JPH04320684A - Production of human asparaginase - Google Patents

Abstract

PURPOSE:To obtain, in significantly high efficiency compared to the method for acquisition from urea, the title enzyme causing no allergic symptoms by incubating human cells stemmed from the lung, stomach or breast and by acquiring human asparaginase from the resultant cultured cells and culture solution. CONSTITUTION:Human cells stemmed from the lung, stomach or breast [e.g. lung adenocarcinomatous cells A549 (FERM P-12189)] are incubated at a pH of 7.0-7.2 at 37 deg.C for 6 days in a medium containing 5% bovine fetal serum, and the resulting cells are removed from the culture vessel and recovered through centrifugation. These cells are then suspended in a phosphoric acid buffer solution, washed, and irradiated with ultrasonic wave for 3-5min, while cooling in ice, to effect disintegration. The resulting liquid is centrifugalized in high speed, insoluble fractions being removed, the supernatant being passed through a filter to effect sterilization. Thence, the filtrate is concentrated with a ultrafiltration equipment, thus obtaining in high efficiency the objective human asparaginase causing no allergic symptoms and useful for medicines, etc.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides human asparaginase (LA
Sparagine amidohydrolase:
EC 3.5.1.1. ).

0002

[Prior Art] Asparaginase is an enzyme that catalyzes the reaction of hydrolyzing L-asparagine to produce L-aspartic acid and ammonia, and is known to exist widely in living organisms such as mammals and microorganisms. , and is currently on the market as a medicine.

[0003] In recent years, it has become clear that asparaginase derived from guinea pig serum and asparaginase produced by microorganisms such as Esserichia, Serratia, and Erwinia are highly effective in treating acute lymphocytic leukemia. The reason for this is explained as follows.

[0004] That is, acute lymphocytic leukemia cells are
- Asparagine is one of the essential nutritional sources, but as they lack asparagine synthetase, they cannot synthesize L-asparagine, so they cannot grow under conditions where L-asparagine is insufficient or absent. It dies out. On the other hand, L-asparagine is not essential for the survival and proliferation of normal cells. Therefore, by hydrolyzing L-asparagine with asparaginase, only the leukemia cells can be selectively killed.

[0005] There have been many reports on the antitumor effect of asparaginase both in vivo and in vitro (Ichita Amaki: Treatment of Acute Leukemia, Clinical Blood, 2
5, 447-459, (1984)).

[0006]

[Problem to be solved by the invention] However, currently,
Asparaginase, which is commercially available as a pharmaceutical, is derived from microorganisms. Therefore, it is presumed that there are some differences in the amino acid sequence from those derived from humans, and it is not immunologically preferable to administer this to the human body.

That is, when asparaginase derived from microorganisms is administered to the human body, the production of antibodies against the asparaginase may be induced, resulting in immediate allergic symptoms such as rash and chills. In particular, if IgE antibodies are produced, long-term administration is not possible because there is a high possibility that dangerous side effects such as systemic anaphylactic shock will occur. For these reasons, treatment with asparaginase derived from microorganisms is currently extremely limited.

[0008] Therefore, a method for producing human-derived asparaginase, which is not immunologically recognized as an antigen, has been desired. A method has been reported (Japanese Unexamined Patent Publication No. 119082/1982) in which the antigen-determining site on the surface of the derived asparaginase molecule is chemically modified with polyethylene glycol to reduce antigenicity. However, in the former case, it is difficult to continuously obtain fresh human urine on an industrial scale, and in the latter case, although the antigenicity has almost disappeared, the residual activity is only about 10%, and the antitumor activity is low. Become something.

[0009] Accordingly, an object of the present invention is to provide an industrially advantageous method for producing human asparaginase, which is non-antigenic and has excellent antitumor activity.

0010

[Means for Solving the Problems] That is, the present invention is characterized in that human cells derived from the lungs, stomach, or breasts such as breasts are cultured, and human asparaginase is obtained from the cultured cells and/or the culture solution. The present invention provides a method for producing human asparaginase.

