JPS59144796A - Monoclonal antibody - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody which has specificity to human beta- type interferon (IFN-beta) and belongs to IgG/kappa as a subclass. USE:The purification of IFN-beta by means of affinity chromatography and the determination of the same by means of immunoassay. PREPARATION:After a different kind of animal is immunized with human IFN- beta, the spleen is isolated and its cells are collected. These cells are mixed with myeloma cells and polyethylene glycol of 1,000-6,000 average molecular weight is added thereto as a cell-fusion accelerator to effect cell fusion, then these cells are cultured in a selective medium to obtain hybridoma. The resultant hybridoma is subjected to cloning by the limitation dilution method to separate the cells producing monoclonal antibody. Then, the cells are given to mice peritoneally and cultured to given the objective monoclonal antibody.

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 "FN-β"). .

In recent years, interferon (hereinafter abbreviated as "IFN") has become the focus of attention in the medical world as it has become clear that it has antitumor effects in addition to antiviral effects9. Three types of IFN are known: α-type interferon (leukocyte interferon, hereinafter abbreviated as rIFN-α), IFN-β, and γ-type interferon (immune interferon, hereinafter abbreviated as rIFN-rj). IFN-α stimulates white blood cells to become infected with Newcastle disease virus (NDV) or Sendai virus (HV).
IFN-β stimulates fibroblasts with double-stranded RNA such as poIJ (I) and (C), or N
IFN-γ can be produced by stimulation with a virus such as DV, or by stimulation of rhinnocytic cells with T cell mitogens or antigens. Furthermore, by applying recent genetic engineering technology, a human IFN-producing gene prepared from mRNA for IFN derived from human cells was incorporated into a plasmid, and this plasmid was then transformed into E. coli.
A method has also been developed in which IFN-e is produced by introducing the gene into the cells of microorganisms such as Bacillus subtilis or yeast to fully express its gene functions (Proc, Natl.

Acad, Set, , 77, 7010 (198
0) ).

However, in cell culture methods, industrial cell culture methods themselves have not yet been established, and the production of IFH is extremely small, leading to the increase in the size of production equipment and the complexity of IFN purification operations. Manufacturing costs are required. Also, in the method using genetically modified microorganisms,
Although mass production of IFH becomes possible, since the microbial culture solution contains a large amount of impurities, a high degree of separation and purification operation is required to fully prepare high-purity IFN.

As one of the advanced separation and purification methods for IFN, a method for purifying 9IFNe by affinity chromatography using an insoluble carrier immobilized with an antibody against IFH is known. In order to establish this antibody affinity chromatography as an industrial purification method, it is necessary to supply antibodies with high specificity and large binding capacity for IFH in large quantities and at low cost. As a means to meet these demands, there is a known method in which several anti-IFN antibody-producing cells and tumor cells are used to prepare a whole hybridoma (fused cell) and then cultured to produce a single clone antibody. ing.

There have also been some reports on monoclonal antibodies against IFH and purification of IFN using affinity chromatography. For example, monoclonal antibodies against IFN-α have been produced by Setcher et al. (Nature, 235.446 (1980)) and Schirin (Proc. . Sechar et al. purified 5000-fold IFN-α by affinity column chromatography using a monoclonal antibody immobilized on whole Sepharose as a t carrier (specific activity of IFN-α: 1,8 X 108U
/■). On the other hand, Shchirin et al. produced approximately 100% IFN-α in one step by genetically engineering Escherichia coli using a carrier bound to a monoclonal antibody on an agarose gel.
It has been purified 00 times.

Nine single-clonal antibodies against IFN-β were developed by Hochkappel A et al.
e, 29L500 (1981), Eur, J.
Blochem, 118.437 (1981), Japanese Unexamined Patent Publication No. 57-194788),
ich) et al. (Hybridoma, 1.223 (1
982)) etc. However, Hochkeppel et al.'s antibody is of the subclass IpM, and its neutralizing titer against IFN-β (the dilution factor of the stock solution that inhibits the antiviral activity of IFN-β by 50%) is 7 in the culture supernatant. It is as low as 70 in mouse ascites, and the antibody of Ehritz et al.
− can only partially neutralize the antiviral activity of β;
None of them were satisfactory for affinity chromatography used for industrial purification of IFN-β.

A monoclonal antibody with high specificity and strong affinity for IFN-β is useful for purifying IFN-β by affinity chromatography, and is not a monoclonal antibody.
It can also be applied to a method for highly sensitively quantifying IFN-β present in trace amounts in biological samples by immunoassay techniques in drug monitoring and the like. Therefore, the creation of such a monoclonal antibody against IFN-β has been long awaited.

