JPH0780914B2 - Antibodies to human tumor necrosis factor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to monoclonal antibodies specific for human tumor necrosis factor.

Conventionally, the acquisition of a specific antibody against a certain antigen has been achieved by obtaining an antiserum from an animal immunized with the antigen.
However, the specific antibody in this antiserum is in a mixed state with other antibody groups, and it is very difficult to separate the necessary specific antibody from other antibody groups. The obtained specific antibody is also a mixture of specific antibodies having different properties of reacting with different antigenic determinants. Furthermore, various specific antibodies with different avidity against one antigenic determinant are produced. For the above reasons, the conventional reaction between the specific antibody from the antiserum and the antigen is a mixed reaction, and has been limited in kind depending on the application.

Research in the field of molecular biology now provides alternative sources of antibodies that can solve the above problems. The fusion between lymphocyte cells and myeloma cells of mammalian (eg, mouse or rat) origin is capable of proliferating in vitro and producing replicable hybrid cells (Koehler and Myrshatin, Nature. , Volume 256, 495
~ 497, see 1975) has been discovered. Such hybrid cells have the property of secreting antibodies with a predetermined specificity. This specificity is the specificity of the antibody produced by the lymphocytes involved in the fusion. By hybridizing and culturing the hybrid cells under stable culture conditions, an infinite number of samples containing an antibody having specificity only for a certain antigenic determinant are continuously produced.
The antibody thus produced is known in the art as a monoclonal antibody.

As a general method for producing the above-mentioned hybrid cells, an antigen is administered to a mammal (usually a mouse or a rat, but not limited to these two) to immunize and secrete an antibody against the antigen. After a sufficient number of doses of antigen and sufficient immunization time to produce many white blood cells, the animal is killed,
Prepare a suspension of spleen cells. Fusion of these cells with myeloma cells is accomplished by contacting them in the presence of a fusion promoting agent (eg, polyethylene glycol). Very few cells fuse to form hybrid cells. As a result of the immune response, multiple different lymphocytes, each secreting antibodies to different antigenic determinants, are formed, and these traits are genetically transmitted to the hybrid cells. By carrying out a careful screening, cells secreting an antibody having a desired specificity can be isolated from the cultured hybrid cell group. Such cells can be cloned and cultured.

An advantage of this technique is that it provides a source of specific antibodies that is free of antibodies corresponding to other antigenic determinants in the antigenic impurities contained in the immunizing antigen or substance.

The present invention relates to human tumor necrosis factor (hereinafter referred to as human TNF)
To a monoclonal antibody against. It is known that human TNF is induced from the macrophages cell group by various substances having an activity of activating the reticuloendothelial system, such as various Gram-positive bacteria and endotoxin.

Induced by various substances that have an activation effect in the reticulum,
The existence of substances having physiological activity such as antitumor cell ability has been reported many times. For example, Carswell et al.
Bacillus Calmette Gurin (BCG) was administered to mice,
Two weeks after that, the serum of the mouse obtained by intravenous injection of endotoxin had a cytocidal effect on the cultured cells, and the tumor was carried by Meth A sarcoma (BA.
LB / C × 5.7BL / 6) F ₁ We found a phenomenon leading to hemorrhagic necrosis in mice and named it TMF [Proc.Nat.Acad.Sc].
i.USA 72 (No.9), page 3666, (1975)]. afterwards,
Reed et al. [J. Immunol. 125 (No. 4), 1671, (1980
Year)) and Mattews et al. [Br. J. Cancer 42, 416, (1
980)] tried to purify TNF from rabbit serum prepared according to the method of Carswell et al., And obtained those purified about 2000 times and about 1000 times, respectively, compared to the original serum. However, in any case, the antitumor effect of the purified product has not been confirmed in animal experiments. Also
Ruff et al. [J. Immunology 115, 395, (1975)]
It has been reported that a factor having TNF-like activity was found in leukemia cells derived from human peripheral blood monocytes and myelomonocytic leukemia patients, but even an antitumor effect in animal experiments has not been confirmed. The body is not clear.

