MX2008004901A - Inhibitor of transplanted islet dysfunction in islet transplantation. - Google Patents

Abstract

An anti-IL-6 receptor antibody is examined on its inhibitory effect against transplanted islet dysfunction produced after islet transplantation. As a result, it is found that the anti-IL-6 receptor antibody can reduce the transplanted islet dysfunction, improve the survival of the transplanted islet, and also improve the hyperglycemic state of a recipient. It is also found that administration of the anti-IL-6 receptor antibody enables to prevent the production of an inflammatory cytokine in an infiltrating cell after the transplantation. Thus, it is now found that transplanted islet dysfunction produced after islet transplantation can be prevented by using the anti-IL-6 receptor antibody.

Description

INHIBITOR OF THE D1SFUNCION OF THE ISLOTE TRANSPLANTED IN TRANSPLANTATION OF THE ISLOTE Field of the Invention The present invention relates to agents for suppressing damage to transplanted islets after islet transplantation, comprising inhibitors of IL-6 as active ingredients, and to their uses Background of the Invention Insulin is a hypoglycemic hormone. Insulin-dependent diabetes is caused by the selective destruction of insulin-producing cells (ß islet cells) by immunological mechanisms, and it is known that its onset results in hyperglycemia, which causes various disorders. In conventional therapy, preparations of insulin (injected), which is usually scarce, are administered to compensate for the lack of insulin and to correct hyperglycemia. However, strict control of blood sugar is difficult in insulin-based therapies, which can lead to excessive administration and can lead to fatal hypoglycaemia. After the onset of diabetes the progress of vascular complications (such as retinopathy, nephropathy and neuropathy) is observed. Conventional therapeutic methods, such as insulin injections, can not stop this progress, which leads to serious therapeutic problems. Under physiological conditions, blood sugar is controlled mainly by the regulatory mechanism of the ß cells of the islets; however, in insulin-dependent diabetes, the elimination of these islets results in violent rise and fall of the blood sugar level, which causes the clinical symptoms described above. In recent years, the clinical application of islet transplants has begun in Europe and the United States, where Langerhaus pancreatic islets (islets) are transplanted as a means to treat diabetes. In this way an attempt is made to carry out a treatment that does not include the administration of insulin, but the implantation of insulin-producing cells. The practical procedure for clinical islet transplantation is as follows: a percutaneous transpulmonary catheterization of the portal vein guided by ultrasound is performed under local anesthesia; and then donor islets are transplanted to the liver through the catheter. The grafted islets survive at the end of the portal vein, and control the level of sugar by secreting insulin. When successful, islet transplantation restores the normal blood sugar level in diabetic recipients, so that insulin treatment is unnecessary. However, to date, successful cases of islet transplantation are limited. In addition, transplantation to a single recipient requires the isolation of islets from the pancreas of two or three donors.

Specifically, as the islet function disorders that appear immediately after transplantation reduce the viability of the graft, the transplantation of islets from a single donor to a single recipient is insufficient, so the transplant is performed from two or three donors to a single receiver. In some reports it is suggested that only between 20% and 30% of the transplanted grafts survive. The details of these functional disorders are not yet clear, but they represent an extremely serious problem in terms of improving clinical islet transplantation. Normally, islet transplantation is performed using islets isolated from the pancreas of brain-dead donors "or donors with cardiac arrest. Recent reports also describe successful cases of islet transplantation from living donors, where the islets are isolated and purified from a portion of pancreas extracted from healthy donors and transplanted to diabetic patients. This transplantation of islets from living donors is invasive and is a burden on donors. Therefore, it is preferable to design treatments that suppress the damage to the transplanted islets just after the transplant, and in those that use less islets of the donor. IL-6 is a cytokine called B 2 cell stimulation factor (BSF2) or β2 interferon. IL-6 was discovered as a differentiating factor involved in the activation of B lymphocyte cells (Non-Patentable Document 1), and then it was revealed to be a multi- functional cytokine that influenced the function of various cells (Non-Patentable Document 2) . It has been reported that IL-6 induces maturation of T lymphocyte cells (Non-Patentable Document 3). IL-6 transmits its biological activity through two types of proteins in the cell. One of the proteins is the IL-6 receptor, which is a ligand binding protein