The present invention was completed based on this technical background, and has extremely useful properties for practical use in purifying IFN-β by affinity chromatography or quantifying IFN-β by immunoassay. The present invention provides monoclonal antibodies against IFN-β having the following properties.

The monoclonal antibody against IFN-β according to the present invention is distinguished from conventionally known antibodies in that its subclass is IyG+/.

The antibodies of the present invention include all antibodies that have a subclass of I f G and/or have practical properties as antibodies. Moreover, as a more preferable embodiment of the antibody of the present invention, one having the following characteristics can be exemplified.

(a) Human I with high affinity for human IFN-β and a neutralizing titer (IOEU (≠100 I U )/ml)
The antibody required to make FN-β to IEU/ml (the dilution factor of the stock solution to obtain the concentration) is 50 or more in the culture supernatant,
It is more than 50,000 in mouse ascites.

(b) h) has high specificity for IFN-β,
It does not show neutralizing activity against human IFN-α and human 1-IFN-γ.

(C) IFN-β and DN produced by fibroblasts
It has parental properties for both human FN-β produced by the A-silk recombination method.

The preparation method and preparation form of the antibody of the present invention are not particularly limited and should be appropriately selected depending on the purpose. A hybridoma producing the antibody of the present invention can be obtained by applying a general cell fusion method. This cell fusion method can be explained as follows.

■ Preparation of antibody-producing cells Antibody-producing cells are prepared by immunizing foreign animals such as mice, rats, rabbits, sheep, horses, and cows with human IFN-β, and then immunizing lung cells, thymocytes, etc. Lymph 9
This is carried out by obtaining all antibody-producing cells such as nodal 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 used. The cell line used is preferably drug-resistant, and preferably has the property that it cannot survive in a selective medium in an unfused state, but can survive in a fused state. The most commonly used are 8-azaguanine-resistant cell lines, which contain hypoxanthine phosphoribosyltransferase (hypoxanthinephosphoribosyltransferase).
ribOsyltransferase) f-deficient, hypoxanthine-aminopterin-thymidine (HAT
) It has the property of not being able to grow in culture medium. Also, the so-called “
Preferably, the cell line is a "non-secreting" cell line. Specific examples of such cell lines include mouse myeloma MOPC.
P3/X63-Ay 8U+ (P
sUl), Ps/X 63-kf8・6・5・3(×
63.6・5・3), Ps/NS I-1-A, 4-
1 (M4-1(,5p210-Ap14(SF3), rat myeloma 210・RCY3・Atl・2・3(Y
3・kf 1.2.3), human myeloma U-26
6-ARs SGM 1500 and the like.

■ Cell fusionCell fusion uses Eagle's minimal essential medium (MEM), RPM
107-1 in animal cell culture medium such as I 1640.
08 myeloma cells and antibody producing cells at a mixing ratio of 1:4~
Mix at 10. As a fusion promoter, the average molecular weight is 1.
000-6000 polyethylene glycol (PEG)
ffi, polyvinyl alcohol, viruses, etc. are used.

(2) Selection of Vibe IJ y-ma in a selective medium To select all hybridomas from cells after cell fusion treatment, selective growth in a selective medium is performed. For example, cells are appropriately diluted with RPMI 1'640 medium containing 15% fetal bovine serum, plated on a microtiter plate at a rate of 10 to 106 cells, and placed in each well with a selective medium (e.g., HAT medium). etc.), and then culture with appropriate replacement of the selection medium. For example, as myeloma cells8
- If HAT medium is used as the azaguanine-resistant selective medium, unfused myeloma cells will die within about 10 days of culture, and normal antibody-producing cells will also die.
It cannot be grown for a long time in vitro. Therefore, all cells that grow after 10 to 14 days of culture become hybridomas.

■ Search for anti-IFN-β antibody-producing hybridomas Anti-IF
N-β antibody-producing hybridomas 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 mixture of total IFN-β and hybridoma culture supernatant for 24 hours, vesicular stomatitis virus (Ve
aicularstomatitis virus,
vsv)k The presence or absence of anti-IFN-β antibody production can be determined by infecting cells for 24 hours and observing plaques on FL cells.

■ Cloning Since there is a possibility that two or more types of hybridomas are growing in each well, cloning is performed by the limiting dilution method to obtain a single clone antibody-producing hybridoma.

■ Production of antibodies The purest single-cloned antibody is produced by all 10-15 bovine fetal serum-containing RPMI 164 antibodies.
It can be obtained from culture medium obtained by culturing in an animal cell culture medium such as 0 medium.

