JPH0659231B2 - Monoclonal antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel monoclonal antibody having specificity for human β-type interferon (fibroblast interferon, hereinafter abbreviated as “IFN-β”). is there.

In recent years, interferon (hereinafter abbreviated as "IFN")
Has proved to have an anti-tumor effect in addition to the anti-viral effect, and has attracted attention in the medical field.
IFN is roughly classified into α-type interferon (leukocyte interferon, hereinafter abbreviated as “IFN-α”), IFN.
-Β, type interferon (immunity interferon, hereinafter abbreviated as "IFN-") are known. IFN
-Α changes white blood cells to Newcastle disease virus (NDV)
Alternatively, a method of stimulating with Sendai virus (HVJ), a method of stimulating IFN-β with double-stranded RNA such as poly (I) / (C) or a virus such as NDV, IFN- is a lymphocyte cell. Can be produced by a method of stimulating T cell with mitogen or antigen. Human IFN prepared from mRNA for human cell-derived IFN by applying the recent gene manipulation technology
A method for incorporating a production gene into a plasmid, introducing the plasmid into the cells of a microorganism such as Escherichia coli, Bacillus subtilis or yeast, and expressing the gene function to produce IFN has also been developed ( Proc . Natl . ．Acad ．Sc
i . , 77 , 7010 (1980)).

However, in the cell culturing method, an industrial cell culturing method itself has not been established, and in addition, the production of IFN is extremely small, which causes an increase in the size of manufacturing equipment or the complexity of the IFN purification operation, resulting in a large amount. Manufacturing costs are needed. In addition, even in the method using a genetically modified microorganism, a large amount of IFN can be produced, but since a large amount of impurities are contained in the microorganism culture solution, high-purity IFN is obtained.
Requires a high degree of separation and purification operation.

As one of the advanced methods for separating and purifying IFN, there is known a method for purifying IFN by affinity chromatography using a carrier obtained by immobilizing an antibody against IFN on an insoluble carrier. To establish this antibody affinity chromatography as an industrial purification method,
It is required to supply an antibody having a high specificity and a high binding ability to IFN in a large amount and at a low cost. As a means to meet such demands, a hybridoma (fused cell) is obtained by fusing anti-IFN antibody-producing cells and tumor cells.
Is known and a method for producing a monoclonal antibody by culturing this is known.

Some monoclonal antibodies against IFN and purification of IFN by affinity chromatography using the same have already been reported. For example, a monoclonal antibody against IFN-α is described by Settia et al. ( Nature , 235 , 466 (1980)), Shuterin ( Proc .
Natl . Acad . Sci , 78 , 1848 (1981)). Settia et al. Have purified IFN-α 5000 times by affinity column chromatography using a single-cloned antibody immobilized on Sepharose as a carrier (specific activity of IFN-α: 1.8 × 10 ⁸ U / mg). On the other hand, Schuteerin et al. Have purified IFN-α produced in Escherichia coli by genetic manipulation about 1000 times in one step using a carrier prepared by binding a monoclonal antibody to an agarose gel.

Regarding the monoclonal antibody against IFN-β, Hochkeppel et al. ( Nature , 291 , 500 (1981),
Eur . J. Biochem . , 118 , 437 (1981), JP-A-57-1947
88), Erlich et al. ( Hybridoma ,
1 , 233 (1982)) and the like. However, the antibodies of Hotsukhotuppel et al.
The neutralization titer against IFN-β (dilution ratio of the stock solution that inhibits the antiviral activity of IFN-β by 50%) was as low as 7 in culture supernatant and 70 in mouse ascites, and the antibody of Erich et al. Has a subclass of IgG ₃ / κ and can only partially neutralize the antiviral activity of IFN-β.
It was not satisfactory for affinity chromatography used for industrial purification of IFN-β.

Monoclonal antibodies with high specificity and high affinity for IFN-β are not only useful for purification of IFN-β by affinity chromatography, but also trace amounts in biological samples such as drag monitoring. It can also be applied to a method for highly sensitively quantifying IFN-β that is present in the mouse by an immunoassay method. Therefore, such an IF
The creation of a monoclonal antibody against N-β has been long awaited.

