JPH03219894A - Antibody against gp130 protein - Google Patents

Abstract

PURPOSE:To enable to prepare the subject antibody useful as a diagnostic or treating agent by culturing a hybridoma originated from a mammarian cell immunized with a gp130 protein antigen relating with the signal transmission of human interleukin-6 (IL-6). CONSTITUTION:A mouse, etc., is immunized with an animal cell (e.g. human IL-6) receptor or myeloma cell strain U266 producing the gp130 protein) as an immunogen, and the splenic cell of the immunized mouse, etc., is fused with a myeloma cell to prepare a fused cell (A). A strain recognizing the gp130 protein is cloned from the cell A to provide a hybridoma (B). The strain B is cultured and the supernatant of the cultured solution is subjected to a separation treatment to provide a gp130 protein-resistant antibody which combines with the IL-6 receptor in the presence of IL-6 but does not combine with the IL-6 receptor in the absence of the IL-6 and which exhibits an apparent molecular weight of 130kDa by a SDS-polycrylamide gel electrophoretic method and combines specifically with the gp130 protein.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to human interleukin-6 (hereinafter referred to as 11L-6).
gp1, a protein involved in signal transduction
The present invention relates to an antibody that specifically binds to No. 30 protein and a method for producing the same.

[Conventional technology]

IL-6 is a protein that has various important physiological activities and is widely involved in cell proliferation and differentiation. Furthermore, IL-6
It has been reported that the abnormal production of the
5suno1. + 6+ p485.Refer to 1988)
.

The IL-6 receptor on the cell membrane, which specifically binds IL-6, was analyzed by Taga et al.
The binding constant etc. with 6 have been reported (J.

ExpoMed,,196. p967.1987). Furthermore, cDNA of the human IL-6 receptor was isolated by Yamazaki et al., and its secondary structure was reported (Scie
nce, 241, 825, 1988), and recently, the extracellular portion (soluble receptor) of the IL-6 receptor has been produced by genetic engineering (Japanese Patent Application No. 1-9774).
(see specification), and is expected to be used as a therapeutic or diagnostic agent for various immune diseases.

Furthermore, the present inventor has discovered a protein called gpt3o protein, which is involved in IL-6 signal transduction, on the cell membrane (see Japanese Patent Application No. 1-200230). That is, this gp130 protein is activated by IL-6 in the presence of IL-6.
It is a protein that binds to IL-6 receptor, but does not bind to IL-6 receptor in the absence of It, -6, and has an apparent molecular weight of 130 k[la] in SDS/PAGE. After IL-6 binds to the IL-6 receptor, in order to transmit the information into the cell, IL-6 must be
Binding to the L-6 receptor is not enough; it must also associate with gp130i white matter, a protein on the cell membrane, and the intracellular region of the IL-6 receptor is not involved in IL-6 signal transduction. revealed.

In order to further analyze the physiological role of IL-6,
Knowledge about the pathways of information transmission into cells is important.

Such knowledge is also important for developing substances that modulate IL-6 action as therapeutic agents. However, since the amount of gp130 protein present in vivo is extremely small, it is necessary to use genetic engineering techniques to obtain a large amount of purified product. In order to grow such genes, anti-gp130 protein can be rapidly identified.
The development of p130 protein antibodies is desired. Also, IL-6
If a monoclonal antibody that competitively blocks the association between the receptor and gp130 protein is developed, it can be used as a therapeutic agent to suppress the biological activity of IL-6.

However, no monoclonal antibody that recognizes this gpt3o protein is known so far.

[Problem to be solved by the invention]

Therefore, the present invention seeks to provide various types of antibodies against gp130 protein.

[Means to solve the problem]

The above-mentioned problem addresses the gp130 protein, which is involved in human interleukin-6 signaling and has the following properties: (1) binds to the IL-6 receptor in the presence of IL-6, but does not act as an IL-6 receptor in the absence of IL-6; -6 receptor; and (2) exhibiting an apparent molecular weight of 130 kDa in SDS-polyacrylamide gel electrophoresis; an anti-gp130 protein antibody that can specifically bind to; a hybridoma that produces the antibody; The problem is solved by providing a method for producing the antibody; and a method for producing the antibody using the hybridoma.