The cell culture method and conditions may be appropriately applied to any conventional animal cell culture method.

On the other hand, as a method for producing a large amount of whole antibodies, using animals syngeneic with the animal from which the parent myeloma cells of the hybridoma were derived,
A method can be adopted in which a mineral oil such as bristan (2,6,10,14-tetramethylpentadecane) is intraperitoneally administered, and then the N. hybridoma is intraperitoneally administered to grow it in large quantities. Hybridomas form ascites tumors in the throat for 10 to 18 days, with high concentrations (approximately 1
-20 η/ml) of antibodies are produced. If purification is required, the ascites is fractionated with ammonium sulfate and then purified by DEAF cellulose ion exchange chromatography, affinity column chromatography using Sepharose 4B fully bound to IFN-β, molecular sieve column chromatography, etc. You can reach your goal by refining it.

As examples of hybridomas suitable for producing the entire antibody of the present invention, hybridoma strain IH12 and hybridoma strain 803 have been successfully obtained. Below, we will provide a more detailed explanation of the methods for preparing these hybridomas and the properties of the antibodies of the present invention produced from them.

(Margins below) ■. Obtaining hybridomas. 700,000 IU (international units) of IFN-β was dissolved in physiological saline and mixed with complete Freund's adjuvant in a ratio of 1:1 to make an emulsion. , 6 weeks of age), administered intraperitoneally several times every two weeks, and finally
00,000 IU of IF'N-β was injected intravenously.

Three days after the final immunization, the mouse tile cells were removed and ME
Washed with M. Mouse myeloma P, U, were washed with MEM, tile cells and P, Ul were mixed at a ratio of 10:1, centrifuged, and the pellet was mixed with 50% PEG100 OMEM solution.
ml was gradually added to perform cell fusion. Further ME
Add M solution gradually and finally lQmA! And so. Centrifuge again and transfer the pellet to RPMI containing 15% fetal bovine serum.
1640 medium as P, U, I x 10
Cell/0.1 mA, suspended at 96
1 ml of Q was sprinkled on a well microplate.

One day later, Q, lrnl of HAT medium was added, and then half of the medium was replaced with fresh HAT medium every 3 to 4 days. Hybridoma growth was observed in some wells from day 9 on day 7.

0.05m supernatant of the well where hybridomas have grown
1 to 9, IFN-β (100 I U/mQO, 051
F'L cells were treated with a mixture of d and F'L cells for 24 hours. In addition, υ300 PFU (plaque forming units) per well
The cells were infected with Sv for 24 hours, and the neutralizing activity of the supernatant against the antiviral activity of IFN-β was examined. As a result, there were 2 to 3 wells secreting antibodies exhibiting neutralizing activity against IFN-β. Select a product with high neutralizing activity, perform cycloning using the limiting dilution method, and create Hybrid Beef 1.
812 strains and hybridoma 8C3 strains were obtained.

Hybridoma strain IH12 and hybridoma strain 803 were each cultured using RPM11640 medium containing 15% fetal bovine serum in a scale-up of 96-well plate → 24-well plate → 25 cm flask, and the supernatant was collected.

The neutralizing titer of anti-IFN-β antibodies obtained in these supernatants was 70. The number was 100 for the hybridoma 8C3 strain. In addition, the neutralization titer is 10E
U (experimental unit)/+nl of IFN-β to IEU/ff1
The antibody concentration required to make 1 liter of antibody was expressed as a dilution factor from the stock solution. In this system, IEU/ml is 10IU/mJ.
It was equivalent to

Next, hybridoma IH12 strain or hybridoma 803 strain was added at least 2x10 each to Bristane O4.
It was administered intraperitoneally to mice that had been previously administered with 5-go to form ascites tumors, and the ascites was collected. The neutralizing titer of the anti-IFN-β antibody in this ascites was 120.0 by the IH12 strain.
00, the number of 803 stocks was 150,000. Furthermore, this antibody did not show neutralizing activity against IFN-α.

Determination of subclasses of L antibody hybrid beef 1812 strain and hybrid beef 12-ma 8
The culture supernatant of C3 strain was salted out with 50 ammonium thiosulfate, 0.1M, ? Dialysis was performed in phosphate buffer (pH 8,0). These dialysates were loaded onto a protein A-Sepharose CL-4B column, washed, and eluted with 0.1 M glycine-hydrochloride buffer (pH 3,0). The purified monoclonal antibody was obtained by dialysis.

