitory activity.
  • (A) represents the polypeptide sequences corresponding to the last 150 amino acids of the protein having tumor growth inhibi ⁇ tory activity.
  • Figure 39 shows whole cell bacterial lysates containing trpE: :TGF-j1 fusion proteins (lanes 1 and 4), trpE: : (A) fusion proteins (lanes 2 and 5) , and the TGF- ⁇ l protein (purchased from R & D Systems) (lanes 3 and 6) were separated on a 12.5% SDS-polyacrylamide gel.
  • the pro ⁇ teins were electrophoretically transferred to a nitro ⁇ cellulose filter (1 ⁇ pore size) and incubated with 100 ug of affinity purified anti-peptide antibody either in the absence (lanes 1, 2 and 3) or presence of a 300 fold molar excess of the antigenic peptide (lanes 4, 5, and 6) .
  • the antibodies were detected using alkaline phosphatase conjugated ' to goat anti-rabbit antibody (Promega) according to the manufacturer's instruc ⁇ tions.
  • Figure 40 shows a schematic diagram of mRNA encoding the protein having tumor growth inhibitory activity with the coding sequence boxed.
  • the relative extension of the cDNA inserts obtained from placenta (1.7 kb) , umbilical cord (1.9 kb) and A673 (1.7 kb) libraries is indicated.
  • the dashed part of the box represents the C-terminal region showing high- homology to TGF- ⁇ s .
  • the 5' EcoRI-Bg II restriction fragment of the placenta cDNA is indicated by a bar.
  • Figure 41 shows the nucleotide sequence encoding the protein having tumor growth inhibitory activity and its deduced amino acid sequence. Putative glycosylation sites and polyadenylation signal are underlined. The start of the mature protein is marked by an asterisk at position 1164.
  • Figure 42 shows a comparison of the nucleotide sequence and predicted amino acid sequence of the gene encoding the protein having tumor growth inhibitory activity with TGF-01 and TGF- ⁇ 2. Identical amino acids are boxed. The mature amino acid sequences start at posi ⁇ tion 315. (A) corresponds to the gene encoding the protein having tumor growth inhibitory activity.
  • Figure 43 shows a homolpgy matrix plot between the gene encoding the protein having tumor growth inhibitory activity and TGF- ⁇ l and TGF-,92.
  • Figure 44 is a schematic representation of the construction of the pCMV-TGF- / 33 expression plasmid from pORFX and pBlue-TGF- ⁇ 3 plasmids.
  • Figure 45 shows the level of TGF- / 53 mRNA expression, determined by Northern hybridization using a TGF- ⁇ 3 specific probe, of parental CHO cells (lane 1) , CHO cells transfected with TGF-03 cDNA (CHO 6.35) (lane 2) and CHO 6.35 amplified with 20nM Mtx (CHO 6.35/20nM) (lane 3)
  • Figure 46A shows the dose response of mink cell growth inhibition using purified TGF- ⁇ l .
  • Cell growth was quantitated by the metabolism of MTT (3-[4,5- Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue) (Mossman, T. (1983) J. Immunol. Methods j55, 55-65) .
  • MTT 3-[4,5- Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue
  • Figure 46B shows the dose response of mink cell growth inhibition using acid activation serum free supernatants CHO 6.35/20nM transfectant and CHO 6.35 transfectant. Cell growth was quantitated by the metabolism of MTT.
  • Figure 47 shows the relative location of the various TGF- 3 peptides used as antigens.
  • Figure 48A shows the immunoblot of TGF- 3 from conditioned media of CHO 6.35/20nM transfectant using
  • Figure 48B shows the immunoblot of TGF-03 from conditioned media of CHO 6.35/20nM transfectant using 3111 and 03V antibodies for detection from gels under non-reducing conditions.
  • Figure 49 shows a Western blot of cell extract (49A) and conditioned media (49B) of the CHO 6.35/20nM transfectant using 03V antibody for detection.
  • Figure 50 shows the immunoprecipitation of native recombinant TGF-03 protein by ⁇ V antibody.
  • Figure 51 shows the staining to paraffin sections of human umbilical cord by ⁇ V antibody ( Figure 51 A,C) and control antibody (51B, D) .
  • Figure 52 shows a silver stained gel of purified TGF-03 and TGF- ⁇ l.
  • Figure 53 shows antibody neutralization of TGF-0 3 inhibition of mink cell growth.
  • This invention provides a protein having tumor growth inhibitory activity comprising the 112 amino acids shown in Figure 29 beginning with alanine at position 1 and ending with serine at position 112.
  • this protein may be a purified protein having 112 amino acids beginning with alanine at position 1 and ending with serine at position 112 as shown in Figure 29.
  • This 112 amino acid protein is the mature form of the protein having tumor growth inhibitory activity.
  • a biologically active derivative of a protein having the tumor growth inhibitory activity is also provided, wherein the derivative has substantially the same amino acid sequence shown in Figure 29 beginning with alanine at position 1 and ending with serine at position 112.
  • the protein may also comprise the 412 amino acids shown in Figure 41 beginning with methionine at nucleotide position 263 and ending with serine at nucleotide position 1496.
  • this 412 amino acid sequence contains the complete precursor sequence of the protein having tumor growth inhibitory activity as well as the complete sequence of the mature protein.
  • a biologically active derivative of the protein comprising the 412 amino acids shown in Figure 41 is provided.
  • the biologically active derivative has substantially the same amino acid sequence as shown in Figure 41 beginning with methionine at nucleotide position 263 and ending with serine at nucleotide position 1496.
  • a protein comprising the 411 amino acids shown in Figure 41 beginning with lysine at nucleotide position 266 and ending with serine at nucleotide position 1496.
  • This invention further provides a nucleic acid molecule encoding the protein having tumor growth inhibitory activity comprising the 112 amino acids shown in Fig- ure 29 beginning with alanine at position 1 and ending with serine at position 112.
  • the nucleic acid molecule may encode the entire protein shown in Figure 41 begin ⁇ ning with methionine at nucleotide position 263 and ending with serine at nucleotide position 1496.
  • the nucleic acid molecule may encode only the 112 amino acids found in the functional protein shown in Figure 29 beginning with alanine at position 1 and ending with serine at position 112.
  • nucleic acid molecules may be cDNA, genomic DNA, or mRNA and may also comprise the entire nucleotide sequence set forth in Figure 41 begining with cytosine at position 1 and ending with guanine at position 2529 or only the 112 amino acid sequence of the mature protein shown in Figure 29 beginning with guanine of the codon at position 1 and ending with cytosine of the codon at position 112.
  • amino acids as well as the nucleic acids encod- ing these amino acids may be varied, thus producing biologically active derivatives, e.g. mutants, without changing the function of the protein.
  • This invention encompasses all variations of the amino acid and nucleotide sequence which produce a functional protein.
  • This invention also provides a plasmid which comprises the nucleic acid molecules of this invention as well as a host vector system comprising the plasmid in a suit- able-host cell.
  • This host vector system comprises any plasmid and vector known in the art which are suitable for producing the proteins of this invention.
  • the suitable host cell may be a bacteria cell or a eucaryo- tic cell.
  • This invention further provides a method for producing a protein comprising growing the host vector system of this invention so as to produce the protein having tumor growth inhibitory activity in the host and recov ⁇ ering the protein so produced.
  • the invention provides a polypeptide derived from the protein having tumor growth inhibitory activity.
  • the polypeptide comprises the 20 amino acids shown in Figure 29 beginning with arginine at position 9 and ending with leucine at position 28.
  • the invention further provides an antibody which specifically binds to an epitope contained within the protein having tumor growth inhibitory activity.
  • the antibody may be monoclonal or polyclonal.
  • This invention also provides an antibody which specifi ⁇ cally binds to an epitope contained within the polypeptide comprising the 20 amino acids shown in Figure 29 beginning with arginine at position 9 and ending with leucine at position 28.
  • the antibody may be monoclonal or a polyclonal.
  • a method for diagnosing a tumor comprises contacting a sample from a human subject with an antibody of the invention under suitable conditions so as to form a complex between the antibody and an epitope contained with the protein and detecting the complex so formed, thereby diagnosing a tumor.
  • suitable conditions applicants contemplate any conditions which would be conducive to the formation of a complex which are known in the art.
  • This invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibodies of this invention and a phar ⁇ maceutically acceptable carrier.
  • a pharmaceutically acceptable carrier includes all carriers known in the art. Merely by way of example, the carrier may be sa ⁇ line.
  • This invention further provides a method of treating a tumor which comprises administering to the subject an effective tumor treating amount of the pharmaceutical composition.
  • This invention also provides a method of treating a proliferative type disorder which comprises administering to the subject an effective proliferative type disorder treating amount of the pharmaceutical composition.
  • the composition may be used to treat various types of proliferative type dis ⁇ orders. Examples of proliferative type disorders of which the composition may be effective include arte ⁇ riosclerosis, inflammation, and psoriasis.
  • the protein having tumor growth inhibitory activity may e used in a pharmaceutical composition which comprise an effective amount of the protein having tumor growth inhibitory activity, or a biologically active derivative thereof, together with a suitable phar ⁇ maceutical carrier.
  • Effective amounts may vary among the various tumor growth inhibitors depending on the indication to be treated, the patient or the stage of tumor development, by methods well known to those skilled in the art.
  • suitable carriers such as saline or other aqueous solutions, gels, creams and the#1ike are well known to those skilled in the art.
  • the protein having tumor growth inhibitory activity may be used in a method to inhibit the growth of human tumor cells, e.g., carcinoma, melanoma or leukemia cells, by contacting the cells with an effective tumor growth inhibiting amount of the pharmaceutical composition which includes the protein having tumor growth inhibitory activity.
