JPH0352480B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the use of transforming growth factors that are specifically present in the urine of cancer patients.
The present invention relates to a method for producing Factor (hereinafter referred to as TGF) in a highly purified and concentrated form. TGF is a general term for polypeptides that are produced by human and rodent cancer cells and cause specific morphological changes in normal cells. Normal human and animal cells do not form colonies on soft agar;
Addition of TGF increases colony forming ability. 1978 Z. Todaro et al. (Proc. Natl. Acad.
Since the existence of this substance was first reported in the culture supernatant of sarcoma cells by Sci 75 , 4001 (1978), numerous reports have been made.
TGF has also been found in human and rodent tissue culture cancer cells and cancer tissue from cancer patients.
(Roberts et B et al. Proc. Natl. Acad. Sci. USA
77, 3494 (1980)). Recently, Dey al-Tajitzk et al. (J. Natl.
Cancer Inst. 69 , 793 (1982)) and Z. Todaro et al. (Cancer Res. 43 , 403 (1983)) detected high molecular weight TGF with a molecular weight of approximately 30,000 to 35,000 from the urine of cancer patients.
However, the exact substance is not yet clear. The present inventors have discovered that high-molecular-weight TGF does not exist in normal human urine, but specifically exists only in cancer patient urine, and that this substance has the above-mentioned biological activity against normal cells. Therefore, we have conducted repeated research to extract this substance, considering it to be an extremely important substance medically, such as in the diagnosis of cancer and the pursuit of immunological problems.
Through separation and purification, we clarified its biological activity and physical properties, and succeeded in establishing a method for producing this product. That is, the urine of various cancer patients is collected and subjected to dialysis to remove inorganic ions and other low-molecular substances, then repeatedly frozen and thawed to remove insoluble matter, and then freeze-dried.
Using the material extracted with weakly acidic water, it was first purified by the gel filtration method as described by Dei al-Tajik and J.Z. Todaro et al. As shown in Tables 1 and 2, the purification efficiency of the obtained active fraction was more than 10 times, but less than 20 times, and a satisfactory result could not be obtained. Therefore, when we attempted gradient elution using neutral salts by pouring dialyzed urine through a cation exchanger, we obtained unexpectedly good results, unlike gel filtration methods. In other words, this method had good results with a purification efficiency of around 100 times (Tables 3 and 4). As a result of further intensive research, we made use of the hydrophobicity of polymeric TGF. SynChropak RP-P (USA,
When reversed phase chromatography was performed using MCI Gel CHP20P (manufactured by Mitsubishi Chemical Industries, Ltd.), a polystyrene-based high porous polymer, the purification was approximately 100 times as high as in the ion exchange chromatography described above. Obtained efficiency (5th and 6th
table). Further, a combination of the above ion exchange chromatography and reversed phase chromatography was carried out. As a result, we surprisingly obtained a purification efficiency of more than 200 times and nearly 300 times (Tables 7 and 8), and based on these findings, we completed the present invention. The present invention involves dialyzing the urine of a cancer patient, and optionally gel-filtering the dialyzed fluid from which low-molecular substances have been removed to collect a fraction exhibiting TGF activity. Ion exchange chromatography in which a factor is adsorbed and then gradient elution is performed using a concentration gradient aqueous solution of a neutral salt; (2) the factor is adsorbed onto a carrier having a hydrophobic group, and then the concentration of a hydrophilic neutral organic solvent is Reversed phase chromatography with gradient elution using a gradient aqueous solution, subject to at least one operation of
TGF characterized by collecting a TGF active fraction
This is the manufacturing method. In the present invention, urine of cancer patients is used as a raw material. Any type of cancer may be present; examples include lung cancer, chorionic tumor, stomach cancer, pharyngeal cancer,
These include colon cancer, breast cancer, melanosarcoma, and ovarian cancer. In order to remove low-molecular impurities from the urine, the urine is first dialyzed using water as an external dialysis fluid. As a dialysis membrane, it permeates low-molecular substances with a molecular weight of about 10,000 or less,
It is desirable to have a membrane that does not transmit polymers exceeding this range, and various such membranes are commercially available. The obtained dialyzed fluid is preferably repeatedly frozen and thawed to precipitate and remove impurities, or is centrifuged or passed through a filter having pores of several μm to remove fine precipitated particles. In addition, if desired, the dialysis fluid may be gel-filtered.