The present invention has been completed based on such a technical background, and is extremely useful for practical use for purification of IFN-β by affinity chromatography or for quantification of IFN-β by immunoassay. The present invention provides a monoclonal antibody against IFN-β having various properties.

The monoclonal antibody against IFN-β according to the present invention is distinguished from conventionally known antibodies in that the subclass is IgG ₁ / κ.

The antibody of the present invention includes any antibody having a subclass of IgG ₁ / κ and having practical properties as an antibody. Further, as a more preferable embodiment of the antibody of the present invention, one having the following characteristics can be exemplified.

(a) High affinity for human IFN-β, neutralizing titer (10 EU
The dilution ratio of the stock solution for obtaining the antibody concentration required to make human IFN-β of (≈100 IU) / ml 1 EU / ml) is 50 or more for the culture supernatant and 50,000 or more for mouse ascites.

(b) has high specificity for human IFN-β, and human IFN-
It shows no neutralizing activity against α and human IFN-.

(c) It has an affinity for both human IFN-β produced by fibroblasts and human IFN-β produced by the DNA recombination method.

The preparation method and preparation form of the antibody of the present invention are not particularly limited and should be appropriately selected according to the purpose. The hybridoma producing the antibody of the present invention can be obtained by applying a general cell fusion method. The cell fusion method will be described below.

Preparation of antibody-producing cells Human IFN-β is prepared by immunizing a heterologous animal such as mouse, rat, rabbit, sheep, horse, or cow, and the immunized spleen cells, thymocytes, lymph nodes. It is performed by obtaining antibody-producing cells of cells and / or peripheral blood cells.

Preparation of myeloma cells As myeloma cells, cell lines of various animals such as mouse, rat, rabbit and human can be applied. The cell line to be used is preferably drug resistant, and it is preferable that it has the property of not being able to survive in the selective medium in the unfused state, but in the fused state. The most commonly used cell line is 8-azaguanine resistant, which is a hypoxanthinephosphoribosyltransferase.
, And has the property that it cannot grow in hypoxanthine-aminopterin-thymidine (HAT) medium. It is also preferably a so-called "non-secreted" cell line. Specific examples of such cell lines include mouse myeloma MO
_P from PC-21 line _{3 / × 63-Ag8U 1 (} P
₃ U ₁ ), P ₃ / × 63-Ag8 ・ 6 ・ 5 ・ 3 (× 63.6 ・
_{5 · 3), P 3 /} NSI-1-Ag4-1 (NS-1), Sp21
0-Ag14 (SP2), Rattomieroma 210 / RCY3 / Ag1
・ 2.3 (Y3 ・ Ag1.2.3), Human myeloma U-266-AR
₁ , GM1500 and so on.

Cell fusion Cell fusion is based on Eagle's minimal basic medium (MEM), RPMI1640
It is performed by mixing 10 ⁷ to 10 ⁸ myeloma cells and antibody-producing cells at a mixing ratio of 1: 4 to 10 in an animal cell culture medium such as. Polyethylene alcohol (PEG) having an average molecular weight of 1000 to 6000, polyvinyl alcohol, virus, etc. are used as the fusion promoter.

Selection of hybridoma in selective medium To select hybridoma from cells after cell fusion treatment, selective growth in selective medium is performed. For example,
The cells are appropriately diluted with RPMI1640 medium containing 15% fetal bovine serum and seeded on a microtiter plate at about 10 ⁵ to 10 ⁶ cells / well, and a selective medium (for example, HAT) is added to each well.
Culture medium) is added, and the selective medium is appropriately exchanged thereafter for culturing. For example, when 8-azaguanine-resistant strains are used as myeloma cells and HAT medium is used as a selection medium, unfused myeloma cells are killed by about 10 days of culture, and antibody-producing cells which are normal cells are also in vitro for a long period of time. I can't grow. Therefore, all cells growing from the 10th to 14th day of culture become hybridomas.