The inventors of the present invention have been earnestly researching a protein-based physiologically active substance having an antitumor effect, which is induced in a tissue culture system containing human-derived activated macrophages.
We found a new protein bioactive substance with excellent antitumor activity of 40,000 and isoelectric point of about 6.0, and completed the invention.
We have already filed an application.

A physiologically active substance having this antitumor activity, that is, human
Since TNF has an extremely small amount and strong physiological activity, it has attracted a great deal of attention, and it has been desired to obtain a specific antibody as a means for its separation / purification and quantitative analysis. It was impossible to obtain a monoclonal antibody having a specific reactivity when TNF having a low purity and a low degree of purification was used as an antigen.

As a result of various studies to obtain an antibody specific to human TNF, the present inventors have purified human TNF.
By immunizing mice with the antigen as an antigen, antibody-producing cells were obtained, and it was found that a monoclonal antibody having specificity for human TNF can be stably produced for the first time by hybrid cells of the cells and myeloma cells. Thus, the present invention has been completed.

That is, the present invention, at least reacts to human tumor necrosis factor having the following properties, rabbit, mouse,
The present invention relates to a monoclonal antibody that does not inhibit LM cell cytotoxicity in the presence of tumor necrosis factor derived from guinea pig and hamster in the presence of each tumor necrosis factor.

1) A protein produced by human macrophage cells.

2) Molecular weight 40,000 ± 4,000 (gel filtration method) 20,000 ± 4,000 (SDS-polyacrylamide gel electrophoresis method) 3) It has cytotoxic activity against LM cells.

4) Causes hemorrhagic necrotic reaction in meth A sarcoma tumor-bearing mice.

The monoclonal antibody according to the present invention can be used for separation and purification of TNF, immunoassay and the like.

Hereinafter, the cell fusion method will be specifically described.

(A) Preparation of antibody-producing cells Preparation of antibody-producing cells may be performed according to a conventional method. That is, a method of immunizing an animal with human TNF, which is an antigen, and obtaining antibody-producing cells of the animal may be used.

Animals include mice, rats, rabbits, guinea pigs,
Examples thereof include sheep, horses, and cows, and splenocytes, lymph node cells, peripheral blood cells and the like are used as antibody-producing cells.

(B) Preparation of myeloma cells The myeloma cells used in the cell fusion method are not particularly limited, and many animal cell lines such as mouse, rat, rabbit and human can be applied. The cell line used should preferably be drug resistant so that unfused myeloma cells cannot survive in the selective medium and only hybrid cells grow. The most commonly used cell line is 8-azaguanine resistant, which lacks hypoxanthine guanine phosphoribosyl transferase.
It has the property that it cannot grow in hypoxanthine-aminopterin-thymidine (HAT) medium. Further, the cell line used is preferably a so-called “non-secretory type”. For example, P ₃ / X63- derived from mouse myeloma line MOPC-21
Ag8U1 (P ₃ U1), P ₃ / X63-Al ・ 6.5 ・ 3, P ₃ / NS1-1
-Ag4-1, Sp2 / O-Ag14, rat myeloma cells 210 / RCY3
-Ag1, 2.3, etc. can be used conveniently.

(C) Cell fusion 1 to 5 × 10 ⁷ myeloma cells and antibody-producing cells 1 to 1 in normal Eagle's minimum basic medium (MEM), Roswell Park Memorial Institute (RPMI) 1640 medium, etc. Mix 5 × 10 ⁸ pieces (mixing ratio is usually 1: 4 to 1:10),
Cell fusion is performed. As the fusion promoter, polyethylene glycol (PEG) having an average molecular weight of 1,000 to 6,000 is preferable, but viruses and the like can also be used. The concentration of PEG used is usually 30 to 50%.