Fractions exhibiting TGF activity may be collected. As a carrier for gel filtration, Biogel P-60,
P-100 (manufactured by Piorad, USA), Sephadex G-50 Super Fine (manufactured by Pharmacia Japan), etc. can be used. The TGCF active fraction can be identified by the test method described below, and has an estimated molecular weight of 28,000~
Exists at position 35000. In the present invention, the above urine dialysis fluid or the active fraction collected by gel filtration is subjected to at least one of ion exchange chromatography and reversed phase chromatography to separate the TGF activity fraction. Collect. Ion exchange chromatography is performed using a cation exchanger. As a cation exchanger,
Materials based on natural or synthetic polymeric materials are used; examples include CM-Sephadex C-25 (manufactured by Pharmacia Japan) and CM-52 (manufactured by Watman, USA). ), Bio-Rex 70 (manufactured by Bio-Rad, USA), SP-Sephadex
(manufactured by Pharmacia Japan), etc. The dialysate solution with or without the gel filtration described above is added to a cation exchanger column, and at that time, TGF
is adsorbed by the exchanger. The adsorbate is then gradient eluted using an aqueous solution of a neutral salt whose concentration is gradually increased. Any neutral salt can be used as long as it is water-soluble and has no adverse effect on the peptide, but sodium chloride is most preferably used. Reversed phase chromatography is performed using a support with hydrophobic groups. Examples of such carriers include carbon chain-modified silica gel such as SynChropak RP-P series (manufactured by SynChrom, USA), MCI Gel CHP20P series (manufactured by Mitsubishi Chemical Corporation), and Amberlite.
Examples include polystyrene-based high-porous adsorption resins such as (Amberlite) XAD series (manufactured by Amberlite, Inc., USA). Among them, the particularly preferred one is Synchropack RP-P [C-
18], MCI CHP20P, Amberlight XAD−
4 etc. The dialyzed fluid, which has or has not been subjected to gel filtration, is added to the carrier column, and TGF is adsorbed onto the carrier.
The adsorbate is then gradient eluted using an aqueous solution of a hydrophilic neutral organic solvent whose concentration is successively increased.
Examples of hydrophilic neutral organic solvents include ethanol,
Examples include lower aliphatic alcohols such as propyl alcohol, lower aliphatic ketones such as acetone, and lower aliphatic nitriles such as acetonitrile, and preferred examples are acetonitrile, methanol, and n-propyl alcohol. The fraction of the eluate obtained by ion exchange or reversed phase chromatography was determined by the test method described below.
TGF activity is tested and active fractions are collected. By ion exchange and reversed phase chromatography
Only one of the TGF purification operations may be used, or one operation may be performed before and the other operation may be performed after. The TGF thus obtained is a white powder, positive for ninhydrin reaction, molecular weight 28,000-35,000 (gel filtration method), single in SDS-electrophoresis, highly stable against acids and heat, and resistant to acid and heat when treated with trypsin and dithiothreitol. become inactive. The highly purified TGF of the present invention is useful, for example, as a reagent for cancer testing, and the product's ability to prevent degeneration of normal cells or normalize degenerated cells is used as an indicator to prevent cancer. It is also expected to be used to screen therapeutic agents. EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited by these in any way. Note that the results regarding the gel filtration method are shown as a reference example. Reference Example 1 750ml of fresh urine from a lung cancer patient was mixed with pure water at 4°C using a dialysis membrane with a molecular weight of 3500 (Spectrapor Por.3, manufactured by Spectrum Medical Ind., USA). 2
I underwent dialysis day and night. After repeating freezing and thawing of the dialysis fluid twice, it was centrifuged at 10,000 xg for 45 minutes to remove insoluble matter, and freeze-dried. 1.5 ml of 0.5 M acetic acid aqueous solution was added to this dry powder, and the mixture was frozen and thawed twice using a homogenizer at room temperature, followed by centrifugation at 12,000 xg for 25 minutes to obtain an extracted supernatant. The centrifuged residue was further washed with 1.0 ml of 0.5M acetic acid water, centrifuged, and the supernatant was combined with the above-mentioned extraction supernatant. Fill a column with polyacrylamide biogel P-60 Fine (manufactured by Bio-Rad, USA) and equilibrate it with 0.5M acetic acid water (capacity 1.8cm x 100cm), place the above extract on top of the resin, and use the descending method to extract 0.5% It was eluted with aqueous M acetic acid (flow rate 12.5 ml/hour, temperature 4°C). The elution curve is shown in FIG. The TGF activity was located in the estimated molecular weight range of 28,000 to 35,000, and an active fraction of 35.0 ml could be collected. In the TGF activity test method described below, 20 μl of this active fraction transformed normal cells and showed colony forming ability. [Table] Reference Example 2 6700ml of urine from a patient with chorioblastic tumor was immediately dialyzed against pure water in the same manner as in Reference Example 1, and then frozen, thawed, freeze-dried, and extracted with a 0.5M acetic acid aqueous solution to obtain an extract of 22.3
