JPH03139292A - Monoclonal antibody against human interleukin 6 - Google Patents

Abstract

PURPOSE:To produce an anti-human IL-6 monoclonal antibody by culturing a hybridoma obtained by cell fusion between a mammal antibody-producing cell taken out form mammal immunized with human IL-6 and a mammal myeloma cell. CONSTITUTION:An antibody-producing cell taken out from mammal immunized with human IL-6 is fused with a mammal myeloma cell and the fused cell capable of producing the objective antibody is cloned to afford a hybridoma. The hybridoma is cultured and subjected to salting out, hydroxyapatite column chromatography, etc., of the supernatant of the culture to provide the monoclonal antibody against purified human IL-6. In the monoclonal antibody, antibody amount required for lowering biological activity of 200pg human IL-6 by 50% is <=4ng.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention relates to human interleukin-6 (hereinafter referred to as human interleukin-6).
(abbreviated as L-6), specifically, human IL-6
2. Concerning a monoclonal antibody that has a high neutralizing effect on the biological activity of cells and hybridomas that produce the same [Prior art]
A substance that is produced by many cells such as endothelial cells and exhibits various physiological activities such as differentiation of B cells into antibody-producing cells, induction of acute phase proteins from hepatocytes, proliferation and differentiation of hematopoietic cells, and differentiation of nervous system cells. (Ann, Rev, Immun
ol, 6. p485 (1988) etc.).

This factor has also attracted attention for its association with various diseases, and has been shown to act as an autocrine growth factor for myeloma (Natur
e, 332. p83 (1988)), tumor cells of atrial myxoma produce large amounts of IL-6! :(Proc.

Natl ＾cad Sci, USA, 89
．． p228 (1987)), with high concentrations in the synovial fluid and serum of patients with rheumatoid arthritis (7) I L
-6 exists (Artt+ritis andR
heumatism, 31. p784 (19
88), Eur, J. Immunol.

18, p1797 (1988)), when a rejection reaction occurs in a kidney transplant patient, IL in the blood and urine is
-6 concentration increases (CIin, Exp,
Immunol, 71. p314 (1988)
) etc. are known.

Obtaining a monoclonal antibody against human IL-6 and quantifying trace amounts of IL-6 in body fluids is considered to be extremely useful for research and clinical diagnosis of these diseases. Furthermore, if the obtained antibody neutralizes the activity of IL6, the antibody can also be used as a pharmaceutical for the treatment of these diseases.

From this perspective, attempts have been made to obtain monoclonal antibodies against IL-6, but few have been reported so far, and two types of antibodies obtained by Matsuda et al. in 1988 are known. (Eur, J
, Immunol, 18. p951 (1
988)).

It has been reported that one of these antibodies has an activity-neutralizing effect, but the activity-neutralizing effect of this antibody is weak, and 0
, the antibody concentration required to reduce the biological activity (antibody production inducing activity) of 1 ng of human IL-6 by 50% is 6
ng or more. In addition, the proliferation activity of IL-6-dependent hybridomas was 0.2 μg even with 2 μg of antibody.
No significant effect on IL-6 of ng.

[Problems to be Solved by the Invention] As mentioned above, the purpose of the present invention is to develop an anti-human IL-6 monoclonal that has a strong activity-neutralizing effect on human IL-6, which is considered to be an etiologic agent of various diseases. The aim is to provide antibodies.

[Means for Solving the Problems] In order to solve the above problems of the present invention, the present invention has the following configuration.

That is, the present invention provides a monoclonal antibody against human interleukin-6, characterized in that the amount of antibody required to reduce the biological activity of 200 pg of human interleukin-6 by 50% is 4 ng or less, and a hybridoma producing the antibody. It is.

The antibody of the present invention can be obtained by fusing antibody-producing cells taken from a mammal immunized with human TL-6 with mammalian myeloma cells, and cloning the fused cells that produce the antibody of interest. Manufactured by culturing hybridomas.