Search for anti-IFN-β antibody-producing hybridoma The anti-IFN-β antibody-producing hybridoma can be determined by measuring the inhibitory activity (neutralizing activity) of the antiviral activity of IFN-β. That is, for example, after treating FL cells (human amniotic cells) with a mixed solution of IFN-β and a hybridoma culture supernatant for 24 hours, vesicular stomatitis virus (Vesicu
larstomatitis virus (VSV) for 24 hours and then FL
Antibody IFN-β by observing plaques on cells
The presence or absence of antibody production can be determined.

Cloning Since two or more hybridomas may grow in each well, cloning is performed by the limiting dilution method or the like to obtain a single clone antibody-producing hybridoma.

Production of antibody The purest monoclonal antibody can be obtained from the culture supernatant of the produced hybridoma by culturing the hybridoma in an animal cell culture medium such as RPMI1640 medium containing 10 to 15% fetal bovine serum. As for the cell culture method and conditions, those of ordinary animal cell culture methods may be appropriately applied.

On the other hand, as a method for producing a larger amount of antibody, in a syngeneic animal derived from the parent myeloma cells of the hybridoma,
A method may be employed in which a mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) is intraperitoneally administered, and then a hybridoma is intraperitoneally administered to proliferate a large amount. The hybridoma forms an ascites tumor in about 10 to 18 days, and has a high concentration (about 1 to 20) in serum and ascites.
(mg / ml) antibody is produced. When purification is required, ascites is subjected to ammonium sulfate fractionation, DEAE cellulose ion exchange chromatography, and IFN-β-bound Sepharose 4
The objective can be achieved by purifying by affinity column chromatography using B or the like, molecular sieve column chromatography, or the like.

Examples of hybridomas suitable for producing the antibody of the present invention include hybridoma 1H12 strain and hybridoma 8
Succeeded in acquiring C3 stock. Hereinafter, a more specific description will be given on the method for preparing these hybridomas and the properties of the antibody of the present invention produced from them.

I. Obtaining hybridoma BALB / C mice (female, 6 weeks old) were prepared by dissolving IFN-β 700,000 IU (international unit) in physiological saline and mixing it with complete Freund's Ajyu band at a ratio of 1: 1. Intraperitoneally several times every 2 weeks and finally 300,000 I
U IFN-β was injected intravenously.

Three days after the final immunization, splenocytes of the mouse were taken out, and ME
Washed with M. The mouse myeloma P ₃ U ₁ was washed with MEM, splenocytes and P ₃ U ₁ were mixed at 10: 1 and centrifuged,
Cell fusion was performed by gradually adding 1 ml of 50% PEG1000MEM solution to the pellet. Further, the MEM solution was gradually added to make the final volume 10 ml. Centrifuge again, and the pellets were added to RPMI1640 medium containing 15% fetal bovine serum as P ₃ U ₁ at 1 × 10 5.
^The cells were suspended at ⁵ cells / 0.1 ml, and 0.1 ml was spread on each 96-well microplate.

One day later, 0.1 ml of HAT medium was added, and then 3-4
Half of the medium was replaced with fresh HAT medium every day. 7
From around the day, growth of hybridomas was observed in some wells.

0.05 ml of supernatant from wells in which hybridomas have grown
And the FL cells were treated with a mixed solution of 0.05 ml of IFN-β (100 UI / ml) for 24 hours. Further, 300 PFU (plaque forming unit) per well of VSV was infected for 24 hours, and the neutralizing activity of the supernatant against IFN-β antiviral activity was examined. As a result, IFN has a 2-3% probability.
There was a well secreting an antibody showing a neutralizing activity against -β. Those with high neutralizing activity were selected and cloned by the limiting dilution method to obtain hybridoma 1H12 strain and hybridoma 8C3 strain.