Got ml. Sephadex G-50 Super Fine packed into a column (capacity 4.8cm x 100cm) 0.5M
After equilibration with aqueous acetic acid, the above extract was placed on the top of the resin and eluted with 0.5M aqueous acetic acid using the descending method (flow rate 65.0
ml/hour, temperature 4°C). TGF active fraction is estimated molecular weight
Eluted at position 28000-35000, active fraction 215ml
When 5 μl of the obtained product was subjected to the TGF activity test method described above, the NRK cells were transformed and formed colonies. [Table] Example 1 450ml of gastric cancer patient urine was added to 0.02M phosphate buffer (PH
7.5) Dialysis was performed on 20 liters at 4°C for 2 days and nights using a dialysis membrane with a molecular weight of 3500. The dialyzed solution was centrifuged at 10,000×g for 40 minutes to remove the precipitate. CM-Sephadex CC-25 (manufactured by Pharmacia Japan) as a column (capacity 3.2cm x 32cm)
and equilibrated with 0.02M phosphate buffer (PH7.5). After passing the dialyzed urine through this column to adsorb TGF, it was thoroughly washed with the same buffer solution, and then gradient elution with 0.02M to 1M sodium chloride in the same buffer solution was performed (flow rate 16.5ml/hour at 4°C). ). The elution curve is shown in FIG. TGF activity is 1.2~
eluted at the position of 1.4M sodium chloride solution,
It was possible to capture 38.5 ml of active fraction. In the TGF activity test method described below, by adding 10 μl of this sample, the cells were altered and exhibited colony forming ability. [Table] Example 2 1985ml of lung cancer patient urine was added to 0.04M phosphate buffer (PH
8.0) Dialysis was performed against 40 liters, and the precipitate was removed by filtration using a membrane filter containing 5 μm cells (manufactured by Nippon Millipore). CM-52 (manufactured by Watmann, UK)
was packed in a column (capacity 4.2cm x 64cm), equilibrated with 0.04M phosphate buffer (PH8.0), the above dialyzed urine was poured into this, and then 0.04M in the same buffer was
A gradient elution was performed with a 1.5M concentration of sodium chloride. TFG has a sodium chloride concentration of 0.7 in the same buffer.
It eluted at ~1.1M position. An active fraction of 132.9 ml was collected. 10 μl of this fraction transformed normal cells and showed colony forming ability. [Table] Example 3 155ml of urine from a nasopharyngeal cancer patient was added to pure water at 4℃ for 2 hours.
After performing dialysis day and night, the dialyzed fluid was frozen and thawed, and insoluble matter was removed by centrifugation at 10,000×g for 30 minutes, followed by freeze-drying. 1.0ml of this dry powder
Add 0.05% trifluoroacetic acid water, homogenize it at room temperature, freeze and thaw it.
The extract was centrifuged at 18,000xg for 20 minutes to obtain an extract. The centrifuged precipitate was further washed with 0.5 ml of 0.05% trifluoroacetic acid water, centrifuged, and the supernatant was combined with the above extract. Silica gel bonded with alkyl group (C18) (Synchropack RP-P, USA,
SynChrom) was packed in a column (4.1 mm x 250 mm) and equilibrated with 0.05% trifluoroacetic acid, and the above extract was
0.25 ml was added dropwise for adsorption, and after washing with 0.05% aqueous trifluoroacetic acid, gradient elution was performed with the same solution of 0-90% acetonitrile. The elution curve is shown in FIG. TGF
The activity was eluted at acetonitrile concentrations of 55-80%. The six active fractions were collected and freeze-dried to obtain 2.3 mg (weight) of dry powder. This product was dissolved in 2.0 ml of physiological saline, and 2 μl of the solution was subjected to the TGF activity test method described later, and it showed colony forming ability. [Table] Sucrose concentration gradient isoelectric focusing was performed using 1.0 ml of this solution. The device is an LKB 110ml capacity isoelectric focusing column (manufactured by LKB, Sweden), and the PH gradient is LKB Ampholine (manufactured by the same company) PH.
Using an equal mixture of PH 3.5-10 and PH 9-11,
Perform electrophoresis at 600V, 0℃ for 48 hours, and after electrophoresis
The samples were fractionated in 1.2 ml portions, and 5 .mu.l of each fraction was used to perform the TGF activity test method described later, and as shown in FIG. 4, it was determined that the TGF activity was at an isoelectric point of 9.5 to 11. This active fraction is collected, dialyzed against pure water, and then lyophilized to form a highly pure white powder of 0.6 mg (weight).