(D) Selective Proliferation of Hybrid Cells Cells that had completed cell fusion were RPMI16 containing 20% fetal bovine serum.
Dilute appropriately with 40 medium etc. and inoculate to a microtiter plate at about 10 ⁵ -10 ⁶ . A selective medium (for example, HAT medium) is added to each well, and then the selective medium is appropriately replaced and cultured. If an 8-azaguanine-resistant strain is used as the myeloma cell, all unfused myeloma cells will be dead in HAT medium by about 10 days, and the antibody-producing cells will be long in vitro because they are normal cells. I can't grow for hours. Therefore, anything that grows after about 10 to 14 days of culture is a hybrid cell.

(E) Search for antibody-producing hybrid cells Screening for hybrid cells may be performed by a conventional method without any particular limitation. For example, a part of the supernatant of a well in which hybrid cells have grown is collected, reacted with human TNF or immobilized human TNF, and then a second antibody labeled with an enzyme, a radioisotope, a fluorescent substance chamber, and a luminescent substance is used. According to the reaction of 1, the amount of labeling can be measured and the presence of anti-human TNF antibody can be assayed.

(F) Cloning Since two or more hybrid cells may grow in each well, cloning is performed by the limiting dilution method or the like to obtain monoclonal antibody-producing hybrid cells.

(G) Obtaining antibody The purest monoclonal antibody yields 10% of desired hybrid cells.
It can be obtained from the culture supernatant by culturing in an appropriate culture medium such as RPMI1640 medium containing about fetal bovine serum.

On the other hand, to obtain a larger amount of antibody, pristane (2,6,10,14-
A large amount of hybrid cells may be grown in vivo by administering a mineral oil such as tetramethylgentadecane) in the retrocavitary cavity and then administering the hybrid cells. In this case, ascites tumors are formed in about 10 to 18 days, and high-purity antibody is produced in serum and ascites.

The antigen human TNF referred to in the present invention has the amino acid composition shown below.

Amino acid content (mol%) Asparagine + aspartic acid 7.8 Threonine 3.6 Serine 8.2 Glutamine + Glutamic acid 12.9 Glycine 7.1 Alanine 8.5 Valine 7.8 Cystine 1.4 Tryptophan 1.4 Isoleucine 5.0 Leucine 11.4 Tyrosine 4.6 Phenylalanine 2.7 Lysine 3.9 Histidine 2.0 Arginine 5.3 Proline 6.5 It is a protein that has a structure that does not depend on the subunit and has the following characteristics.

a) Molecular weight 40,000 ± 4,000 (gel filtration method) 20,000 ± 4,000 (SDS-polyacrylamide electrophoresis method) b) Isoelectric point 6.0 ± 0.5 (isoelectric focusing method) c) Use LM cells as defined in the text The specific activity in the evaluation was 1 × 10 ⁶ units / mg protein. Further, the substance of the present invention was the same as Meth A sarco described in Experimental Example 4.
In the biological evaluation using ma tumor-bearing BALB / C mice, the activity when 100 to 200 units per mouse was intratumorally administered was (+) or more.

The antigen human TNF in the present invention is derived from human-derived activated macrophages. Human-derived activated macrophages were established from compositional macrophages such as spleen or lung tissue macrophages, peripheral blood monocytes cultured in the co-presence of substances capable of inducing differentiation or leukemia cells of patients with certain myelomonocytic leukemias. A cell line or the like can be used. Examples of the cell line include HL-60 cells [Nature 270, p. 347 (1977)] THP-1 cells (Int. J. Cancer 26, p. 171 (1980)] and the like. Is 12-O-tetradecanoylphorbol-13-
Phorbol esters such as acetate, phorbol-12,13-didecanoate, phorbol-12,13-dibenzoate, diterbenes such as mezerein, vitamin A derivatives such as teleocidin, vitamin A acid, vitamin A alcohol, dimethyl sulfoxide, peptone Alternatively, casein hydrolyzate and the like are preferably used.

The activated macrophages in the present invention produce a larger amount of human TNF by culturing endotoxin derived from Gram-negative bacterium or Gram-positive bacterium or yeast cell wall in the coexistence.

