JPH0532033B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a monoclonal antibody specific for a new lymphokine and a method for producing the same.

For monoclonal antibodies, Cesar Milstein, Scientific American
Vol. 243, No. 4, pp. 56-64 (1980) provides a detailed explanation.

Lymphokines, especially lymphokines that have damaging activity against malignant tumors, include lymphotoxin derived from animals including humans, and tumoa necrosis factor (hereinafter referred to as
known as ``TNF'').

Lymphotoxins are co-authored by Ryuichi Aoki et al., “Lymphokines,” New Immunology Series 6, pp. 87-105 (1979)
Igaku Shoin, Bloom B.R.
R.) and Glade PR
Co-edited with ``In Vitro Methods in Cell Medication and Immunity''
Methods in Cell-Mediated Immunity” Academic Press (1971)
), and Cellular Immunology (Cellular
Immunology) Vol. 38, pp. 388-402 (1978), and TNF is
Carswell EA, etc., Proceedings of the National Academy of
Proceeding of the National Academy of Science
Science of the USA) Vol.72, No.9, 3666~
3670 pages (1975) and E.Pick
Tumor Necrosis Factor in
Lymphokines) Vol., pp. 235-272, Academic Press (1981), etc.

Furthermore, recently, Haruo Onishi et al. have disclosed an antitumor glycoprotein, which is a type of lymphokine, in Japanese Patent Application Laid-Open No. 146293/1983.

The inventors have been studying lymphokines for many years. As a result, these previously known lymphokines were found to have completely different physicochemical properties described at the end of the literature, and the existence of new lymphokines was discovered.
Establishing the monoclonal antibody and its production method,
The invention has been completed.

That is, the present invention has the following physical and chemical properties: Molecular weight 20000±2000 Isoelectric point pI=6.2±0.3 Mobility Rf=0.29±0.02 in Disc-PAGE Ultraviolet absorption spectrum Maximum absorption around 280 nm Solubility in solvent Water; Soluble in physiological saline or phosphate buffer Slightly soluble or insoluble in ethyl ether, ethyl acetate or chloroform Color reaction Shows a positive protein reaction by the Lowry method or microbiuret method, and shows a positive reaction for protein by the phenol sulfuric acid method or anthrone sulfuric acid method. Effect: Shows cytotoxic activity against L ₉₂₉ cells, KB
Substantially exhibits no cell proliferation inhibitory activity or interferon activity against cells. Activity stability in aqueous solution: Stable up to 60℃ when kept at PH7.2 for 30 minutes; PH40 to 11.0 when kept at 4℃ for 16 hours. A monoclonal antibody that exhibits immunological neutralizing activity against a new lymphokine (hereinafter referred to as "new lymphokine" throughout this specification) that is stable for more than one month when stored frozen at -10°C, and its production method Regarding.

First, the method for producing the new lymphokine will be described.

The new lymphokine may be produced by, for example, treating human-derived cells capable of producing the new lymphokine, such as leukocytes, lymphocytes, cultured cells, etc., with an inducing agent.

It may be prepared by separating human-derived white blood cells, lymphocytes, and blood collected from humans.

Cultured human-derived cells and cells grown in vitro according to conventional methods can be used.

However, in the case of the present invention, when growing cells in culture, they are directly transplanted into the body of a warm-blooded animal other than humans, or inoculated into a diffusion chamber, and the supply of body fluids of the warm-blooded animal is reduced. It is preferable to multiply while receiving.

In other words, unlike in vitro cell proliferation, not only is there no need for, or can significantly save, nutrient media containing expensive serum, etc., but maintenance and management during cell proliferation is also extremely easy. Moreover, the new lymphokine activity produced by induction from the obtained cells is high.

Methods using warm-blooded animals other than humans include transplanting cultured human-derived cells into the body of a warm-blooded animal other than humans, or creating a diffusion chamber that can receive the animal's body fluids. If the cells are implanted inside an animal and reared normally, the cells can easily proliferate using the nutrient-containing body fluids provided by the warm-blooded animal.

Furthermore, compared to in vitro proliferation, the proliferation of these cells is stable, the proliferation rate is high, the amount of cells obtained is large, and the new lymphokine per cell is Another major feature is that the yield increases significantly.