  • the protein having tumor growth inhibitory activity may also be used in a method to treat burns or to facilitate the healing of wounds by contacting the burn or wound with a pharmaceutical composition which includes an effective amount of the protein having tumor growth inhibitory activity and a suitable pharmaceutical carrier.
  • This invention also provides a method of treating a proliferative type disorder in a subject which compris ⁇ es administering to the subject an effective amount of the composition which includes the protein having tumor growth inhibitory activity comprising the 112 amino acids shown in Figure 29 beginning with alanine at position 1 and ending with serine at position 112 in a suitable pharmaceutical carrier effective to treat the proliferative type disorder.
  • a proliferative type disorder may be treated using the proteins of the invention. Examples of proliferative type disorders include arteriosclerosis, inflammation, and psoriasis.
  • the various proteins of this invention may further be used as an immune modulator.
  • a pharmaceutical composition comprising an effective amount of the protein comprising the 412 amino acids shown in Figure 41, or a biologically active derivative thereof, in a suitable pharmaceutical carrier.
  • the pharmaceutical composition may be used in a method of inhibiting the growth of human tumor cells. The method comprises contacting the cells with an effective tumor growth- inhibiting amount of the pharmaceutical composition.
  • a method of treating a proliferative type disorder with the pharmaceutical composition comprising the 412 amino acid protein comprises administrating to a subject an amount of the pharmaceutical composition effective to treat the proliferative disorder.
  • the pharmaceutical composition comprising the 412 amino acid protein may also be used in a method for treating a burn or healing a wound.
  • the method comprises contacting the burn or wound with an effective amount of the pharmaceutical composition.
  • a method for detecting the presence of a tumor is dis- closed.
  • the method comprises quantitatively determin ⁇ ing the amount of the protein having tumor growth inhbitory activity present in a sample, e.g., blood, amniotic fluid, peritoneal fluid, ascites fluid, cere- brospinal fluid or urine, from a subject and comparing the amount so determined with the amount present in a sample from a normal subject, the presence of a sig ⁇ nificantly different amount, e.g. a significantly higher amount, indicating the presence of a tumor.
  • a sample e.g., blood, amniotic fluid, peritoneal fluid, ascites fluid, cere- brospinal fluid or urine
  • the method comprises separately quantita ⁇ tively determining both the amount of the protein having tumor growth inhibitory activity and of transforming growth factor alpha (TGF-alpha) present in a sample from a subject, determining the ratio of the amount of the protein having tumor growth inhibitory activity present in the sample to the amount of TGF- alpha present in the sample from a subject, determining the ratio of the amount of the protein having tumor growth inhibitory activity present in the sample, determining the comparable ratio for a sample from a normal subject and comparing the ratio for the sample from the subject to the ratio for the sample from the normal subject, a significant variation in the ratio indicating the presence of a tumor.
  • TGF-alpha transforming growth factor alpha
  • a method for typing tumors comprises quantitatively determining for a sample from a subject with a tumor the amount of each of TGI-1, TGI, TGI-2, the protein having tumor growth inhibitory activity, CM-I, or the polypeptide recoverable from conditioned media of A431 cells present in the sample, the presence of specific amounts or relative amounts thereof, e.g, a significant increase in the amount of TGI or a signif ⁇ icant variation in a ratio such as the ratio of TGI-1 to CM-I, being indicative of a specific tumor type.
  • This invention further provides a method of inhibiting the activity, for example, immunosuppressive activity, of the protein having tumor growth inhibitory activity, or any biologically active fragment thereof, which comprises contacting the cells with an effective amount of the antibody which specifically binds to an epitope contained with the protein having tumor growth inhibitory activity comprising the 112 amino acids shown ' in figure 29 beginning with alanine at position 1 and ending with serine at position 112.
  • a method of inhibiting the activity for example immunosuppressor activity, of the protein having tumor growth inhibitory activity, or any biologically active fragment thereof.
  • the method comprises contacting the cells with an effective amount of the antibody which specifically binds to an epitope contained within the 20 amino acid polypeptide shown in Figure 29 beginning with arginine at position 9 and ending with leucine at position 28.
  • This invention further provides a method of producing TGF- B3 which comprises:
  • a eucaryotic host cell preferably a mammalian host cell.
  • a CHO cell e.g. a DHFR " CHO cell, is particularly preferred.
  • the preferred promoter is an inducible promoter, e.g. a promoter associated with the dhfr gene. - 37B -
  • a activating agent such as an acid
  • Those skilled in the art will readily appreciate the types of acids which may be so employed.
  • Each series of experiments comprises a means of isolating proteins exhibiting tumor growth inhibitory activity.
  • TGI tumor growth in ⁇ hibitory activity.
  • TGI-1 tumor growth in ⁇ hibitory activity.
  • TGI-2 discrete pro ⁇ teins are purified that demonstrated tumor growth in ⁇ hibitory activity.
  • CM I-IV CM I-IV.
  • the second and third series of experiments are improvements of the purification process resulting in more purified proteins demonstrat ⁇ ing tumor growth inhibitory activity.
  • TGF-01 was cloned and used to isolate a related gene encoding a protein having tumor growth inhibitory activity.
  • TGIs Tumor Growth Inhibitors
  • the Jsuffer for extraction consisted of 375 ml of 95% (v/v) ethanol (punctilious, 190 proof, U.S. Industrial Chemicals, #UN1170) , 7.5 ml of concentrated HCl, 33 mg of phenylmethylsulfonyl fluoride (PMSF) (Sigma P-7627) and 1 ml of Aprotinin (Sigma A6012 with 19.8 Trypsin inhibitor units per ml in 0.9% NaCl and 0.9% benzyl alcohol) mixed with 192 ml of distilled water at 4 ⁇ C.
  • PMSF phenylmethylsulfonyl fluoride
  • Aprotinin Sigma A6012 with 19.8 Trypsin inhibitor units per ml in 0.9% NaCl and 0.9% benzyl alcohol
  • natant changed from brown to an orange solution.
  • the supernatant was trans ⁇ ferred to large 6 liter flasks to which four volumes of anhydrous ether (-20 C) (Baker 9244-3) and two vol ⁇ umes of 95% ethanol (4 * C) were added.
  • the mixture was allowed to stand undisturbed at -22°C for 48 hours to allow the resulting precipitate to settle.
  • the etherized material was brought to ambient temperature in a fume- hood. Warming of the acidified, ethanol extract to ambient temperature enhances the aggregation of the precipitate. The clear organic phase of ether and ethanol was removed by a water aspirator and the pre ⁇ cipitate was left in the fume hood for several hours to allow the residual organic phase to evaporate.
  • the "dried" precipitate was dissolved in 1.0 M acetic acid and dialyzed extensively against 1.0 M acetic acid (Baker #9507-5) using dialysis membranes with a molecu ⁇ lar cutoff of 3500 (Spectropor 3, Spectrum Medical Industries, Los Angeles, CA) . The dialyzed acidified ethanol extract was lyophilized in 250 ml Corning coni ⁇ cal centrifuge tubes (Corning 25350) and stored as crude acidified, ethanol extract.
  • the column (14 x 100 cm) (Amicon; #86012) contained 13.8 liters of equilibrated and degassed Bio-Gel* P10 in 1.0 M acetic acid at either 23°C or 4'C.
  • the void volume was determined by the addition of 50 ml of blue dextran (Sigma #D5751) at 2 mg/ml in 1.0 M acetic acid.
  • the column was "conditioned" with 100 ml of bovine serum albumin (Sigma #A-4503) at 100 mg/ml in 1.0 M acetic acid followed by extensive wash ⁇ ing with 1.0 M acetic acid.
  • TGI activity from the Bio-Gel* P-10 column were lyophilized and resuspended in 1 to 10 ml of 0.05% trifluoracetic acid (TFA) (Pierce #28901) depending upon the amount of protein in each fraction.
  • TFA trifluoracetic acid
  • Water used for HPLC was generated using a Milli-Q water purification system. Starting buffer in all HPLC chromatography runs consisted of Milli-Q water containing 0.05% TFA. Prior to injection, the sample was centrifuged in a Beckman tabletop centrifuge (Beckman TJ-6) at 3000 rpm for 20 min to remove insol ⁇ uble material.
  • the supernatant was injected into ei ⁇ ther a Waters uBondapak* analytical C lg column (0.39 x 30 cm) (Waters PN27324) or semipreparative column (0.78 x 30 cm) (Waters PN84176) as specified in individual experiments.
  • a Waters automated gradient controller (Waters Model 510) was utilized for column elution monitored by a variable wavelength u.v. detectors (Wa ⁇ ters Lambda-Max, Model 481) set at 206 nm.
  • the solvent used for elution was either acetonitrile (Baker 9017-3) or 2-propanol (Fisher, A452) containing 0.05% TFA.
  • the resins as aqueous suspensions, were suspended in an equal volume of 0.1 M ammonium acetate, pH 4.0, con- taining 1.0 M NaCl.
  • the resin was allowed to equili ⁇ brate for at least 3 hours and was degassed at 4"C. Twenty ml of resin was packed into a 1.6 x 20 cm column (Pharmacia; #19-0362-01) and washed with 2 column vol ⁇ umes of 1.0 ammonium acetate, pH 4.0, followed by 0.01 M ammonium acetate, pH 4.0. The column was washed until the effluent exactly matched the conductivity of the equilibrating buffer (i.e., 0.01M ammonium acetate.
