JPS60208924A - Antigen against human tumor necrosis factor - Google Patents

Abstract

PURPOSE:Purified human tumor necrosis factor (TNF) is used as an antigen to form hybrid cells to give a monoclonal antibody which can be used in isolation and purification or determination of TNF, because it has a specific reactivity to human TNF. CONSTITUTION:The culture mixture of activated macrophage is concentrated by, e.g., ultrafiltration, then subjected to, e.g., (i) electrophoretic isoelectric fractionation, (ii) basic anion-exchange chromatography, (iii) gel filtration, (iv) affinity chromatography and (v) gel filtration to give purified human TNF. Animals are immunized said human TNF as an antigen to collect antibody-producing cells such as spleen or lymph node cells. The cells are fused with myeloma cells and the resultant hybrid cells are screened and cloned to give the hydrid cells producing monoclonal antibody. The resultant hybridoma is cultrated in an appropriate medium or proliferated in animal's abdominal cavity.

Description

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

Traditionally, the acquisition of specific antibodies against a certain antigen has been achieved by obtaining antiserum from animals immunized with the antigen.However, the specific antibodies in this antiserum are in a mixed state with other antibody groups. -It is very difficult to separate the necessary specific antibodies from other antibody groups.In addition, the specific antibodies obtained also react against VC.
It is a mixture of specific antibodies with different properties. Furthermore, a) various specific antibodies with different binding strengths are produced even for a single antigenic determinant. For the above reasons, conventional reactions between specific antibodies derived from antiserum and antigens are mixed reactions, and have been subject to various limitations in terms of use.

Research in the field of molecular biology now provides alternative sources of antibodies that may solve the above-mentioned problems. Fusion between lymphoid cells and bone marrow lung cells derived from mammals (e.g. mice or rats) can produce hybrid cells that can grow and reproduce in vitro (
Cora and Milstein, Nature, vol. 256, 4.
95-497, 1975) was discovered.

Such hybrid cells have the property of secreting antibodies with a predetermined allergenic property. This percentage of opposite sex is
This is the specificity of the antibodies produced by the lymphocytes involved in the fusion. By cloning the hybrid cells and continuing to culture them under stable culture conditions, certain antigens can be determined.
Samples containing antibodies specific only for iK will continue to be produced indefinitely. Antibodies produced in this manner are known in the art as monoclonal antibodies.

The general method for creating the above-mentioned hybrid cells is to administer an antigen to a mammal (usually a mouse or a rat, but not necessarily limited to these two), immunize it, and collect white blood cells that secrete antibodies against the antigen. After a sufficient number of antigen administrations and sufficient immunization time to produce a large number of cells, the animal is sacrificed and a suspension of lung cells is prepared. Fusion of these cells with myeloma cells is achieved by contacting them in the presence of a fusion promoter (eg, polyethylene glycol). Only a very small number of cells fuse to form hybrid cells. As a result of the immune response, a plurality of different cells are generated, each secreting antibodies against different antigenic determinants, and these traits are genetically transmitted to the hybrid cells. By performing careful screening, cells secreting antibodies with the desired specificity can be isolated from a cultured hybrid cell population. Such cells can be cloned and cultured.

The advantage of this technique is that it provides a source of specific antibodies that is free of antibodies directed against other antigenic determinants in the antigenic impurities contained in the immunizing antigen or immunizing agent.

The present invention relates to a monoclonal antibody against human tumor necrosis factor (hereinafter referred to as human TNF). H) TNF is known to be induced from macrophage cell groups by various substances that activate the reticuloendothelial system, such as various Durum-positive bacteria and todoxin.