Human cells used in the present invention include epithelial cells derived from the lung, stomach, or breast, such as lung adenocarcinoma cells A549, gastric cancer cells MKN-45/K, and breast cancer cells Z.
Examples include R-75-1. Characteristics of these cells,
The properties and acquisition route are as follows.

(1) Lung adenocarcinoma cells A549 (AT
CC CCL185, Microtechnical Research Institute No. 12189) 58
An established cell line derived from lung cancer tissue of a young man, which proliferates uniformly in a monolayer. Morphologically Epithelial
-Like, the number of chromosomes is distributed between 53 and 67. American Type Culture Col.
lection (Rockville, Maryl
and), but since 1976 M. L
ieber (Department of Urol
Mayo Clinic, Rockville
LE, Maryland). (2) Breast cancer cell ZR-75-1 (ATCC C
RL1500, Microtechnical Research Institute No. 12188) An established cell line derived from malignant ascites exudate of a 63-year-old woman, with a chromosome number of 60 to
75, and the doubling time is about 80 hours. These cells have receptors for estrogen and other steroid hormones. Although stored and passaged at ATCC, L. W. Engel (NCI, NIH, Be
thesda, Md. ) Provided by. (3) Gastric cancer cells MKN-45
No. 2187) This is a cell line cultured from liver metastasis of a 62-year-old gastric cancer patient, and the doubling time is approximately 26 hours. These cells adhere to and proliferate in small round or spindle-shaped petri dishes, but they often tend to clump together and form clusters of cells like clusters of grapes. In addition, these cells do not exhibit a cobblestone-like arrangement, but instead spread as cancer cells fall off singly or in groups of several cells from a cell mass at the center of the colony. For this reason, there are no clear boundaries around the colony. Cultured cells have large nuclei and distinct nucleoli, and are relatively lacking in cell bodies (Haruto Hojo: Niigata Medical Society Journal, No. 9)
Volume, No. 11, 1978).

[0013] In order to obtain human asparaginase using these cells, the cells may be inoculated into a medium and cultured. Examples of the culture medium include ERDF medium, Dulbecco's modified Eagle's medium (DME), and RPMI16.
Examples include basic media such as Hams F12 medium and Hams F12 medium to which 5 to 20% of fetal bovine serum, bovine serum, etc. are added. In addition, as nutritional sources, carbon sources, nitrogen sources, inorganic salts,
Amino acids, nucleic acids, vitamins, etc. are required, and basically those described in "Tissue Culture" (edited by Junnosuke Nakai et al., published by Asakura Shoten in 1976) are added. Furthermore, various growth factors (insulin, transferrin, ethanolamine, selenium, egg yolk lipoprotein, etc.) can be added to replace a portion of serum. For other culture methods, methods used in normal cell culture, such as the method described in "tissue culture" mentioned above, can be applied. For example, the pH and temperature of the medium can be adjusted to The conditions normally used for growth are generally pH 7.0 to 7.2 and temperature 35 to 37°C.

[0014] Culture vessels are all those commonly used for animal cell culture, such as ordinary culture dishes, roller bottles, plastic bags, spiral films, hollow fibers, capillaries, glass beads, multilayer plates, and spinner culture vessels. Can be applied to inventions. After culturing, asparaginase can be obtained by salting out (ammonium sulfate fraction), organic solvent precipitation (acetone),
It is purified by conventional enzyme purification methods using a combination of chromatography (cationic, anionic), gel filtration, centrifugation, ultrafiltration, etc.

The activity of the obtained asparaginase was measured by J.
．． Bacteriol. : Volume 122, No. 1017-
1024 (1975) and Cancer Research
It can be carried out by the method described in Vol. 30, No. 929-935 (1970). That is, L-
L-aspartic acid is produced from asparagine by the action of asparaginase, and this L-aspartic acid is converted to malic acid via oxaloacetate by the enzymatic action of L-glutamate-oxaloacetate transaminase (GOT) and L-malate dehydrogenase. It is decomposed until At this time, by monitoring the amount of NADH (reduced form of the coenzyme NDA involved in redox reactions) consumed, it is possible to quantify the L-aspartic acid produced, and thereby the activity of asparaginase can be measured. can. More specifically, this can be done as follows.

