JP2528549B2 - Novel monoclonal antibody that binds to human IL-2 receptor-heavy chain - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial application] Binding of human IL-2 receptor heavy chain, binding of human IL-2 to human IL-2 receptor heavy chain, and physiological activity of human IL-2 The present invention relates to a monoclonal antibody having an activity of inhibiting both. The monoclonal antibody of the present invention is a substance useful as an immunosuppressive agent that can be applied to the prevention of rejection at the time of organ transplantation or the treatment of autoimmune diseases by using alone or in combination with an antibody that binds to the human IL-2 receptor light chain. Is. Incidentally, in the present invention, interleukin 2 is IL-
2 and the interleukin 2 receptor will be abbreviated as IL-2 receptors.

[Conventional technology]

Now that the surgical technique for organ transplantation has been remarkably improved, success or failure of organ transplantation has been translated into how postoperative transplant rejection can be suppressed. Rejection occurs when the living body recognizes the graft as a foreign body and a series of immune reactions is elicited to eliminate it. Therefore, conventionally, administration of so-called immunosuppressive agents such as steroids, azathioprine, methotrexate and 6-mercaptopurine has been performed as anti-rejection agents. However, the survival rate was not significantly improved due to the narrow safety margin or weak effect. However, with the advent of the recently developed cyclosporin A, the engraftment rate has markedly improved. However, it has been revealed that cyclosporin A has serious nephrotoxicity, and its use has been restricted. Therefore, development of safer and more effective immunosuppressive agents has been desired.

By the way, IL-2 is a protein produced from helper T cells, and has a wide range of functions such as proliferation and differentiation induction of killer T cells in vivo, differentiation induction of B cells, and so on. It is an important factor. In organ transplantation and bone marrow transplantation, it is activated by IL-2 in host-to-graft reaction (HVG reaction) or graft-to-host reaction (GVH reaction), which is considered to be the key to graft engraftment. It has been shown that humanized killer T cells are deeply involved. On the other hand, autoimmune diseases are thought to develop when the immune system in the body is out of balance and attack the body itself, and in particular, overproduction of factors such as IL-2 that are involved in the immune system. It is highly likely that the over-reaction to it, etc. is one of the major causes.

From these, it seems that if the biological response involving IL-2 can be selectively and effectively suppressed, it will be possible to prevent rejection at the time of organ transplantation and to treat autoimmune diseases. Became. In fact, when IL-2 conjugated with a cytotoxin that selectively damages IL-2 responsive cells having an IL-2 receptor is administered to adjuvant arthritis rats, which is one of animal models of autoimmune disease, , The onset of arthritis is delayed and the symptoms are alleviated (Proceedings of the N
ational Academy of Science of USA, 86, 287 pages,
1989), and when administered at the time of allogeneic heart transplantation in mice, the rejection of transplanted heart was suppressed (Proceedings of the Nationa).
l Academy of Science of USA, 86, 1008, 1989
Year). However, IL-2 to which a cytotoxin is bound has a short half-life in blood, and it is necessary to administer a large amount of IL-2 in order to be effective, and side effects associated therewith are concerned. Therefore, there is a demand for the development of a more safe and effective drug capable of controlling the IL-2 response.