with which IL-6 binds, and has a molecular weight of approximately 80 kDa (Non-Patentable Documents 4 and 5). In addition to a membrane bound form that penetrates and is expressed in the cell membrane, the IL-6 receptor is present as a soluble IL-6 receptor, which mainly consists of the extracellular region of the membrane-bound form. The other is the gp130 membrane protein, which has a molecular weight of approximately 130 kDa and participates in the transduction of non-ligand binding signals. The biological activity of IL-6 is transmitted to the cells by the formation of the IL-6 / IL-6 receptor complex by IL-6 and the IL-6 receptor, and the subsequent binding of the complex to gp130 (Document No Patentable 6). Inhibitors of IL-6 are substances that inhibit the transmission of the biological activity of IL-6. Until now there were known antibodies against IL-6 (anti-IL-6 antibodies), antibodies against IL-6 receptors (anti-IL-6 receptor antibodies), antibodies against gp130 (anti-gp130 antibodies), variants of IL-6, partial peptides of IL-6 or IL-6 receptors, and the like. There are several sources in which anti-IL-6 receptor antibodies are described (Non-Patentable Documents 7 and 8, Patentable Documents 1-3). A humanized antibody PM-1, which was obtained through the transplantation of the Complement Complement Region (CAR) of mouse PM-1 antibody (Non-Patentable Document 9), which is one of the anti-receptor antibodies, is known. of IL-6 (Patentable Document 4). The information in the prior art documents relating to the present invention is described below: [Non-Patentable Document 1] Hirano, T. et al., Nature (1986) 324, 73-76 [Non-Patentable Document 2] Akira, S. et al., Adv. in Immunology (1993) 54, 1-78 [Non-Patentable Document 3] Lotz, M. et al., J. Exp. Med. (1988) 167, 1253-1258 [Non-Patentable Document 4] Taga, T. et al. ., J. Exp. Med. (1987) 166,967-981 [Non-Patentable Document 5] Yamasaki, K. et al., Science (1988) 241, 825-828 [Non-Patentable Document 6] Taga, T. et al., Cell (1989) 58, 573-581 [Non-Patentable Document 7] Novick, D. et al., Hybridoma (1991) 10 , 137-146 [Non-Patentable Document 8] Huang, YW et al., Hybridoma (1993) 12,621-630 [Non-Patentable Document 9] Hirata, Y. et al., J. Immunol. (1989) 143,2900-2906 [Patentable Document 1] Publication of International Patent Application No. WO 95/09873 [Patentable Document 2] French Patent Application No. FR 2694767 [Patentable Document 3] United States Patent No. 5216128 [Document Patentable 4] WO 92/19759 Description of the invention Problems to be solved by the invention In islet transplantation for the treatment of diabetes, it is important to improve the viability of the islets by suppressing damage to the transplanted islets at the time of transplantation. . However, to date there are no effective methods. In addition, it has not previously been evaluated whether the anti-IL-6 receptor antibody, which is an inhibitor of IL-6, has the effect of suppressing damage in the transplanted islets after islet transplantation. The present invention was conceived taking into account the aforementioned background, and is intended to provide agents for suppressing damage to transplanted islets, comprising inhibitors of IL-6 as active ingredients, which are used in the transplantation of islets. Another objective of the present invention is to provide methods for suppressing damage to transplanted islets to subjects, wherein the methods comprise the step of administering IL-6 inhibitors to subjects. Means for solving the problems In order to achieve the objectives previously described, the present inventors evaluated whether anti-IL-6 receptor antibodies exerted the effect of suppressing the damage to the transplanted islets after the islet transplantation. First, the present inventors prepared diabetic mice as receptors by administering streptozotocin to male C57BL / 6 mice intravenously. Then, the recipient diabetic mice received a transplant of islets isolated from the pancreas of two mice (400 islets) or from a single mouse (200 islets). The results indicate that transplantation of islets from the pancreas of two mice restored normal levels of blood sugar, so it presented a therapeutic effect on diabetes, while transplantation from a single mouse did not restore normal levels of blood sugar, so the state of hyperglycemia was maintained (Figures 2 and 3). Meanwhile, when 500 pg of an anti-IL-6 receptor antibody (M16-1) was administered intraperitoneally three times after an islet transplant from a single mouse (200 islets), normal levels of blood sugar in all recipients after transplantation (Fig. 4). Alternatively, when an equal dose of an anti-IL-6 receptor antibody was administered on one occasion, normal blood sugar levels were restored in three-quarters of the recipients (FIG. 5). When 200 g of anti-IL-6 receptor antibody was administered on one occasion, normal blood sugar levels were restored in one third of the receptors (FIG. 6). It was also discovered that administration of the anti-IL-6 receptor antibodies of the present invention suppressed the production of inflammatory cytokines in infiltrating cells