It is induced by various substances that have the effect of activating the reticuloendothelial system,
The existence of many substances having physiological activities such as antitumor cell ability has been reported. For example, Carswell et al.
-I 5w1ss? Bacillus Cal in mice
administering mette Guerin (B CG );
Two weeks later, the serum of the mouse obtained by intravenous injection of Cx/Cx had a cytocidal effect on the cultured cells, and the cells were loaded with Meth A sarcoma (BALB/Cx 5.7 BL/6)
We discovered a phenomenon that caused tumors in Fl mice to undergo hemorrhagic necrosis and named them TMF (Proc, Nat, Ac
ad, Sci, USA vol. 72 (A9), 366
6 pages, (1975)]. Subsequently, 1eed et al. [J-I
nimunol, vol. 125 (/164), 1671 negative, (1980)] A and Mat t ews et al. [B
r, J, Cancer vol. 42, 416 negative, (198
0)] attempted to purify TNF from rabbit blood soak prepared according to the method of Carswell et al., and the results were approximately 20,130 times and approximately 10
I am getting one made of 00x A7. However, in any case, antitumor effects of the purified product 7 have not been confirmed in animal experiments.

Also, Ruff et al. J-Immunology Vol. 115, p. 395, (1975)] C. '1
'Although it has been reported that a factor with NF-like activity has been discovered, its true nature is not clear, as no antitumor effect has even been confirmed in animal experiments.

The present inventors have been conducting intensive research on a proteinaceous physiologically active substance with anticancer activity that is prevented by a tissue culture system containing human-derived activated macrophages. discovered a new proteinaceous physiologically active substance with an isoelectric point of approximately 6.0 and excellent antitumor activity, completed the invention, and has already filed an application.

This bioactive substance with antitumor activity, i.e.
TNIII has attracted a lot of attention because it has extremely weak vibrations and strong physiological activity, and it has been desired to obtain specific antibodies as a means for its separation and purification and quantitative analysis.
has low antigenicity due to low species % isomerism, and when 1゛NF with a low degree of purification was used as an antigen, it was not possible to obtain a monoclonal antibody with specific reactivity.

The present inventors conducted various studies to obtain antibodies specific to human TNF, and as a result, purified human T
Antibody-producing cells are obtained by immunizing mice with NF as an antigen, and hybrid cells between these cells and myeloma cells are used to produce antibody-producing cells.
The present invention was completed by discovering for the first time that monoclonal antibodies having specificity for human TNF can be stably produced.

That is, the present invention relates to a monoclonal antibody having specificity for human tumor necrosis factor. The monoclonal antibody according to the present invention can be used for separation and purification of TNF, immunoassay, etc.

The cell fusion method will be specifically explained below.

(a) Preparation of antibody-producing cells Antibody-producing cells may be prepared according to conventional methods. That is, the best method is to immunize an animal with human TNF, which is an antigen, and obtain antibody-producing cells from that animal.

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

(b) Regulation 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 used.

The cell line used should preferably be drug resistant so that unfused myeloma cells cannot survive in the selective medium and only hybrid cells will proliferate. The most commonly used cell lines are 8-azaguanine-resistant cell lines, which are deficient in hypoxanthinloganine phosphoribosyl transferase and are unable to grow in hypoxanthine-aminopterin-themid (HAT) medium. has. Furthermore, it is preferable that the cell line used is a so-called "non-secreting" cell line. For example, P3/X63 Ag8Ul derived from mouse bone marrow seed strain MOPC-21
(P3U1), P3 /X 63-Ag・6・5・3,
P3/N81-N3l-1-A, Sp 210-Ag
14, rat myeloma cells 210-RCY3-Ag1-2
-3 etc. can be suitably used.

(C) Cell fusion Normal Eagle Minimal Essential Medium (MEM), Roswell Burke Memorial Institute/Stituate (Itl) Mt.
) in a medium such as 1640 medium.
J7 myeloma cells and 1-5 antibody-producing cells X 108
Cell fusion is performed by mixing the cells (mixing ratio is usually 1:4 to 1:10). As a fusion promoter, an average molecular weight of 1.0
00-6,000 polyethylene glycol (PEG)
is preferred, but other viruses and the like can also be used. PEG
The concentration used is usually 30-50%.