The cultured human-derived cells used in the present invention may be cells that can be transplanted into the body of a warm-blooded animal other than humans and easily proliferate, and that have the ability to produce new lymphokines. Various human-derived cell lines described in "Protein Nucleic Acid Enzyme Vol. 20, No. 6", pages 616-643 (1975) can be used. Among others, the Journal of Clinical Microbiology
Namalwa cells described in "Clinical Microbiology" Vol. 1, pp. 116-117 (1975); described in I. Miyoshi's "Nature" Vol. 267, pp. 843-844 (1977) BALL-1 cells, TALL-1 cells,
NALL-1 cells, “The Journal of Immunology”
Vol. 113” pages 1334-1345 (1974) M-
7002 cells, B-7101 cells, "Tissue Culture" Volume 6,
No. 13, pp. 527-546 (1980).
JBL cells, EBV-Sa cells, EBV-Wa cells,
EBA-HO cells, MOLT-3 cells, and others
BALM2 cells, CCRF-SB cells (ATCC CCL
120) Established lymphoblastoid cell lines such as CCRF-CEM cells and DND-41 cells, as well as normal mononuclear cells and granular leukocytes, were infected with various viruses,
Cells that have been treated with drugs, radiation, etc. and cultured are suitable.

In addition, the genes that have the ability to produce new lymphokines in these human-derived cells can be used, for example, by means of cell fusion using polyethylene glycol or Sendai virus, or by using enzymes such as DNA ligase, restriction enzymes (nucleases), and DNA polymerases. It may be used by treating it by genetic recombination means to increase its proliferation rate or increase its ability to produce new lymphokines per cell, and is limited to the established cell lines described herein. It's not a thing. In the process of inducing the production of new lymphokines, which will be discussed later, these cells
They can be freely used alone or in combination of two or more. If necessary, leukocytes, lymphocytes, etc. collected and prepared from humans, for example, can be used in combination.

The warm-blooded animals used in the present invention may be any animal in which human-derived cells can proliferate, such as birds such as chickens and pigeons, dogs, cats, monkeys, rabbits, goats, pigs, horses, cows, guinea pigs, Mammals such as rats, nude rats, hamsters, normal mice, and nude mice can be used.

Transplanting human-derived cells into these animals may cause an unfavorable immune reaction, so in order to suppress such reactions as much as possible, the animals used should be kept as young as possible, i.e. eggs, embryos, fetuses, or newborns. Preferably from childhood.

In addition, these animals may be subjected to pretreatment such as irradiation with X-rays or gamma rays at a dose of about 200 to 600 rem, or injection of antiserum or immunosuppressants, etc., to weaken the immune response before transplantation. .

When the animal used is a nude mouse or nude rat, the immune response is weak even when the animal is grown, so there is no need for these pretreatments, and cultured human-derived cells can be used. It is particularly advantageous because it can be transplanted and rapidly propagated.

In addition, human-derived cells that have been cultured are first transplanted into hamsters and grown, and then these cells are then transplanted into nude mice. It is also possible to further stabilize the transplantation of the derived cells and further increase the amount of new lymphokines induced and produced from them.

In this case, transplantation can be done not only between the same species and the same genus, but also between the same class and phylum. The site within the animal body to which human-derived cells are transplanted may be any site where the transplanted cells can proliferate, such as the allantoic cavity, vein, abdominal cavity, or subcutaneous site.

In addition, porous filtration membranes capable of blocking the passage of animal cells without transplanting human-derived cells into the animal body, such as membrane filters ^{, ultrafiltration} membranes, or Diffusion chambers of various shapes and sizes known in the art, each equipped with a fluorophore or the like, are buried in the animal's body, for example, in the abdominal cavity, and the above-mentioned culture is carried out within the chamber while being supplied with body fluids containing nutrients from the animal's body. Any established human-derived cell line can be grown.

If necessary, a chamber that connects and perfuses the solution containing nutrients within the chamber with body fluids within the animal's body is attached to the surface of the animal's body, for example, to check the proliferation status of human-derived cells within the chamber. By making it possible to see through the window provided on its surface, and by making only this chamber part removable and replaceable, cells can be multiplied to the full lifespan of the animal without having to be slaughtered, and the amount of cells produced per individual animal can be increased. It can also be increased further.

These diffusion chamber-based methods do not allow direct contact between human-derived cells and animal cells;
Not only can only human-derived cells be easily collected, but there is also little risk of causing undesirable immune reactions, so there is no need for pretreatment to suppress immune reactions, and various warm-blooded animals can be used freely. There is.