The essence of the preferred method for purifying human TNF for obtaining the antigen in the present invention is to subject the culture solution of activated macrophages to the next step.

(1) Electrophoretic isoelectric point fractionation method (2) Basic anion exchanger chromatography (3) Gel filtration (4) Affinity chromatography (5) Gel filtration Human TNF purified as an antigenic substance The physical properties are measured by each of Experimental Methods 1 to 8 described below.

Experimental method 1) Molecular weight measurement method A) Gel filtration with 0.1 M sodium chloride / 50 mM phosphate buffer (pH 7.4) using a column (1.5 × 100 cm) of Cefacrylic S-200 (Falmatia, Sweden). As a result, the molecular weight was 40,000 ± 4,000.

B) The method of Segrest et al. [Method in Enzymology 28-B
Vol. 54 (1972)], Tris / Glycine / SDS
When 10 μg of sample was applied to SDS / polyacrylamide gel at (pH 8.3) and electrophoresis was performed, the molecular weight was 20,000 ±
At 4,000 positions, a stained band and activity were shown.

2) Isoelectric point measuring method Isoelectric focusing device (SJ-1071EC
Type, using Fumarulite (Pharmacia, pH4)
.About.6.5) and 5% polyacrylamide slab gel containing glycerol (thickness 1 mm, length 10 cm, width 10 cm) was prepared.
0.04M on the anode side, DL-glutamic acid, 0.2M, L on the cathode side
-Histidine was used to perform a pre-migration at 700V for 50 minutes. Then, add 50μg of sample, 700V for 1 hour, 500V
The electrophoresis was performed for 16 hours. After completion of the electrophoresis, cut out the gel with a width of 2.5 mm, and then cut each gel piece with 0.02M containing 0.15M sodium chloride.
Extraction with 0.2 ml of Tris-hydrochloric acid buffer solution (pH 8.2) and activity evaluation using LM cells for each extract were carried out.
The isoelectric point of human TNF was 6.0 ± 0.5.

3) Activity evaluation using LM cells The activity evaluation using LM cells is performed by Ruff [Lymphokine Rep
Volume 2, orts, edited by E. Pick, Academic Press, page 235 (1980
Or (J. Immunol. 126, 235 (1981)]
According to the method of 1. above, the present inventors have improved and measure the effect of the physiologically active substance according to the present invention to kill LM cells (American type culture collection, CCL1.2). That is, 0.1 ml of a sample serially diluted with a medium and 0.1 of a medium suspension of LM cells at a concentration of 10 ⁵ co / ml
ml was added to a 96-well tissue culture microplate (Flow Laboratory). The medium used was Eagle's Minium-Etsusenshiyaru medium containing 1 V / V% fetal bovine serum (the composition is described in, for example, "Tissue Culture" edited by Junnosuke Nakai et al., Asakura Shoten, 1967). The microplate was incubated at 37 ° C. for 48 hours in air containing 5% carbon dioxide. After the culture was completed, 20 μl of glutaraldehyde was added to fix the cells. After fixation, the microplate was washed and dried, and 0.1 ml of 0.05% methylene blue solution was added to stain the surviving cells. The excess methylene blue was washed off and dried, and the remaining methylene blue was extracted with a 0.36N hydrochloric acid solution, and the absorbance at 665 nm was measured by Titertec Multisky (Flow Laboratory). This absorbance is proportional to the number of surviving cells. L-
1 unit / m of bioactive amount required to kill 50% of M cells
It is defined as l, and the dilution rate of the sample corresponding to 50% of the absorbance of the control without addition of the sample is obtained by graph or calculation, and the reciprocal of the dilution rate is calculated as the reciprocal of the dilution rate (unit / ml of sample).
Will be written as).

On the other hand, the protein amount was determined by the method of Branford et al. [Anal. Biochem.
2, p. 248 (1976)], and calculated from the dye binding method using Coomassie Brilliant Blue G250.