(d) Selective proliferation of hybrid cells Add 20% fetal bovine serum proprietary RP to cells that have completed cell fusion.
Appropriately dilute it with M11640 medium or the like and plant it in a microtiter plate at a concentration of about 105 to 106. Add selective medium (e.g. II A i' medium) to each well;
Thereafter, the selection medium is appropriately replaced and cultured. If an 8-azaguanine resistant strain is used as myeloma cells, the unfused σ) myeloma cells will grow after 10 days in IIAT medium.
By the time leprosy occurs, all the cells have died, and since the antibody-producing cells are normal cells, in vitro testing does not take a long time. Therefore, all cells that grow after about 10 to 14 days of culture are hybrid cells.

(e) Search for antibody-producing hybrid cells Screening for hybrid cells may be performed by any conventional method and is not particularly limited. For example, a part of the supernatant of a well in which hybrid cells have been grown is collected and reacted with TNF or immobilized TNF, and then an enzyme, a radioisotope, a fluorescent substance,
By reaction with a second antibody labeled with a luminescent substance, the amount of label can be measured and the presence of anti-human TNF antibody can be assayed.

(f) Cloni/G Since there is 8J potential in which hybrid cells of two or more types of V'i are growing in each well, monoclonal antibodies can be produced by cloni/g using limiting dilution method, etc. Obtain hybrid cells.

(g) Antibody Obtain The purest monoclonal antibody is obtained from the desired hybrid cells by 10
It can be obtained from the culture supernatant obtained by culturing in a suitable culture medium such as RPM11640 medium containing about 10% fetal bovine serum.

On the other hand, in order to obtain even larger amounts of antibodies, it is necessary to inject blister/(2H6r t
The hybrid cells may be grown in large quantities in vivo by intravenously administering a mineral oil such as ol 14-tetramethylpentadecane and then administering the hybrid cells.

In this case, ascites tumors are formed in about 10 to 18 days, and high concentrations of antibodies are produced in serum and ascites.

Here, the antigen TNF referred to in the present invention has the amino acid composition shown below.

Amino acid content ill<mo/L%) Asparagi/+Asparagi/Acid 7.8 Threonine 3
．． 6 Serine 8,2 Glutami/+Glutamic acid 12.9 Glycine 7.1 Aranino8.5 Valine 7,8 Cystine 1.4 Tryptophan 1.4 Isoleucine 5.0 Leucif 11.4 Tyrocy/4.6 Phenylalanine 2.7 Lysine 3.9 Histidine 2.0 Argini 15.3 Proli/6.5 It is a protein that has a structure consisting of polypeptide subunits and has the following properties.

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)
The specific activity in the evaluation using LM cells as defined in the text is I
X 10' units/m9 protein Furthermore, the substance of the present invention:
In the biological plan using Metl+ A sarcoma tumor-bearing 13 AL B/C mice described in Experimental Example 4, the activity is (+) or higher when 100 to 200 units per mouse is administered intratumorally.

The antigen human TNF in the present invention is derived from human-derived activated macrophages. Activated macrophages are derived from tissue macrophages such as lung or lung tissue macrophages, peripheral blood monocytes cultured in the presence of substances capable of inducing differentiation, or leukemia cells from certain myelomonocytic leukemia patients. Established cell lines can be used. The cell line is HL-60 cell CNatu.
re vol. 270, p. 347 (1977)] T 11
P-1 cells (Int, J, Cancer vol. 26,
171 Sada (1980), etc. Substances that increase the ability to induce differentiation include 12-0-tetrazokalphorbol-13-acetate and phorbol-12,1.
3-didecanoate, phorbol nisdels such as phorbol-12,13-dibenzoate, diterpenes such as mezerein, vitamin A derivatives such as vitamin A acid, vitamin A alcohol, dimethyl sulfoxide, pepto/or casei/ Hydrolyzates and the like are preferably used.