By the way, the human IL-2 receptor on human IL-2 responsive cells is composed of two glycoprotein molecules, a heavy chain having a molecular weight of approximately 75,000 and a light chain having a molecular weight of approximately 55,000, both of which have the ability to bind to human IL-2. Is known (Proceedings of the
National Academy of Science of USA, Volume 84, 2002
Page, 1987). The dissociation constants of the binding between each molecule and human IL-2 are 10 ^-8 M in the case of light chain and 10 ^-9 M in the case of heavy chain. It has been clarified that when a tripartite association with -2 is formed, the dissociation constant is 10 ^-12 M with high affinity. Also, in cells expressing only the light chain, no response reaction occurs even if human IL-2 binds, but in cells expressing the heavy chain or both the heavy chain and the light chain, Since various physiological activities are induced by the binding of human IL-2, human IL-2
It has come to be considered that the presence of the heavy chain of the human IL-2 receptor is essential for the expression of the physiological function of -2 (Jikken Igaku, Vol. 6, p. 606, 1988). That is, if the binding between human IL-2 and heavy chain can be inhibited, human IL-
It is estimated that the response to 2 does not occur. Human
As an inhibitor of the binding between the heavy chain of human IL-2 receptor and IL-2, an antibody having binding activity to the heavy chain of human IL-2 receptor is considered. In fact, it binds to the heavy chain of human IL-2 receptor and
Some antibodies that have the activity of inhibiting the binding to -2 have already been reported (Proceedings of the National Acad.
emy of Science of USA, 86, 1982, 1989, Jou
rnal of Experimental Medicine, 169, 1323, 1989
Year).

The present inventors also previously reported an antibody (monoclonal antibody TU27 (a monoclonal antibody produced by the hybridoma FERM BP-2510)) having an activity of inhibiting the binding of the heavy chain of human IL-2 receptor to human IL-2. (See Japanese Patent Laid-Open No. 2-200198). However, it was revealed that addition of these antibodies at a normal concentration does not inhibit the physiological activity of human IL-2, although the binding between human IL-2 and human IL-2 receptor heavy chain is inhibited. Became. The reason for this is that the binding affinity of these antibodies to the heavy chain molecule is low, and the high affinity due to the association of the heavy chain molecule and the light chain molecule that are successively generated on the cell surface during culturing under physiological conditions. This is probably because it cannot inhibit the formation of affinity receptors. Therefore, when attempting to administer these antibodies to a patient, it is necessary to keep the blood concentration at a remarkably high concentration, side effects are a concern, and clinical application is actually impossible. To solve this problem, an antibody that binds to the heavy chain molecule of human IL-2 receptor with very high affinity is used, or more preferably, such an antibody and human are used.
It is necessary to inhibit the high-affinity binding of human IL-2 by co-using an antibody that binds to the light chain of IL-2 receptor. However, an antibody that binds to the human IL-2 receptor heavy chain with high affinity and has an activity of independently inhibiting the physiological activity of human IL-2 is not known until now,
Furthermore, there is no known therapeutic agent that uses such an antibody in combination with an antibody that binds to the human IL-2 receptor light chain.

[Problems to be Solved by the Present Invention]

Therefore, the object of the present invention is to obtain human IL with high affinity.
It is intended to provide a monoclonal antibody having an activity of binding to the -2 receptor and inhibiting not only the binding between human IL-2 and human IL-2 receptor heavy chain but also the physiological activity of human IL-2. The monoclonal antibody of the present invention can be expected to be useful as an immunosuppressive agent useful for preventing rejection at the time of organ transplantation or treating autoimmune diseases, when used alone or in combination with an antibody that binds to human IL-2 receptor light chain.

[Means for solving the problem]

In order to solve the above-mentioned problems, the present inventors
A large number of monoclonal antibodies that bind to the heavy chain of the 2 receptor were prepared, and among them, (a) human with high affinity
The present invention has been completed by selecting an antibody capable of binding to the heavy chain molecule of the IL-2 receptor and (b) independently inhibiting the physiological activity of human IL-2 on human T cells. That is, according to the present invention, human IL with high affinity
Provided are a hybridoma that produces a monoclonal antibody that binds to a heavy chain molecule of the -2 receptor and that can inhibit the physiological activity of human IL-2, and a monoclonal antibody with uniform properties produced by the hybridoma.

The hybridoma of the present invention comprises myeloma cells and human IL-
It binds to the heavy chain molecule of the human IL-2 receptor from a hybridoma with the antibody-producing cells present in the cells of the spleen or lymph node of an animal immunized with human cells highly expressing the heavy chain of the 2 receptor. , A clone producing a monoclonal antibody capable of inhibiting the physiological activity of human IL-2.