The transplanted animal can be maintained and managed simply by continuing the normal care and management of the animal, and is convenient because no special handling is required even after transplantation.

The purpose can usually be achieved in a period of 1 to 10 weeks for growing human-derived cells.
It has also been found that the number of human-derived cells obtained in this manner reaches about 10 ⁷ to ^{10 12} or more per animal.

In other words, the number of human-derived cells grown in an animal is approximately the same as the number of cells transplanted per animal.
10 ² to 10 ⁷ times, or even more, compared to about 10 ¹ to 10 ⁶ when grown in vitro in nutrient media.
twice or even more, making it extremely convenient for the production of new lymphokines.

Any method can be used to induce and produce new lymphokines from human-derived cells grown in this manner. It is also possible to cause the new lymphokine inducer to act within the animal's body after it has grown. For example, a new lymphokine inducer is directly applied to human cells grown in suspension in ascites in the peritoneal cavity or tumor cells generated subcutaneously to induce the production of new lymphokines, and then the serum, ascites or New lymphokines can be purified and collected from tumors.

Furthermore, proliferating cells derived from humans can be removed from the body of a non-human animal and a new lymphokine inducing agent can be applied to induce the production of new lymphokines in vitro. For example, human-derived cells grown in ascites are collected, or tumors containing human-derived cells generated subcutaneously are excised and dispersed, and the resulting cells are placed in a nutrient medium kept at approximately 20 to 40 degrees Celsius to achieve a cell concentration. is about
The cells may be suspended at a concentration of 10 ⁵ to ^{10 8} /ml, treated with a new lymphokine inducer to induce production of new lymphokines, and purified and collected.

Furthermore, when human-derived cells are grown in a diffusion chamber, the grown cells can be left in the chamber or removed from the chamber and treated with a new lymphokine inducer to induce the production of new lymphokines. can.

In addition, as mentioned above, human-derived cells obtained using warm-blooded animals other than humans can be further cultured in vitro for about 1 to 4 days to synchronize the cell proliferation generation, if necessary. It is also possible to induce the production of new lymphokines by applying a lymphokine inducer.

In addition, for example, new lymphokines can be induced and produced in the animal's body by first proliferating human-derived cells, and then human-derived cells collected from a specific part or the whole of the same animal are injected into the animal's body. A method of inducing the production of new lymphokines, a method of using cells once used for the production of new lymphokines for induction production of new lymphokines more than once, or a method of implanting or replacing the chamber to be connected in the animal body. It is also possible to further increase the amount of new lymphokine produced per individual animal by using methods such as increasing the number of cells obtained.

The new lymphokine inducer of the present invention includes α
- Viruses, nucleic acids, nucleotides, etc. known as interferon inducers, phytohemagglutinin, concanavalin A, porkweed mitogen, lipopolysaccharide, endotoxin, polysaccharides, etc. known as γ-interferon inducers, Bacteria and the like are used as appropriate. Also,
For sensitized cells, antigens are also inducers of neolymphokines.

Furthermore, when producing new lymphokines from human-derived cells, it is also possible to increase the production amount of new lymphokines by using α-interferon inducers and γ-interferon inducers together as new lymphokine inducers. It is.

Furthermore, it has been found that not only new lymphokines are produced by these induced productions, but also human interferon, which is highly species-specific, is also produced at the same time.

This makes it possible to simultaneously produce two or more valuable human physiologically active substances, and also to make advanced use of human-derived cells, which is extremely important in terms of supplying new lymphokines and human interferon in large quantities at low cost. It's convenient.

The new lymphokine thus induced can be purified using known purification and separation methods such as salting out, dialysis,
It can be easily purified and separated and collected by filtration, centrifugation, concentration, freeze-drying, etc. If a higher level of purification is required, for example, adsorption to ion exchanger-elution, gel filtration and isoelectric point fractionation, electrophoresis, ion exchange chromatography, high performance liquid chromatography, column chromatography. By combining known methods such as E and Affinity chromatography, the specific activity can be reduced to approx.
It is also possible to obtain the highest purity new lymphokines of ¹⁰⁹ units/mg protein.

The method for producing a monoclonal antibody of the present invention involves immunizing a non-human warm-blooded animal with the new lymphokine obtained as described above as an antigen, collecting antibody-producing cells from the animal, and combining these cells with myeloma cells. and select fused cells from the resulting fused cells that have the ability to produce antibodies specific to the new lymphokine, proliferate these selected cells, and exhibit immunological neutralizing activity against the generated new lymphokine. All you need to do is collect monoclonal antibodies.