The activated macrophages of the present invention produce a larger amount of human TNF by culturing in the coexistence of bacterial cells derived from Gram-negative bacteria, todoxy, or Durum-positive bacteria, or yeast.

The gist of the preferred method for purifying TNF to obtain the antigen in the present invention is to pass the culture solution of activated macrophages to the next step.

(i) Electrophoretic isoelectric focusing (2) Basic anion exchange chromatography (3)
Gel filtration (4) Affinity chromatography (5) Gel filtration The physical properties of TNF purified as an antigen-substance were measured by each of Experimental Methods 1 to 8 described below.

Experimental method 1) Molecular weight measurement method A) Using a column (1.5 x 100 cm) of 77 Acrylic S-200 (manufactured by Pharmacia, Sueten),
When Kell filtration was performed with 0.1 M sodium chloride/50 m M phosphate gauze solution (pH 7.4), the molecular fi
t was 40,000±4.000.

B) Segrest et al.'s method CMethod in
Tris/chrysin/81)S(pi(8,
3) to SDS/polyacrylamide gel with 10 μg O)
When a sample was applied and electrophoresis was performed, the molecule i 20
A dyed band and activity were observed at the 4,000 to 4,000 position.

2) Iso-focused measurement method Ato-Isoelectric focusing device manufactured by Co., Ltd. (8, 1-1 (
Pharmalite (manufactured by Pharmacia, 5% containing -(4-6.5) and glycerol) was used.
Polyacrylamide flat plate gel (thickness l mm, length 10
cm, medium 10 cm) was created.

0.04M, DL-glutamic acid on the anode side, 0 on the cathode side
．． Aeroboresis was performed at 700 μm for 50 minutes using 2M, L-histidine. Subsequently, 50 μg of sample was applied, and 7
Electrophoresis was performed at 00v for 1 hour and at 500v for 16 hours. After the electrophoresis was completed, the gel was cut into a 2.5-width piece, and each gel piece was extracted with 0.2 ml of 0.02 M Lis-HCl buffer (pH 8,2) containing 0.15 M sodium chloride.
When the activity of each extract was evaluated using LM cells, the isoelectric point i1t of human TNF was 6.0±0.5.

3) Activity evaluation using LM cells Activity evaluation using LM cells is performed using Ruff [Lymph
Okine Reports Volume 2, E-Pic
Edited by K, Academic Press, 235 pages (
1980)] or (J, Immunol, 1
26, p. 235 (1981)], the present inventors have improved the method, and the physiologically active substance according to the present invention is used in LM cells (American Type Culture Collection, CCL1, 2). It measures the effectiveness of killing. That is, K jt! sequentially diluted in medium! lO
, i mt and 0.1 mt of 0 degree L-M cell culture medium suspension of 105+/m was added to a 96-well tissue culture microslate (
Flow Laboratory, Inc.). Medium is IV/V%
Mueagle's Minimum Esse/Shall medium containing fetal bovine serum (the composition of which is described in "Tissue Culture" by Junnosuke Nakai et al., MA Han, Kyokusho Shoten, 1967) was used. The microplates were incubated at 37°C for 48 hours in an atmosphere containing 5% carbon dioxide. After culturing, cultaraldehyde was added to kltiffl the cells.

Fixed wave, wash and dry microplate, 0. (
Add 15% methylene blue solution to Q. Add 1 mA,
Surviving cells were stained. After washing off the excess methylene flue and drying, remove the remaining methylene flue with 0.36
It was extracted with N hydrochloric acid solution, and its absorbance at 665 nm was measured using Titertic Multiscan (Flow Laboratory). This absorbance is proportional to the number of surviving cells. The bioactive cap required to kill 50% of LM cells is defined as 1 unit/mA.

Determine by graph or calculation the dilution rate of the sample that corresponds to 50% of the absorbance of the control without adding the sample,
The reciprocal of the dilution rate was defined as the physiological activity amount (expressed in units/ml) of the sample.