  • the sample was applied to the resin (1 gm/20 ml resin) at a flow rate of 1 ml/min, the column was washed with equilibration buffer until the optical density leveled (e.g., approaching zero optical density) and 200 ml of an ascending molarity linear gradient (Pharmacia gradient mixer GM-1, #19- 0495-01) was applied through a column flow adaptor of - 44 -
  • CM- and SP- TRISACRYL* chromatography The chro atographic resin preparation and procedure was performed exactly as described for CM- and SP- TRISACRYL* chromatography, except the equilibration buffer used was 0.1 M ammonium acetate, pH 6.0, the gradient elution ranged from 0.1 M to 1.0 M ammonium acetate, pH 6.0, and the sample was equilibrated in the above mentioned equilibration buffer.
  • Test cells were sub-cultured on 96-well tissue culture plates (Nunc 167008) in 50 ul of Dulbecco's modified Eagle's medium (Whittaker M.A. Bioproducts 12-6143) containing 10% fetal bovine serum (Whittaker M.A. Bio- products 14-501B) , 2% L-glutamine (Whittaker M.A. Bio ⁇ products 17-605-A) , 1% penicillin and 1% streptomycin.
  • Human lung carcinoma cells, A549, and normal human fibroblasts (HuF) required a seeding density of 5 x 10 cells per well.
  • Mink cells (ATCC: CCL 64) required - 45 -
  • 125 IUdR was collected using the Titertek Supernatant 5 Collection System* (Skatron Inc. , 7072) .
  • the amount of cell growth is approximated by the extent of 125 IUdR incorporated into the DNA of cells in the log phase of growth. Before the assay was harvested each well was observed using a Zeiss* inverted microscope to visually 0 note the amount of cell growth. Inhibition or stimula- tion of growth was expressed as a ratio of 125IUdR incorporated by test cells (e.g. human tumor cells) containing the test aliquots relative to 125IUdR incor ⁇ porated by the untreated control cells. The inhibition 5 - 46 -
  • the molecular weight calculations are based on the elution profiles of molecular weight standards (i.e., carbonic anhydrase - 29,000; RNase - 14,400; insulin - 6,000) chromatographed on 1 liter of resin in a column of 4 x 100 cm.
  • molecular weight standards i.e., carbonic anhydrase - 29,000; RNase - 14,400; insulin - 6,000
  • Extracts from human placental material showed a greater overlap of TGI with the major protein peaks than was observed with material from human umbilical cords (data not shown) .
  • Aliquots of identical volumes from gel filtration chromatography electrophoresed by SDS-PAGE on a 5-20% polyacrylamide gradient also il ⁇ lustrated that by fraction 4, considerably less protein is found than in fractions 1 to 3.
  • fractions 5 an 6 major protein bands of 5,600 and 14,000 band are observed and by fraction 7 very little protein remains, although inhibitory activity extends into fraction 10 as shown in Figure 2.
  • the obvious advantage of the majority of activity eluting in regions of less protein is that it facilitates further purification of TGIs.
  • Mink lung (CC1 64) Undiluted 91 43 1/5 90 13 1/25 70 9 1/125 31 2
  • Figure 4 illustrates an elution profile of the tumor growth inhibitory activity obtained by HPLC using a linear acetonitrile gradient of lyophilized fraction 4 (19.8 mg/3ml 0.05% triflur- oacetic acid) derived from the Bio-Gel* P-10 chromato- graphic step.
  • the first peak of tumor growth inhibitory activity was designated TGI-1 and the second TGI-2.
  • Figure 5 demon ⁇ strates the elution profile and tumor growth inhibitory activity of TGI-1 (Figure 4).
  • the concentration of injected material was 1.5 mg/1.5 ml of 0.05% trifluro- acetic acid (TFA) .
  • TGI-1 activity elutes between 17- 23% using a linear gradient of 2-propanol ( Figure 5) .
  • Figure 6 indicates that TGI-2 (0.8 mg/1.8 ml 0.05% TFA) rechromatographed between 23-27% (fraction 17-23) using a linear gradient of 2-propanol.
  • the tumor growth inhibitory activity presented in Figures 4 and 5 are consistently 20% higher against the mink cells than against the A549 human carcinoma cells.
  • Acid ethanol extracts of human placenta contained TGI activities which, following a gel filtration chromato- graphic step, also eluted between 26-34% acetonitrile - 52 -
  • CM-TRISACRYL* 0.01 M ammonium acetate, pH 4.0.
  • a linear gradient was applied from 0.01 to 1.0 M ammonium acetate, pH 4.0.
  • Figure 7 demonstrates at least 4 separate tumor growth inhibitory activities designated CM-I, CM-II, CM-II, and CM-IV.
  • CM-I was presently inhibited only the A549 human carcinoma cells at 60% inhibition (Table 2) .
  • CM peaks II and III have similar levels of growth inhibiting activity against both A549 human carcinoma (80 and 63%, respectively) and mink cells (61 and 76%, respectively) .
  • the last peak of activity (CM-IV) demonstrates a specificity in activi ⁇ ty against mink (i.e. mink cells were more inhibited (95%) than were the A549 human carcinoma cells (69%)).
  • CM-I was not retained and CM-II was slightly retarded by the negatively charged resin since they both were eluted before the gradient was started by 0.01 M ammo ⁇ nium acetate, pH 4.0.
  • CM-III and IV are probably slightly more basic since they bind more tightly to the CM-TRISACRYL* resin (eluting at greater than 0.5M ammonium acetate). This is substanti ⁇ ated by the fact that no TGI activity was retained by a - 53 -
  • Protein concentrations for the fractions tested for TGI activity ranged from 15-300 ⁇ g.
  • CM-III and CM-IV eluted (greater than 0.5 M ammonium acetate) in exactly the same position as did the original column fractions from which they were derived ( Figure 10) .
  • the higher tumor growth inhibito ⁇ ry inhibitory activity against mink cells was preserved and the difference between the inhibitory activity against the two cell lines remained exactly the same at 25-30% around the peak of activity.
  • TGIs tissue derived tumor cell growth inhibitory activity
  • Protein concentrations for the fractions tested from TGI activity ranged from 15-300 ⁇ g.
  • TGIs Tumor Growth Inhibitors
  • Veins and arteries were removed from human umbilical cord tissues and the remaining tissues were extensively washed to remove blood prior to acid/ethanol extraction as described under First Series of Experiments.
  • the buffer for washing and homogenizing the tissue consisted of 2 liters of water containing 16 gm NaCl, 2.5 gm Na 2 HP0 4 .H 2 0, 0.4 gm NaH 2 P0 4 * 7H 2 0, 116 mg phenylmethylsulfonyl fluoride (PMSF) (Sigma P7627) and 3.3 ml Aprotinin (Sigma A6012 with 19.8 units Try- psin inhibitor per ml in 0.9% NaCl and 0.9% benzyl alcohol), adjusted to pH 7.4 with HCl and NaOH.
  • the extraction buffer consisted of 375 ml of 95% (v/v) ethanol (punctilious, 190 proof, U.S.
  • The._iissue was placed in a 4°C chilled Cuisinart food processor (Model DLC-7-PR0) and suspended in 200 ml of 4'C PBS-PA.
  • the suspended tissue was homogenized by the food processor. After the first minute of homoge ⁇ nization, an additional 200 ml of 4'C PBS-PA was added.
  • the tissue suspension was homogenized for a total of 10 min. at 4'C.
  • the pellet was washed by repeated centrifugation and resuspension as described until the supernatant fluid was clear with no tint of red from contaminating blood or blood products.
  • the resulting washed pellet was white.
  • the washed pellet was resuspended in the buff ⁇ er for extraction to a final volume of 6 ml per gram of original dissected tissue.
  • the homogenate was trans ⁇ ferred to a large 4 liter beaker with a 3 inch stir bar and stirred at half of the maximum stirring capacity of a LAB-line Multimagnestir* multimixer. Model #1278.
  • the material was brought to ambient temperature in a fumehood. Warming of the acidified, ethanol extract to ambient tempera ⁇ ture enhances the aggregation of the precipitate.
  • the clear organic phase of ether and ethanol was removed by a water aspirator and the precipitate remained in the fume hood for several hours to allow the residual or ⁇ ganic phase to evaporate.
  • a gentle stream of dried nitrogen gas over the extract accelerated the evapora ⁇ tion of the remaining organic solvent present with the precipitate.
  • the "dried" precipitate was dissolved in 1.0 M acetic acid and dialyzed extensively against 1.0 M acetic acid (Baker #9507-5) using dialysis membranes with a molecular weight cutoff of 3500 (Spectropor 3*, Spectrum Medical Industries, Los Angeles, CA) .
  • the dialyzed acidified extract was lyophilized in 250 ml Corning conical centrifuge tubes (Corning 25350) and stored as crude acidified, ethanol extract or dialyzed extensively against 20 mM NH.O-C-H-, pH 4.5.
  • the modified procedure yielded 9700 units of tumor growth inhibitory activity per gram of umbilical cord (wet weight) .
  • the overall efficiency of extrac ⁇ tion was improved 5-fold by the modified procedure.
  • the volumes of ether and ethanol required to precipitate the extracted proteins are less.
  • the amounts of protein and the numbers of different proteins extracted by the modified proce ⁇ dure are fewer and therefore the subsequent purifica ⁇ tion procedures to be employed will require less chro- - 61 -
  • FIG. 10 shows that following adjustment of the pH to 8.0 as described in the figure legend and application of the extract (an identical amount to that used in Figure 9) to the anion exchange - 62 -
  • CM-TRISACRYL* Large amounts of sample can be reproducibly fractionat ⁇ ed by CM-TRISACRYL*, thus furnishing more TGI for sub ⁇ sequent purification procedures.
  • Figure 11 9.9 mg of tissue extract were applied to a CM-TRISACRYL* col ⁇ umn (15 ml) under the same chromatographic conditions as shown in Figure 10 for a smaller sample size (2.65 mg protein) on a smaller CM-TRISACRYL* column (5 ml) .