The specific activity of the antigen human TNF in the present invention is 1 × 10 ⁶ units / m 2.
It was g protein.

4) Activity evaluation using Meth A sarcoma tumor-bearing mice 2 × 10 ⁵ Meth A sarcoma in the abdominal skin of BALB / C mice
7 days after transplanting cells, the size of the transplanted tumor was 7
Select a mouse with a tumor that is 8 mm and has a good blood circulation without hemorrhagic necrosis, inject a 0.05 ml sample diluted with physiological saline into the tumor, and after 24 hours, test the necrosis reaction according to the following criteria. I took a measurement.

(-): No change (+): Slight hemorrhagic necrosis (): Moderate hemorrhagic necrosis (necrosis 50% or more from the center of transplanted cancer surface) (): Marked hemorrhagic necrosis (transplanted cancer) In the state where the central part of the mouse was severely necrosed and the surrounding cancer tissue was slightly left) In addition, 20 days after the sample administration, it was observed whether the cancer had completely regressed, and the complete cure rate was calculated.

The activity of human TNF measured by the above method is shown in the table below.

5) Amino acid composition determination The following studies were performed using the human TNF of the present invention.

An SDS-polyacrylamide slab gel containing 15% polyacrylamide having a thickness of 1 mm, a length of 11 cm, and a width of 15 cm was prepared for sample preparation. The details of the preparation method were based on the pan frets of "Slab-type SDS-acrylamide gel electrophoresis" manufactured by Atto Co., Ltd. The apparatus used was SJ-1060.SDH type manufactured by ATTO CORPORATION.

400 μg of human TNF of the present invention is added to each plate gel.
Granted. 1% SDS aqueous solution 1 as pretreatment before application
00 μl and 40% sucrose water solution 200 μg of human TNF was applied to each plate gel. Upon application, as a pretreatment, 100 μl of 1% SDS aqueous solution and 50 μl of 40% sucrose aqueous solution were mixed with 500 μl of an aqueous solution containing 200 μg of human TNF, and heat treatment was performed at 50 ° C. for 30 minutes. Electrophoresis took 150 hours at a constant voltage of 4 hours and 30 minutes. This operation was repeated 5 times. After the electrophoresis, a part was stained with Coomassie Brilliant Blue G250 for 5 minutes, and the stained band clearly visible after decolorization was cut into 5 pieces at 2.5 mm intervals. Then 1.5 ml of 1% ammonium bicarbonate
The gel piece was dipped in and extracted at 4 ° C. for 24 hours. After extraction, remove the extract with a pipette and wash with the same solution to extract 2.5 ml of each extract for 5 slices.
I got it. The obtained extract was evaluated for activity using LM cells for confirmation. Most of the activity was recovered in the central band.

The extracted sample solution was applied to a column of Sephadex G25 (0.9 × 10
cm) and desalted. Then made by Tommy Seiko,
A centrifugal vacuum dryer concentrator (EC-10) was used to perform drying at 1,500 rpm at 25 ° C. for 2 hours to obtain an amino acid composition analysis sample.

The sample was hydrolyzed in vacuum with 4M methanesulfonic acid at 110 ° C for 24 hours, and then it was used as a high-speed amino acid analyzer (Hitachi, 835
Amino acid composition (mol%) was measured using the (type).

In addition, cysteine was hydrolyzed and then air-oxidized to be converted into cystine.

The amino acid composition of human TNF was blood as shown below.

Amino acid content (mol%) Asparagine + Aspartic acid 7.8 Threonine 3.6 Serine 8.2 Glutamine + Glutamic acid 12.9 Glycine 7.1 Alanine 8.5 Valine 7.8 Cystine 1.4 Tryptophan 1.4 Isoleucine 5.0 Leucine 11.4 Tyrosine 4.6 Phenylalanine 2.7 Lysine 3.9 Histidine 2.0 Arginine 5.3 Proline 6.5 or higher As described above, the human TNF in the present invention has an extremely excellent antitumor effect, its action has little species specificity, and has a wide range of effects, and in addition, since toxicity is hardly observed, it is extremely useful. It can be expected as an antitumor agent.