after transplantation. The above findings suggest that anti-IL-6 receptor antibodies reduce damage to transplanted islets, improve islet viability and correct hyperglycemia at the receptors. Specifically, the present inventors discovered for the first time that damage to transplanted islets after islet transplantation using anti-IL-6 receptor antibodies according to the present invention could be suppressed, and thus they completed the present invention. More specifically, in the present invention the following items [1] to [23] are provided: [1]. An agent for suppressing damage to the transplanted islets after islet transplantation, comprising an inhibitor of IL-6 as an active ingredient. [2]. The agent to suppress damage to the transplanted islets of [1], where the IL-6 inhibitor is an antibody that recognizes an IL-6. [3]. The agent to suppress damage to the transplanted islets of [1], where the IL-6 inhibitor is an antibody that recognizes an IL-6 receptor. [4]. The agent to suppress damage to the transplanted islets of [2] or [3], where the antibody is a monoclonal antibody. [5]. The agent for suppressing damage to the transplanted islets of any of [2] to [4], wherein the antibody is a human anti-IL-6 antibody or an anti-human IL-6 receptor antibody. [6] The agent to suppress damage to the islet transplanted from any of [2] to [5], where the antibody is a recombinant antibody. [7] The agent to suppress damage to the transplanted islets of [6], where the antibody is a humanized or humanized chimeric antibody. [8] The agent to suppress damage to the transplanted islets of any of [1] to [7], which is used to treat diabetes. [9] A method for suppressing damage to transplanted islets in a subject undergoing islet transplantation, comprising the step of administering an IL-6 inhibitor to the subject; [10] A method for improving the viability of an islet transplant in a subject, comprising the step of administering an IL-6 inhibitor to the subject; [eleven]. The method of [9] or [10], wherein the inhibitor of IL-6 is an antibody that recognizes an IL-6; [12] The method of [9] or [10], wherein the IL-6 inhibitor is an antibody that recognizes an IL-6 receptor; [13] The method of [11] or [12], where the antibody is a monoclonal antibody; [14] The method of any of [11] to [13], wherein the antibody is a human anti-IL-6 antibody or an anti-human IL-6 receptor antibody; [fifteen]. The method of any of [11] to [14], wherein the antibody is a recombinant antibody; [16] The method of [15], wherein the antibody is a chimeric, humanized or human antibody; [17] The use of an IL-6 inhibitor to produce agents to suppress damage to transplanted islets after islet transplantation; [18] The use of [17], where the inhibitor of IL-6 is an antibody that recognizes an IL-6; [19] The use of [17], where the inhibitor of IL-6 is an antibody that recognizes an IL-6 receptor; [twenty]. The use of [18] or [19], where the antibody is a monoclonal antibody; [twenty-one]. The use of any of [18] to [20], wherein the antibody is a human anti-IL-6 antibody or an anti-human IL-6 receptor antibody; [22] The use of any of [18] to [21], wherein the antibody is a recombinant antibody; and [23]. The use of [22], where the antibody is a chimeric, humanized or human antibody. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph illustrating changes in blood sugar levels in diabetic recipient mice that were not subjected to an islet transplant. Figure 2 is a graph illustrating changes in blood sugar levels in diabetic recipient mice that received a transplant of isogenic islets from the pancreas of two mice (400 islets). Figure 3 is a graph illustrating changes in blood sugar levels in diabetic recipient mice that received an isogenic islet transplant from a pancreas of a single mouse (200 islets). Figure 4 is a graph illustrating changes in blood sugar levels in diabetic recipient mice that received an isotonic islet transplant from a single mouse pancreas (200 islets), and that received three administrations Intraperitoneal 500 pg of anti-IL-6 receptor antibody after transplantation. Figure 5 is a graph illustrating changes in blood sugar levels in diabetic recipient mice that received an isogenic islet transplant from a single mouse pancreas (200 islets), and that received a single intraperitoneal administration of 500 pg of anti-IL-6 receptor antibody after transplantation. Figure 6 is a graph illustrating changes in blood sugar levels in diabetic recipient mice that received a transplant of isogenic islets from a single mouse pancreas (200 islets), and that received a single intraperitoneal administration of 200 pg of anti-IL-6 receptor antibody after transplantation. Figure 7 is a graph illustrating that administration of the anti-IL-6 receptor antibody suppresses post-transplant production of inflammatory cytokines by infiltrating cells. Figure 8 is a graph showing the changes in blood sugar levels in diabetic recipient mice that received an allogeneic islet transplant from a single mouse pancreas (200 islets) and received three intraperitoneal administrations of 200 pg of rat IgG after transplantation. Figure 9 is a