  • Resolution of tumor growth inhibitory activity from the majority of proteinaceous material by a linear gradi ⁇ ent of NaCl was essentially the same in both experi ⁇ ments.
  • Figure 12 shows fractionation of pooled samples from a CM-TRISACRYL column by HPLC on a ⁇ BONDAPAK* C18 column. Following application of the sample, no significant inhibitory activity was observed by linearly increasing acetonitrile concentrations from 0-25%. However, tu ⁇ mor growth inhibitory activity against both A549 (human - 63 -
  • lung-carcinoma and CCL 64 (mink lung, 0-0) eluted in a single peak between 28-34% acetonitrile (fractions 21- 31) while the majority of material absorbing at 206 nm was eluted at lower (fractions 11-19) and at higher (fractions 37-50) acetonitrile concentrations.
  • TGI-1 and CM-III and CM-IV in the initial procedure were deter ⁇ mined by gel filtration chromatography (Sephadex G-50, data not shown) using suitable protein standards of known molecular weights.
  • cer ⁇ tain interfering proteins e.g., hemoglobin
  • the appar ⁇ ent molecular weight of TGI has been determined to be between 20 kDa and 30 kDa under nondenaturing condi ⁇ tions.
  • the modified procedure detailed herein describes a powerful and simple procedure for removing inert or interfering compounds from the TGI extracts prepared as described in the initial procedure. Furthermore, the modified procedure improves the efficacy of the various chromatographic steps employed in the isolation of TGI by reducing the amount of chromatographic mate ⁇ rials required thus reducing the preparation time of TGI. In addition, and as shown, extraction of TGI from the umbilical cord as described herein allows TGI and other proteins to chromatograph more reproducibly than in the procedure previously described.
  • TGI isolated according to the modified procedure has been characterized with respect to the chromatographic features on both reverse phase high performance liquid chromatography and CM-TRISACRYL* ion exchange chroma ⁇ tography. TGI has been found to behave similarly to or identically with TGI-l (compare Figures 5 and 12) by - 64 -
  • TGI as isolated in the modified procedure and TGI-1 and CM-III and CM-IV are similar or identical compounds having similar or identical ionic and hydro ⁇ phobic properties and thus are of similar or identical composition. Therefore, the modified procedure de- scribed herein provides a more efficacious method of obtaining a purer form of TGI for further analysis and characterization.
  • the buffers and equipment used were exactly as de ⁇ scribed in the second series of experiments, for each relevant step in the procedure.
  • Two hundred to four hundred grams (200-400 gr.) of human umbilical cord, either dissected free of vasculature or left intact and chopped into 1/2 inch pieces were washed free of the majority of blood in PBS-PA at 4'C.
  • the cord was drained by gravity through a sieve and transferred to a chilled food processor at 4'C for homogenization in a maximum volume of 200 ml of PBS-PA.
  • the tissue was homogenized for fifteen minutes and washed free of blood by repeated centrifugation in 200 ml plastic bottles at 5,000 rpm using an RC-5B centrifuge
  • the pellet was trans ⁇ ferred to a 2 liter glass beaker and suspended in ex ⁇ traction buffer, as described in the first series of experiments, at a final volume of 3 ml per gram of the original wet weight of tissue and stirred for twenty- four hours at 4°C.
  • the suspension was centrifuged in a 1.0 liter plastic centrifuge bottle using a RC-3B cen ⁇ trifuge (Sorvall) equipped with a H6000A rotor (Sorvall) for 30 minutes at 3,500 rpm.
  • the resulting supernatant was transferred to a 2 liter beaker and the pH adjusted first to 5.0 with concentrated ammonium hydroxide, and then to 5.2 by the addition of 2 M ammo ⁇ nium acetate to a final concentration of 1% of the total volume.
  • the solution retained a clear or very slightly yellow tinted appearance.
  • the supernatant was siphoned from the flask to within 3/4 of an inch above the bottom of the flask containing the flocculent precipitate.
  • the precipitate and remaining ether/ethanol solution was centrifuged in a GSA rotor at 5,000 rpm for 20 minutes in 250 ml plas ⁇ tic conical bottles (Corning #25350) in a Sorvall RC-5B centrifuge. This step in the procedure was designed to decrease the loss of TGI's from the ether/ethanol su- peraatants immediately above the precipitate.
  • the resulting pellet was suspended in 1.0 M acetic acid and the flask containing the ether/ethanol precipitate was also washed with 1.0 M acetic acid to remove any TGI protein remaining on the wall of the flask.
  • the opti ⁇ cal density at 280 nm was between 0.5 and 1.0 and the final volume did not exceed 100 ml for each prepara ⁇ tion.
  • the TGI containing protein solution was dialyzed - 66 -
  • tumor growth inhibitory activi ⁇ ty can also be obtained from acidified ethanol extrac ⁇ tion of the tissue with omission of the ether-ethanol precipitation step.
  • specific activity of these preparations is 50% less and the total yield of activity 10-30% less than "standard" preparation uti ⁇ lizing the ether-ethanol precipitation.
  • the dialyzed protein was subjected to hydrophobic in ⁇ teraction chromatography using phenyl-Sepharose* (Phar ⁇ macia) as the chromatographic resin.
  • the phenyl-Sepha ⁇ rose* was equilibrated with 4.0 M ammonium acetate, pH 4.5.
  • dialysis at least 24 to 48 hours
  • the conductivity and pH of the protein solution was mea ⁇ sured and dialysis terminated when the conductivity of the dialysate and equilibration buffer were the same.
  • the protein was pumped onto (Microperplex* pump #2132 - LKB) the resin contained in a 1.6 x 2.0 cm chromatogra- phy column (K-20-Pharmacia) using 1 ml of resin per 2.0 mg of protein, at 1.0 ml per minute.
  • the column was washed until the OD 280 was zero and tumor growth inhibitory activity eluted from the column using a decreasing gradient from 4.0 M to 0.04 M ammonium ace- tate, pH 4.5 containing an increasing concentration of ethylene glycol (Mallinkrodt) from 0 to 50%.
  • the total volume of the eluting gradient was 10 times the total volume of the resin used for each individual prepara ⁇ tion.
  • the bound protein was eluted over approximately - 67 -
  • the tumor growth inhibitory activity began eluting from the column at 1.5 M ammoni ⁇ um acetate, 31% ethylene glycol and was completely eluted from the column by 40 mM ammonium acetate and 50% ethylene glycol.
  • the biologically active fractions were pooled, dialyzed against 0.1 M acetic acid, and lyophilized in a polypropylene 50 ml tube (Scientific Products #C2390-50) or siliconized glass lyophilization flask (Virtis) .
  • the lyophilized biologically active material was dilut ⁇ ed in 1.0 to 3.0 ml of 0.05% trifluoracetic acid (TFA) containing 10% acetonitrile, placed in a 16 x 100 mm siliconized disposable glass tube, sonicated for two minutes, centrifuged at 3,000 rpm for 10 minutes (Beck ⁇ man Model TJ-6) to remove insoluble material, and sub ⁇ jected to reverse phase, high pressure liquid chro ⁇ matography (RPHPLC) using a ⁇ Bondapak* C18 resin (Waters Assoc. 0.39 x 30 cm, PN 27324).
  • TFA trifluoracetic acid
  • Tumor growth inhibitory activity eluted initially at 27% acetonitrile and continued to elute at 28-30% ace ⁇ tonitrile as shown in Figure #14A & 14B.
  • the biologically active fractions were pooled and subsequently assayed for total tumor growth inhibitory activity by removing an aliquot and multiplying the activity obtained in the assay by the appropriate dilution factor.
  • the quantity of tumor growth inhibitory activity present in the pool was compared to an aliquot of starting material. Thus, column recoveries of activity and protein (where mea ⁇ surable) could be obtained.
  • Lane 1 contains a purified preparation of TGF- ⁇ derived from platelets (provided by Bruce Magun, Oregon State Health Science University, Portland, Oregon) .
  • the biological activity that has been ascribed to this protein is the ability to confer anchorage independent growth to normal rat kidney cells (NRK) in a soft agar assay, only in the presence of a growth factor, such as EGF at 2.0-2.5 ng/ l.
  • NRK normal rat kidney cells
  • EGF EGF at 2.0-2.5 ng/ l.
  • its growth promoting ac ⁇ tivity is directly dependent on other bioactive pro ⁇ teins (Roberts et al., Cold Spring Harbor Conf. Cell Proliferation, 9_: 319-332 (1982)); Anzano et al., Anal. Bioche . 125: 217-224 (1982) ; Cancer Research 42: 4776-4778 (1982).
  • TGF- ⁇ was shown to posses 1-30 units of inhibitory activity per ng of protein.
  • TGF-0 Figure 15B (lane 1 also changed its migratory position to 13 kDa following reduction.
  • TGI biologically active fractions derived from the RPHPLC C18 chromatographic procedure was accomplished by RPHPLC using a CN ⁇ BONDAPAK* column (0.39 x 30 cm Waters PN 84042) ( Figure #16) .
  • the biologically active fractions were lyophi ⁇ lized in 16 x 100 mm siliconized glass tubes, dissolved in 1.0-3.0 ml 0.05% TFA containing 10% propanol and applied to the column. Column elution was achieved at 1.0 ml/minute by using a linear gradient of 2-propanol from 10 to 20% in ten minutes, followed by 20 to 50% in fifteen minutes (0.6% /min.), and finally from 50-100% in 20 minutes.