[Specific explanation]

The antibodies of the present invention specifically recognize the gp130 protein involved in human IL-6 signal transduction, and include monoclonal antibodies and polyclonal antibodies. Monoclonal antibodies include those that competitively inhibit the binding between human IL-6 receptor and gp130 protein and those that do not competitively inhibit this. Examples of the former include the AM64 monoclonal antibody produced by M64 to the hybridoma of the present invention;
On the latter side is the Al'1277 monoclonal antibody produced by hybridoma AM277.

As the immunogen used for producing the antibody of the present invention, animal cells expressing gp130 protein on the cell surface can be used. Such cells include human IL
-6 receptor and gp130 protein, such as human myeloma cell line U266.
, or a host cell transformed with DNA encoding gp130 protein, such as an animal cell line, such as a host cell transformed with DNA encoding gp130 protein.
Examples include mouse T cells transformed with a plasmid containing DNA. However, such cell lines cannot be said to be efficient immunogens due to the lack of gp130 protein expressed on the cell surface.

However, even using such immunogens,
- Once we have an antibody against the gp130 protein, we can use it to prepare a more efficient immunogen and use it to produce a wider variety of antibodies. For example, gp
An antibody against the 130 protein is bound to a suitable solid support, while cells producing the gp130 protein, such as the above-mentioned cells, are cultured and lysed, and this cell lysate is conjugated with the above-mentioned anti-gp130 protein antibody. By contacting with a solid carrier, the gp130 protein in the cell lysate is adsorbed and concentrated onto the carrier, and this can be used as an immunogen. There are no particular restrictions on the solid carrier as long as it can bind antibodies and does not seriously affect the growth of the animal to be immunized. For example, a solid support based on Sepharose or the like as described in detail in the present invention can bind antibodies with a simple operation and does not affect the growth of animals. It is suitable as

Alternatively, a peptide constituting a portion of the gp130 protein can be prepared and used as an immunogen by binding it to a suitable polymeric carrier such as ovalbumin.

Furthermore, it is also possible to use a vaccinia virus in which the gp130 protein is expressed after infection. Any of these immunogens can be used as an immunogen for producing polyclonal antibodies and as an immunogen for preparing hybridomas.

Polyclonal antibodies can be produced in accordance with conventional methods, for example, using any of the above-mentioned immunogens in mice, rabbits, sheep,
This can be done by immunizing a goat or the like.

Hybridomas can also be produced according to conventional methods. For example, a mammal such as a mouse is immunized with any of the immunogens described above, lung cells are obtained from the animal, and these are fused with established myeloma cells. Next, a hybridoma producing a monoclonal antibody having the desired reactivity is cloned.

To produce a monoclonal antibody, the hybridoma grown as described above is cultured, and the monoclonal antibody is collected from the culture supernatant. Alternatively, the hybridoma can be intraperitoneally inoculated into an animal to obtain ascites, from which monoclonal antibodies can be isolated. Antibodies in hybridoma cell supernatants or antibodies in ascites can be concentrated according to conventional methods, for example, by salting out with ammonium sulfate, and further purified by affinity chromatography, for example, affinity chromatography using a carrier immobilized with gp130 protein. be able to.

The above-mentioned methods for producing polyclonal antibodies, methods for producing hyfridomas, methods for preparing monoclonal antibodies, and methods for collecting and purifying antibodies can all be performed by methods well known in the art.

〔Example〕

Examples will be shown below to explain the present invention in more detail, but the present invention is not limited to these Examples.

For the purpose of producing a mouse monoclonal antibody against gp130 protein, human gp130 protein was extracted as an immunogen by the following method.

3 x 10' cells of human myeloma cell line U266
By reacting with IL-6 of n/idl at 37 °C for 30 minutes, the IL-6 receptor present on the cells and gp
After associating with 130 protein, the thus formed rlL-6 receptor/gp130 protein complex was treated with 11d of 1% digitonin (Wako Pure Chemical Industries, Ltd.), 10IIIM.
Triethanolamine buffer y-(pH 7,4),
It was solubilized with 0.15M NaCl 1°1 a+M pAPMsF (manufactured by Wako Pure Chemical Industries, Ltd.). On the other hand, MT1B antibody (reference example; see Japanese Patent Application No. 1-186016), which is an anti-IL-6 receptor antibody, was bound to Sepharose 4B activated with bromcyan according to a conventional method. Mix this with the above-mentioned solubilized cell supernatant, and transfer the solubilized IL-6 receptor onto the resin. The gp130 protein was bound to the t718 antibody via the IL-6. After washing away non-specific binders with the 1% digitonin solution described above, this resin was used as an immunogen.