The desired monoclonal antibody can be recovered from the supernatant obtained by culturing such a clone by purification operations such as salting out and ion exchange chromatography. When a large amount is obtained, the obtained hybridoma is inoculated into the abdominal cavity of a histocompatibility animal, athymic nude mouse, or the like and allowed to grow, and the monoclonal antibody produced in the ascites of the animal is recovered. Then, it may be purified by the same operation.

The method for preparing a monoclonal antibody capable of binding to the human IL-2 receptor heavy chain molecule and inhibiting the physiological activity of human IL-2 is described below.

Hybridomas are produced by fusing myeloma cells with antibody-producing cells. Human as antibody-producing cells
Using spleen or lymph node cells from animals such as mice and rats immunized with TL-Mor cells, which is a human adult T-cell leukemia virus-infected T cell line highly expressing the heavy chain molecule of IL-2 receptor, Good. Any cells may be used as the cells to be immunized as long as they are human cells expressing a human IL-2 receptor heavy chain molecule. Further, the heavy chain molecule itself purified from those cells may be used as an immunogen.

The species of the animal from which the antibody-producing cells and the myeloma cells are derived may be different as long as both cells can be fused, but it is usually better to use cells of the same species. One preferred hybridoma for practicing the present invention is a hybridoma between mouse spleen cells or lymph node cells immunized with TL-Mor cells, which is a human adult T-cell leukemia virus-infected T cell line, and mouse myeloma cells. Is. For example, hybridomas between spleen cells of Balb / c mice and myeloma cells SP2 / 0-Ag14 of Balb / c mice immunized with TL-Mor cells suspended in physiological saline were used as shown in the Examples below. Excellent results have been obtained. As myeloma cells, in addition to SP2 / 0-Ag14, X63-Ag8-6.5.3, P3-X63-Ag8-U1, P3-X63-
Ag8, P3-NSI / 1-Ag4-1, MPC11-4.5.6.TG.1.7, (above mouse cells), 210.RCY.Ag1.2.3, (rat cells), SKO-0
A cell line resistant to 8 azaguanine such as 07, GH15006TG-A12 (above human cells) may be used. Preparation of a hybridoma and selection of a clone producing a monoclonal antibody capable of inhibiting the physiological activity of human IL-2 by binding to the heavy chain molecule of human IL-2 receptor from the hybridoma are performed, for example, as follows. You can do it. The antibody-producing cells are fused with myeloma cells using polyethylene glycol, Sendai virus, or the like. Only the fused hybridomas contained medium containing hypoxanthine, thymidine, and aminopterin (HA
T medium). Not all the obtained hybridomas produce the antibody, and not all the antibody-producing hybridomas produce the desired antibody. A hybridoma clone producing a monoclonal antibody capable of binding to the heavy chain molecule of the receptor and inhibiting the physiological activity of human IL-2 must be selected.

The selection can be performed using the following method, for example. That is, ¹²⁵ I-labeled human IL-2 was prepared as a primary screen and
^The inhibition of binding between ¹²⁵ I-labeled human IL-2 and human IL-2 receptor heavy chain molecule and TL-Mor cells expressing both light chain molecules is measured. As a secondary screening, ¹²⁵ I-labeled human IL-2 by the hybridoma culture supernatant and MT-1 cells, which is a human adult T cell leukemia virus-infected T cell line expressing only the light chain of human IL-2 receptor, were used. The binding inhibitory activity of is measured. TL-Mor cells and human IL-2
And a hybridoma producing an antibody against the heavy chain molecule of the IL-2 receptor, if the hybridoma has an activity of inhibiting the binding of MT-1 cells and human IL-2. Become. ^{As a} method for producing ¹²⁵ I-labeled human IL-2, as long as human IL-2 has physiological activity,
Bolton-Hunter method (Biochemical Journal, 133, 5
29, 1973), Lactoperoxidase method (Nature, 2)
69, 309, 1977), Chloramine-T method (Nature, 19)
Vol. 4, p. 495, 1962), etc. may be used. The cells used for the primary screening may be any cells as long as they are human cells expressing both the heavy chain and the light chain. The cells used in the secondary screening may be any cells as long as they are human cells expressing either the heavy chain or the light chain. Human IL thus obtained
Culture supernatant of a hybridoma clone producing a monoclonal antibody against the C-2 receptor heavy chain is further grown together with human IL-2 in a human IL-2 dependent T cell leukemia virus-infected T cell line ILT- In addition to Mat cells, the growth inhibitory activity of ILT-Mat cells is measured. If it has a growth inhibitory activity, it means that the hybridoma clone is a hybridoma clone producing the target monoclonal antibody. As the hybridoma thus obtained, for example, the hybridoma TU25 (FERM
There is a cell called P-11858).