The above-mentioned human TNF was poor in antigenicity due to its low species specificity, and it was difficult to obtain an antibody.However, by removing impurities as much as possible and using highly purified human TNF, human It was possible to make a monoclonal antibody against TNF.

According to the present invention, a monoclonal antibody characterized by having specificity for human tumor necrosis factor (human TNF) is provided by a hybrid cell line.

The present invention will be described with reference to the following experimental results, but it should be understood that these are merely examples and the present invention is not limited thereto.

Example Purification of Antigen THP-1 cells were dispersed in RPMI-1640 medium containing 5% fetal bovine serum at a concentration of 1.5 × 10 ⁶ cells / ml, and then dispensed in 20 ml aliquots in a plastic sheathe having a diameter of 10 cm. . To the cell solution heated to 37 ° C., 12-O-tetradecanoylphorbol-13-diacetate and vitamin A acid were added so that the final concentrations were 1 μg / ml and 0.5 μg / ml, respectively.
The cells were cultured at 37 ° C. for 1 hour in a% carbon dioxide incubator.

After 1 hour, the culture solution in the dish was removed and replaced with 20 ml of 5% fetal bovine serum-RPMI-1640 medium containing 0.5 μg / ml vitamin A acid and 100 mg / ml polypeptone, and then for another 24 hours 37
After culturing at ℃, the medium was removed and 20 ml of RPMI-16
The medium was replaced with 20 ml of 40 medium and the culture was further continued for 72 hours.

After that, the culture solution was collected and centrifuged at 3000 rpm for 30 minutes to remove the cell precipitate, and then the cytotoxicity to LM cells was examined to obtain a culture solution having an activity of 180 units / ml. It was

Next, an example of an experiment for purifying 4 liters of the culture solution will be illustrated.

Purification step 1 (isoelectric focusing) 4 l of the culture solution was concentrated 10 times by ultrafiltration and then
pholine [Carrier / Am
pholytes)) (LKB, Sweden) -Sucrose density control
After adding to the distribution column (440 ml), energize for about 42 hours,
After running the electrophoresis, use a tube thinner than the lower end of the column.
Fractionation was performed at a flow rate of about 2 ml / min without disturbing the chamber.

After collecting the pH 5.5-6.5 fractions, adjust the pH to about 7.4.
After that, on dialysis, Ampholine And remove sucrose
It was

Purification step 2 (anion exchange chromatography) 2 liters of 0.02 M Tris-hydrochloric acid buffer solution (pH 7.8) per 0.5 liter of the fractionation liquid
And 0.02M Tris-containing 0.02M sodium chloride.
It was gradually applied to a column (10 × 90 cm) of a base anion exchanger DEAE-Sepharose CL-6B (manufactured by Pharmacia) fully equilibrated with a hydrochloric acid buffer (pH 7.8). Then 1.5l
Column equilibration buffer (0.02M containing 0.02M sodium chloride)
After washing with 2M Tris-HCl buffer (pH 7.8), 0.02M Tris-acetate buffer (pH 7.8) containing 0.05M sodium chloride
After washing with 1, elution was performed using 0.02M Tris-hydrochloric acid buffer (pH 7.2) containing 0.1M sodium chloride. The flow rate was 0.5 l / hr, the eluate was fractionated by 50 ml, and the active fractions were collected. The active fraction was diluted with the same volume of 0.02 M Tris-HCl buffer (pH 7.8) and then diluted with DEAE at a flow rate of 0.2 l / hr.
-Applied to a column of Sepharose CL-6B (10 x 13 cm).

Then, using 2 l of 0.02 M Tris-hydrochloric acid buffer solution (pH 7.8) containing 0.05 M sodium chloride, washing was performed at a flow rate of 20 ml / hr, and then 0.02 M Tris-hydrochloric acid buffer solution containing 0.1 M sodium chloride ( Elution was carried out using pH 7.2) 1 at a flow rate of 20 ml / hr. The eluate was fractionated in 25 ml portions to collect active fractions. At this stage, the recovery rate of activity was 60% and the degree of purification was 300 times.

