JPH0720438B2 - Antibodies to interleukin-1 - Google Patents

Description

TECHNICAL FIELD The present invention relates to an antibody against interleukin-1 (IL-1), and more specifically, a novel human IL-1 which enables immunological purification and measurement of IL-1. To an antibody against.

2. Description of the Related Art Human IL-1 is known to be produced not only by macrophages / monocytes but also by many cells and exhibit various molecular forms and biological activities. At present, two types of human IL-1 having different isoelectric points, that is, IL-1α and IL-1β, are known, and their primary structures have been clarified respectively [Nature, 315 , p641]. (1985); J. Exp. Med., 164 , p237.
(1986) etc.].

The above-mentioned IL-1 has been variously studied for its application as a medicine, and its measurement in clinical samples, especially for clinical diagnosis of various immunodeficiency diseases and abnormal immune responses and clinical diagnosis. Attention is paid.

At present, a bioassay (biological assay method) is known as a technique for measuring the IL-1. In this method, IL-1 is measured as the active amount of a test sample. However, this method is inferior in operability and accuracy, and it is necessary to always consider the presence of a component that interferes with the measured value. Moreover, in the above method, since IL-1α and IL-1β exhibit the same activity, there is a major drawback that they cannot be measured separately.

Problems to be Solved by the Invention An object of the present invention is to provide an antibody against the above human IL-1β.

Another object of the present invention is to provide an antibody against human IL-1 which can be used in a new immunological measurement method capable of measuring human IL-1β separately from human IL-1α.

Another object of the present invention is to provide an antibody against human IL-1 that can be used for suppressing (neutralizing) the action of IL-1 in various diseases accompanied by abnormal production of IL-1.

Another object of the present invention is to provide a technique for producing the above antibody.

Means for Solving the Problems According to the present invention, a human IL-1β antibody obtained by using human IL-1β obtained by natural or genetic recombination method as an antigen, wherein the following fragment of human IL-1β (1 ) And (7) and (1), (5), (6) and (7),
(1), (3), (4), (5), (6) and (7)
Or a human IL-1β monoclonal antibody, which binds to all of (1) to (7).

(1) A polypeptide consisting of the 24th to 153rd sequences.

(2) A polypeptide consisting of the 1st to 82nd sequences.

(3) A polypeptide consisting of the 1st to 102nd sequences.

(4) A polypeptide consisting of the 1st to 120th sequences.

(5) A polypeptide comprising the 1st to 140th sequences.

(6) A polypeptide consisting of the 1st to 144th sequences.

(7) A polypeptide consisting of the 1st to 148th sequences.

According to the use of the antibody of the present invention, human IL-1β is converted into human IL-1.
Provided is a new immunoassay method (immunoassay method) which is highly sensitive and accurate and can be easily measured in distinction from α.

Since the antibody of the present invention is specific to human IL-1β,
The use thereof also provides a means for specifically purifying them, for example, by a technique such as affinity chromatography.

Furthermore, the antibody of the present invention includes a type of antibody having a neutralizing activity with respect to the biological activity of human IL-1, and such antibodies include various diseases associated with abnormal production of IL-1, such as rheumatoid arthritis, In chronic inflammatory diseases such as thyroiditis, hepatitis, nephritis, arteriosclerosis, vasculitis such as Kawasaki disease, generalized intravascular coagulation syndrome, blood cancer, etc., enhanced bioactivity of IL-1 based on its abnormal production A drug which is useful for suppressing or neutralizing the above diseases and which is extremely valuable in the treatment of such various diseases is provided.

The antibody of the present invention can be produced by using human IL-1β as an immunogen. More specifically, for example, a fused cell (hybridoma) of a mammalian plasma cell (immune cell) immunized with the above-mentioned immunogen and a mammalian plasmacytoma cell is prepared, and human IL-1β is recognized therefrom. It can be produced by selecting a clone producing a desired antibody and culturing the clone.

Human IL-as an immunogen used in the above method
1β is not particularly limited, and a known culture supernatant containing human IL-1β induced in vitro or a purified preparation thereof, human IL-produced according to gene recombination technology
Any of synthetic peptides having 1β or a part of the amino acid sequence thereof may be used.

The mammal to be immunized with the immunizing antigen in the above method is not particularly limited, but is preferably selected in consideration of compatibility with the plasmacytoma cells used for cell fusion, and is generally mouse or rat. Etc. are advantageously used.