In the monoclonal antibody of the present invention, the amount of antibody required to reduce the biological activity of 200 pg of human interleukin-6 by 50% is 4 ng or less, preferably 2.5 ng.
It is as follows.

Hereinafter, the present invention will be explained according to the steps.

(a) Preparation of antibody-producing cells To obtain the hybridoma of the present invention, a mammal is first immunized with human IL-6. The human IL-6 used is
Either natural IL-6 produced from human-derived cells or recombinant IL-6 created by recombinant DNA technology can be used.

Immunization can be carried out by administering the above-mentioned immunizing antigen to a mammal by intraperitoneal, subcutaneous, intradermal, intravenous injection, etc. using a conventional method.

As the mammal, commonly used animals such as mice, rats, rabbits, and guinea pigs can be used. When using mice as mammals, inoculation is 5 to 5 times per dose.
Approximately 0 μg/mouse is administered in combination with a conventional adjuvant or alone, and administration is repeated several times at intervals of one week or more. Three to four days after the final immunization, the spleen or lymph nodes are removed and used as antibody-producing cells.

(b) Cell fusion The fusion reaction between the above antibody-producing cells and myeloma cells can be carried out using known methods, such as the method of Milstein et al.
EnBmol, 73. p3 (1981)
), etc.

There are no particular restrictions on the myeloma cells (sieroma) used here, and various known myeloma cells, such as P3X63-Ag
8, P3-X53-Ag8-Ul, SP2O-Ag14
, X63-Ag8-6.5°3, etc. are used.

Antibody-producing cells and myeloma cells are mixed at a ratio of about 5:1 to 10:1, and fusion is performed in the presence of a fusion promoter such as polyethylene glycol (PEG) or Sendai virus (HVJ).

(C) Cells after HAT selection fusion of hybridomas were added to RPM11640 containing 15% tallow serum.
Float in a medium used for normal cell culture, such as a medium,
105 ~ microplate (usually 96 well type)
Plant at approximately 106 cells/100 AID/well. Add HAT selection medium to each well and
By culturing for 14 days, only hybridomas can be grown selectively.

During this HAT selection culture, it is desirable to add human or mouse IL-6 at a concentration of 0.2 to Long/ml in order to increase the efficiency of hybridoma formation and the incidence of inducing antibody production.

In addition, some hybridomas require IL-6 for proliferation, so until the target antibody-producing cells are established as a single clone and proliferation is confirmed to be non-IL-6 dependent, culture media should not be used. It is desirable to always add human or mouse T L-6.

(d) Screening purpose Screening for antibody-producing cells is carried out using the culture supernatant of each well as a sample, according to various methods generally used for detecting antibodies, such as enzyme-linked immunosorbent assay (ELISA). In addition, for more rigorous confirmation of reaction specificity,
Western plot method etc. are used.

(e) Cloning Cells are isolated from the positive wells as a result of the screening, and cloning is performed by a limiting dilution method or the like to finally obtain a single clone producing the antibody of interest. It is desirable to repeat the cloning operation two or more times. The hybridoma thus obtained can be subcultured in a conventional medium and can be stored in liquid nitrogen for a long period of time.

(f) Obtaining monoclonal antibodies Monoclonal antibodies can be obtained from the above hybridomas by culturing the hybridomas according to conventional methods and obtaining them from the culture supernatant, or by administering the hybridomas to compatible mammals and proliferating them. A method is used in which the ascites is collected from the ascites.

When obtaining the culture supernatant, it is desirable to use a serum-free medium (such as Cell Blocker-H, manufactured by Sumitomo Pharmaceuticals) to facilitate purification. The antibody thus obtained can be purified by conventional methods such as salting out, hydroxyapatite column chromatography, affinity chromatography, and gel filtration.

(g) Selection of antibodies with activity-neutralizing effect The monoclonal antibodies against human 1L-6 obtained by the above method include both antibodies with activity-neutralizing effect and antibodies without activity-neutralizing effect. These antibodies were IL-6
Antibodies with a strong activity-neutralizing effect can be selected by adding them to a biological activity measurement system and examining their effects on IL-6 activity.