Got TGF. Stability tests were conducted on this product based on the method of Z. Todaro (Natl, Cancer Inst, 69 (4) 793 (1982)). In other words, this product is
It does not lose activity even after 24 hours at 37°C in 1M acetic acid and repeated freeze-drying and thawing. However, the activity was lost when incubated in trypsin and dithiothreitol solution. In addition, SDS-electrophoresis was performed on the physiological food aqueous solution of the freeze-dried powder obtained in the first period using PREP-DISC (manufactured by Seikagaku Corporation) pD-2/70. 10% acrylamide/0.03% bisacrylamide/0.1
A %SDS gel column (capacity 70 mm ² × 2.7 cm) was used, and 0.1% SDS-0.1M phosphate buffer (PH7.2) was used as the migration buffer, and the same buffer was used for elution. . The elution rate was 0.5 ml/min, and electrophoresis was carried out at a constant current of 10 mA to obtain the results shown in FIG. BPB in the same figure
is promophenol blue (molecular weight 670), and this dye was used as an indicator of low molecules. Example 4 After dialysis of 250 ml of gastric cancer patient urine against pure water,
It was treated in the same manner as in Example 1 to obtain an extract with 1.0M aqueous acetic acid. The above extract was placed on the resin surface of a Pyogel P-100 (manufactured by Bio-Rad, USA) column (capacity 1.5 cm x 42 cm) equilibrated with 1.0 M acetic acid and eluted with 0.5 M acetic acid using the descending method. (Flow rate 3.4
ml/hour, 4℃) TGF activity has an estimated molecular weight of 28,000 ~
It was eluted at position 35000. This fraction showed colony forming ability in TGF activity test method. This fraction was freeze-dried, dissolved in 1.0 ml of 0.02% triflic acid water, and repeatedly frozen and thawed.
Centrifugation was performed at 15,000×g for 20 minutes to obtain an extracted supernatant.
The precipitate was further washed with 0.5 ml of 0.02% trifluoroacetic acid, centrifuged, and the supernatant was combined with the above extract. Polystyrene-based highly porous polymer
MCIGel CHP20P column (capacity 5mm x 400mm)
After equilibrating with 0.02% trifluoroacetic acid water, the above extract solution is poured onto the top of the resin to adsorb it, and the resin is washed with the same solution. Then 0-90 in 0.02 trifluoroacetic acid
% n-propanol gradient extraction was performed, and TGF was eluted at a position where the n-propanol concentration was 30 to 60%. This fraction was collected and freeze-dried to obtain 8.3 mg (weight) of dry powder. When this was dissolved in 3.5 ml of physiological saline and 2.5 μl of the solution was subjected to the TGF activity test described below, normal cells were altered and colonies were formed. [Table] Example 5 5600 ml of urine from a patient with nasopharyngeal cancer was dialyzed against 0.01M phosphate buffer (PH7.0) using a dialysis membrane with a molecular weight of 3500 at 4°C for 2 days and nights. 5μm for dialysis fluid
The mixture is filtered using a membrane filter having pores of 100 mL, and TGF is adsorbed in a batch method using Biorex 70 (manufactured by BioRad, USA). The adsorbed resin
After washing with 0.01M phosphate buffer (PH7.0), TGF was eluted in batches using 0.5M phosphate buffer (PH9.0) containing 1M sodium chloride. The extract was dialyzed against pure water and freeze-dried. 1/10 (weight) of this dry powder was dissolved in 1.0 ml of 0.05% trifluoroacetic acid water and centrifuged at 15,000 xg for 20 minutes to obtain an extracted supernatant. Add another 0.5 ml of centrifugal sediment.
The supernatant obtained by extraction with 0.05% trifluoroacetic acid water and centrifugation was combined with the previous supernatant. Silica gel with alkyl groups (C4) bonded (Synchropack RP-
Column (capacity: 4.1
mm x 250 mm) was equilibrated with 0.05% trichloroacetic acid water, and then adsorbed through the above extract solution from the resin surface. After washing with the above acidic solution, gradient elution was performed with 0 to 75% acecetonitrile in the same solution, and the acetonitrile concentration was 30%.
2.8 ml of TGF activity fraction was collected at ~50% position. This operation was repeated 10 times to obtain the active fraction. This was lyophilized, dissolved in 2.5 ml of physiological saline, and subjected to the TGF activity test described below.