Next, for the large-scale preparation method of monoclonal antibody, TU
25 (FERM P-11858) cells were inoculated into the abdominal cavity of histocompatibility animals, athymic nude mice, etc., and allowed to grow, and the antibodies produced in the ascites of the animals were collected, salted out, and ionized. It may be purified by an operation such as exchange chromatography.

The monoclonal antibody thus obtained has the following properties.

Anti-human IL-2 produced by TU25 (FERM P-11858) cells
Receptor heavy chain monoclonal antibody (hereinafter referred to as monoclonal antibody TU25) (a) Type of immunoglobulin: IgG1 (b) Molecular weight: 150,000 daltons (c) Molecular extinction coefficient: E _280nm = 14.0 (d) Human IL-2 receptor weight It binds specifically to the chain.

(E) Inhibits the binding between human IL-2 and human IL-2 receptor heavy chain.

(F) Inhibits the physiological activity of human IL-2.

The monoclonal antibody TU25 produced in this manner is
Since it is possible to inhibit the physiological activity of human IL-2 alone, an antibody having a binding activity to the light chain of human IL-2 receptor alone, for example, a commercially available monoclonal antibody 2R
12 (manufactured by T-cell Sunense Co., Ltd.) may be present together. Furthermore, other immunosuppressive agents such as cycloporin A and various stabilizers,
Various excipients may be included in the immunosuppressive agent of the present invention.

These drugs can be used as immunosuppressive agents for preventing rejection at the time of organ transplantation or for treating autoimmune diseases.

In the human IL-2 receptor heavy chain monoclonal antibody of the present invention and an immunosuppressant, it is usually 0.1 to 100% by weight, preferably 0.5.
It is enough to contain up to 70% by weight. Of course, it is not limited to the above value.

Further, the administration method of the immunosuppressive agent is oral, subcutaneous injection,
The administration may be performed by any method such as intravenous injection and intravenous drip, but it is preferable to administer by intravenous injection and intravenous drip.

When used as an injection or drip, for example, the lyophilized antibody of the present invention containing no bacteria or pyrogen is dissolved in distilled water for injection in the Japanese Pharmacopoeia at the time of use. After confirming complete dissolution, the drug is administered to patients suffering from autoimmune diseases, or to patients undergoing organ transplantation or bone marrow transplant surgery or who have completed surgery.

The route of administration is preferably intravenous injection or infusion as described above, and the concentration and dose differ depending on the patient or the situation of the patient, but when an antibody that binds to the heavy chain of human IL-2 receptor is used as a single agent Is 5 to 250 mg / ml at a concentration of 1 to 50 mg / kg / day, and when co-administered with the antibody that binds to the light chain of human IL-2 receptor,
It is suitable to administer the antibody at a concentration of ˜250 mg / ml as a combination drug so as to be about 1 to 50 mg / kg / day. To reiterate, the above dose is a tentative standard and may be appropriately selected according to the condition of each patient.

The immunosuppressive agent of the present invention is formulated according to a method generally used for protein preparations. That is, gelatin, which is a substance that does not react with the immunosuppressive agent of the present invention, which is commonly used in the field of pharmaceuticals, as an excipient for human serum albumin, glutathione, various amino acids and the like as a stabilizer, and if necessary, further. A buffering agent, a preservative and the like may be contained.