Immunization can be carried out by a general method, for example, by administering the above-mentioned immunizing antigen to a mammal by intravenous, intradermal, subcutaneous, or intraperitoneal injection. More specifically, the immune antigen
It is preferable that the test animal be administered several times every 2 to 14 days in combination with an ordinary adjuvant so that the total dose is about 100 to 500 μg / mouse. As the immunizing antigen, it is preferable to use spleen cells extracted about 3 days after the final administration.

Further, as mammalian plasmacytoma cells as the other parent cell fused with the above-mentioned immune cells, various known cells such as p3 (p3 / x63-Ag8) [Nature, 256 , 495-49] are used.
7 (1975)], p3-U1 (Current Topics in Microbiolog
y and Immunology, 81 , 1-7 (1978)], NS-1 [Eur.
J. Immunol., 6 , 511-519 (1976)], MPC-11 [Cell,
8 , 405-415 (1976)], SP2 / O [Nature, 276 , 269-270]
(1978)], FO [J. Immunol. Meth., 35 , 1-21 (198
0)], × 63.6.5.3. (J. Immunol., 123 , 1548-1550 (197
9)], S194 [J. Exp. Med., 148 , 313-323 (1978)] and R210 [Nature, 277 , 131-133 (197) in rats.
9)] and the like myeloma cells can be used.

The fusion reaction between the immune cell and the plasmacytoma cell is a known method, for example, the method of Milstein et al. [Method in Enzymolog
y, Vol.73, pp3 (1981)] and the like. More specifically, the above fusion reaction is carried out in an ordinary medium in the presence of an ordinary fusion promoter such as polyethylene glycol (PEG), Sendai virus (HVJ), etc. An auxiliary agent such as dimethyl sulfoxide may be added as necessary to increase the amount. The ratio of immune cells to plasmacytoma cells used is not different from the usual method, and for example, it is common to use about 1 to 10 times more immune cells than plasmacytoma cells. Examples of the medium at the time of the fusion reaction include various ones usually used for the growth of plasmacytoma cells, for example, RPMI-1640 medium, MEM medium, and other commonly used ones for this kind of cell culture. It is advisable to remove serum replacement fluid such as fetal calf serum (FCS) from the iso-medium. For the fusion, a predetermined amount of the above immune cells and plasmacytoma cells is mixed well in the above medium, and the mixture is pre-heated to 37 ° C.
PEG solution heated to a moderate degree, for example, average molecular weight 1000-6000
It is carried out by adding about 1 to about 30 to 60 W / V% of the concentration to a medium and mixing them. After that, a desired hybridoma is formed by repeating the operation of sequentially adding an appropriate medium, centrifuging and removing the supernatant.

Isolation of the desired hybridoma obtained is carried out by culturing in a usual selection medium, for example, HAT medium (medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT medium may be carried out for a time sufficient to kill cells other than the target hybridoma (unfused cells etc.), usually for several days to several weeks. The hybridoma thus obtained is subjected to a search for a desired antibody and a monocloning by a usual limiting dilution method.

The target antibody producing strain can be searched by, for example, the ELISA method [Engvall, E.,
Meth. Enzymol., 70 , 419-439 (1980)], plaque method,
Spot method, agglutination method, octellony
y) method, radioimmunoassay (RIA) method, and other various methods generally used for antibody detection [[Hybridoma method and monoclonal antibody], published by R & D Planning, pages 30-53, 1982. March 5]], and the above immunogen can be used for this search.

The thus obtained hybridoma that produces the desired monoclonal antibody that recognizes human IL-1β can be subcultured in an ordinary medium and can be stored in liquid nitrogen for a long period of time.

The desired antibody is collected from the hybridoma by a method of culturing the hybridoma according to a conventional method to obtain a culture supernatant thereof, or a method of administering the hybridoma to a mammal having compatibility with the hybridoma and proliferating it to obtain ascites. Etc. are adopted. The former method is suitable for obtaining high-purity antibody, and the latter method is suitable for mass production of antibody.

The antibody obtained as described above can be further purified by ordinary means such as salting out, gel filtration, affinity chromatography and the like.

The monoclonal antibody of the present invention thus obtained is human.
It has a specific reactivity to IL-1β.

The antibody of the present invention also includes a type of antibody having a neutralizing activity against the biological activity of human IL-1. Such an antibody is suitable for specifically measuring human IL-1 having biological activity. Further, antibodies of such a type having neutralizing activity, particularly those of the type recognizing the site involved in the binding to the IL-1 receptor on the IL-1 molecule are various types of antibodies which are accompanied by abnormal production of IL-1 described above. It is suitable for application to diseases.