Purification step 3 (gel filtration) 0.005M phosphate buffer containing 0.1M sodium chloride (pH 7.
The concentrated solution was applied to a column (5 × 80 cm) of Cefacrylic S-200 (manufactured by Pharmacia) fully equilibrated in 4), and gel filtration elution was carried out with the same buffer solution. Flow rate is 4ml / hr, 4m
The active fractions were collected by fractionation by l, and the active fractions were concentrated by ultrafiltration. The activity recovery rate through the purification steps 1 to 3 was 45%, and the degree of purification was 4 × 10 ³ times.

Purification Step 4 (Affinity Chromatography) The concentrated solution of the active fraction obtained by gel filtration was applied to a Zn ²⁺ chelate sepharose column. Column packed with chelate sepharose (iminodiacetic acid-immobilized resin) (1.6
X 10 cm), 60 ml of 1 mg / ml zinc chloride aqueous solution at a flow rate of 10 ml / h
I washed away with r. After equilibration with 0.05M phosphate buffer (pH7.4) containing 0.1M sodium chloride, apply the concentrate obtained in the previous step at a flow rate of 10 ml / hr, and then add 60 ml of the same buffer. The eluted non-adsorbed fraction was collected. The damaging activity on LM cells was recovered only in this fraction.

The activity recovery rate through the purification steps 1 to 4 was 30%, and the degree of purification was 1 × 10 ⁴ times.

Purification Step 5 (Gel Filtration) Toyopearl HW-55 (Toyo Pearl HW-55, concentrated by concentration of the active fraction obtained in the previous purification step and fully equilibrated with 0.005M phosphate buffer (pH7.4) containing 0.15M sodium chloride) It was applied to a column (1.5 × 90 cm) of Soda Co., Ltd. Flow rate 4 ml with the same buffer
Gel filtration elution was performed at / hr to collect the active fraction. 20% recovery of activity and 2 × purification through all purification steps
It was 10 ⁴ times. The specific activity of the human TNF thus obtained was 8 × 10 ⁵ units / mg protein.

Human TNF purified as described above was electrophoresed on the γ-globulin region by cellulose acetate membrane electrophoresis. In addition, human TNF includes various lectin-immobilized resins (ConA-Sepharose, WGA-Sepharose (all manufactured by Pharmacia) and R
CA-I-gel, UEA-I-gel, LPA-I-gel (above
EY Laboratory))).

Furthermore, the human TNF thus obtained exhibited excellent antitumor effect against various syngeneic transplant mouse cancers and nude mouse transplant human cancers by intratumoral and intravenous administration. That is, B transplanted with mouse-derived colon cancer Colon26
In a test in which human TNF 5,000 to 10,000 units were intravenously administered once to three times to ALB / C mice or A mice transplanted with neuroblastoma Neuro2a, in comparison with the control group (saline administration group) The significant suppression of cancer growth and regression were observed.
In addition, human-derived lung cancer PC-10 and neuroblastoma GOTO were transplanted.
In a test in which 10,000 to 30,000 units of human TNF were intravenously administered to BALB / C nude mice 1 to 7 times, significant suppression of cancer growth and regression were observed as compared with the control group (saline administration group). Admitted.

Immunization of animals 100 μg of human TNF purified as described above was added to Freund's
Emulsify into complete adjuvant, mouse BALB
/ C♂ group was subcutaneously administered at intervals of 2 weeks for immunization.

Of these mice, the mouse with the highest serum anti-human TNF antibody titer was injected intravenously with 100 μg of human TNF to complete immunization.