graph illustrating changes in blood sugar levels in diabetic recipient mice that received an allogeneic islet transplant from a single mouse pancreas (200 islets), and who received a single intraperitoneal administration of 200 pg of anti-CD4 antibody after transplantation. Figure 10 is a graph illustrating changes in blood sugar levels in recipient diabetic mice that received an allogeneic islet transplant from a single mouse pancreas (200 islets), and that received three administrations Intraperitoneal 500 pg of anti-IL-6 receptor antibody, and a 200 pg administration of anti-CD4 antibody after transplantation. BEST MODE FOR CARRYING OUT THE INVENTION The present inventors discovered that anti-IL-6 receptor antibody can suppress damage to transplanted islets after islet transplantation. The present invention is based on these findings. The present invention relates to agents for suppressing damage to transplanted islets after islet transplantation, comprising an inhibitor of IL-6 as an active ingredient. In the present documentation, an "IL-6 inhibitor" is a substance that blocks the transduction of signals mediated by IL-6 and inhibits the biological activity of IL-6. Preferably, the inhibitor of IL-6 is a substance that has an inhibitory function against the binding of IL-6, the IL-6 receptor, or gp130. The IL-6 inhibitors of the present invention include, without limitation, for example, anti-IL-6 antibodies, anti-IL-6 receptor antibodies, anti-gp 130 antibodies, IL-6 variants, soluble variants of the IL-6 and partial peptides of IL-6 or IL-6 receptor, and low molecular weight compounds that exhibit similar activities. Preferred IL-6 inhibitors of the present invention include antibodies that recognize IL-6 receptors. In the present invention, the source of the antibody is not particularly restricted; however, the antibody preferably derives from mammals, and more preferably derives from humans. The anti-IL-6 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody through known means. In particular, monoclonal antibodies derived from mammals are preferred as the anti-IL-6 antibody used in the present invention. Monoclonal antibodies derived from mammals include those produced from hybridomas and those produced from hosts transformed with an expression vector comprising an antibody gene, according to genetic engineering methods. Upon binding to IL-6, the antibody inhibits the binding of IL-6 to an IL-6 receptor, and blocks the transmission of the biological activity of IL-6 in the cell. These antibodies include MH166 (Matsuda, T. et al., Eur. J. Immunol. (1988) 18, 951-956), SK2 antibody (Sato, K. et al., Transaction of the 21st Meeting of the Japanese Society of Immunology (1991) 21, 166), and the like. Basically, it is possible to prepare hybridomas that produce the anti-IL-6 antibody using known techniques, as indicated below. Specifically, these hybridomas can be prepared using IL-6 as the sensitizing antigen to perform the immunization, according to a conventional immunization method, by fusing the obtained immune cells with known progenitor cells, according to a conventional cell fusion method, and searching for cells producers of monoclonal antibodies with a conventional analysis method. More specifically, it is possible to produce anti-IL-6 antibodies as indicated below. For example, it is possible to obtain human IL-6, used as a sensitizing antigen to obtain antibodies, using the gene and / or the amino acid sequence of IL-6 described in Bur. J. Biochem. (1987) 168, 543-550; J. Immunol. (1988) 140, 1534-1541; and / or Agr. Biol. Chem. (1990) 54, 2685-2698. After transforming an appropriate host cell with a system of known expression vectors with a genetic sequence of inserted IL-6, the desired IL-6 protein is purified by a known method, from the interior of a host cell or from the supernatant of culture. This purified IL-6 protein can be used as a sensitizing antigen. Alternatively, one fusion protein of the IL-6 protein and another protein can be used as a sensitizing antigen. The anti-IL-6 receptor antibodies used in the present invention can be obtained as polyclonal or monoclonal antibodies according to known methods. In particular, the anti-IL-6 receptor antibodies used in the present invention are preferably monoclonal antibodies derived from mammals. Monoclonal antibodies derived from mammals include those produced from hybridomas and those produced from hosts transformed with an expression vector comprising the antibody gene, according to genetic engineering methods. By binding to an IL-6 receptor, the antibody inhibits the binding of IL-6 to the IL-6 receptor and blocks the transmission of the biological activity of IL-6 in the cell. These antibodies include the MR16-1 antibody (Tamura, T. et al., Proc, Nati, Acad. Sci. USA (1993) 90, 11924-11928), the PM-1 antibody (Hirata, Y et al., J Immunol. (1989) 143, 2900-2906), the antibody AUK12-20, the antibody AUK64-7 and the antibody AUK146-15 (SVO 92/19759), and the like. Among them, the PM-1 antibody can be mentioned as an example of a preferred monoclonal antibody against the human IL-6 receptor, and the R16-1 