II. Production of Monoclonal Anti-IFN-β Using RPMI1640 medium containing 15% fetal bovine serum for each of hybridoma 1H12 strain and hybridoma 8C3 strain
A 96-well plate → 24-well plate → 25 cm ² flask and a scale-up culture, and the supernatant was collected. The neutralizing titer of the antibody IFN-β antibody obtained in these supernatants was 70 for hybridoma 1H12 strain, and hybridoma 8C3.
The stock was 100. The neutralization titer was indicated by the dilution ratio from the stock solution to the concentration of antibody required to make 10 EU (experimental unit) / ml IFN-β 1 EU / ml. In this system, 1 EU / ml corresponds to 10 IU / ml.

Next, hybridoma 1H12 strain or hybridoma 8C3 strain, 2 × 10 ⁶ or more each, pristane 0.5 ml
Was intraperitoneally administered to a mouse to which was previously administered to form an ascites tumor, and ascites was collected. The neutralizing titer of the anti-IFN-β antibody of this ascites was 120,000 for the 1H12 strain, which was 8C.
The number of three strains was 150,000. In addition, this antibody
It showed no neutralizing activity against N-α.

III. Determination of Subclass of Antibody Hybridoma 1H12 Strain and Hybridoma 8C
The culture supernatants of the three strains were salted out with 50% ammonium sulfate and dialyzed in 0.1 M phosphate buffer (pH 8.0). These dialysates were loaded onto a Protein A-Sepharose CL-4B column, washed, and then washed with 0.1 M glycine hydrochloride buffer (pH
3.0) and elute them with phosphate buffered saline (PB
S) was dialyzed in to obtain a purified monoclonal antibody.

Next, a 96-well soft microplate was coated with the above purified monoclonal antibody, and 1% bovine serum albumin (BS
A) After blocking with PBS containing MONOABI
D EIA KIT (ZYMED Inc.) by _{anti-I g} A antibody, _anti-I g _{G 1} antibody, _anti-I g _{G 2} a antibody, _anti-I g _G 2 b
Antibody, _anti-I g _{G 3} antibodies, the reactivity with the anti _{I g} M antibody was seen. As a result, it was divide any hybridoma antibody produced also I _g G _1. Further, as a result of examining the reactivity of the L chain with the anti-κ antibody and the anti-λ antibody, it was revealed to be of the κ type.

IV. Molecular Weight and Neutralization Titer of Purified Antibody Hybridoma BC3 strain was cultured in a serum-free medium, and the culture supernatant was concentrated by ultrafiltration with a molecular weight cutoff of 50,000 and further by ultrafiltration with a molecular weight cutoff of 125,000. This concentrated solution was applied to a Sephadex G-200 column, and a section having a molecular weight of 150,000 was collected. When this was subjected to SDS-polyacrylamide gel electrophoresis in the presence of β-mercaptoethanol, only two bands were observed. From the calibration curve obtained by plotting the molecular weight against the relative mobility of proteins of known molecular weight, it was found that these bands have molecular weights of 52,500 and 23,700, respectively, and are the H chain and L chain of the antibody. From this result, hybridoma 8C
The molecular weight of the single clone anti-IFN-β antibody produced by the three strains was determined to be 152,400.

Next, the neutralizing titer of this purified antibody was measured by VS using FL cells.
Dye uptake variant of Fuinta the V as Chiya range virus was determined by (J. Gen. Virol. ( 1969) 5 419-427). As a result, this antibody showed a concentration of IF of 1 μg / ml.
N-β10 EU / ml was neutralized to 1 EU / ml. In this experimental system, 1 EU / ml corresponded to 11,7 IU / ml.

V. Purification of IFN-β by the antibody column of the present invention (1) The hybridoma 8C3 strain was intraperitoneally administered to a mouse previously administered with 0.5 ml of pristane to form an ascites tumor,
Ascites was collected. The neutralizing titer of the monoclonal anti-IFN-β antibody in this ascites fluid was 100,000.