Next, a 96-well soft microplate was coated with the purified single clone antibody, and 1% bovine serum albumin (BSA) was added.
) After blocking with PBS containing MONOABID
υ anti-IA using EIA KIT (manufactured by ZYMED)
Antibody, anti-1. G.

Antibody, anti-IG, a antibody, anti-IgG, b antibody, anti-I,
, G, antibody, and the reactivity with anti-gM antibody was examined.

As a result, the antibodies produced by any hybridoma are
It turned out to be gGI. Furthermore, as a result of examining the reactivity with an anti-L chain antibody and an anti-λ antibody, it was revealed that the antibody was of type 2.

(2) Molecular weight and neutralization titer of purified antibody Hybridoma strain 803 was cultured in a serum-free medium, and the culture supernatant was concentrated by ultraviolet filtration with a molecular weight fraction i of 50,000. ,000 by ultrafiltration. This concentrated solution was applied to a Sephadex G-200 column, and a fraction with a molecular weight of 150,000 was collected.

When this was subjected to 5DS-polyacrylamide del electrophoresis in the presence of β-mercaptoethanol, only two bands were observed. From a calibration curve obtained by plotting the molecular weight against the relative mobility of proteins with known molecular weights, these bands have molecular weights of 52,500 and 23.70.
It was found that these were the H and L chains of the antibody. From this result, the molecular weight of the single clone anti-IFN-β antibody produced by No. 8C3 strain is 152,400.
It was decided.

Next, the neutralizing titer of this purified antibody was determined using F T cells.
Modified Finter's dye uptake method using ■S■ as a challenge virus (J, Gen, Virol.

(1969) 5 419-427).

As a result, at a concentration of 1 μg/mε, this antibody
10 EU/ml was neutralized to IEU/ml. In this experimental system, IEU/me is 11.7 IU/m
It corresponded to l.

■1 Purification of IFN-β using the antibody column of the present invention (1)
The hybridoma 8C3 strain was intraperitoneally administered to mice that had been previously administered 0.5 ml of pristane to form ascites tumors, and the ascites was collected. This ascites monoclonal anti-IFN
The neutralization titer of -β antibody was 100,000.

This ascites was diluted 2 times with PBS, an equal volume of saturated ammonium sulfate solution was added and centrifuged, the precipitate was washed with 50% ammonium sulfate solution, centrifuged again, and the precipitate was diluted with PBS.
Dissolved in 0.1M hydrogen carbonate) IJum buffer (
The mixture was dialyzed overnight (pH 8.5). of this solution. D.2
80 nm was measured, and the tannock mass was 50 to 0.
．． 1M hydrogen carbonate) IJum buffer (pH 8,5)
diluted to 6r/ll with
Gel 10) 2*g was combined overnight at 4°C.

After filling the column with the coupling carrier, it was thoroughly washed with PBS, and the partially purified IFN-β10 produced by fibroblasts was washed with PBS.
00,000 IU in PBS containing 0.1 T BSA (pH 7,4
) and the volume was adjusted to 20 rnl, and the solution was passed at 5V=1. P.B.
After washing with S, elution was carried out with 0.2M glycine-hydrochloride buffer (containing 0.5M sodium chloride, pH 3.0).

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

(2) Ascites 81 obtained by intraperitoneal administration of hybridoma 8C3 strain to BALB/C mice was salted out with ammonium 45 sulfate, and after centrifugation, the precipitate was dissolved in 0.1 M sodium bicarbonate buffer (0 , contains 5M sodium chloride,
pH 9,0) and dialyzed against the same buffer. This ammonium sulfate fraction-purified antibody (pnzQ protein content 11.9rng/
8 mJ was reacted with Sepharose 4B 9-9 activated with bromcyan at 4°C for 18 hours. After the reaction, remove the buffer solution by filtering it on a glass filter, and then add PBS.
Washed with. The protein content in the buffer is 1.44 m
y/rnl, the coupling efficiency was 88%.

This gel was packed into a column, and crude IFN-β produced by fibroblasts (specific activity 2.5×10'IU/n1g)
was loaded at 4.4×10 IU. After washing the column with PBS, remove non-specific adsorbates with a 1.0 M sodium chloride + 0.1% Tween 80 solution, and add 4° 1, 0 M sodium chloride 10, 1 Tween 80 + 0.
2M phosphoric acid + 50% ethylene glycol solution (pH 2,
0) was eluted.