As a system for measuring biological activity of IL-6, methods for measuring induction of antibody production from B cells (Proc, Na1Acad,
Sci,, 82. p5490 (1985))
, a method using IL-6-dependent proliferating hybridomas (
J, Exp,~1ed,,' 165. p64
1 (1987)) may be used.

[Example] Example 1 Preparation of anti-human IL-6 monoclonal antibody (a) Immunization of animals Intraperitoneally, 11 female BALB/C mice aged 7 to 9 weeks were
5 to 15 μg/mouse of purified human IL-6 (recombinant IL-6 expressed in night robber moth cells) was administered.

Immunization was performed 4 times a week at intervals of 1 to 10 weeks. The first immunization was carried out in Freund's complete adjuvant or aluminum adjuvant supplemented with killed B. pertussis, the second immunization was carried out in incomplete Freund's adjuvant or aluminum adjuvant, and the third and subsequent booster immunizations were carried out in PBS (8 mM disodium hydrogen phosphate, 1 The test was carried out using a solution containing (.5mM potassium dihydrogen phosphate, 137mM sodium chloride, 2.7mM potassium chloride).

(b) Cell fusion and HAT selection Pile cells isolated from mice 3 days after the first immunization were combined with mouse myeloma cells (P3-X63-Ag8-Ul) for 10
The cells were mixed at a cell count of 1, and cell fusion was performed using 50% polyethylene glycol 1500. 2 as tile cells in RPML 1640 medium containing 15% tallow serum.
, suspended to around 5 x 106/ml,
After adding HAT and human IL-6 (final concentration 2 ng/ml), 200 μU/well was dispensed into a 96-well microplate. One week later, hybridomas had grown in almost all the wells.

(C) Screening and cloning of antibody-producing cells Human IL-6 was incubated in 50 mM sodium carbonate buffer (
pH 9.6) and adjusted to 0.3 μg/ml.
100 μα aliquots were dispensed into a 6-well microplate. 4°
After being left overnight at C, the solution was removed and blocking was performed with a PBS solution containing 1% bovine serum albumin (BSA). Next, after washing with PBS (PBS-T) containing 0.05% Tween20, 100 μα of the HAT selection culture supernatant (8 to 12 days after cell fusion) was dispensed into each well and incubated at room temperature for 1 hour. Made it react. Below, PBS-T washing, biotin-labeled rabbit anti-mouse IgG reaction at room temperature for 1 hour, PB
S-T washing, streptavidin-horseradish peroxidase reaction room temperature 0.5 hours, PBS-T washing, 0.0
31% hydrogen peroxide, 0.1M phosphate citrate buffer containing 0.1% ABTS (pH 4゜0) Reaction was performed at room temperature for 1 hour, and the reaction was stopped by adding 100μ0 of 1% oxalic acid. Thereafter, the color development of the reaction product was measured by absorption at 414 μm. For wells in which strong antibody activity was observed,
Cloning was performed by the limiting dilution method, and the above screening and cloning operations were repeated twice to establish 12 antibody-producing hybridomas as single clones.

(d) Confirmation of reaction specificity 12 obtained by screening using the above ELISA method
As a result of Western blotting using the culture supernatants of several hybridomas, three clones were identified as human I
It was confirmed that it specifically reacts with L-6. The monoclonal antibodies produced by these clones were tested at IC6.
7, IF14, and 1G61. The hybridoma producing lG61 has been deposited with the Microbial Research Institute under Microbiological Research Institute No. 10713.

(e) Obtaining monoclonal antibodies As described above (the three types of hybridomas obtained were respectively grown in the peritoneal cavity of BALB/c mice, and the obtained ascites was subjected to 50% ammonium sulfate precipitation, hydroxyapatite column chromatography, protein A column chromatography) Antibodies were purified using graphical techniques.

Example 2 Determination of antibody subclass Mouse monoclonal antibody subclass fixation kit (zy
As a result, IC67, IF1
4. The subclasses of IC61 antibody are IgGl and IgGl, respectively.
, IgM, and IgG1.