Furthermore, the antibody of the present invention recognizes different sites of human IL-1 molecule, does not have steric hindrance with each other, and at the same time, human IL-1
An antibody of a type capable of binding to a molecule is also included, and such an antibody is extremely useful for use in an immunoassay such as a sandwich method.

In addition, the antibody of the present invention includes an antibody of a type which is particularly excellent in reactivity in a liquid phase system or a solid phase system. They are highly preferred for application in liquid phase and solid phase immunoassays.

EFFECTS OF THE INVENTION According to the present invention, a monoclonal antibody specific to human IL-1β is provided, and by adopting the antibody of the present invention, the measurement sensitivity is extremely high and the specificity is excellent. Is provided, which provides an assay method by an immunoassay method capable of accurately measuring the human IL-1β in a sample containing a very low concentration of human IL-1β.

Examples Hereinafter, examples will be given to explain the present invention in more detail, but the present invention is not limited to these.

Example 1 Human Human IL-l [beta] prepared according to manufacturing recombinant art antibodies against IL-l [beta] [Biochemistry, 58, No. 8, p840 (1986); No. EPO187991] The 10μg of, B
ALB / C mice were intraperitoneally administered with complete Freund's adjuvant. Immunization was performed by boosting the same amount twice with incomplete adjuvant every 3 to 4 weeks. After 3 to 4 weeks, 30 µg of a human IL-1β saline solution was intravenously administered as the final immunization. Three to four days after the final immunization, according to the usual method,
Cell fusion was performed [Method in Enzymology, 73 , pp3 (1
981) etc.]. That is, the cell fusion involves the immunized splenocytes and myeloma cells [P3U1, Current Topics in Microb
iology and Immunology, 81 , 1-7 (1978)] at a ratio of 10: 1 and polyethylene glycol (PEG-4000).

After the hybridomas were selected in HAT medium, the supernatant was mixed with the human IL-1β-coated 96-well microplate and a peroxidase-labeled goat anti-mouse globulin antibody [E. Wai. [Manufactured by Lab (EYLab.)] Was used to detect an antibody-producing strain against the target human IL-1β.

Cloning was repeated by the limiting dilution method to obtain 7 desired antibody-producing clones.

The characteristics of each antibody obtained from each clone were determined according to the following methods.

Antibody Subclass Mouse Antibody Subclass Detection Kit (Bio-Rad (Bi
o-Rad)).

Antibody production level It was shown by the amount of IgG (μg / ml) in the culture supernatant when the hybridoma reached the maximum cell density.

The titer was determined according to the following.

RIA: 100 μl of human IL-1β labeled with ¹²⁵ I according to the iodogen method [BBRC, 80, 849-857 (1978)] (about 10,000 cp
m), 100 μl of diluted solution of hybridoma culture supernatant,
100 μl of PBS containing 0.01% NaN ₃ , 5 mM EDTA and 0.1% BSA
Were mixed and reacted (4 ° C), 50 μl of normal goat serum was added as a carrier protein, and 20% PEG in PBS was added.
After 600 μl was added and mixed, the mixture was allowed to stand in ice melt for 20 minutes, then centrifuged and the radioactivity of the precipitate was measured with a γ-counter.
¹²⁵ % I-IL-1β in 15% sediment
The RIA titer was used.

EIA: Human IL prepared at 20 μg / ml in 96-well microplate
-1β was dispensed (100 µl / well) and left at room temperature for 1 hour. After washing with PBS, the cells were blocked with BSA to prevent nonspecific adsorption and washed with PBS-Tween 20. Aliquot the diluted hybridoma culture supernatant (100 μl / well) and
After reacting for 2 hours at ℃, washed, and further peroxidase-labeled anti-mouse immunoglobulin (X5000 manufactured by Capel)
Was added at 100 μl / well and reacted in the same manner. After washing
The bound enzyme activity was colorimetrically analyzed, and the EIA titer was determined by using the dilution ratio of the culture supernatant at which OD ₄₉₂ = 1.0.

RIA / IgG and EIA / IgG: etc. are the values obtained by dividing the above RIA titer and EIA titer by the antibody production level, respectively.

In addition, RIA / IgG is reactive in liquid phase [ ¹²⁵ I-labeled human IL
-1β with the antibody of the present invention], and EIA / IgG shows the reactivity in a solid phase system [reactivity of human IL-1β immobilized with the antibody of the present invention].