Immunization and hybridoma production were performed as follows.

The above-mentioned immunogen was administered once a week, a total of 4 times, to BALB/
C mice were immunized intraperitoneally. Next, lung cells from the mouse and myeloma cell line P3U1 as a parent strain were fused according to a conventional method using polyethylene glycol.

Screening was performed as follows. First, the upper slide of the bilidoma and 0.01 d protein G Sepharose (
(manufactured by Pharmacia) was mixed, and the immunoglobulin in the supernatant was adsorbed onto the resin. On the other hand, IL-
After reacting 6 at 37°C for 30 minutes, the 3SS-labeled IL-6 receptor/gp130 protein complex was affinity purified using the M71B antibody-conjugated Sepharose 4B described above. This was immunoprecipitated using the aforementioned protein G Sepharose in a conventional manner, and analyzed by SDS/PAGE and autoradiography.

As a result, one hybridoma clone producing an antibody that specifically binds to gp130 was isolated.

This was named AM277. In addition, the monoclonal antibody produced by this hybridoma was used with AM277.
We will call them antibodies.

Using this AM277 antibody, a further monoclonal antibody against gp130 protein was produced as follows.

3×10′ U266 cells were solubilized with the 1% digitonin solution described above. On the other hand, AM277 antibody was activated with bromcyan and bound to Tasepharose 4B according to a conventional method. Mix this with the ultracentrifuged supernatant of the solubilized cells described above,
The solubilized gp130 protein was bound to the AM277 antibody on the resin. Non-specific binders were washed away with the 1% digitonin solution described above. Then, using this resin as an immunogen,
A hybridoma was prepared in the same manner as HachiM277 described above.

Screening was performed as follows. First, AM277
Immunoglobulin in the supernatant of the hybridoma was adsorbed to protein G Sepharose in the same manner as in the case of . on the other hand,
U266 cells internally labeled with methionine 353
After reacting with X10'trg/dl of rL-6 at 37°C for 30 min,
In B, 353-labeled gp130 protein was purified. This was immunoprecipitated using the aforementioned protein G Sepharose in a conventional manner, and analyzed by SDS/PAGE and autoradiography. As a result, one biploidoma clone producing an antibody that specifically binds to gp130 protein was isolated. This was named AM64. In addition, the monoclonal antibody produced by this hybridoma was
It will be called M64 antibody. These hybridomas AM64 and AM277 were submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, respectively, as part of the Microbiological Research Institute No. 11194 (FERM).
P-11194) and Microtechnical Research Institute No. 11195 (F
ERM P-11195).

In order to confirm that AM64 antibody and He111277 antibody specifically recognize and immunoprecipitate gp130 protein, internally labeled U266 cells were solubilized with detergent, and then immunoprecipitated with AM64 antibody or total 277 antibody. SDS/PAGE and autoradiography were performed.

The results are shown in FIG.

As a result, A164 antibody and Al'+277 antibody
It was confirmed that the protein specifically recognizes the 130 protein.

Using a method of fluorescently staining the antigen exposed on the cell membrane, it was confirmed whether the AM64 antibody recognizes the extracellular portion of the gp130 protein.

The aforementioned u266 cells and ^M64 antibody were mixed, and the AM64 antibody bound to gp130 protein on the cell membrane was detected using fluorescence (F).
ITC) labeled anti-mouse immunoglobulin antibody (F
It was fluorescently stained with ITCanti-mIg).

FIG. 2 shows that the AI'+64 antibody binds to the extracellular portion of the gp130 protein expressed in 0266 cells. That is, in Fig. 2, ■ indicates AM in U226 cells.
After reacting with 64 antibody, AM64 bound on the cell membrane
It shows the fluorescence intensity distribution of cells when the antibody was fluorescently stained with the above-mentioned FITCanti-+w1g. Compared to I, which represents a control containing only the medium, the fluorescence intensity has shifted to the stronger side.
As a result, it was confirmed that the AM64 antibody recognized the extracellular portion of the gp130 protein.

Using the same method as in Example 2, it was confirmed that the recognition site of the HeM64 antibody was the part of the gp130 protein that interacted with the Ru6 receptor.