  • Active, lyophilized fractions 56, 58, 59-65, 66-68, illustrated in Figure #18, and approximately 4 ng of TGF-0 were iodinated by the chloramine T method (McConahey, P.J. and Dixon, F.J. (1966) Int. Arch, of Allergy 2_9, 185-189) .
  • Each fraction was resuspended in 100 microliters of 0.1 M acetic acid, and 3 microliters of 1.5 M Tris, pH 8.8 was added to adjust the pH to 7.0.
  • Ten microcuries of carrier-free sodium iodide I Na was added, followed by 2 microliters of chlora ⁇ mine T (Sigman #C9887) at 1.0 mg/ml.
  • the -remainder of the sample was diluted in 0.05 ml of twice concentrated sample buffer and approximately 200,000 TCA precipitable radioactive counts were ap ⁇ plied to individual lanes for SDS-PAGE, Figure #17) .
  • the gel was stained with 0.125% Coomassie Blue for 10 minutes to fix the protein in the gel, and exhaustively destained to remove free radioactive iodine.
  • the destain solution did not contain detectable label as judged by counting 1.0 ml of destain solution in a gamma counter (Beckman, Riaga ma #1294), the gel was dried using a gel dryer (Hoeffer-SE1150) and exposed to x-ray film (Kodak-XAR) for autoradiography (one week) .
  • Lane 1 In lanes 1, 2, 3 and 5 containing approximately 180, 2,000, 46 and 408 units of tumor growth inhibitory activity respectively, the M 25 kDa band was observed while lanes 4 and 6, which did not possess tumor growth inhibitory activity, did not contain this protein band. Lane 2, which contained the most active fractions (from Figure 16) , showed two faint bands at M 26 kDa and 30 kDa. Lane 3 appears to have only one band of M 26 kDa.
  • TGI demonstrated at least one log more inhibitory activity than TGF- ⁇ .
  • Figure #18 illustrates the elution profile of the CN column of active fractions pooled at 27% acetonitrile (Pool I) from the previous C18 column.
  • the most active fraction (fraction #14) eluted at 40-41% 2-propanol. A lower amount of activity was seen eluting after this - 73 -
  • Pool I from the C18 column contained 82% more total inhibitory activity than Pool II.
  • Figure #20 is a tracing of the peaks of activity from the two separate chromatographs Figures 20 (Pool I) , and 21 (Pool II). This figure (#20) illustrates two distinct peaks of inhibitory activity as the different active fractions from the C18 column Pool I and Pool II.
  • An erythroleukemia cell line, K562 was grown in sus ⁇ pension to a cell density of 10 cells per ml and one liter of serum-free conditioned media was collected. Cellular debris was removed from the conditioned media (RC-5B GSA rotor-Sorvall) by centrifugation at 800 rpm for 60 min. at 4'C. The supernatant was treated with l ml of 1 M acetic acid per 100 ml of conditioned media, extensively dialyzed in Spectropor 3 dialysis tubing (Spectrum Medical Laboratories) against multiple chang ⁇ es of 1 M acetic acid, and lyophilized.
  • the lyophi ⁇ lized, acid-treated conditioned media was resuspended in 4 mm HCl at a volume of 5.0 ml for A431, A673 and T24, and 1.5 ml for K562 derived media.
  • Insoluble material was removed by centrifugation in a RC-5B cen ⁇ trifuge (Sorvall, SA 600 rotor) at 3400 rpm for 15 min. at 4'C and the supernatants transferred to 1.5 ml microfuge tubes. Following centrifugation in an Eppendorf microfuge for 15 min. at 4'C, the superna ⁇ tants were transferred to 1.5 ml microfuge tubes for - 75 -
  • the effect of DTT on TGI activity derived from conditioned media from the A431, A673, K562 and T24 cell lines using mink cells, CCL 64, and A549 cells as target cells is summarized in Tables 6 and 7, respectively.
  • the table shows the tumor growth inhibitory activity from conditioned media from A673, K562, and T24 against both mink and A549 cells was lost following reduction (Table 6) , whereas the tumor growth inhibitory activity from the conditioned media of A431 cells, which showed preferential inhibitory activity against A549 cell, was only slightly reduced following reduction (First column. Table 7) .
  • the volume of acidified ethanol per gram of tissue used for extraction was 50% less than described for both the initial procedure and modified procedure.
  • the total volume of extracted protein was less, therefore requiring 1/2 the amount of ether and ethanol used for precipitation. This minimized the amount of protein that would remain on the flask walls.
  • the amount of 1.0 M acetic acid necessary to dissolve the precipitate and wash the flask was smaller so that final volumes were kept to a minimum.
  • the obvious advantage is the minimization of protein/activity loss, thereby creating a more efficient method of extraction, including less reagents required. Also, chopping whole cord rather than dissecting cord shortened the tedious preparation time considerably.
  • Table 8 summarizes the current procedure utilized to obtain active TGI from human umbilical cord. Between 60 to 100% recovery of units of activity was observed through the first two steps of purification (HIC and RPHPLC on C18) . This represents a 40,000 increase in specific activity of 1. x 10 units/microgram. (2.3 x 10 total units from 300 g wet umbilical cord) . It was observed that contaminating proteins probably aided in the stabilization of biological activity of TGI, be ⁇ cause as the purification ensued, activity became more labile. The greatest loss of recovery occurred follow ⁇ ing lyophilization of the active fraction obtained after RPHPLC on the C18 column. This greatly reduced the total number of units applied to the CN column in the final step of purification. This loss was amelio ⁇ rated by concentrating the active fractions by lyophi ⁇ lization, but not to completion. The recovery of units from this final step of purification was between 60- 100%.
  • Fraction #58 lane 7 is active but contains at least 5 bands.
  • Fraction #56 which is the major peak of protein and is not biologically active contained all of the protein bands in fraction #58 except that 26 kDa band ( Figure 17, lane 1) . - 82 -
  • TGI demonstrates a similar qualitative activity to an ubiquitous protein derived from platelets and other tissues designated as TGF- ⁇ , in that it migrates by SDS-PAGE as a protein of M 25 kDa as shown in Figures 15B and Figure 17.
  • a purified protein, derived from platelets, designated as TGF- ⁇ , is biologically active in our inhibitory assay but consistently possesses 10-100 fold less ac ⁇ tivity than Pool I. Since activity in all cases. Pool I, Pool II, and TGF-0 is consistent with presence of a protein band of M 26 kDa ( Figures 15 and 17), one can assume that all these proteins may be similar or belong to a family of growth inhibitory and/or growth modulat ⁇ ing proteins. Alternatively, because of the differen ⁇ tial elution of these proteins on both C18 and CN res ⁇ ins, and the greater specific activity of the TGIs, the TGIs may be entirely different than TGF- ⁇ (elevation of TGF- ⁇ profile not shown) .
  • the conditioned media from A431 contained two types of growth inhibitory activity.
  • One TGI elutes at approxi ⁇ mately 25% acetonitrile and inhibits both A549 and CC1 64 mink cells.
  • the selectivity of inhibition of this TGI is similar to what is observed for TGI-1 and TGI-2 in human umbilical cord extracts.
  • the second TGI elut ⁇ ing between 30-36% acetonitrile shows a greater speci ⁇ ficity for inhibiting A549 cells over mink cells.
  • Each family member is a polypeptide dimer, bound by disulfide bonds, with a molecular weight of 26,000 - 84 -
  • TGI-1 and TGI-2 are the same polypeptide which may be distinct from both TGI-1 and TGI-2. TGI-1 and TGI-2 being discrete cannot both be the same as TGF-0 . TGI-1 and TGI-2 each have a specific activity greater than TGF- ⁇ . Both TGI-1 and TGI-2 elute differently from TGF-0 on high pressure liquid chromatography on a CN column with 2-propanol. Further, TGI-1 and TGI-2 elute differently from each other on high pressure liquid chromatography on a CN column with 2-propanol.
  • CM-1 Two separate factors CM-1 and a polypeptide derived from conditioned media of human tumor cell line (A549) are also disclosed. Because both have the property of substantially inhibiting the growth of a human tumor cell line (A549) but hot of an established mink lung cell line (CCL 64) it is contemplated that CM-1 may be the same as the TGI derived from conditioned media from A431 cells. It is also contemplated that CM-1 may be similar to TIF-2 of an earlier patent. - 85 -
  • TGF-01 cDNA The sequence of TGF-01 cDNA is published (Derynck, R. , et al.. Nature, 316, 701-705). Based on this sequence, we synthesized a 25 mer oligonucleotide probe (TGGTGTCCAGGGCTCGGCGGTGCCG) which was used to isolate a TGF-01 cDNA from a commercial lambda-gtll human placen ⁇ ta library (Clonetech*) . For these, and the following experiments, standard molecular biological techniques were employed (e.g., Maniatis, T. , et al. (1982) Molec- ular Cloning, a laboratory manual. Cold Spring Harbor Lab) .
  • the clone was shown to contain the complete coding sequence for the 390 amino acid TGF- ⁇ l precur ⁇ sor but to lack some untranslated sequences from both the 5' and 3' ends (439 bp from the 5' end and approxi ⁇ mately 200 bp from the 3' end).
  • TGF- ⁇ l was expressed in E_-_ coli as trpE::TGF- l fusion proteins using two related in- ducible expression vectors: pATH II (Spindler et al. (1984) J. Virol. 49: 132-141) and pKS-1 (a pATH 11 derivative).
  • the pATH ll::TGF- ⁇ l construct was made by cloning a Bal I-Sal I fragment into the multiple cloning site of pATH 11.
  • the Bal I-Sal I fragment encodes amino acid residues 249-391 of TGF- ⁇ l.