Normal cells were transformed with 1 μl and showed colony forming ability. [Table] Example 6 1100ml of lung cancer patient urine was added to 0.01M phosphate buffer (PH
8.0) was subjected to dialysis for 2 days and nights at 4°C using a dialysis membrane with a molecular weight of 10,000 (manufactured by Sanko Pure Chemical Industries, Ltd.).
The dialysed fluid was centrifuged at 12,000 xg for 60 minutes to remove the precipitate. Column (capacity 4.5cm x 33cm) using SP-Sephadex (Pharmacia, manufactured by Japan Co., Ltd.)
After equilibration with 0.01M phosphate buffer (PH8.0), the above dialysis solution was allowed to flow down and the active ingredient was adsorbed, and then thoroughly washed with the same buffer. A gradient elution of sodium chloride from 0.01M to 1.5M was performed. (Flow rate 22.5 ml/hour, temperature 4°C, using 4.8 liters of sodium chloride gradient concentration buffer). TGF was eluted at a sodium chloride concentration of 0.6 to 1.0 M in the same buffer, and 62.0 ml of active fraction was obtained. In the TGF activity test method described below, normal cells were transformed and colonies were formed in 5 μl of this active fraction. Diaflow molecular sieve membrane PM-10 (Amicon,
Desalting and concentration were repeated using Far East Limited (Japan) to make 1.2 ml, and trifluoroacetic acid was added thereto to obtain a 0.05% trifluoroacetic acid solution. Synchropack RP-P (manufactured by Synchrome), which uses silica gel with alkyl groups (C18) as a carrier, is packed in a column (capacity 4.1 cm x 2250 cm).
This column 1 was equilibrated with 0.05% trifluoroacetic acid.
After pouring 1/3 of the above TGF and trifluoroacetic acid solution into the book and adsorbing it, washing with 0.05% trifluoroacetic acid water, perform gradient elution of 0 to 90% acetonitrile in the same solution, and elute at the acetonitrile concentration position of 55 to 75%. Take the active fraction. The above operation was repeated three times, and the entire volume of the concentrated solution was treated with the molecular sieve membrane, and 7.9 ml of the TGF active fraction was collected. After freeze-drying this fraction, it was dissolved again in 3.0 ml of physiological saline, and this solution was added to TGF
In 0.4 μl of the test method, normal cells were transformed and showed colony-forming ability. [Table] TGF activity test method materials (1) NRK-cells (49F strain) (2) Dulbecco's modified MEM medium supplemented with 5% fetal bovine serum and 1% non-essential amino acids (3) Purified agar (Agar Noble, manufactured by Difco) ) (4) Operation using a Petri dish with a diameter of 60 mm Pour 5 ml of the medium in (1) containing 0.5% agar into each Petri dish to form the lower layer medium. 0.3% containing 1 x ¹⁰³ NRK cells/ml on top of this
2 ml of agar-containing medium was layered on each Petri dish to form an upper layer medium, on top of which 0.5 ml of a sample dissolved in physiological saline was added, and the mixture was placed in a carbon dioxide incubator.
Incubate at 37°C. Removed from the incubator after 14 days,
Obtain a 2 mm x 2 mm field of view on a Petri dish using a 40x Kenbi mirror.
Randomly select 10 locations, count the number of colonies consisting of 10 or more cells, and use the total number as the result.

[Brief explanation of drawings]

Figure 1 is the elution curve of gel filtration in Reference Example 1, Figures 2 and 3 are the elution curves of ion exchange chromatography in Example 1 and reversed phase chromatography in Example 3, respectively. FIG. 5 shows the results of sucrose concentration gradient isoelectric focusing and SDS electrophoresis in Example 3, and BPB in FIG. 5 is an abbreviation for promphenol blue.

Claims (1)

[Claims] 1 Urine from a cancer patient is dialyzed, and the dialysed fluid from which low-molecular substances have been removed is (1) adsorbed to a cation exchanger, and then treated with a neutral salt concentration gradient aqueous solution. Ion exchange chromatography using gradient elution; (2)
The cell-transforming growth factor is subjected to at least one of the following operations: reverse phase chromatography, in which the factor is adsorbed onto a carrier having a hydrophobic group, and then gradient elution is performed using a concentration gradient aqueous solution of a hydrophilic neutral organic solvent. A method for producing a cell-transforming growth factor, which comprises collecting an active fraction. 2. The production method according to claim 1, wherein the dialysis fluid is subjected to gel filtration to collect fractions exhibiting cell-transforming growth factor activity, and then subjected to predetermined operations.