On the other hand, the protein amount was determined using the method of Branford et al. (Ana
l.

Biochem. 7 vol. 2, p. 248 (1976)
] by Coomasie Prioria/To Blue 0250
Calculated from the dye binding method using

Antigen in the present invention) 'I' N FO specific activity H,
IX 106 units/~protein.

4) Activity evaluation using Meth A sarcoma-infected mice 2 x 105 Met cells were placed in the abdomen of BALB/C mice.
hA. After 7 days of transplantation with sarcoma cells, the size of the transplanted tumor was 7 to 8 mm in diameter, and a mouse was selected that had a tumor with good blood circulation without hemorrhagic necrosis. diluted with , osmJ samples were injected, and 24 hours later, the necrotic reaction was evaluated according to the following criteria.

(-): No change (+): Faint hemorrhagic necrosis (-1+): Moderate hemorrhagic necrosis (necrosis extending over 50% from the center of the transplanted cancer surface) (+14): Significant hemorrhagic necrosis (transplant (The central part of the cancer was severely necrotic, with only a small amount of surrounding cancer tissue remaining.) Furthermore, on the 20th day after the sample was used, it was observed whether the cancer had completely regressed to determine the complete cure rate.

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

(Margins below) 100 6 2130 2/6 200 6 0123 3/6 1.000 6 0 0 1 5 6/6 Control (normal saline) 6 6000 0/6 *Complete cure rate: Number of mice with complete regression of cancer /Number of experimental mice 5) Determination of amino acid composition The following studies were conducted using TNF in the present invention.

A 5DS-polyacrylamide plate gel containing 15% of acrylamide and a boria of 15% in diameter and a thickness of 1n and a length of 11crIL for sample preparation was prepared. For details on the creation method, please refer to the
According to the pamphlet of "Slab Type 5DS-Acrylamide Gel Electrophoresis" Co., Ltd. The equipment is made by ATO Co., Ltd. $
1!5J-1060・SDH type was used.

One plate of the gel contains 40% of the human TNF according to the present invention.
0 μg was given. 1% SD as pre-treatment when applying
200 μg of human TNF was added to each gel plate containing 100 μg of S aqueous solution and 40% sucrose aqueous solution. 1% SDS aqueous solution 10% as a pretreatment when administering
Mix 0 μl of a 50 μl aqueous solution containing 200 μg of human TNF with 50 μl of a 40% sucrose aqueous solution, and incubate at 50°.
Heat treatment was performed for 0.30 minutes. 'Electrophoresis rJl 50
The test time was 4 hours and 30 minutes at a constant voltage of V. This operation was repeated five times. After electrophoresis, a portion was stained with Coomassie Brilliant Blue 0250 for 5 minutes, and after decolorization, five sections were cut out at 2.5n intervals centering on the clearly visible stained band. The gel pieces were then immersed in 1% ammonium bicarbonate 1.5 rnlO to 4°024
Time extraction was performed. After extraction, the extract was taken out with a pipette and washed with the same solution to obtain 5 slices of 2.5 ml each of the extract. The obtained extract was
For confirmation, activity evaluation using LM cells was performed. Most of the activity was recovered in the central band.

The extracted sample solution was passed through a Sephadex G25 column (0,
9×10aa) for desalting.

Then, using a centrifugal vacuum dryer co/centrator (EC-10) manufactured by Tomy Seiko Co., Ltd., at 25°C for 2 hours at 1.50°C.
It was dried at 0 rpm and used as an analysis sample for amino acid composition.

The sample was treated in vacuo with 4M meta/sulfonic acid at 110°C.
Hydrolysis was carried out for 24 hours, and the amino acid composition (mol %) was measured using a high-speed amino acid analyzer (Hitachi, model 835).

After hydrolysis, cysteine is oxidized in the air to form cysteine/cysteine.
Converted to .

h) Amino acid composition of TNF U The values were as shown below.