Molecular weight After culturing the hybridoma in the abdominal cavity of the mouse, using an IgG purification kit [MOPS Kit, Bio-Rad], Ig
The molecular weight of the purified product is shown in G ₁ [the sum of the molecular weights of the heavy chain and the light chain by SDS-PAGE is the molecular weight of the antibody].

Western blotting [Western blotting] Using test proteins (both human) produced by gene recombination technology
blotting; Proc.Natl.Acad.Sci., USA, 76 , 4350 (1979); A
nal.Biochem., 112 , 195 (1981)]. "-"
Indicates that there is no cross-reactivity.

Coupling constant (kd) Scatchard plot analysis (Scatchard pl
ot analysys).

Neutralizing activity This is the result obtained by LAF activity [J. Immunol., 116 , 1466 (1976)], and "+" indicates that it has neutralizing activity.

Table 1 below shows the results of obtaining the above-mentioned properties.

Determination of Binding Site Each fragment of human IL-1β shown below was produced according to the production of human IL-1β [EPO187991].

<Fragment> N-10: Polypeptide consisting of amino acid numbers 11 to 153 of human IL-1β N-16: Polypeptide consisting of amino acid numbers 17 to 153 of human IL-1β N-23: Amino acid number of human IL-1β Polypeptide consisting of 24-153 C-82: Polypeptide consisting of amino acid numbers 1 to 82 of human IL-1β C-102: Polypeptide consisting of amino acid numbers 1 to 102 of human IL-1β C-120: Human IL- 1-Polypeptide consisting of amino acid numbers 1-120 of C-140: Polypeptide consisting of amino acid numbers 1-140 of human IL-1β C-144: Polypeptide consisting of amino acid numbers 1-144 of human IL-1β C-148 : Polypeptide consisting of amino acid Nos. 1-148 of human IL-1β. Each of Escherichia coli expressing human IL-1β and each of the above fragments was treated with 6.25 mM Tris buffer (pH 6.8, 2% S
DS, 5% 2ME, 10% glycerol, 0.001% BPB included) 1
00 μl was added and dissolved, and boiled at 100 ° C. for 2 minutes to prepare a sample for electrophoresis, and the reactivity with the antibody of the present invention was tested by Western blotting.

The results obtained are shown in Table 2 below.

From Table 2 above, the antibodies ANOC201, 206 and 207 are all in the sequence of amino acid numbers 121 to 140 of human IL-1β, and the antibodies ANOC202 and 204 are in the sequence of 24 to 82,
Antibody ANOC203 contains antibody ANOC205 in the sequence from 83 to 102.
It is understood that each has a recognition site in the sequence from 145 to 148.

Steric hindrance test of the antibody of the present invention The antibody of the present invention was labeled with ¹²⁵ I by the iodogen method to obtain a labeled antibody.

Further, the antibody of the present invention, a physical adsorption method [Clin.Chem.Acta.,
135 , 263 (1983)], polystyrene beads (6.4m
m) was adsorbed to obtain an insolubilized antibody.

The insolubilized antibody obtained above and 20 ng of human IL-1β were adjusted to 0.
After reacting in 0.5 ml of PBS solution containing 1% BSA and 0.01% thimerosal for 2 hours at 37 ° C. and washing the beads, 0.5 ml of the above-obtained PBS solution of about 100000 cpm of the labeled antibody was added, and 37 The reaction was carried out at 0 ° C. for 2 hours with shaking.

After washing the beads, the radioactivity bound to the beads was counted and the reactivity between the antibodies (insolubilized antibody-human IL-1β-
Whether a sandwich of labeled antibodies is formed).

As a result, a sandwich was not formed between the antibodies ANOC201, 206 and 207, and these antibodies showed IL-1
It was presumed to be an antibody that recognizes almost the same site of β.

No interference was observed in the reaction among all the tested antibodies (excluding the same antibody) other than the above.

Test for inhibition of binding to IL-1 receptor on fibroblasts I BALB / c3T3 fibroblasts (ATCC CCL-163, 1 × 10 ⁶ cells / well) which were grown evenly over one surface on a 6-well plate And 8500 cpm / well of IL-1β labeled with ¹²⁵ I (specific activity of 250 μCi / μg protein or more by the Bolton-Hunter method [Biochem. J., 133 , 529 (1973)]), and added with 10% FCS in advance. A predetermined amount of the antibody of the present invention (anti-IL-1β monoclonal antibody) incubated in -MEM at 37 ° C was added and reacted at 37 ° C for 4 hours. Then, 50 μl of rat serum and 250 μl of 20% PEG were added to the reaction solution, and the mixture was kept at 4 ° C.
After reacting for 20 minutes and centrifuging (12000 rpm for 15 minutes) to separate the precipitate (bound product) and the supernatant (unbound product), the radioactivity (bound radioactivity) was measured with a γ-counter.