The antibody used in the present invention may be the antibody itself,
Fab fragments, F (a
b) ' ₂ fragments, fragments of V region only, etc. may be used, chimeric antibodies in which C region is replaced with those of other species, antibodies to other polypeptides or cytotoxic agents, metal conjugates, etc. It may be a combination of.

Hereinafter, the present invention will be described in more detail based on examples.

(Example 1, Preparation of Hybridoma and Monoclonal Antibody) TL-Mor cells suspended in physiological saline are intraperitoneally administered to 6 to 8 week-old female Balb / c mice at 1 × 10 ⁷ cells / mouse. I was immunized. Ten days later, booster immunization was performed by the same procedure, and further five days later, blood was collected from the orbital vein of the mouse, and ¹²⁵ I-labeled human IL-2 TL- was prepared by the following method.
The antibody titer was measured by examining the binding inhibitory activity against Mor cells. Mice with high antibody titers were finally immunized by the same procedure, and 3 days later, the spleens were extracted and spleen cells and mouse myeloma cells (SP2 / 0-Ag14) were mixed with 50% polyethylene glycol # 4000 ( (Manufactured by Hanai Chemical Co., Ltd.) were mixed at a ratio of 10: 1 in terms of the number of cells to fuse the cells. The fused cells were suspended in RPMI1640 medium (manufactured by Gibco) containing 10% fetal calf serum (manufactured by Gibco) at 5 × 10 ⁶ cells / ml, and 5 × 10 ⁵ mouse thymus per well. 9 containing cells
100 μl each was dispensed into a 6-well flat bottom plate (manufactured by Corning). After 1 day, 2 days, 3 days, and 6 days, half of the medium was replaced with a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium), and the same operation was repeated every 3 days. Approximately 2 weeks after the fusion, the culture supernatant of each well in which the fused cells (hybridomas) grew was analyzed by the method described in Reference Example below to inhibit ¹²⁵ I-labeled human IL-2 binding to TL-Mor cells. , And the binding inhibitory activity against MT-1 cells was measured, and the hybridoma contained in the hole having the binding inhibitory activity against TL-Mor cells and not the binding inhibitory activity against MT-1 cells was subjected to limiting dilution. Cloned by the method. Further, the inhibitory activity of the culture supernatant column of each hybridoma clone was measured to obtain an anti-human IL-2 receptor heavy chain antibody-producing hybridoma. Furthermore, the culture supernatant of the obtained anti-human IL-2 receptor heavy chain antibody-producing hybridoma was examined for its ability to suppress the physiological activity of human IL-2 by the following method. 2 × 10 ⁵ cells / m with RPMI1640 medium (Gibco) containing 10% beef tallow serum (Gibco)
ILT-Mat cell suspension suspended to have a concentration of l was dispensed into 96-well flat-bottom microplate Corning (100 μl per well), and 50 μl of sample culture supernatant was added,
Incubated at 37 ° C for 30 minutes.

The ILT-Mat cell is a human cell line that proliferates dependently on human IL-2. In addition, PRMI1640 containing 10% fetal bovine serum
Human recombinant IL adjusted to 200 units / ml in culture medium
-2 solution (50 ul each), 48% at 37 ℃ in the presence of 5% CO _2.
Cultured for hours. For the last 4 hours, 1 μCi of ³ H-thymidine (manufactured by DuPont) was added and cultivated, and the amount of radioactivity incorporated into cells was measured by a scintillation counter (manufactured by Packard). Human
The ability to inhibit the physiological activity of IL-2 was examined. As a hybridoma clone selected by such a method, TU25 (FE
RM P-11858).