antibody can be mentioned as a preferred monoclonal antibody against the mouse IL-6 receptor. Basically, it is possible to prepare hybridomas that produce an anti-IL-6 receptor monoclonal antibody using known techniques, as indicated below. Specifically, these hybridomas can be prepared using IL-6 as a sensitizing antigen to perform the immunization, according to a conventional immunization method, by fusing the immune cells obtained with known progenitor cells, according to a conventional cell fusion method, and looking for cells producing monoclonal antibodies with a conventional analysis method. More specifically, it is possible to produce anti-IL-6 antibodies as indicated below. For example, it is possible to obtain a human IL-6 receptor or a mouse IL-6 receptor, used as a sensitizing antigen to obtain antibodies, using the genes and / or the amino acid sequences of IL-6 receptors described in FIG. European Patent Publication No. LP 325474 and Japanese Patent Application Publication No. Kokai (JP-A) H03-155795 (unexamined, Published Patent Application), respectively. There are two types of IL-6 receptor proteins, i.e., the proteins that are expressed on the cell membrane and the proteins separated from the cell membrane (soluble IL-6 receptor) (Yasukawa, K. et al., J. Biochem. (1990) 108, 673-676). The soluble IL-6 receptor consists essentially of the extracellular region of the membrane-bound IL-6 receptor, and differs from the membrane-bound IL-6 receptor because it lacks the transmembrane region, or because it lacks the transmembrane and intracellular regions . It is possible to employ any IL-6 receptor as an IL-6 receptor protein, provided that it can be used as a sensitizing antigen to produce the anti-IL-6 receptor antibody used in the present invention. After transforming an appropriate host cell with a system of expression vectors with an inserted IL-6 receptor gene sequence, the desired IL-6 receptor protein is purified by a known method, from the interior of a host cell or from the culture supernatant. This purified IL-6 receptor protein can be used as a sensitizing antigen. Alternatively, a cell expressing the IL-6 receptor, or a fusion protein of the IL-6 receptor protein, and another protein as a sensitizing antigen can be used. The ariti-gp130 antibodies used in the present invention can be obtained as polyclonal or monoclonal antibodies according to known methods. In particular, the anti-gp130 antibodies used in the present invention are preferably monoclonal antibodies derived from mammals. Monoclonal antibodies derived from mammals include those produced from hybridomas and those produced from hosts transformed with an expression vector comprising the antibody gene, according to genetic engineering methods. Upon binding to gp130, the antibody inhibits the binding of gp130 to the complex of I L-6 / IL-6 receptor, and blocks the transmission of the biological activity of IL-6 in the cell. These antibodies include antibody AM64 (JP-A (Kokai) H03-219894), antibody 4B11 and antibody 2H4 (US 5571513), antibody B-S12 and antibody B-P8 (JP-A (Kokai) H08- 291199), and the like. Basically, it is possible to prepare hybridomas that produce the anti-gp130 antibody using known techniques, as indicated below. Specifically, these hybridomas can be prepared using gp130 as the sensitizing antigen to perform the immunization, according to a conventional immunization method, by fusing the obtained immune cells with known progenitor cells, according to a conventional cell fusion method, and looking for cells that produce monoclonal antibodies with a conventional analysis method. More specifically, the monoclonal antibody can be produced as indicated below. For example, it is possible to obtain gp130, used as a sensitizing antigen to obtain antibodies, using the gene and / or amino acid sequence of gp130 described in European Patent Application Publication No. EP 411946. After transforming an appropriate host cell with a system of known expression vectors with a genetic sequence of gp130 inserted, the desired gp130 protein is purified with a known method, from inside a host cell or from the culture supernatant. This purified gp130 protein can be used as a sensitizing antigen. Alternatively, a cell expressing gp130, or a fusion protein of the gp130 protein and another protein as a sensitizing antigen can be used. Mammals that can be immunized with a sensitizing antigen are not particularly limited, but are preferably selected taking into account compatibility with the progenitor cell used for cell fusion. In general rodents are used, such as mice, rats and hamsters. Immunization of the animals with a sensitizing antigen is carried out according to known methods. For example, as a general method, it is carried out by injecting the sensitizing antigen intraperitoneally or subcutaneously in mammals. Specifically, the sensitizing antigen is preferably diluted or suspended in an appropriate amount of phosphate buffered saline (PBS), physiological saline or the like, mixed with an appropriate amount of a general adjuvant (eg, Freund's complete adjuvant), it is emulsified and then administered