Cell fusion 3 days after the final immunization, the mouse was killed, the spleen was removed,
Spleen cells obtained by shredding and then filtering with a stainless mesh were washed three times with Eagle's minimal ethical medium (MEM) to prepare mouse myeloma cells (P ₃ / X63-A).
g8U1) was washed once with MEM and splenocytes and myeloma cells were mixed 10: 1.
After mixing and centrifuging, 44% polyethylene glycol 2000 and 1 ml of MEM solution were gradually added to the pellet, and the centrifuge tube was gently rotated for 1 minute for cell fusion. After 1 minute, 1 ml of MEM was added and the mixture was gently rotated, and 1 ml of MEM was further added every 30 diseases to make a final volume of 8 ml. Centrifuge again and put 20 pellets on it.
Roswell Park Memorial with% fetal bovine serum
Myeloma cells were suspended in Institute (RPMI) 1640 medium at 5 × 10 ⁴ cells / 0.1 ml, and 0.1 ml each was seeded in a 96-well microplate.

One day later, 0.1 ml of HAT medium was added to each well, and half of the amount was replaced with HAT medium every 3 to 4 days.
Growth of hybrid cells was observed in some wells from around the day, and after 2-3 weeks, hybrid cells proliferated in almost all wells.

Search and cloning of antibody-producing cells Take 0.1 ml of the culture supernatant from the wells in which hybrid cells have grown, incubate it with human TNF immobilized on a polystyrene 96-well microplate, and search for those that bind to it. As a result, it was possible to recognize wells secreting an antibody capable of binding to human TNF with a probability of 5 to 10%. Several clones having high binding ability were selected, and cloning was performed by a limiting dilution method in which cells were seeded in a 96-well microplate at a concentration of 1 / well, and 8 clones were obtained.

RPMI-16 containing 10% fetal calf serum
96 well plate using 40 medium → 24 well plate →
The cells were cultured in a 25 cm ² flask and scaled up, and the culture supernatant was collected.

Each supernatant was mixed with 1,000 units / ml of human TNF solution, and the activity was evaluated using LM cells.
Only clones inhibited the activity of human TNF. This hybrid cell was named P3TT. The cells can be stored at liquid nitrogen temperature, and the present inventor always stores them. (Furthermore, I applied for a deposit to the Institute of Microbial Technology, Ministry of International Trade and Industry, but was refused.) Next, mouse BALB pretreated with 0.5 ml of pristane containing 2 × 10 ⁶ cells or more. / C was administered intraperitoneally to form an ascites tumor and ascites was collected. This ascites completely suppressed the cytotoxic activity of 100 units of human TNF against LM cells even when diluted 10,000 times.

The antibody derived from P3TT suppresses the activity of antitumor active substances obtained from human lung macrophages (evaluated by LM cytotoxicity), whereas TNF derived from rabbit, mouse, guinea pig and hamster. Did not suppress the LM cell damaging activity.

As described above in detail, the P3TT-derived monoclonal antibody according to the present invention specifically reacts with human TNF and is not only applicable to separation and purification of human TNF. It can be widely applied to quantitative analysis of human TNF by immunoassay using enzyme and radioisotope.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/577 (C12P 21/08 C12R 1:91) (56) References 1. Korean J. Biochem m. , 11 [1] (1979) P. 1-9 2. Infect. Immun. , 42 [1] (1983) p. 385-393 3. Proc. Natl. Acad. S ci. USA, 80 [17] (1983) P. 5397-5401 4. Nature, 256 (1975) P. 495-497

Claims (1)

[Claims] 1. A human tumor necrosis factor having at least the following properties, and a tumor necrosis factor derived from rabbit, mouse, guinea pig and hamster in the presence of each tumor necrosis factor. A monoclonal antibody that does not inhibit LM cytotoxicity. 1) A protein produced by human macrophage cells. 2) Molecular weight 40,000 ± 4,000 (gel filtration method) 20,000 ± 4,000 (SDS-polyacrylamide gel electrophoresis method) 3) It has cytotoxic activity against LM cells. 4) Cause Meth A sarcoma tumor-bearing mice to undergo hemorrhagic necrotic reaction.