  • the pKS-1: :TGF- ⁇ l construct was made by cloning a Nae I-Sal I fragment into the multiple cloning site of pKSl. - 86 -
  • The. ⁇ Nae I-Sal I fragment encodes the TGF- ⁇ l amino acid residues 25-391 ( Figure 23) .
  • Bacteria E ⁇ coli RRI Bacteria E ⁇ coli RRI containing the expression plasmids were grown overnight into l ml M9 media (for 1 liter: 10 g Na 2 HP0 4 & H 2 0, 3 g KH 2 P0 4 , 0.5 g NaCl, 1 g NH.C1, 5 g casamino acids, 1 ml MgS0 4 , 0.2 ml 0.5 M CaCl 2 , 5 ml 40% glucose, 10 ml 1 mg/ml thiamine B.) supplemented with 50 ⁇ g/ml ampicillin and 20 ng/ml tryptophan.
  • l ml M9 media for 1 liter: 10 g Na 2 HP0 4 & H 2 0, 3 g KH 2 P0 4 , 0.5 g NaCl, 1 g NH.C1, 5 g casamino acids, 1 ml MgS0 4 , 0.2 ml 0.5 M CaCl 2 , 5
  • a half ml of the overnight culture was diluted into 5 ml M9 media supplemented with ampicillin and grown for one hour at 30"C with great aeration.
  • the expression of the protein was induced by adding 12.5 ⁇ l of 2 mg/ml indole acylic acid (IAA) and grown another 2 hours at 30'C.
  • IAA indole acylic acid
  • One ml was centrifuged (su- pernatant is the soluble fraction) and the pellet was resuspended in 100 ⁇ l TEN buffer (50 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 0.3 M NaCl). Then subsequently were add ⁇ ed:
  • the insoluble fraction was collected by spinning 5 minutes in a microfuge.
  • the pellet was washed twice with 100 ⁇ l TEN buffer and finally dissolved in 50 ⁇ l 0.01 M Na phosphate pH 7.2, 1% -mercaptoethanol, 1% SDS, 6 M urea and incubated 30 minutes at 37*C.
  • the strongly hybridizing band is likely TGF-01, and the weakly hybridizing band is a related gene which also encodes a protein having tumor growth inhibitory ac ⁇ tivity.
  • the nucleotide sequence encoding this protein having tumor growth inhibitory activity and its amino acid sequence are shown in Figure 29.
  • the Pvu I-Pvu II probe from the TGF-01 clone was used to screen a human phage library constructed from the DNA of a chronic myelocytic leukemia cell line (K562) .
  • K562 chronic myelocytic leukemia cell line
  • Two genomic loci, which correspond to TGF- ⁇ l and the relat ⁇ ed gene encoding a protein having tumor growth inhibi- tory activity ( Figure 29) were cloned and the pUC subclones of phages were mapped by restriction enzyme analysis ( Figure 25 and 26) . Construction of the K562 library, screening and isolation of recombinant clones was carried out essentially according to the procedures - 88 -
  • the phage DNA clone containing the sequence encoding the protein related to TGF-01 and with tumor growth inhibitory activity was cut with Sau 3A and the re ⁇ striction fragments cloned into M13.
  • the recombinant plaques were screened with the Sma I-Pvu II probe of TGF-01.
  • Six hybridizing genomic clones were sequenced by the method of Sanger, et al. (1977) Proc. Natl. Acad. Sci. 74: 5463-5467, and a region of approximately 130 bp was found to be homologous to TGF-01 cDNA (Fig ⁇ ure 27) . When the amino acid sequence of TGF- ⁇ l and the related gene cloned in these experiments were com ⁇ pared they were found to be 82% homologous.
  • the Bam HI-Taq I unique probe of the sequence encoding the protein having tumor growth inhibitory activity was used to screen the lambda-gtll human placenta cDNA li ⁇ brary (Clonetech*) .
  • Two strongly hybridizing clones, as well as four weakly hybridizing clones, were isolat ⁇ ed.
  • the weakly hybridizing clones were shown to correspond to the TGF-01 ( Figure 29) .
  • One strongly hybridizing clone was isolated and a 1.7kb EcoRI insert was subcloned into pUC 8. The - 89 -
  • a 17 kb genomic DNA fragment containing the sequence of the gene encoding the protein having tumor growth in ⁇ hibitory activity has been cloned (see Figure 26) .
  • Hybridizing 5' and 3' portions of the 1.7 kb cDNA clone which encodes the protein having tumor growth inhibito ⁇ ry activity with the genomic locus of the protein hav ⁇ ing tumor growth inhibitory activity revealed that the 1.7 kb cDNA sequence is completely contained in the genomic clone.
  • additional 5' and 3' flanking sequences may be isolated to obtain the com- plete gene. This is done by screening genomic phage and cosmid libraries with probes unique to the gene encoding the protein having tumor growth inhibitory activity.
  • the sequence R-R (as indicated at position -l and -2 in Figure 31) represents the proteolytic cleav ⁇ age site which generates the mature protein.
  • the sequence R-K-K-R likely represents the corre- sponding cleavage site.
  • TGF- ⁇ l and the related gene encoding the protein having tumor growth inhibitory activity exhibit only 7% homology. Both proteins, however, contain the se ⁇ quence R-G-D-L in this region which may be recognized by the fibronectin receptor.
  • Northern hybridization was car ⁇ ried out using a 5' terminal Eco RI-Bgl II probe (Fig ⁇ ure 32). This revealed a mRNA of approximately 3.5 kb in A673 (a rhabdomyoscarcoma) , A498 (a kidney carcino ⁇ ma) and a faintly hybridizing signal in A549 (a lung adenocarcinoma) .
  • a genomic probe from the 3' region of the related gene encoding the protein having tumor growth inhibitory activity was then used to screen the same Northern blot.
  • Three strong hybridization signals were observed in both A673 and A498, corre ⁇ sponding to TGF-01 (2.5kb), the related protein having tumor growth inhibitory activity (3.5kb) and, another related gene (4.2kb) (Figure 33). These results are consistent with the fact that this probe would be pre ⁇ dicted to cross react with sequences homologous to the protein having tumor growth inhibitory activity. - 91 -
  • Southern blot analysis was performed on a variety of different tumor DNAs digested with Eco RI and hybrid ⁇ ized with a Sma I- Ava I cDNA fragment of the TGF- ⁇ l related gene encoding the protein having tumor growth inhibitory activity as a probe at low (2.5 X SSC, 65'C) and high (0.3 X SSC, 65'C) stringency washes.
  • Southern blot analysis indicates the possible presence of other loci related to the gene encoding the protein having tumor growth inhibitory activity, as the probe hybridizes with two bands (3 kb and 12 kb) which are only observed under conditions of washing at low stringency.
  • a Okayama-Berg cDNA library blot of human fibroblasts was screened with the 5' Eco RI-Bgl II probe of the 1.7 kb cDNA clone of the gene encoding the protein having tumor growth inhibitory activity.
  • a hybridizing band of 3.2 kb is visible at moderate wash stringency 0.3 x SSC, 65'C.
  • Chimeric bacterial proteins have been constructed that contain the C terminal 150 amino acids of the related protein having tumor growth inhibitory activity fused to a small region of the trpE gene. Such a fusion protein was found to be recognized by an antibody that was produced against a peptide derived from amino acid numbers 9 to 28 of the mature form of the protein hav ⁇ ing tumor growth inhibitory activity. The antibody recognizes the trp: :protein having tumor growth in- - 93 -
  • hibi-tory activity fusion protein to a much higher de ⁇ gree than a trp: :TGF-0 fusion protein and the peptide specifically competes with the protein having tumor growth inhibitory activity for the binding of the anti ⁇ body.
  • DNA sequences that code for the the TGF-01 related protein having tumor growth inhibitory activity were cloned into a pKS vector.
  • This vector is a pATH II derivative that contains the inducible trp promoter and a multiple cloning site.
  • the resulting constructs produce a chimeric protein consisting of the first 22 amino acids of the trpE gene, the C-terminal 150 amino acids of the protein having tumor growth inhibitory activity.
  • Transformants containing these clones were screened primarily by restriction endonuclease analysis and ultimately for production of the chimeric protein by SDS polyacrylamide gel electrophoresis.
  • the protein products of 3 clones, pll6, pl34, and pl35, are shown in Figure 37.
  • FIG. 37 is a photograph of one such gel that had been stained with Coomassie blue. As can be seen, lysates pll6 and pl35 produce a protein of about 19,000 Dalton molecular weight whose relative abundance increases in the pres ⁇ ence of IAA. In contrast, pl34 does not produce this protein species.
  • pll6 and pl35 contain plasmids that, by restriction analysis, have the sequences of the protein having tumor growth inhibitory activity cloned in the orientation that should produce a 19,500 Dalton molecular weight fusion protein.
  • plasmid was found to have the sequences of the protein having tumor growth inhibitory activity in the opposite orientation.
  • the trpE: :protein having tumor growth inhibitory activ ⁇ ity fusion protein was used to test the specificity of an antibody that used a peptide homologous to part of the protein having tumor growth inhibitory activity as an antigen.
  • a polypeptide was synthesized correspond ⁇ ing to residues 9 through 28 of the mature protein having tumor growth inhibitory activity, except that residue 9 in the sequence, arginine, was replaced by serine.
  • the peptide was purified by RPHPLC and cou ⁇ pled to keyhole limpet hemocyanin for use as an immunogen in rabbits.
  • the antisera were screened by standard ELISA using lOOng of peptide per well.
  • One rabbit demonstrated a signal of 1.0 OD units at a 1:25 dilution of the anti- body.
  • the fol ⁇ lowing bleed 20 days after the first bleed showed a 20- fold increase in antibody response to the peptide anti ⁇ gen.