The monoclonal antibody TU25 produced by the hybridoma TU25 (FERM P-11858) was prepared as follows. Pre-injected with 0.5 ml of pristane (manufactured by Wako Pure Chemical Industries, Ltd.) intraperitoneally 1 week before, 8 weeks old female
1 x 10 ⁷ hybridoma T per Balb / c mouse
U25 (FERM P-11858) was injected intraperitoneally. Approximately 10 days later, ascites was collected from the abdominal cavity of the mouse, and cells were removed by centrifugation. Salt out with 0.4 saturated ammonium sulfate to obtain 0.15M NaC
It was dialyzed against 10 mM phosphate buffer (pH 7.5) containing l. 10 mM phosphate buffer containing 0.15 M NaCl in advance (p
H7.5) was added to Protein A Sepharose (Pharmacia) equilibrated to obtain a flow-through fraction.
Elute with 0.5M acetic acid. Immediately elute fractions with 0.15 MN
The monoclonal antibody TU25 was obtained by dialysis against 10 mM phosphate buffer (pH 7.5) containing aCl.

(Example 2, monoclonal antibody TU25 alone, or
Growth inhibitory effect of IL-2 dependent cell line ILT-Mat cells in the presence of IL-2 receptor light chain antibody) PRMI1640 medium (GBco) containing 10% fetal calf serum (Gibco)
ILT-Mat cell solution suspended at a concentration of 2 × 10 ⁵ cells / ml (manufactured by ibco) was dispensed into 96-well flat low microplates (manufactured by Corning) at 100 μl per well. Monoclonal antibody TU25, mouse monoclonal anti-human IL-2
RPMI 1640 containing a receptor light chain antibody, a known mouse monoclonal anti-human IL-2 receptor heavy chain antibody, and a control mouse monoclonal antibody in 10% fetal calf serum
Prepared to 50 μg / ml in medium, and monoclonal antibody TU25,
50 μl of each of the control mouse monoclonal antibody and the mixture of the monoclonal antibody TU25 and the mouse monoclonal anti-human IL-2 receptor light chain antibody was added, and the mixture was incubated at 37 ° C. for 30 minutes. Furthermore, 50 μl of human recombinant IL-2 solution prepared at various concentrations in PRMI1640 medium containing 10% fetal bovine serum was used.
I was added to each well and cultured at 37 ° C. for 48 hours in the presence of 5% CO ₂ . For the last 4 hours, 1 μCi of ³ H-thymidine (manufactured by DuPont) was added and cultivated, and the amount of radioactivity incorporated into the cells was measured by a scintillation counter (manufactured by Packard), and human IL-2 by the antibody was measured. The ability to inhibit the physiological activity of was investigated. As a result, as shown in FIG.
Mouse monoclonal anti-human IL-2 receptor heavy chain antibody TU with known physiological activity of human IL-2 by addition of TU25 alone
It was significantly inhibited as compared to 27 (a monoclonal antibody produced by FERM BP-2510). Also monoclonal antibody TU2
It was revealed that the physiological activity of IL-2 was completely inhibited even at a high concentration by adding 5 and a mouse monoclonal anti-human IL-2 receptor light chain antibody together.

(Effects of the Invention) The present invention uses a monoclonal antibody alone or in human.
When used in combination with an antibody that binds to the IL-2 receptor light chain, it can be used as an immunosuppressive agent useful for preventing rejection at the time of organ transplantation or treating autoimmune diseases.

[Brief description of drawings]

FIG. 1 shows that the monoclonal antibody TU25 has an activity of inhibiting the physiological activity of IL-2 alone or in the presence of a mouse monoclonal anti-IL-2 receptor light chain antibody.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-2-13371 (JP, A) Lymphokine Res. , 7 [3] (1988) p. 313 J. Exp. Med. , 169 [4] (1989) P. 1332-1332 Immunology, 70 [1] (1990) P. 111-115 Blood. 76 [10] (1990) P. 2080-2085 Proc. Natl. Acad. Sc i. USA, 86 (1989) P. 1982-1986

Claims (2)

(57) [Claims] 1. A monoclonal antibody TU-25 which binds to a human IL-2 receptor heavy chain having the following properties. (A) binds to human IL-2 receptor heavy chain (b) inhibits binding of human IL-2 to human IL-2 receptor heavy chain (c) isolates human IL-2 physiological activity against human T cells Interfere with 2. An immunosuppressive agent comprising the monoclonal antibody according to claim 1.