several times every 4 to 21 days to a mammal. In addition, it is possible to use a suitable vehicle for immunization with a sensitizing antigen. After this immunization, an increased level of the desired antibody in serum is confirmed, and then immune cells are obtained from the mammal to effect cell fusion. Preferred immune cells for cell fusion include, in particular, spleen cells. To use mammalian myeloma cells as progenitor cells, i.e., to be able to fuse a progenitor cell with the above immune cells, various strains of cells are appropriately used, for example, P3X63Ag8.653 (Keamey, JF et al., J. Immunol. (1979) 123, 1548-1550), P3X63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler, Cz and Nilstein, C, Eur. J. unol. (1976) 6, 511-519), MPC-11 (Margulies, A, H. et al., Cell (1976) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978 276, 269-270), FO (from St. Groth, S. E et al., J. Immunol. Methods (1980) 35.1-21), 5194 (Trowbridge, IS, J. Exp. Med. 1978) 148, 313-323), R210 (Ufre, G et al., Nature (1979) 277, 131-133), and the like. Basically, it is possible to perform cell fusion of the immune cells and myeloma cells mentioned previously using known methods, for example, the method of Nilstein et al. (Kohler, G. and Nilstein, C, Methods Enzymol. (1981) 73, 3-46), and the like. More specifically, the cell fusion mentioned above is generally carried out in a nutrient culture medium, in the presence of an agent that promotes cell fusion. For example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used as agents that promote cell fusion. In addition to improving the efficiency of the fusion, auxiliary agents, such as dimethyl sulfoxide, can be added for use as needed. The ratio between the immune cells and myeloma cells used preferably is, for example, between 1 and 10 immune cells per each myeloma cell. The culture medium used for the cell fusion mentioned above is, for example, culture medium RPMI1640 or MEM, which is appropriate for the proliferation of the aforementioned myeloma cells. A general culture medium used to grow this type of cells can also be used. In addition, serum supplements may be used in combination, such as fetal calf serum (FCS). For cell fusion, the fusion cells (hybridomas) of interest are formed by appropriately mixing predetermined amounts of the immune cells and the aforementioned myeloma cells in the aforementioned culture medium, and then adding and mixing a concentration of between 30 and 60% (w / v) of PEG solution (for example, a PEG solution with an average molecular weight of between about 1000 and 6000) preheated to about 37 ° C. Subsequently, it is possible to eliminate cell fusion agents and the like, which are not suitable for the growth of the hybridoma, by repeating the steps of successive addition of an appropriate culture medium and removal of the supernatant by centrifugation. The above hybridomas are selected by culturing the cells in a general selection culture medium, for example, HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). The culture in the HAT culture medium is continued for a sufficient period of time, generally for several days to several weeks, to kill the cells other than the hybridomas of interest (unfused cells). Then a conventional limited dilution method is applied to separate and clone the hybridomas that produce the antibody of interest. In addition to the method for immunizing a non-human animal with an antigen to obtain the aforementioned hybridomas, it is possible to obtain a desired human antibody, having antigen binding activity or a cell expressing a desired antigen, stizing a human lymphocyte with a desired antigenic protein or a cell expressing an antigen in vitro, and fusing the stized B lymphocyte with a human myeloma cell (e.g., U266) (see Japanese Patent Application Publication Kokoku No. (JP-B) H01- 59878 (Japanese patent application examined and approved, published for opposition)). Moreover, it is possible to obtain a desired human antibody by administering the antigen or cell expressing the antigen to a transgenic animal having a repertoire of human antibody genes, and then implementing the above-mentioned method (see the Publications of Requests for International Patents No. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735). Hybridomas prepared in this way, which produce monoclonal antibodies, can be subcultured in a conventional culture medium and stored in liquid nitrogen for a prolonged period. To obtain monoclonal antibodies from the aforementioned hybridomas, the following methods can be employed: (1) a method in which the hybridomas are cultured according to conventional methods and the antibodies are obtained as a culture supernatant; (2) a method in which the hybridomas are proliferated by administering them to a compatible mammal, and obtaining the antibodies as ascites; and similar. The first method is used to obtain antibodies with a high purity, and the second method is more appropriate for the production of antibodies on a large scale. For example, the preparation of hybridomas producing anti-IL-6 receptor antibodies can be carried out according to the method