Methods for immunizing non-human warm-blooded animals with new lymphokines include, for example, chickens, pigeons, dogs,
Cat, monkey, goat, pig, cow, horse, rabbit,
Injecting a new lymphokine as an antigen into a non-human warm-blooded animal such as a guinea pig, rat, hamster, or mouse, for example, intravenously, peritoneally, or subcutaneously, in the form of an aqueous solution, emulsion, or suspension, and then rearing the animal for 3 days or more. to induce antibody production. Also,
It is also advantageous to convert the new lymphokine into a carbohydrate conjugate of the new lymphokine by the method described in Japanese Patent Publication No. 58-23847, and then use it as an antigen.

The antigen may be injected only once or, if necessary, twice or more at intervals of about 3 to 30 days.

The spleen cells of animals immunized in this way to induce antibody production are combined with myeloma cells derived from the same or xenogeneic animal, as described by G. Kohler and others in ``Nature''. ” Vol. 256, pp. 495-497 (1975), and “European Journal of Immunology”
Vol. 6, pp. 511-519 (1976), the resulting fused (hybrid) cells were selected and cloned, and then in vitro (in vitro).
In vitro) or in vivo, monoclonal antibodies with high immunological neutralizing activity are collected from this culture. Among these, in vivo culture not only does not require expensive serum compared to in vitro culture, but also allows the fused cells to proliferate at a faster rate and can produce large amounts of monoclonal antibodies. Extremely advantageous.

When culturing in vivo, the fused cells are transplanted into a warm-blooded animal of the same or different species as the animal that was immunized to induce antibody production, or they are inoculated into a diffusion chamber and supplied with body fluids from that warm-blooded animal. Monoclonal antibodies can be collected from body fluids such as ascites and serum, or the fused cells grown in vivo can be cultured for a short period of about 1 to 5 days in a serum-free medium. The monoclonal antibody may be collected from the culture.

As the antibody-producing cells used in the present invention, spleen cells of a warm-blooded animal other than humans immunized with an antigen are used. 1983), page 11, lines 2 to 7, in addition to spleen cells, lymph node cells and lymph cells in peripheral blood can also be used.

The monoclonal antibodies produced as described above can be easily purified and separated and collected by conventional methods such as salting out, dialysis, filtration, centrifugation, concentration, and freeze-drying. I can do it. If a higher level of purification is required, for example, adsorption to ion exchanger-elution, gel filtration, isoelectric point fractionation, electrophoresis, ion exchange chromatography, high performance liquid chromatography, column chromatography. It is also possible to collect monoclonal antibodies of the highest purity by combining known methods such as chromatography and affinity chromatography.

In addition, highly purified new lymphokines, e.g.
It can also be advantageously used to immobilize monoclonal antibodies on a carrier such as Promcyan-activated Sepharose, purify the immobilized new lymphokine gel, and collect monoclonal antibodies in high yield.

The monoclonal antibodies obtained in this way can be used as diagnostic agents for human diseases by showing immunological neutralizing activity against new lymphokines that have inhibitory activity against malignant tumors. It can also be used as a measuring reagent for diagnosing diseases by comparing it with that of normal people.
It can be advantageously used as a ligand for affinity chromatography to produce new lymphokines.

The activity of new lymphokines as target cells
It was measured using KB cells or _L929 cells. That is, when using KB cells, Cancer Chemotherapy Reports
Reports) Parts 3, Vol.3, No.2,
The growth inhibitory activity of KB cells was measured according to the description in September 1972, and when using L ₉₂₉ cells,
Edited by E.Pick, Tumor Necrosis Factor in Lymphokines
Vol., pp. 245-249, as described by Academic Press (1981).
Cytotoxic activity against _L929 cells in the presence of actinomycin D was measured. At this time, the activity when inhibiting the proliferation of _L929 cells by 50% was defined as 50 units. In this specification, unless otherwise specified, an activity measurement method using _L929 cells was adopted.

Examples of the present invention will be described below.