Example 3 Measurement of neutralizing activity The biological activity of IL-6 was measured using human B cell line 5KW6-C1-4 (Proc, N
atl, Acad, Sci, USA, 82.
p5490 (1985)). RPM
5KW6-C1-4 cells suspended in 11640 medium (
4 x 104 cells/ml) in a 96-well microplate.
0 μU was dispensed. Monoclonal antibody 50 μα at a concentration of 20 μg/ml and human hIL-650 at various concentrations
μa was added. After culturing for 3 days, take 100μα of the supernatant,
IgM concentration was measured by ELISA method.

Figure 1 shows the results. Among the three types of monoclonal antibodies obtained, the IC61 antibody showed a strong activity-neutralizing effect, and 6
The antibody production inducing activity of 4 μg/ml IL-6 was
The activity was suppressed to an amount equivalent to g/ml.

Additionally, 4 μg for the same amount of 5KW6-C1-4 cells.
The results of a similar experiment performed with the addition of 50 μfl/ml of human IL-6 and 50 μa of monoclonal antibody at various concentrations are shown in FIG. 1 ng/ml human IL
The concentration of lG61 antibody, which is the monoclonal antibody of the present invention, required to reduce the activity of -6 by 50% is about 10 n
g/mi.

That is, the activity of 200 pg of human IL-5 was reduced to 50%.
The amount of antibody required for reduction was approximately 2 ng. The activity neutralizing effect of this lG61 antibody is much stronger than that of anti-IL-6 monoclonal antibodies reported so far.

Example 4 Analysis of epitope The recognition site (epitope) on the IL-6 molecule was analyzed for clone IG61, a monoclonal antibody of the present invention having a strong activity-neutralizing effect.

(1) Isolation and analysis of peptide fragments that react with antibodies lG6
1 antibody (0.5 mg) was reacted with 0.5 ml gel of Affigel 10 (manufactured by Bio-Rad) to prepare an antibody-immobilized gel. 12 μg of human IL-6 was reacted with 120 ng of glue endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.) in 4 opn of 20 mM) Lis-HCl buffer (pH 8°0) to give 37 μg of human IL-6.
After peptide digestion was carried out at °C for 6 hours, it was diluted to 1 ml and adsorbed onto the above-mentioned lG61 antibody-immobilized gel at room temperature for 30 minutes. The gel after peptide adsorption was packed into a small column, and 5 ml of 0.1M Tris-HCl buffer (pH 8) was added.
゜0) to wash away unreacted peptides, then 0.
The adsorbed peptide was eluted with 1M glycine-hydrochloric acid (pH 2, 4). Samples before reaction with antibody-immobilized gel after lysyl endopeptidase treatment, and 0.5 ml to 1.0 ml at pH 2.4 elution. The results of analyzing the sample eluted in 0 ml by C18 reverse phase HPLC are shown in FIGS. 3A and 3B, respectively.

As a result of analyzing the amino acid sequence of the peak detected in B (retention time 39 minutes), a sequence of 21 residues corresponding to amino acids 151 to 171 of the IL-6 molecule, Leu-Gln
-Ala-Gin-Asn-Gin-Trp-Leu-
Gin-Asp-Met-Thr-Thr-His-L
eu-I le-Leu-Arg-8e r-Phe-
Lys was discovered.

(2) Effect of inhibiting antigen-antibody reaction by synthetic peptide IL-6 molecule contains the above peptide sequence, and further contains two peptides at both the amino terminal and the carboxyl terminal.
Peptide IL of 25 residues corresponding to the region extended by residues
-6Thr149-Phe173 (Thr-Lys-
Leu-Gln-Ala-Gln-Asn-Gln-T
rp-Leu-Gln-Asp-Met-Thr-Th
r-His-Leu-11e-Leu-Arg-8er
-Phe-Lys-Glu-Phe) and EIA
The effect on the system was investigated.