That is, after stimulating U266 cells with IL-6 to cause the IL-6 receptor on the cells to associate with gp130 protein, AM64 antibody was further mixed, and the above-mentioned FITCan
Fluorescent staining was performed with ti-IIlig. The fluorescence intensity of the cells at that time is shown in Fig. 2 as ■. Comparing this ■ with ■, we can see that the binding of AM64 antibody to gp130 protein is
It can be confirmed that the inhibition is caused by the stimulation of IL-6 receptor, that is, by the association between IL-6 receptor and gp130 protein.

Next, the M64 antibody was applied to IL-6-induced IL-6.
It was confirmed as follows whether the association between 6 receptor and gp130 protein could be inhibited. By simultaneously adding AM64 antibody and IL-6 to a suspension of U266 cells surface-labeled with ItJ, MA64 antibody and IL-6 on υ266 cells were examined for binding to gp130 protein on υ266 cells in the presence of IL-6. competition with receptors.

These [1266 cells were solubilized with a 1% digitonin solution, and the supernatant was immunoprecipitated with MT18 antibody in the same manner as in Example 1, and analyzed by SO5/PAGE. The results are shown as lane 2 in FIG. The results of a similar experiment conducted in the absence of AM64 antibody are shown in lane 1 of FIG.

In lane 1, bands were detected at both the 130 kDa and 80 kDa positions, indicating that the IL-6 receptor (molecular weight 80 kDa) associated with gp130 protein (molecular weight 130 kDa) (both labeled by ItJ) had a force <M71B. This shows that it was precipitated by the antibody. In contrast, in lane 2, 130
The density of the band at the kDa position decreased significantly, which indicates that the IL-6 receptor (molecular weight 80 kDa)
It was precipitated by T18 antibody, which means that the association between gp130 protein and IL-6 receptor was inhibited by the presence of AM64 antibody.

A similar operation was performed for AM277 antibody. The results are shown as lane 3 in FIG. As a result, AM27
7 antibody does not inhibit the association between IL-6 receptor and gp130 protein.

Proliferation test of IL-6-dependent human T-cell leukemia line KT-3 according to the method of S. Shimizu et al.
, 72(5) p1826.1988), A.
It was confirmed whether the M64 antibody inhibits the function of IL-6. That is, 1 x 104 KT-3 cells were injected with IL-6 (50
7 at 5 × 104 cells/− in the presence of ρg/d)
It was cultured for 2 hours. At this time, AM64 antibody was added at a concentration of 40 n/d. 3H-thymidine (
After culturing, the radioactivity incorporated into the KT-3 cells was measured.

In addition, M277 antibody was added instead of AM64 antibody,
A proliferation test was conducted in the same manner.

These results are shown in FIG. As a result,
It was confirmed that the M277 antibody did not inhibit IL-6-induced proliferation of KT-3 cells, whereas the AM64 antibody inhibited the proliferation.

Furthermore, 5KW6-CL according to the method of T. Hirano et al.
Method for observing enhancement of IL-6-dependent IgM antibody production in 4 cell lines (Proc, Natl, Acad, Sci
, USA, 82. p5490.1985) to confirm whether the AM64 antibody inhibits the function of IL-6. That is, 1 × 104 5KW6~CL4 cells were incubated with 5X1 in the presence of IL-6 (200 pg/d).
Cultured for 72 hours at 0' cells/d. At this time, AM64
The antibody or ^h277 antibody was added to the culture medium at a concentration of 5 μ/d.

After culturing, IgM antibodies contained in the supernatant were measured by enzyme antibody method.

The results are shown in FIG. As a result, AM277
The antibody was isolated from the 5KW6-CL4 cell line by IL-6.
It was confirmed that the AM64 antibody inhibited gM antibody production, whereas it did not inhibit its production.

For the purpose of producing a mouse monoclonal antibody against human IL-6 receptor, a mouse T cell line expressing human IL-6 receptor on its membrane surface was produced as an immunogen by the following method. That is, pBSF2R described in Japanese Patent Application No. 1-9774.

236 and pSV2neo to mouse T cell line CTLL-
2 (ATCC.

TlB214) was introduced in a conventional manner and screened in a conventional manner using G-418. Finally, a strain expressing approximately 30,000 IL-6 receptors per cell was established, and this was combined with Cr3C2. It's a name.

Immunization was performed as follows. After culturing in the usual manner using RPM11640, C was washed four times with PBS buffer.
r3C2 at lXl0' per C57BL6 mouse.
Individual cells were immunized intraperitoneally once a week for a total of 6 times.

Lung cells from the immunized mice were fused with the parental myeloma cell line P301 according to conventional methods using polyethylene glycol.