That is, after culturing the above cells, at 37°C,
Cells were detached from the chalet by trypsinization for 5 min (final concentration 0.2%) and centrifuged (10 min).
The cultured cells obtained were suspended in phosphate buffer (pH 7.4), the cells were broken by ultrasonication, and the resulting cell debris was centrifuged (10,000 rpm x 10
(min) and use the supernatant as a sample for activity measurement. Add 900μl of 10mM to 100μl of this sample.
- Add asparagine and react for 60 minutes at 37°C (reaction solution-1). Immediately after the reaction, immerse in boiling water at 100°C for 5 minutes to stop the enzyme reaction, and then cool in ice water. Then α-ketoglutarate, NADH, GOT and L-
Add 400 μl of the above reaction solution-1 to 600 μl of the reaction solution containing malate dehydrogenase, react at 37° C. for 30 minutes, then add 200 μl of 2N NaOH to stop the reaction, and measure the absorbance at 340 nm. At this time, the activity (potency) of asparaginase is at pH 7.
．． 4 (phosphate buffer), the amount of enzyme capable of releasing 1 μmole of NH3 from L-asparagine in 1 minute at 37°C was 1
Define it as a Unit.

[0017]

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 A549 cells were grown in a plastic Petri dish (BE) with a diameter of 9 cm using 5% fetal bovine serum/DME as a medium.
CTON-DICKINSON), 1 x 105
Cells/dish were seeded at a cell density of 7.
0 to 7.2, and cultured for 6 days at a temperature of 37°C. Cells cultured in these three dishes were cultured in a roller bottle (100
ml) in a 10% FBS/DME medium for one week (pH 7.0 to 7.2, 37°C). The cells were then detached from the bottle and centrifuged (2000 rpm).
×5min). 10 m of the obtained cells
After suspending and washing the cells in 1 liter of phosphate buffer, the cells were centrifuged again to collect the cells. This was suspended in 1 ml of phosphate buffer and crushed by ultrasonic irradiation for 3 to 5 minutes while cooling on ice. This crushing liquid was heated at 10,000 rpm.
Centrifuge for 5 minutes at
It was passed through a filter and sterilized by filtration. The obtained filtrate was concentrated about 4 times using an ultrafiltration device, and the asparaginase activity was measured using this as a sample. As a result, the activity is
It was 0.002 Unit/ml. On the other hand, a sample was prepared by concentrating the culture fluid separated from the cells using an ultrafiltration device, and its asparaginase activity was measured, and the activity was found to be 0.005 Unit/100ml.

Example 2 MKN-45 cells were grown in a 9 cm diameter plastic Petri dish using 5% FBS/ERDF as a medium.
Seed at a cell density of ×105 cells/dish,
The cells were cultured for 5 days in the same manner as in Example 1. Transfer the cells cultured in these three dishes to a roller bottle (100ml).
The cells were cultured for one week in a 5% FBS/ERDF medium in the same manner as in Example 1, and the culture solution was collected and the cells were crushed to prepare samples. Asparaginase activity was measured and found to be 0.001 Unit/ml in the cell fraction.
The culture solution was 0.002 Unit/100ml.

Example 3 ZR-75-1 cells were treated with 10% FBS/RPMI 1
640 was used as a medium and seeded in a plastic Petri dish with a diameter of 9 cm at a cell density of 6 x 105 cells/dish, and cultured for 4 days in the same manner as in Example 1. The cells cultured in these three dishes were mixed with 10% FBS/RPMI 1 using a roller bottle (100 ml).
640 medium for one week in the same manner as in Example 1, the culture solution was collected, the cells were crushed, and samples were prepared. When asparaginase activity was measured, it was found to be 0.0 in the cell fraction.
008 Unit/ml, 0.001 Un in culture fluid fraction
It/100ml.

[0021]

According to the present invention, human asparaginase that does not cause allergic symptoms can be obtained more efficiently than the conventional method of obtaining it from urine.

Claims (1)

[Claims] 1. A method for producing human asparaginase, which comprises culturing human cells derived from lung, stomach, or chest, and obtaining human asparaginase from the cultured cells and/or the culture solution.