Amino acid content (Mo/L%) Asparagium/+Aspartic acid 7.8 Threonine 3.6 Serine 8.2 Glutamine + Glutamine/Acid 12.9 Glyci/7.1 Alania 8.5 Valine 7.8 Cysti/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 proly/6.5 As explained above, the present invention Human TNF has extremely excellent anti-tumor effects, has little species-specificity, has a wide range of effects, and has almost no toxicity, making it an extremely useful anti-tumor agent. This is something that can be expected.

The above-mentioned human TNFt: has low species specificity and antigenicity, making it difficult to obtain antibodies. It has become possible to create monoclonal antibodies against TNF.

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

The present invention will be explained with reference to the following experimental results, but these are merely examples, and it goes without saying that the present invention is not limited thereto.

Purified THP-1 cells of the example antigen were prepared at a concentration of 1.5 x 10 cells/1.
After dispersing in RPMI-1640 medium containing % beef fat serum, 20aJ
Dispensed in portions. 12-
0-tetradecanoylphorbol-13-diacetate and vitamin A acid were added to the solution to give a final concentration of 1 μg/- and 0.5 μg/vtd, and incubated at 37°C in a 5% carbon dioxide incubator. , and cultured for 1 hour.

After 1 hour, remove the culture solution in the petri dish and collect 0.5 μg.
5% Tallow Baby Serum Containing /WLl Hitami/A Acid, 10 gq/1nl Polypeptone - RP M l-1640
After replacing the medium with RPMI-16 of 20111 and culturing at 37°C for another 24 hours, the medium was removed.
40 medium was replaced with 2011 tl, and culture was continued for an additional 72 hours.

Afterwards, the culture solution was collected and incubated at 30 U rpm for 30 min.
After centrifuging for 1 minute to remove cell sediment,
- Examining cytotoxicity to M cells, 180 units/I
A culture solution having Ll activity was obtained.

Next, an example of an experiment for purifying four culture solutions will be illustrated.

Purification Step 1 (Isoelectric Focusing) After concentrating the culture solution 41 10 times by ultrafiltration, Ampholine ■ [Carrier °ampholytes] (
L KB, Sweden/)-Sucrose density gradient column (
440WLl), and after running electricity for about 42 hours and performing electrophoresis, a tube with a diameter smaller than the bottom of the column was inserted at a rate of about 21Ll/min so as not to disturb the flow.

Fractionation was performed by flow rate.

After collecting the fraction with pH 5.5 to 6.5, the pH was adjusted to about 7.
．． After adjusting 4IC, Ampholine was used in dialysis.
■ and sucrose were removed.

Purification step 2 (anion exchange chromatography) After adding 0.02 M of 21) Lis-HCl buffer (pH 7,8) to 0.51 of the fractionated solution, 0.02 M1 containing 0.02 M sodium chloride was added. -Lis-hydrochloric acid buffer (FJ(
Basic anion/exchanger DE fully equilibrated with 7,8)
It was gradually applied to a column (10×90ffi) of AE-Sepharose CL-6B (manufactured by Pharmacia). then 1
．． After washing with 57 column equilibration buffer (0.02 M Tris-HCl buffer containing 0.02 M sodium chloride, ...7.8), 0.02 M containing 0.05 M sodium chloride
2M Tris-HCl buffer (pH 7,8) IA! After washing, elution was performed using 0.02 M Tris-HCl buffer (pH 7.2) containing 0.1 M sodium chloride.

The flow rate was 0.5 l/br, and the eluate was fractionated into 5 OWLl portions to collect active fractions. Add the same volume of 0 to the active fraction.
．． After diluting by adding 02M Tris-HCl buffer (pH 7, 8), it was applied to a DEAE-Cefbroth CI, -6B column (10×13cx) at a flow rate of 0.21/hr.