This ascites fluid is diluted twice with PBS, an equal volume of saturated ammonium sulfate solution is added, and the mixture is centrifuged. The precipitate is washed with 50% ammonium sulfate solution and then centrifuged again.
Dissolved in 0.1M sodium bicarbonate buffer (pH 8.5)
Dialyzed overnight. O.V. of this solution D. At 280 nm, 50 mg of protein was diluted to 6 ml with 0.1 M sodium hydrogen carbonate buffer (pH 8.5), and coupled with 2 ml of agarose gel carrier (Affi gel 10) overnight at 4 ° C.

After the coupling carrier was packed in a column, it was thoroughly washed with PBS to produce partially purified IFN-β10 produced from fibroblasts.
20,000 IU made up to 20 ml with PBS (pH 7.4) containing 0.1% BSA was passed at SV = 1. 0.2M after washing with PBS
Glycine hydrochloric acid buffer (containing 0.5M sodium chloride, pH 3.
It was eluted at 0).

As a result, 10 to 20% of the passed amount of IFN-β passed, but the rest was adsorbed on the column, and 80% of the adsorbed amount was obtained by elution.
~ 90% could be recovered. Therefore, the antibody of the present invention has a property of dissociating the adsorbed IFN-β at pH 3.0, and the IFN-β can be purified in a pH range in which IFN-β is more stable.

(2) 8 ml of ascitic fluid obtained by intraperitoneally administering the hybridoma 8C3 strain to BALB / C mice was salted out with 45% ammonium sulfate, centrifuged, and the precipitate was precipitated with 0.1M sodium hydrogen carbonate buffer (0.5M sodium chloride). Contained, pH 9.0)
And was dialyzed against the same buffer. 8 ml of this ammonium sulphate-purified antibody (protein content 11.9 mg / ml) was reacted with 9 ml of sepharose 4B activated with bromocyan at 18 ° C. for 18 hours. After the reaction, the buffer solution was removed by filtration on a glass filter and further washed with PBS. The protein content in the buffer was 1.44 mg / ml and the coupling efficiency was 88%.

This gel was packed in a column, and crude IFN-β (specific activity 2.5 × 10 ⁵ IU / mg) produced by fibroblasts was added to 4.4 × 10 ⁷ IU.
Loaded After washing the column with PBS, the non-specific adsorbate was removed with 1.0 M sodium chloride + 0.1% Tween 80 solution, and 1.0 M sodium chloride + 0.1% Tween 80 + 0.2 was added.
M citric acid + 50% ethylene glycol solution (pH 2.0)
Was eluted by.

As a result, the recovery of the eluted IFN-β was 45%, and the specific activity was 3.5 × 10 ⁷ IU / mg, which was 140 times that of IFN-β before purification. In the peak fraction, the specific activity reached 1.0 × 10 ⁸ IU / mg.

(3) (2) in the same manner as in the section method I _{g G} fraction was partially purified by salting-out from the mouse ascites 8.5ml obtained from hybridoma 1H12 strain, cyanogen bromide activated Sepharose 4B
It is reacted with 9ml was produced above I _g antibody column the _G fraction is bound carrier 1ml per 12 mg.

This antibody column was loaded with 8.1 × 10 ⁶ IU of IFN-β partially purified product (specific activity 1.8 × 10 ⁵ IU / mg) produced by Escherichia coli in which genetic information for human IFN-β was incorporated. PB
After washing the column with S, 1 M sodium chloride and 50
PH of 0.2M citrate buffer containing 10% ethylene glycol
Elution was performed with 5.0 solution and then with pH 2.0 solution.

As a result, the activity recovery rate of the eluted fraction at pH 2.0 was 34%.
The average specific activity is 6.3 × 10 ⁷ IU / mg, which is about 3 in one step.
It was purified 50 times. The specific activity of peak fraction is 1.
It was 1 × 10 ⁸ IU / mg.