Example 1-(a) Preparation of partially purified new lymphokine Newborn hamsters were injected with antiserum prepared from rabbits by a known method to weaken the hamster's immune response, and then BALL-1 cells were subcutaneously injected into the hamsters. were transplanted and then reared for 3 weeks in the usual manner. The tumor formed under the skin was removed, cut into small pieces, and dispersed and loosened in physiological saline. The obtained cells were added with serum.
The cells were washed with RPMI1640 medium (PH7.2) and suspended in the same medium at a concentration of approximately 2×10 ⁶ /ml. Sendai virus with a hemagglutination titer of about 400 per ml was added to this cell suspension and kept at 37°C for 24 hours to induce the production of new lymphokines.

This was centrifuged at about 4°C and about 1000g to remove the precipitate, and the resulting supernatant was dialyzed for 20 hours with physiological saline containing PH7.2 and 0.01M phosphate buffer.
Furthermore, the filtrate obtained by microfiltration is passed through an antibody column on which anti-interferon antibodies are immobilized, the non-adsorbed fraction is collected, and the active fraction is collected by chromatography-e-cutting method. death,
Concentration and lyophilization yielded a powder containing new lymphokine activity.

The specific activity of this powder was approximately 10 ⁶ units/mg protein. Furthermore, the yield of the new lymphokine was approximately 20 million units per hamster.

Example 1-(b) Preparation of anti-neo-lymphokine antibody The new lymphokine obtained by the method of Example 1-(a) was dissolved in physiological saline to a protein concentration of approximately 0.05 w/v%, and the protein concentration was approximately 0.05 w/v%. Mix equal amounts of Freund's complete adjuvant emulsion and add 0.2
ml was injected subcutaneously into mice, and 7 days later, the same injection was repeated to immunize the mice. Spleen cells and mouse myeloma cells P ₃ -X63- are obtained by inducing anti-neo-lymphokine antibodies to be produced in cells that have the antibody-producing ability, removing the spleen from this mouse, and dispersing it into small pieces.
Ag8 “Flow Laboratories (Llow
manufactured by ``Faboratories'' into a 50w/v% polyethylene glycol-1000 solution (PH7.2, temperature 37℃) prepared in serum-free Eagle's minimal basic medium.
They were suspended at a concentration of 10 ⁴ /ml and kept for 5 minutes, then diluted 20 times with the above-mentioned basic medium.
Cell Genetics (somatic Cell
Genetics), Vol. 2, pp. 175-176 (1976), fused cells that can proliferate in hypoxanthine-aminopritene-thymidine culture solution were collected, and anti-neolymphokine was extracted from these fused cells. Fusion cells capable of producing antibodies were selected. Approximately 10 ⁶ of the obtained fused cells were transplanted into the peritoneal cavity of each mouse and kept for 2 weeks. After that, the mice were sacrificed and body fluids such as ascites and blood were collected, centrifuged, and the supernatant was diluted with ammonium sulfate. The precipitated fraction with a saturation of 30-50% was collected, then dialyzed, and this solution was further reacted with the new lymphokine obtained by the method of Example 1-(a) with Promcyan-activated cephalose at room temperature. Affinity chromatography was performed using the immobilized new lymphokine gel obtained in this manner to obtain an anti-new lymphokine antibody fraction, which was dialyzed, concentrated, and lyophilized to collect a new lymphokine monoclonal antibody powder. .

This product showed immunologically strong neutralizing activity against the cytotoxic activity of new lymphokines.

The stability of this monoclonal antibody in an aqueous solution was investigated by measuring the neutralizing activity. As a result, when kept at pH 7.2 for 30 minutes, more than 80% of the activity remained at 60°C, and more than 90% of the activity remained at 70°C. activity has been lost. In addition, under the condition of holding at 4℃ for 16 hours, PH4.0 ~ 11.0
It was stable within the pH range, and more than 90% of its activity was lost at pH 2.0.

Further, as a result of investigating the properties of this monoclonal antibody, it was found that it is unstable to 2-mercaptoethanol and exhibits a specific antigen-antibody reaction with anti-mouse immunoglypurin M antibody.

Therefore, this monoclonal antibody is an antibody classified into the immunoglobulin M class.

Example 1-(c) Preparation of highly purified new lymphokine and its physicochemical properties The partially purified product of new lymphokine prepared by the method of Example 1-(a) was purified by the method of Example 1-(b). Affinity chromatography was performed using the gel immobilized with the monoclonal antibody prepared in 1. The active fraction of the new lymphokine was collected, dialyzed, concentrated, and lyophilized.

This product is a highly purified new lymphokine, and its specific activity was approximately 10 ⁹ units/mg protein.