Screening was performed as follows. IL-6 receptor negative human T cell line JURKAT (ATCC, CRL
8163), pBsF2R, 236 and pSV2neo
As a result of screening, a strain expressing approximately 100,000 IL-6 receptors per cell was established, which was named NJBC8. Recognizes NJBC8 solubilized with NP40, and J solubilized with NP40
ul? One hybridoma clone producing an antibody that does not recognize KAT was isolated and named M71B. Furthermore, the monoclonal antibody produced by this hybridoma is called MT18 antibody. The bilidoma MT1B was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology, as part of the Microbiology Research Institute No. 10840 (FERM P-1084).
0).

〔Effect of the invention〕

The monoclonal antibody that specifically recognizes gp130 protein provided by the present invention is a monoclonal antibody that specifically recognizes gp130 protein, which is involved in IL-6 signal transduction and is expected to be used as a diagnostic or therapeutic agent.
30 proteins are useful for producing and purifying large quantities.

Furthermore, by using the antibodies provided by the present invention, it becomes possible to analyze various properties of the gp13o protein that may be possessed by various cells that have not been previously known. This has great significance for research on ontogeny and the immune system, as well as for the development of therapeutic agents, diagnostic agents, etc. based on these results.

[Brief explanation of drawings]

FIG. 1 shows the results of internally labeled U266 cells solubilized with a detergent, immunoprecipitated with M64 antibody or AM277 antibody, and subjected to SO5/PAGE and autoradiography, respectively. FIG. 2 shows the fluorescence intensity distribution when AM64 antibody bound to U266 cell membrane was fluorescently stained with FITC-labeled anti-mouse immunoglobulin antibody. In the figure, I is a control using only the medium without AM64 antibody, ■ is when AM64 was reacted, and ■ is A after pre-reacting with IL-6.
The fluorescence intensity distribution when reacting with M64 antibody is shown. Figure 3 shows that internally labeled U266 cells were stimulated with IL-6 (lane 1) and stimulated with IL-6 in the presence of AM64 antibody.
6 (lane 2) or stimulated with IL-6 in the presence of M277 antibody (lane 3), cells were then solubilized with digitonin, immunoprecipitated with MT1B antibody, and SOS/
The results of PAGE and autoradiography are shown. FIG. 4 shows the inhibitory effect of AM64 antibody and AM277 antibody on the proliferation of KT-3 cells induced by IL-6. FIG. 5 shows the suppressive effects of AM64 antibody and AM277 antibody on 1 gM production from 5KW6-CL4 cells by IL-6.

Claims (1)

[Claims] 1. gp130 protein involved in human interleukin-6 signal transduction and having the following properties: (1) IL-6
(2) exhibits an apparent molecular weight of 130 kDa in SDS-polyacrylamide gel electrophoresis; An anti-gp130 protein antibody capable of specifically binding. 2. The antibody according to claim 1, which is a monoclonal antibody. 3. The antibody according to claim 2, which competes with human interleukin-6 receptor in binding to gp130 protein involved in human interleukin-6 signal transduction. 4. The antibody according to claim 3, which is an AM64 antibody. 5. The antibody according to claim 2, which does not compete with human interleukin-6 receptor in binding to gp130 protein involved in human interleukin-6 signal transduction. 6. The antibody according to claim 5, which is AM277 antibody. 7. The antibody according to claim 1, which is a polyclonal antibody. 8. A hybridoma producing the monoclonal antibody according to claim 2. 9. Sensitize a mammal with a gp130 protein antigen involved in human interleukin-6 signal transduction, collect immune cells from the mammal, fuse the immune cells with a myeloma cell line, and extract the gp130 protein from the fusion line. A method for producing a hybridoma, which comprises growing a strain that recognizes a protein. 10. The method according to claim 9, wherein the gp130 protein antigen involved in human interleukin-6 signal transduction is gp130 protein bound to a solid carrier. 11, the hybridoma according to claim 8, or claim 9
Alternatively, the hybridoma produced by the method described in 10 is cultured, and human interleukin-6 is extracted from the culture.
Anti--
Method for producing gp130 protein antibody. 12. Anti-gp13, characterized by immunizing a mammal with a gp130 protein antigen involved in human interleukin-6 signal transduction, and collecting a polyclonal antibody that recognizes the gp130 protein from the animal.
0 protein polyclonal antibody production method.