The radioactivity measurement value in a control not using the antibody of the present invention is (A), the nonspecific adsorption radioactivity value on the plate is (B), and the measurement value of the antibody of the present invention is used as (C). The inhibitory ability (%) of the binding of IL-1β to the IL-1 receptor on fibroblasts by the antibody of the present invention was calculated by the following formula.

The A value in the above test was 3895 cpm / well, and the C value was 301 cpm.

The results obtained using ANOC203 and ANOC205 as the antibody of the present invention are shown in Table 3.

From Table 3 above, the antibody ANOC203 of the present invention inhibits the binding of ¹²⁵ I-IL-1β to the IL-1 receptor, and thus IL-1β
It is found that it recognizes the IL-1 receptor binding site. Further, the antibody ANOC205 of the present invention does not substantially have the above-mentioned inhibitory ability, and therefore it is understood that this recognizes a site different from the IL-1 receptor binding site of IL-1β.

IL-1 Receptor Binding Inhibition Test on Fibroblasts II In each well in which fibroblasts were grown in the same manner as in the above binding inhibition test I, IL-1β was labeled with ¹²⁵ I-labeled IL-1β.
10 ng / ml and a predetermined amount of the antibody of the present invention are added and reacted,
Then, after centrifugation in the same manner, the radioactivity in each well was measured, and the IL-1 receptor on fibroblasts by the antibody of the present invention was detected.
The inhibitory ability (%) of the binding of IL-1β was calculated.

The A value in the above test was 8278 cpm / well, and the C value was 410 cpm.

The results obtained using ANOC203 and ANOC205 as the antibodies of the present invention are shown in Table 4.

From Table 4 above, although the binding of ¹²⁵ I-IL-1α to the IL-1 receptor is inhibited by IL-Iβ, the antibody of the present invention ANOC20
3 recognizes the IL-1 receptor binding site of IL-1β, and the IL-
Since the binding of 1β to the IL-1 receptor is inhibited, recovery of the binding inhibitory ability is observed. The antibody ANOC205 of the present invention is IL-1β
Since it does not recognize the IL-1 receptor binding site of
It is clear that recovery of the binding-inhibiting ability is not observed with its use.

As typical examples of the hybridomas obtained in the above-mentioned examples, the following three types have been deposited at the Institute for Microbial Industry (MIC), Ministry of International Trade and Industry, and their labeling and deposit number are as follows: Is.

○ Antibody ANOC203 producing hybridoma indication: KOCO203 Mikokenjoyori No. 1551 (FERM BP-1551) ○ Antibody ANOC205 producing hybridoma indication: KOCO205 Mikokenkiyose No. 1552 (FERM BP-1552) ○ Antibody ANOC206 producing hybridoma : KOCO206 Miko Kenjoyori No. 1553 (FERM BP-1553)

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // C07K 1/16 8318-4H 16/18 8318-4H (C12P 21/08 C12R 1:91) (56) References Journal of Leukocyte Biology, 40 [3] (1986. 9) P. 276-277

Claims (2)

[Claims] 1. A human interleukin-1β antibody obtained by using human interleukin-1β obtained as a natural or gene recombination method as an antigen, which comprises the following fragments (1) and (7) of human interleukin-1β: ,
(1), (5), (6) and (7) and (1),
A human interleukin-1β monoclonal antibody, which binds to (3), (4), (5), (6) and (7) or all of (1) to (7). (1) A polypeptide consisting of the 24th to 153rd sequences. (2) A polypeptide consisting of the 1st to 82nd sequences. (3) A polypeptide consisting of the 1st to 102nd sequences. (4) A polypeptide consisting of the 1st to 120th sequences. (5) A polypeptide comprising the 1st to 140th sequences. (6) A polypeptide consisting of the 1st to 144th sequences. (7) A polypeptide consisting of the 1st to 148th sequences. [Claim 2] KOCO to the Institute of Microbial Industry, Ministry of International Trade and Industry
203 (Microtechnical Laboratory Article 1551, FERM BP-1551), KOCO205
(Ministry of Industrial Technology Article 1552, FERM BP-1552) and KOCO206
The human interleukin-1β monoclonal antibody according to claim 1, which is produced by the antibody-producing cells deposited under the designation (Microtechnology Research Institute No. 1553, FERM BP-1553).