The physical and chemical properties of this product were investigated.

Molecular Weight K.Weber and M.Osborn, Journal
Journal of Biological Chemistry,
According to the description in Vol. 244, page 4406 (1969),
It was investigated by SDS-polyacrylamide gel electrophoresis. That is, approximately 10 μg of the sample was loaded onto a 10% acrylamide gel column in the presence of 0.1% SDS,
After running for 4 hours at 8 mA per column, staining with Coomassie blue gave a single sharp band with a molecular weight of 20000±2000. This single band had neolymphokine activity.

Isoelectric point Gel for isoelectric focusing, manufactured by LKB, Sweden, product name: AMPHOLINE PAGPLATE
As a result of electrophoresis at 25W for 2 hours using (PH3.5-9.5), the isoelectric point pI was 6.5±0.3.

Electrical Mobility BJDavis, Annuals of the New York Academy of Sciences
Approximately 10 μg of the sample was loaded onto a 7.5% acrylamide gel column according to the description in Academy of Sciences) Vol. 121, p. 404 (1964), and after electrophoresis at pH 8.3 and 3 mA per column for 2 hours, the activity of When the electrical mobility was determined from the measurement,
Rf was 0.28±0.02.

Ultraviolet absorption spectrum Spectrophotometer manufactured by Shimadzu Corporation, product name
As a result of examining the absorption spectrum in the ultraviolet region using UV-250, the maximum absorption was found around 280 nm.

Solubility in solvents Soluble in water, saline or phosphate buffer.

It was sparingly soluble to insoluble in ethyl ether, ethyl acetate, or chloroform.

Color reaction A positive protein reaction was shown by the Lowry method or microbiuret method, and a positive carbohydrate reaction was shown by the phenol sulfuric acid method or anthrone sulfuric acid method.

Effect: Showed cytotoxic activity against _L929 cells.
Substantially no cytostatic activity or interferon activity against KB cells was shown.

Activity stability in aqueous solution (i) Thermal stability Approximately 1 × 10 ⁵ units/ml of sample at each temperature
After holding at pH 7.2 for 30 minutes, the remaining activity was measured and found to be stable up to 60°C.

(ii) PH stability 0.1 ml of a sample of approximately 1×10 ⁶ units/ml was added to each PH buffer (PH2-7...Mcllvaine buffer, PH7-8...Phosphat buffer, PH8～11...Glycine-sodium hydroxide buffer (Glycine-
After adding 1 ml of NaOH buffer) and keeping it at 4℃ for 16 hours, 0.1 ml of this was adjusted to PH7.2 with 0.05M phosphate buffer and the remaining activity was measured. It was stable in the range of 0 to 11.0.

(iii) Stability against dispase (DISPASE) Dispase (protease derived from Bacillus bacteria manufactured by Godo Shusei Co., Ltd.) was added to a sample of approximately 1 × 10 ⁵ units/ml at a concentration of 100 units/ml, pH 7.2, The reaction was carried out at a temperature of 37° C. for 0 to 2 hours, samples were taken over time, and the reaction was stopped by adding bovine serum albumin to a concentration of 1 w/v%. As a result of measuring the residual activity of the new lymphokine in this solution, it was found that the new lymphokine was unstable due to dispase treatment, and the activity of the new lymphokine was lost as the reaction progressed.

Stability due to frozen storage An aqueous solution of pH 7.2 was frozen at -10°C, stored for one month, thawed, and measured for activity. As a result, no decrease in activity was observed.

From the above results, the new lymphokine has the physicochemical properties described in the literature that are clearly different from the previously known lymphokines such as lymphotoxin, TNF, and interferon.

Therefore, the monoclonal antibody of the present invention, which exhibits cytotoxic activity and immunologically specific neutralizing activity of new lymphokines, is a novel monoclonal antibody.

Example 2 Spleen cells and mouse bone marrow obtained by immunizing a mouse in the same manner as in Example 1-(b) except that the highly purified new lymphokine obtained by the method in Example 1-(c) was used as the antigen. tumor cells P3-NS-1/1-Ag4-1 (manufactured by Dainippon Pharmaceutical Co., Ltd.) in 140mM NaCl and 54mM
The cells were suspended in a saline solution containing KCl, 1mM NaH ₂ PO ₄ , and 2mM CaCl ₂ at a concentration of 10 ⁴ /ml, and the saline solution containing Sendai virus, which had been previously inactivated by ultraviolet rays, was added to the saline solution. Mix under ice-cooling, and after 5 minutes transfer the mixture to RPMI at 37℃.
The mixture was diluted approximately 20 times with a medium, and then fused cells having the ability to produce anti-neolymphokines were selected in the same manner as in Example 1-(b).