Human IL-6 was added to a 96-well microplate at 0.3 μg/m in 50 mM sodium carbonate buffer (pH 9,6).
Dispense 100 μα/well of the prepared solution into 4
After adsorption overnight at °C, PB containing 1% BSA was added.
Blocking was performed with S solution. After washing each well with PBS-T, the peptide IL-6Thr14 synthesized above
9-Phe173, and as a control human) IL-6 synthesized in a similar manner.
゜Residue peptide (Val-G I n-A ]
a-A 1a-I 1e-Asp-Tyr-11e-A
sn-G IV) at 1 mg/ml each.
From 0. 50 μf at a concentration of 25 μg/ml and 0.1 g/ml of biotin-labeled lG61 antibody.
f and was allowed to react at room temperature for 1 hour. Hereinafter, PBS-T washing, strepavidin/HRP reaction at room temperature 0. 5 hours, PBS-T wash, 0.031% hydrogen peroxide, 0. 1
0.1M phosphate citrate buffer (pH 4) containing %ABTS
, 0) Reaction The reaction was carried out at room temperature for 2 hours, and after the reaction was stopped by adding 100 μa/well of 1% oxalic acid, the color development of the reaction product was measured by absorption at 414 nm. As a positive control, an experiment was also conducted in which human IL-5 was added instead of the synthetic peptide, and the inhibitory effects were compared.

As shown in the results in Figure 4, the peptide Thr149-P
For he173, a decrease in color development was observed at concentrations of 8 μg/ml or higher, and almost complete inhibition of the reaction was observed at 5ool1 g/ml. No inhibitory effect was observed with the control peptide.

(3) More detailed study of the effect of synthetic peptides on inhibiting antigen-antibody reactions.
Eight types of peptides (Peptide 1 to Peptide 82, see Table 1) corresponding to the region containing the 1-residue sequence (Leu151-Lys171) were synthesized, and the same EI as in Example 4 (2) was performed.
It was added to system A to investigate its effect.

As shown in Table 1 and the results in Figure 5, peptide 1 (T
hr149-Phe173), Peptide 2 (A1a15
3-Phe173) and peptide 6 (Ala153
-Thr162) showed a dose-dependent inhibitory effect, and these peptides commonly contained a region Al
It can be concluded that a153-Thr162 is the epitope of the lG61 antibody.

[Effects of the Invention] The present invention provides a monoclonal antibody that has specific reactivity against human TL-6 and strongly neutralizes its biological activity. The antibody of the present invention can be used to treat various diseases associated with abnormal production of IL-6, such as autoimmune diseases such as rheumatoid arthritis, myeloma, and atrial myxoma.
It becomes possible to suppress the biological activity of TL-6, which is enhanced due to its abnormal production, and can be expected to be applied to extremely valuable pharmaceuticals for the treatment of various diseases.

At the same time, we are developing immunoassay methods that can specifically, highly sensitively, and easily measure human IL-6, as well as methods for specific purification of human IL-6 using methods such as affinity chromatography. It becomes possible to provide.

[Brief explanation of the drawing]

Figure 1 shows the measurement results of the neutralizing activity of antibodies against IL-6 at various concentrations, Figure 2 shows the measurement results of the neutralizing effects of antibodies at various concentrations against L-6 at a constant concentration, and Figure 3 is lG
FIG. 4 shows the inhibitory effect of the synthetic peptide on the antigen-antibody reaction, and FIG. 5 shows the peptide concentration dependence of the inhibition by the synthetic peptide.

Claims (6)

[Claims] (1) A monoclonal antibody against human interleukin-6, characterized in that the amount of antibody required to reduce the biological activity of 200 pg of human interleukin-6 by 50% is 4 ng or less. (2) The monoclonal antibody according to claim 1, wherein the antibody class is IgG_1. (3) The monoclonal antibody according to claim 1 or 2, which binds to human interleukin-6 between amino acids 149 to 173. (4) The monoclonal antibody according to claim 1 or 2, which binds to human interleukin-6 between amino acids 153 to 162. (5) A hybridoma producing a monoclonal antibody against human interleukin-6 according to claims 1 to 4. (6) The hybridoma according to claim 5, wherein the hybridoma is derived from a mouse.