described in JP-A (Kokai) H03-139293. The preparation can be carried out according to a method comprising injecting a hybridoma producing PM-1 antibody into the abdominal cavity of a BALB / c mouse, obtaining ascites, and then purifying the PM-1 antibody from the ascites, or the method comprising culturing the hybridoma in an appropriate medium (eg, RPMI1640 medium containing 10% fetal bovine serum and 5% BM-Condimed H1 (Boehringer Mannheim), medium for SFM hybridomas (GIBCO-BRL), medium PFHM -II (GIBCO-BRL), etc.), and then the PM-1 antibody can be obtained from the culture supernatant. A recombinant antibody can be used as a monoclonal antibody of the present invention, wherein the antibody is produced by genetic recombination techniques, by cloning an antibody gene from a hybridoma, inserting the gene into an appropriate vector, and subsequent introduction of the vector into a host (see, for example, Borrebaerck, CAK and Larrick, J. W, THERAPEUTIC MONOCLONAL ANDTIBODIES, published in the United Kingdom by MACMILLAN PUBLISHERS LTD., 1990). More specifically, mRNA encoding the variable region (V) of the antibody is isolated from a cell that produces the antibody of interest, such as a hybridoma. Isolation of the mRNA can be effected by preparing total RNA according to known methods, such as the guanidine ultracentrifugation method (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299) and the AGPC method (Chomczynski, P et al., Anal. Biochem. (1987) 162, 156-159), and the preparation of the mRNA can be carried out with a set of elements for purifying mRNA (Pharmacia), and the like. Alternatively, the mRNA can be prepared directly using the set of elements for purifying QuickPrep mRNA (Pharmacia). The cDNA of the V region of the antibody is synthesized from the mRNA obtained using reverse transcriptase. The synthesis of the cDNA can be carried out using the set of first strand reverse transcriptase cDNA synthesis elements, and the like. In addition, to synthesize and amplify the cDNA, the 5'-RACE method (Frohman, MA et al., Proc. Nati. Acad. Sei. USA (1988) 85, 8998-9002; Belyavsky, A. et al. ., Nucleic Acids Res. (1989) 17, 2919-2932), using the set of elements 5'-AmpliFINDER RACE (Clontech) and PCR. The DNA fragment of interest is purified from the obtained PCR products, and then it is ligated with a vector DNA. A recombinant vector is then prepared using the above DNA, and it is introduced into Escherichia coli or the like, and its colonies are selected to prepare the desired recombinant vector. The nucleotide sequence of the DNA of interest is confirmed, for example, by the dideoxy method. When a DNA encoding the V region of an antibody of interest is obtained, the DNA is ligated with a DNA encoding a desired antibody constant region (region C) and inserted into an expression vector. Alternatively, the DNA encoding the V region of the antibody can be inserted into an expression vector comprising the DNA of an antibody C region.

To produce an antibody that can be used in the present invention, as will be described below, the antibody gene is inserted into an expression vector, so that it is expressed under the control of the expression regulatory region, eg, a enhancer and a promoter. The antibody can then be expressed by transforming a host cell with this expression vector. In the present invention, to reduce the heteroantigenicity against humans and the like, artificially genetically modified recombinant antibodies can be used, for example, chimeric antibodies, humanized antibodies or human antibodies. These modified antibodies can be prepared using known methods. It is possible to obtain a chimeric antibody by binding the DNA encoding the V region of the antibody, described above, with a DNA encoding a C region of a human antibody, inserting the DNA into an expression vector and introducing it into a host to produce it ( see European Patent Application Publication No. EP 125023, International Patent Application Publication No. WO 92/19759). This known method can be used to obtain chimeric antibodies useful for the present invention. Humanized antibodies are also known as reformed human antibodies, and are antibodies in which regions of complementarity determination (CDR) of an antibody from a mammal other than human (e.g., a mouse antibody) are transferred to the CDR of a human antibody. General methods for this genetic recombination are also known (see European Patent Application Publication No. EP 125023 and International Patent Application Publication No. WO 92/19759). More specifically, a DNA sequence, designed so that the CDRs of a mouse antibody are bound to the framework regions (FR) of a human antibody, is synthesized by PCR from several oligonucleotides that have been produced from so that they contain portions superimposed on their ends. The DNA obtained is linked to a DNA encoding a C region of a human antibody, and then inserted into an expression vector. The expression vector is introduced into a host to produce the humanized antibody (see European Patent Application Publication No. EP 239400 and International Patent Application Publication No. WO 92/19759).