  • Forty days after the initial bleed (3rd bleed) a signal of 1.0 OD unit was achieved at a 1:8000 dilution of the antisera, a 16-fold increase in antibody titer over the second bleed.
  • TGF- 1 derived peptide consisted of amino acid numbers 4 to 19 of the mature TGF- 1 protein. Of the 11 common amino acids, residues 9-19, 7 are conserved between the protein having tumor growth inhibitory activity and TGF- ⁇ l. - 95 -
  • the antibody was used in Western blot analy ⁇ sis against a fusion protein of the protein having tumor growth inhibitory activity and a TGF- ⁇ l fusion.
  • the anti-peptide antibody reacted strongly with the fusion protein of the protein having tumor growth inhibitory activity while it reacted only weakly with a trp: :TGF-01 fusion protein. Both fusion proteins were recognized by a commercially available anti-TGF-01 antibody (R and D systems) ( Figure 38) .
  • the anti-peptide antibody recognizing the protein having tumor growth inhibitory activity also has a high level of background reactivity to bacterial proteins.
  • the purified peptide antibody was then tested by Western blot analysis for its cross- reactivity with TGF- ⁇ l. The results are shown in Fig ⁇ ure 39.
  • the purified antibody reacts very strongly with the fusion protein of the protein having tumor growth inhibitory activity (lane 2)and with a higher molecular weight protein species, while the hybridiza ⁇ tion to other bacterial proteins was found to be greatly reduced compared to the unpurified antibody ( Figure 38) .
  • the purified antibody exhibits negligible reactivity with either the TGF-01 fusion protein (lane 1) or purified, TGF-01 obtained commercially (R and D systems) (lanes 3 and 6) .
  • a competition experiment was also performed where the purified antibody was - 96 -
  • the anti- peptide antibody specifically recognizes proteins con ⁇ taining sequences of the protein having tumor growth inhibitory activity.
  • Human recombinant TGF-01 has been expressed in monkey COS cells. Sequences encoding the complete precursor of the TGF- ⁇ l cDNA were cloned down stream from a SV40 promoter using the pSVL* eukaryotic expression vector (obtained from Pharmacia) . This construct was trans- fected into COS cells using a standard calcium phos ⁇ phate precipitation method, Graham and van der Eb (1973) Virology 52, 456-467. After transfection, ap ⁇ proximately 4x10 cells were grown in serum free media for two days. The conditioned media was then collect- ed, acidified and tested for biological activity.
  • Conditioned media from TGF- ⁇ l transfected cells was found to inhibit the growth of a monolayer mink lung test cell line (CCL 64) by 59% as compared to condi ⁇ tioned media from COS cells tranfected with the pSVL vector alone which inhibited growth of CCL 64 cells by only 32%.
  • CCL 64 monolayer mink lung test cell line
  • a ch-i ⁇ eric TGF- 1::protein having tumor growth inhibito ⁇ ry activity fusion construct was made by substituting 3' sequences of the TGF- ⁇ l precursor with sequences encoding the protein having tumor growth inhibitory activity. Given the homology between these two pro ⁇ teins and the conserved position of their cysteine residues, when such a construct is transfected into COS cells the novel fusion protein may be processed into the biologically active mature protein having tumor growth inhibitory activity.
  • TGF-01 was cloned and used to isolate a related gene encoding a protein having tumor growth inhibitory activity. Although it has not yet been determined which of TGI-1 or TGI-2 corresponds to the protein related to TGF- ⁇ l and having tumor growth inhibitory activity, one skilled in the art would understand that such a correspondence exists although the exact nature of this corresponding remains to be clarified. - 98 -
  • mRNA was isolated from A673 cells and a cDNA library prepared. Starting with 5 ⁇ g poly (A) + RNA, a random primed cDNA library of approximately 2 x 10 clones was constructed in lambda gtlO, using the Amersham cDNA synthesis system plus according to the . manufacturer's procedures. Approximately 0.7 x 10 - 99 -
  • unamplified cDNA clones were screened with a 25-mer oligonucleotide probe (5'-A T A T A G C G C T G T T T T G G C A A T G T G C T -3') corresponding to a sequence near the 5' end of the 1.9 kb cDNA clone and a single positive clone containing a 1.7 kb insert was identified.
  • TGF-01 and TGF-02 are produced in precursor forms of 390 and 414 amino acid residues respectively (Derynck, et al. (1985) Nature 3_16, 701-705; de Martin, et al.
  • Fig. 41 contains an open reading frame coding for 412 amino acids, with the first ATG preceeded by a stop codon, 162 nucleotides upstream. As found with TGF- ⁇ l (Derynck, et al. (1985)
  • TGF- ⁇ l and -02 have stretches of 4 and 5 basic residues respectively.
  • the N-terminal domain of the precursor portion of the gene encoding the protein having tumor growth inhibito ⁇ ry activity exhibits approximately 35% homology to TGF- ⁇ l and 45% homology to TGF-02.
  • the corresponding sequences of the TGF- ⁇ l and -02 precur ⁇ sors have 33% sequence homology (Fig. 42) (Derynck, et al. (1985) Nature 316, 701-705; de Martin, et al. (1987) EMBO J. 6, 3673-3677) .
  • a homology matrix plot clearly illustrates the greater similarity between the gene encoding the protein having tumor growth inhibito ⁇ ry activity and TGF-02, compared to TGF- ⁇ l (Fig. 43).
  • Four potential glycosylation sites are contained in the N-terminal part of the gene encoding the protein having tumor growth inhibitory activity, one of which is con- - 101 -
  • both TGF- ⁇ l and the gene encoding the protein having tumor growth inhibitory activity (but not TGF- 02) contain the fibronectin binding sequence RGD (Ruoslahti and Pierschbacher (1986) Cell 44, 517-518).
  • RGD fibronectin binding sequence
  • the protein having tumor growth inhibitory activity is likely to be synthesized as a 412 amino acid precursor which undergoes proteolytic cleavage to produce the mature polypeptide.
  • the protein having tumor growth inhibi ⁇ tory activity Based on the functional and structural homology to TGF- ⁇ l and -02, the protein having tumor growth inhibi ⁇ tory activity likely has therapeutic activity in can ⁇ cer therapy, wound healing and i ir. osuppression.
  • a 1500 bp Alul-Hgal restriction fragment of TGF-03 cDNA (sites are indicated in Figure 41) which encodes the complete TGF-03 protein was cloned into the Bluescript plasmid (Strategene, La Jolla, CA) , to yield the plasmid pBlue-TGF-03.
  • the fl intergenic region of this vector allows the production of single stranded DNA via infection of its host bacteria with fl helper phage.
  • the predicted initiation codon of TGF-03 does not form part of a Kozak consensus sequence (CCACC[ATG]G; Kozak, Cell 44:283-292, 1986), which has been shown to influence the efficiency of translation.
  • TGF-03 cDNA sequence is transcriptionally regulated b y the,, cytomegalovirus immediate early promoter (see Figure 44) .
  • Stable transformants expressing TGF-03 were obtained by cotransfecting the pCMV-TGF-03 construct ( Figure 44) with the dihydrofolate reductase (DHFR) gene (the pDCHIP plasmid containing hamster DHFR minigene driven by its own promoter) into Chinese Hamster Ovary (CHO) cells, which lack the DHFR gene (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).
  • DHFR dihydrofolate reductase
  • TGF-03 expression levels were measured by RNA cytodot hybridization normalizing the expression of TGF-03 mRNA to that of actin.
  • Two of the three clones with initial high expression (clones CHO 6.35 and CHO 9.1) showed increased TGF-03 mRNA - 104 -
  • CHO 6.35/20 nM primary transfectant CHO clone 6.35 at 20nM Mtx, which had the highest level of expression, was chosen for initial protein purification from conditioned media and for further gene amplification.
  • CCL 64 a cell line derived from Mink lung (American Type Culture Collection, Rockville, MD) , was found to be extremely sensitive to the naturally occurring TGF-03 isolated from umbilical cord. This cell line was initially chosen, therefore, to test conditioned media for biological activity of the recombinant TGF-03 protein according to the method of Iwata et al. , Cancer Res. 45:2689-2694, 1985. Growth inhibition of CCL 64 mink lung cells produced by TGF- ⁇ l (purified) or TGF-03 (from conditioned media) is shown in Figure 46.
  • Figure 46A shows a dose response of growth inhibition using purified TGF- ⁇ l (Calbiochem) ; a 50% inhibition was obtained with 0.1 ng TGF- ⁇ l. An increase in mink cell growth inhibitory activity was found comparing conditioned media from the transfectant selected at 20nM Mtx versus media from the parental transfectant.
  • Figure 46B shows the biological activity of acid activated serum free supernatants of CHO 6.35/20 nM - 105 -
  • Peptides corresponding to various partial amino acid sequences of the TGF-03 protein were synthesized on an Applied Biosysterns peptide synthesizer (Model 430A) using tBoc chemistry (see Figure 47) . Peptides were coupled to keyhole limpet hemocyanin with glutaraldehyde and used for immunization of rabbits. Enzyme-linked immunosorbent assays were used initially to characterize the antibody titers (see Table 9). For this, and the following immunological experiments, standard techniques were employed as described by Harlow and Lane, 1988, in Antibodies, A Laboratory Manual.
  • High titer antisera from immunized rabbits injected with 03V or 03III peptides were purified using an affinity matrix composed of the respective peptide 03 antigen conjugated to Affi-prep ⁇ o (Bio Rad, Richmond, CA) . - 106 -
  • the affinity purified 0 III antisera exhibits greater than 300 fold specificity for the 03III pepti d e compared to the cognate peptide sequences from either the TGF- ⁇ l or -02. Furthermore, no significant cross reactivity of this antisera has been observed against either the TGF- ⁇ l or -02 proteins.