Approximately 10 ⁷ of the obtained fused cells were transplanted into the abdominal cavity of 7-day-old hamsters whose immune response had been weakened using a known method, and monoclonal antibodies were collected in the same manner as in Example 1-(b). did.

This product, like the monoclonal antibody prepared in Example 1-(b), showed immunologically specific neutralizing activity against the cytotoxic activity of the new lymphokine.

The stability of this monoclonal antibody in aqueous solution was investigated by measuring its neutralizing activity, and the PH
Under the conditions of holding at 7.2 for 30 minutes, more than 80% of the activity remained at 60°C, and more than 90% of the activity was lost at 70°C. In addition, under the condition of holding at 4℃ for 16 hours, the pH
It was stable in the range of 2.0 to 11.0.

Furthermore, as a result of investigating the properties of this monoclonal antibody, it was found to be stable in 2-mercaptoethanol.
It was found that it showed a specific antigen-antibody reaction with anti-mouse immunoglobulin G antibody.

Therefore, this monoclonal antibody is an antibody classified into the immunoglobulin G class.

Example 3 Among the methods of Example 2, mouse myeloma cell P3
-NS-1/1-Instead of Ag4-1, SP2/0-
Except for using Ag14 (manufactured by Dainippon Pharmaceutical Co., Ltd.),
A monoclonal antibody against a new lymphokine was produced in the same manner as in Example 2.

When this product was examined for cytotoxic activity and immunologically specific neutralizing activity of the new lymphokine, the same results as in Example 2 were obtained.

Therefore, this monoclonal antibody is an antibody classified into the immunoglobulin G class.

Claims (1)

[Claims] 1 Physical and chemical properties include: Molecular weight 20000 + 2000 Isoelectric point pI = 6.2 + 0.3 Mobility Rf = 0.29 ± 0.02 in Disc-PAGE Ultraviolet absorption spectrum Maximum absorption around 280 nm Solubility in solvents Water, Soluble in physiological saline or phosphate buffer Slightly soluble or insoluble in ethyl ether, ethyl acetate or chloroform Color reaction Shows a positive protein reaction by the Lowry method or microbiuret method, and shows a positive reaction for protein by the phenol sulfuric acid method or anthrone sulfuric acid method. Effect: Shows cytotoxic activity against L ₉₂₉ cells, KB
Substantially shows no cell proliferation inhibitory activity or interferon activity against cells Activity stability in aqueous solution Stable up to 60℃ when kept at PH7.2 for 30 minutes, PH4.0 when kept at 4℃ for 16 hours
A monoclonal antibody that exhibits immunological neutralizing activity against a new lymphokine that is stable in the range of 11.0 to 11.0 and is stable for 1 month or more when stored frozen at -10°C. 2 Physical and chemical properties: Molecular weight 20000±2000 Isoelectric point pI=6.2±0.3 Mobility Disc-PAGE, Rf=0.29±0.02 Ultraviolet absorption spectrum Maximum absorption around 280 nm Solubility in solvents Water, physiological saline or phosphorus Easily soluble in acid buffers Slightly soluble or insoluble in ethyl ether, ethyl acetate, or chloroform Color reaction: Shows a positive protein reaction by the Lowry method or microbiuret method, and shows a positive carbohydrate reaction by the phenol sulfuric acid method or anthrone sulfuric acid method. Action: Shows cytotoxic activity against L ₉₂₉ cells, KB
Substantially shows no cell proliferation inhibitory activity or interferon activity against cells Activity stability in aqueous solution Stable up to 60℃ when kept at PH7.2 for 30 minutes, PH4.0 when kept at 4℃ for 16 hours
Stable in the range of 11.0 to 11.0 A non-human warm-blooded animal is immunized with a new lymphokine that is stable for more than 1 month when stored frozen at -10°C as an antigen, antibody-producing cells are collected from the animal, and these and myeloma cells are and select fused cells capable of producing anti-neo-lymphokine antibodies from the resulting fused cells, and then proliferate these selected cells to produce monoclonal antibodies that exhibit immunological neutralizing activity against neo-lymphokines. Featured monoclonal antibody production method.