Then, using 2 l of 0.02M Tris-HCl buffer (,117,8) containing 0.05M sodium chloride,
After washing at a flow rate of 20 ml/l+r, 0.
Using 0.02 M) Lis-HCl buffer (PI 37.2) 1/containing 1 M sodium chloride at a flow rate of 20
Elution was performed at m/V/hr. The eluate was fractionated into 25ml portions and the active fractions were collected. At this stage, the recovery rate of activity was 60% and the degree of purification was 300 times higher.

Purification step 3 (gel filtration) 13. A column (5X80
The concentrated solution was applied to cIn, ) and gel filtration elution was performed using the same buffer. At a flow rate Vi4 ml/hr, 4 rn
The active fraction was collected by fractionation in liter portions, and the active fraction was concentrated by ultrafiltration. The activity recovery rate through purification steps 1 to 3 was 45%, and the degree of purification was 4×103 times.

Purification step 4 (affinity chromatography) The concentrated solution of the active fraction obtained by gel filtration is
It was applied to a chelate sepharose column.

Into a column (t, 6xloc1n) packed with chelate sepharose (iminodiacetic acid immobilized resin), 60rnl of a 1/d zinc chloride aqueous solution was passed at a flow rate of 10ml/hr. Next, after sufficient equilibration with 0.05M phosphate buffer (pH 7.4) containing 0.1M sodium chloride, the concentrate obtained in the previous step was applied at a flow rate of 10 m/hr to 60 m/hr.
The non-adsorbed fraction eluted with the same buffer as in step d was collected. L-M
Damage to cells (1/lower stiffness) was recovered only within 1 iln.

Through purification steps 1 to 4 (7 active t ← recovery rate 130%,
The degree of Nell's fissure was I x 10'.

Purification step 5 (gel filtration) Completely concentrate the Wada active fraction (prepared in the AiJ purification step) to 0.1
o, uo 5M phosphate buffer containing 5M sodium chloride (
p) Toyopearl fully equilibrated with 47.4)]W-
55 (Toyo Soda Co., Ltd.) column (1.5X90c)
rn) was in season. Flow rate of 4 mε/! with the same buffer solution! Gel filtration elution was carried out at 1r, and the active fraction was collected at 1:J'.Throughout the entire purification process, the recovery rate of oil was 20%, and the purity was f-
It was i2×104 times. Hi1 obtained in this way
-The specific activity of TNF is 8X] (1511 imon/m
It was y protein.

'fN I' is a type of lectin as described above (in particular, the human rNF f: i cellulose acetate membrane's body moved into the γ-cloprine region due to the movement of the cellulose membrane). Identification fl (1iar (Con A-Sepharose, WGA-Sepharose (manufactured by Pharmacia)
, RCA-I-gel, UE A-1-gel, LP
A-■-Gel (E, Y, manufactured by Laboratory Co., Ltd.)
It showed no adsorption property against.

Furthermore, each human TNT-1 at
It showed excellent anticancer effects when administered intratumorally and intravenously against syngeneic transplanted mouse cancer and nude mouse transplanted human cancer. That is, mouse-derived colon cancer Co1on 26
BALB/C mouse or neural valve kNe transplanted with
h) TNF5 to A-thread mice transplanted with uro2a.
In a test in which ,000~to,ouo units were intravenously administered once to three times, the burn group (physiological saline immersion-IEjr
Significant cancer growth inhibition and regression were observed compared to R).

In addition, human-derived lung cancer PC-10, salmonoblastoma GOT
BALB/C nude mice transplanted with human T
In tests in which NF 10.0 (10-3 (J, 000 units) was administered intravenously once to seven times, significant cancer growth inhibition and regression compared to the control group (physiological saline administration! 11') was observed. was recognized.

Animal Immunity 100 μg of human TNF purified as described above was emulsified in Freund's complete adjuvant and administered subcutaneously to mice in the 13ALI3/C♂ group at 2-week intervals.
The mouse with the highest serum anti-human TNF antibody titer was intravenously injected with 100 μg of human i'NF to complete the immunization.