  • this antibody shows only a very limited ability to immunoprecipitate the native recombinant TGF-03 protein from conditioned media.
  • the affinity purified 03V antisera exhibits at least a 400-fold selectivity for the 03V peptide compared to the corresponding peptide sequence from TGF-01. This antibody can also efficiently immunoprecipitate the native TGF-03 protein (see figure 50) . However, this polyclonal sera appears to contain a significant population of antibodies (approximately 30-50%) which react with both the TGF-02 cognate peptide sequence and the TGF-02 protein.
  • Figure 48 shows an immunoblot of TGF-03 in conditioned media produced by the CHO 6.35/20 nM transfectant using 03III and 03V antibodies for detection.
  • the antibody was preincubated with 80-fold molar excess of peptide prior to incubation with the blot.
  • alkaline phosphata ⁇ e Zymed, San Francisco, CA
  • Figure 48A shows a western blot of a gel where the sample was subject to reduction prior to electrophoresis while Figure 48B shows the Western blot of the sample under non-reducing conditions.
  • lanes 1-3 and 4-6 corresponds to conditioned media immunoblotted with 03V and 03III antisera, respectively, lanes 2 and 5 immunoblots carried out in the presence of excess cognate peptide, while lanes 3 and 6 represent immunoblots in the presence of an - 108 -
  • FIG. 49 shows a Western blot of cell extract (Fig. 49A) and conditioned media (Fig. 49B) of the CHO 6.35/20 nM transfectant using 03V antibody for detection.
  • This protein may correspond to either the non-glycosylated precursor or a proteolytic breakdown product.
  • the 03V antibody proved to be much more efficient in immunoprecipitation of TGF-03 protein than the 03III antibody.
  • the specificity of the immunoprecipitation was determined by preincubating the antibody with a 80-fold molar excess of either the cognate peptide or an unrelated peptide sequence. The specific peptide showed complete competition of all three bands whereas the unrelated peptide did not.
  • the 50 kDa contains significantly more S 35 label. - 110 -
  • the 0V affinity purified antibody was also used in paraffin sections of human umbilical cord (see Figure 51) . Fibroblasts and epithelial cells stained (Fig. 51A) as did the smooth muscle fibers of the cord vasculature (Fig. 51C) whereas neither ;he connective tissue nor the extracellular matrix stained with this antisera.
  • a control rabbit polyclonal antisera Ig against P210 phl/abl :OSI catalog #PC02
  • showed no staining Figs. 51B and D
  • Conditioned media was prepared from CHO 6.35/20nM cells grown to confluence in the presence of 20nM methotrexate. The cells were washed with phosphate buffered saline and incubated with serum free medium for 2 hours to eliminate carryover of serum proteins. Conditioned media was derived from cells incubated with fresh serum-free media for 48 hours. The conditioned media was centrifuged, acidified and dialyzed in (Spectrapor 3 membrane (3.500 MW, cut off) Thomas Philadelphia, PA) against 1 M acetic acid and subsequently lyophilized. The acid-soluble material was applied to a BioGel P-60 column (4 x 100 cm) , equilibrated with 1 M acetic acid. Fractions containing 10 ml were collected and aliquots of selected column fractions analyzed by Western blot analysis using the 03III antibody for detection. Two peaks of cross-reactive bands were found which - Ill -
  • TGF-03 correspond to precursor and mature forms of TGF-03, respectively.
  • Fractions containing the mature TGF-0 3 protein were pooled and partially lyophilized. This pool was neutralized with 2M Tris to pH 7 and passed through an affinity column of 03V antibody coupled to protein A agarose with dimethylpimelimidate using standard procedures as described by Harlow and Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988) .
  • TGF-03 protein was eluted with 50 mM glycine -HCl pH 2.0.
  • Figure 52 shows a silver stain of purified TGF-03 and TGF-01 (Calbiochem) .
  • TGF-03 100 ng
  • TGF- ⁇ i 10 mM DTT in the loading buffer.
  • Fractions were analyzed by silver stain and Western blot analysis and peak fractions pooled.
  • the silver stained gel showed a single band of 12kDa and 24 kDa under reducing and non- reducing conditions, respectively ( Figure 52). The detection of a single silver staining band indicates that the preparation is greater than 90% homogeneous.
  • TGF-03 The effect of TGF-03 on the growth of various cell lines is shown in Table 10. Growth was determined using a modification of the monolayer assay for tumor growth inhibitory activity described in the Materials and Methods section of the "First Series of Experiments". Cells were subcultured on 96-well tissue - 112 -
  • TGF-03 has only minimal effect on normal human fibroblasts while significantly inhibiting mink lung cells (CCL 64) and human tumor cells from lung, skin, colon and breast tumor tissue.
  • TGF-03 Purified recombinant TGF-03, at concentrations from 3.125 to 0.049 ng/ml, was incubated with 5 ug/ ml of affinity purified polyclonal rabbit antibodies (03III and 03V antisera) for 3 hours at 37*C. Control TGF-03 was incubated without antibodies. Growth inhibition of mink cells by antibody treated and control untreated TGF-03 was determined as described above.
  • Figure 53 shows that the 03V antisera (closed squares ) decreases the growth inhibitory activity of TGF-03 on mink cells relative to the growth inhibitory activity of identical concentrations of TGF-03 in either the absence of antibody (closed circles) or treated with 03III antisera (open squares) . Neither antisera had any significant effect on the growth of CCL 64 cells in the absence of TGF-03. Antibodies against the TGF-03 - 113 -
  • peptide 0 V are apparently neutralizing the growth inhibitory activity of TGF-03.

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Abstract

Cette invention concerne une protéine ayant une activité inhibitrice de la croissance de tumeurs et comprenant les 112 acides aminés illustrés dans la figure 29 commençant par l'alanine en position 1 et se terminant par la sérine en position 112. La protéine peut également inclure les 412 acides aminés illustrés dans la figure 41 commençant par la méthionine en position nucléotide 263 et se terminant par la sérine en position nucléotide 1496. Ainsi, cette séquence de 412 acides aminés contient la séquence précursseur complète de la protéine ayant l'activité inhibitrice de la croissance des tumeurs ainsi que la séquence complète de la protéine mature illustrée dans la figure 29 commençant par l'alanine en position 1 et se terminant par la sérine en position 112. Finalement, l'invention divulgue une protéine comprenant les 411 acides aminés illustrés dans la figure 41 commençant par la lisine en position nucléotide 266 et se terminant par la sérine en position nucléotide 1496.
PCT/US1990/002753 1989-05-17 1990-05-17 Inhibiteurs de la croissance de tumeurs derivees de tissus, procedes de preparation et leurs utilisations WO1990014360A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0538398A1 (fr) * 1990-06-25 1993-04-28 Oncogene Science, Inc. Inhibiteurs de croissance de tumeurs derives de tissus, procedes de preparation et utilisations desdits inhibiteurs
WO1993016099A2 (fr) * 1992-02-12 1993-08-19 Biopharm Gesellschaft Zur Biotechnologischen Entwicklung Von Pharmaka Mbh Sequences d'adn codant de nouveaux facteurs de croissance/differentiation
WO1997039023A1 (fr) * 1996-04-12 1997-10-23 Astra Aktiebolag Peptides contenant de la cysteine ou de la methionine et possedant des effets immunomodulateurs

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4886747A (en) * 1985-03-22 1989-12-12 Genentech, Inc. Nucleic acid encoding TGF-β and its uses

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NZ222168A (en) * 1986-10-20 1991-05-28 Oncogene Science Inc Tumour growth inhibiting protein related to tgf-#b#1 and tgf-#b#2, preparation by genetic engineering methods, antibodies and pharmaceutical compositions

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4886747A (en) * 1985-03-22 1989-12-12 Genentech, Inc. Nucleic acid encoding TGF-β and its uses

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Bio/Technology "Growth Factors Speed Wound Healing" Volume 6, No. 1, page 25-29. VAN BRUNT et al. January 1988. See page 25, 3rd column. *
HARLOW et al. "Artibodies, A laboratory Manual" published 1986, Cold Spring Harbor Laboratory (New York) page 148-157. See all pages cited. *
Proc. Natl. Acad. Sci. USA "Identification of another member of the transforming growth factor type B gene family" Volume 85, page 4715-4719. TEN DYKE et al., July 1988. See entire article, esp. figure 2. *
Science, "Transforming Growth Factor-B: Biological Function and Chemical Structure", Volume 233, page 532-534. SPORN et al. 01 August 1986. See page 532, section entitled "Effects of Cell Proliferation". *
See also references of EP0508983A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0538398A1 (fr) * 1990-06-25 1993-04-28 Oncogene Science, Inc. Inhibiteurs de croissance de tumeurs derives de tissus, procedes de preparation et utilisations desdits inhibiteurs
EP0538398A4 (en) * 1990-06-25 1993-12-29 Oncogene Science, Inc. Tissue-derived tumor growth inhibitors, methods for preparation and uses thereof
WO1993016099A2 (fr) * 1992-02-12 1993-08-19 Biopharm Gesellschaft Zur Biotechnologischen Entwicklung Von Pharmaka Mbh Sequences d'adn codant de nouveaux facteurs de croissance/differentiation
WO1993016099A3 (fr) * 1992-02-12 1993-09-30 Bioph Biotech Entw Pharm Gmbh Sequences d'adn codant de nouveaux facteurs de croissance/differentiation
WO1997039023A1 (fr) * 1996-04-12 1997-10-23 Astra Aktiebolag Peptides contenant de la cysteine ou de la methionine et possedant des effets immunomodulateurs

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