VI IFN-β by enzyme immunoassay using the antibody of the present invention
(1) Hybridoma 1H12 strain was treated with BALB. Mouse ascites obtained by intraperitoneal administration to C mice was diluted 100-fold with 20 mM PBB (pH 7.4) containing 0.15 M sodium chloride, and 0.2 ml each was commercially available 96-well immunomicroplate for immunoassay. Each well was placed in each well and coated at room temperature for 1 hour. Then PB containing 0.05% Tween 20
Each well of the microplate was washed once with S and twice with PBS. 0.3 ml / well of PBS containing 2% BSA was added, and the protein-engaging site on the plastic surface was BS.
Blocked with A. The plate can be stored in the refrigerator for a long time in this state.

After blocking, the plate was washed with PBS in the same manner as above, and a human IFN-β preparation was added as a measurement sample to a P containing 0.2% BSA.
It was dissolved diluted with BS 0.1~10 ⁵ IU / ml of solution 0.2 ml /
Wells were added and left at room temperature for 2-4 hours or at 4 ° C. overnight to allow IFN-β to bind to the primary antibody.

After washing, an immunoglobulin fraction purified from a rabbit antiserum obtained by sensitizing with the above IFN-β preparation using a protein A-Sepharose column was used to obtain N- (4-carboxycyclohexylmethyl) maleimide N-hydroxysuccinimide. A fixed amount (1 to 20 μg) of a labeled antibody cross-linked with β-galactosidase derived from Escherichia coli using an ester was added to 0.2
As a PBS solution containing% BSA, 0.2 ml was added to each well and left at room temperature for 2 to 4 hours or at 4 ° C for 2 nights to obtain IFN.
Bound to β.

After washing with PBS containing Tween 20 two to three times and twice with PBS, the β-galactosidase activity of the bound second antibody was adjusted to p.
-Nitrophenyl β-D-galactopyranoside as a substrate, pH 7.2 phosphate buffer (0.1 M sodium chloride, 1 m
M magnesium chloride) to give 40
Absorbance at 5 nm was measured using a Multiscan of Titertec. As a reference, human serum albumin and bovine serum components were measured by the same method as test substances. These measured values are shown in Table 1.

As is clear from the results in Table 1, this assay system did not react at all with anything other than IFN-β, and the specificity of using the monoclonal antibody as the primary antibody was fully utilized. Moreover, IFN-β itself is measured with extremely high sensitivity. Of course, in this measurement system, IFN-α and IFN-
Does not react at all.

(2) Monoclonal antibody produced by the hybridoma 1H12 strain and monoclonal antibody produced by the hybridoma 8C3 strain were diluted with a 1000-fold dilution of mouse ascites.
By the same procedure as in (1), the cell extract containing IFN-β produced by Escherichia coli in which the genetic information for human IFN-β was incorporated was measured. The measurement results are shown in Table 2.

As is clear from the results in Table 2, the E. coli extract containing no IFN-β did not interfere with the present measurement system at all, and human IFN-β derived from E. coli was measured with extremely high sensitivity.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI technical display location (C12P 21/08 C12R 1:91) (72) Inventor Hirohisa Kato 2-chome Shinsei-cho, Choshi-shi, Chiba No. 1 at Yamasa Shoyu Co., Ltd. in the laboratory (72) Inventor Masao Fujimoto 2-10 Shinsei-cho, Choshi-shi, Chiba No. 1 at Yamasa Shoyu Co., Ltd. in the laboratory (72) Inventor Hirohiko Shimizu 1111 Tehiro, Kamakura, Kanagawa Address: Toray Co., Ltd. Basic Research Laboratory (72) Inventor Kazuo Hosui, 1111 Tehiro, Kamakura City, Kanagawa Prefecture Toray Co., Ltd. Basic Research Laboratory (72) Inventor, Masahiko Hirano, 1111, Tehiro, Kamakura City, Kanagawa (56) References Hybridoma, 1,233 (1982)

Claims (2)

[Claims] _{1. A} monoclonal antibody belonging to IgG ₁ / κ derived from a hybridoma 1H12 strain or 8C3 strain, which specifically reacts with human β-type interferon by an antigen-antibody reaction. 2. The monoclonal antibody according to claim 1, which has the ability to neutralize 10 EU / ml of human β-type interferon to 1 EU / ml at an antibody concentration of 1 μg / ml.