3 hours after the final cell fusion immunization, the mice were killed and the lungs were removed.
Bonded cells obtained by shredding and filtering through a stainless steel mesh were washed three times with Eagle's Minimal Esse/Shall medium (MEM), and mouse myeloma cells (P3/
X63-Ag 8 U 1 ) was washed once with MEM,
Liver cells and bone marrow knee group 1ji! After mixing 10=1 and centrifuging, add 44% polyethylene glycol 2000 to a pellet.
A 1M solution of ゜MEM was gradually added, and the centrifuge tube was slowly rotated for 1 minute to perform engineered cell fusion. 1 minute later MEM
Add 1 mε and rotate slowly, then add 111Ll of MEM every 30 seconds until finally 811Ll
And so. Centrifuge again and transfer the pellet 5X as myeloma cells to Roswell Park Memorial Institute (RPMI) 1640 medium containing 2υ% fetal bovine serum.
Suspend to 10' pieces/0.1 ml, 96
A 0.1 d deep well was planted in a well microplate.

One day later, 0.11M of IIAT medium was added to each well, and half of the volume was replaced with HAT medium every 3 to 4 days. As a result, hybrid cells started growing in some wells from around 71 cells onwards. Recognized, 2-3 weeks later Vr:, tI
'i' Hybrid cells grew in all wells.

Search for antibody-producing cells and culture supernatant of wells in which Cloni/G hybrid cells have grown 0, 1 ml
When we incubated the sample with human TNF immobilized on a 96-well polystyrene microplate and looked for something that would bind to it, we found that there was a 5-10% chance that it would bind to human TNF.
Wells secreting antibodies capable of binding to NF could be observed. A number of clones with high binding ability were selected, and 8 clones were obtained by reflow analysis using the limiting dilution method in which cells were inoculated into a 96-well microplate at a concentration of 1 cell/well.

Cloned cells were added to RPM containing 10% fetal bovine serum.
Using I-1640 medium, the cells were cultured in a 96-well plate → 24-well plate → 25-flask and scaled, and the culture supernatant was collected.

After each supernatant was mixed with 1,000 units/vertical human i' NF solution, activity evaluation using LM cells was performed, and only one clone inhibited the activity of human TNF. This hybrid cell was named P3TT. These cells can be stored at liquid nitrogen temperature and are kept by the inventor at all times. ”A-
, I sent a deposit request by mail (registered mail ＾11.
．． RejJ% b :4 fi U No.) Next, 2M
At least 106 cysts were intraperitoneally administered to BAL B/C mice that had been pretreated with 0.5 ml of Gulistan to form ascites tumors, and the ascites was collected. Even when diluted 10,000 times, this ascites contains 100 units of H) TNF L-
Cytotoxic activity against M cells was completely suppressed.

In addition, antibodies derived from P3TT suppress the activity of antitumor active substances obtained from human lung macrophages (evaluated by LM cytotoxic activity), whereas antibodies derived from rabbits, mice, guinea pigs, and hamsters The LM cell damaging activity of TNF was not inhibited at all.

As described above in detail, P3T according to the present invention
Monoclonal antibodies derived from H) specifically react with TNF, and can be applied not only to the separation of TNF but also to immunoassays using enzymes and radioisotopes. It is widely applicable to quantitative analysis of human TNF.

Procedural amendment: July 26, IF159 Manabu Shiga, Commissioner of the Patent Office 1 Indication of the case Japanese Patent Application No. 1987-65255 2 Title of the invention Antibody against human tumor necrosis factor 9 3 Person making the amendment Relationship to the case/special characteristics TR Applicant (OOX) Asahi Kasei Kogyo Co., Ltd. 4 Agent Mail (Saradzu No. 105 Toranomon Sangyo Pill 5, Toranomon-chome-2ti29, Minato-ku, Tokyo)
v@6 Amendment content Clear and pure writing d (no change in content)

Claims (1)

[Claims] Monoclonal antibody specific for human tumor necrosis factor