As a result, the recovery rate of eluted IFN-β was 45%, and the specific activity was 3.5×10 IU/m, which was lower than that of IFN-β before purification.
140 times more purification was achieved compared to

In addition, the specific activity of the peak fraction is 1.0X10
1 for IU/fng.

(3) Hybridoma I was prepared in the same manner as in (2).
The IgG fraction was partially purified from 8.5μ of mouse ascites obtained from the H12 strain by salting out, and reacted with 9ml of Bromsia Y-activated Sepharose 4B.
An antibody column to which the above I□G fraction was bound was prepared.

8.1×10 I
U negative load. After washing the column with PBS, IM-0.2M containing sodium chloride and 50 thiethylene glycol
It was eluted with a pH 5.0 solution of citrate buffer, followed by a pH 2.0 solution.

As a result, the activity recovery rate of the elution fraction at pH 2.0 was 34
The average specific activity was 6.3 x 107 IU/mg, which was produced approximately 350 times in one step. The specific activity of the peak fraction is 1. ], X108 IU/my.

Quantification (1) Mouse ascites obtained by intraperitoneal administration of hybridoma I H12 strain to BALB/C mice was mixed with 20 m MPBS (pH 7,4) containing 015 M sodium chloride.
diluted 100 times with
Coating was carried out for 1 hour at room temperature. Each well of the microplate was then washed once with PBS containing 0.0596 Tween 20 and twice with PBS. Contains 2% BSA
.

0,:11J/well of PBS was added, and the tannin binding site on the plastic surface was blocked with BSA. The plate can be stored in this state for a long period of time in the refrigerator.

After blocking, it was washed with PBS in the same manner as above, and used as a measurement sample.H) IFN-β preparation was dissolved and diluted in PBS containing 0.2% BSA at a concentration of 0, 1 to 10' IU/
Add 0.2 ml of solution per well and leave to incubate for 2-4 hours at room temperature or overnight at 4°C to infuse the primary antibody with IFN.
−β was combined.

After washing, the immunogloupulin fraction purified on a protein A-Sepharose column from the rabbit antiserum obtained by sensitization with the above IFN-β preparation was treated with N-hydroxysufushiimide ester of N-(4-carboxycyclohexylmethyl)maleimide. A certain amount (1 to 10 μg) of a labeled antibody cross-linked with E. coli-derived β-galactosidase using 0.
2Q,'l in each well as BSA-containing PBS solution.
The mixture was added in ml portions and allowed to stand at room temperature for 2 to 4 hours or overnight at 4°C to bind to IFN-β.

After washing 2-3 times with PBS containing Tween 20 and 2 times with PBS, the β-galacto/tase activity of the bound second antibody was determined.
- Phosphate buffer (0.1M chloride) at pH 7.2 using nitrophenyl β-D-galactopicinoside as a substrate I
Jum, containing 1mM magnesium chloride),
The absorbance of the product at 405 nm was measured using a Titertec Multiscan. As a reference, human serum albumin and bovine serum components were measured in the same manner as the test substances. These measured values are shown in Table 1.

Table 1 H) As is clear from the results of enzyme immunoassay cloth 1 for IFN-β, this assay system does not react at all to anything other than IFN-β, and a monoclonal antibody was used as the first antibody. The uniqueness of the tank is fully utilized, and the IF
N-β itself has been measured with very high sensitivity. Of course, in this measurement system, IFN-α and IFN-γ do not react at all.

(2) Regarding the monoclonal antibody produced by hybridoma IH12 strain and the monoclonal antibody produced by hybridoma strain 803, follow the same procedure as in the previous section (1) using a 1000-fold dilution of mouse ascites. A bacterial cell extract containing IFN-β produced by E. coli into which genetic information for β had been incorporated was measured. The measurement results were as shown in Table 2.

Table 2 Enzyme immunoassay of IFN-β produced by Escherichia coli Antiviral activity measured using FL cell/VSVO system (37°C) IFN-β international unit conversion value As shown in Table 2, The E. coli extract containing no IFN-β did not interfere with this measurement system at all, and human IFN-β derived from E. coli was measured with very high sensitivity.

Representative Patent Attorney Moto Imamura -Edict- Kamakura Kata Tehiro 1111 Higashi Co., Ltd. Inside the company basic research institute ■Initiator: Masahiko Hirano Kamakura Kata Tehiro 1111 Higashi Co., Ltd. Inside the company basic research institute ■Applicant: Toshi Co., Ltd. Nihonbashi Muromachi 2-chome, Chuo-ku, Tokyo No. 2

Claims (1)

[Claims] It has specificity for human β-type interferon, and its subclass is I! G+/only C=-on antibody.