The FRs of the human antibodies that are desired to bind through the CDRs are selected so that the CDRs form an appropriate antigene binding site. The amino acid (s) within the FR of the variable regions of the antibodies can be replaced as necessary, so that all CDRs of the human antibody reformate form an appropriate antigen binding site (Sato, K, et al., Cancer Res. (1993) 53, 851-856). The C regions of the human antibody are used for the chimeric and humanized antibodies, and include Cy. For example, Cy1, Cy2, Cy3 or Cy4 can be used. In addition, to improve the stability of the antibody or its production, it is possible to modify the C regions of the human antibody. Chimeric antibodies consist of the variable region of an antibody derived from non-human mammals and a C region derived from a human antibody; and humanized antibodies consist of the CDRs of an antibody derived from non-human mammals and regions of framework and regions C derived from a human antibody. Both have a reduced antigenicity in the human body, so they are useful as antibodies for use in the present invention. Preferred specific examples of humanized antibodies used in the present invention include a humanized antibody PM-1 antibody (see International Patent Application Publication No. WO 92/19759). Additionally, in addition to the method for obtaining a human antibody mentioned above, techniques for obtaining human antibodies comprising the use of a human antibody library are also known. For example, it is possible to express the variable regions of human antibodies on the phage surface as single chain antibodies (scFv) with the phage display method, and then select the phages that bind to antigens. By analyzing the genes of the selected phages, it is possible to determine the DNA sequences encoding the variable regions of the human antibodies that bind to the antigen. Once the DNA sequence of a scFV that binds to the antigen has been revealed, an appropriate expression vector comprising the sequence can be constructed in order to obtain a human antibody. These methods are known, and it is possible to use the publications of WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388 as reference. The antibody gene constructed in advance can be expressed according to conventional methods. When a mammalian cell is used, the antibody gene can be expressed using a DNA in which the antibody gene to be expressed is functionally linked to a useful common promoter, and to a poly A signal downstream of the antibody, or a vector comprising the DNA. Examples of a promoter / enhancer include the early promoter / enhancer of human cytomegalovirus. In addition, other promoters / enhancers that can be used to express the antibody to be used in the present invention include promoters / enhancers of retroviruses, polyoma viruses, adenoviruses, simian viruses (SV40) and the like.; and promoters / enhancers derived from mammalian cells, such as human elongation factor 1a (HEF1 a). For example, when the SV40 promoter / enhancer is used, expression can be easily performed according to the method of Mulligan et al. (Mulligan, R C. et al., Nature (1979) 277, 108-114). Alternatively, in the case of an HEF1a promoter / enhancer, the method of Mizushima et al. (Mizushima, S. and Nagata S., Nucleic Acids Res. (1990) 18, 5322). When E. coli is used, it is possible to express the antibody gene by functionally binding a conventional useful promoter, a signal sequence to secrete the antibody and the antibody gene that is desired to be expressed. Examples of promoters include the lacZ promoter, the araB promoter and the like. When the lacZ promoter is used, expression can be effected according to the method of Ward et al. (Ward, E. S. et al., Nature (1989) 341, 544-546; Ward, E. S. et al., FASEB J. (1992) 6, 2422-2427); and the araB promoter can be used according to the method of Betten et al. (Better, M, et al., Science (1988) 240, 1041-1043). When the antibody is produced in the periplasm of E. coli, the signal sequence pei B (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379-4383) can be used as a signal sequence to secrete the antibody. The antibody produced in the periplasm is isolated and then used after properly folding the structure of the antibody (see, for example, WO 96/30394). As origins of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can be used. In addition, to improve the copy quantity of the gene in the host cell system, the expression vector may comprise the aminoglycoside phosphotransferase (APH) gene, the thymidine kinase (TK) gene, the xanthine-guanine phosphoribosyltramferase gene of E. coli (Ecogpt), the dihydrofolate reductase (dhfr) gene, or a similar gene, as a selection marker. Any production system can be used to prepare the antibodies that will be used in the present invention. Production systems for preparing the antibodies include in vitro and in vivo production systems. In vitro production systems include those in which eukaryotic cells or prokaryotic cells are used. Production systems in which eukaryotic cells are used include those in which animal cells, plant cells or fungal cells are used. Animal cells include (1) mammalian cells, eg, CHO, COS, myeloma cells, neonatal hamster kidney cells (BHK), HeLa, Vero and the like, (2) amphibian cells, eg, oocytes from Xenopus; and (3) insect cells, for example, sf9, sf21, Tn5 and the like. Known plant cells include cells derived from Nicotiana tabacum, which can be grown as callus. Known fungal cells include yeasts, such as Sacchcromyces (e.g., S. cerevisiae), mold fungi, such as Aspergillus (e.g., A. niger), and the like. Production systems in which prokaryotic cells are used include those in which bacterial cells are used. Known bacterial cells include E. coli and Bacillus subtilis. Antibodies can be obtained by introducing a gene of an antibody of interest into these cells by transformation, and culturing the transformed cells in vitro. The cultivation is carried out according to known methods. For example, DMEM, ME, RPMI1640, IMDM can be used as the culture medium, and it is possible to use serum supplements, such as FCS, in combination. In addition, a cell into which an antibody gene has been introduced can be transferred to the abdominal cavity, or to a similar part of an animal, to produce an antibody in vivo. On the other hand, in vivo production systems include those in which animals or plants are used. Production systems in which animals are used include those in which mammals or insects are used. Mammals that can be used include goats, pigs, sheep, mice, bovines and the like (Vicki Glaser, SPECTRUM Biotechnology Applieations, 1993). In addition, the insects that can be used include silkworms. When plants are used, for example, tobacco can be used. In these animals or plants an antibody gene is introduced, an antibody is produced in the body of animals or plants, and then recovered. For example, the antibody gene is prepared as a fusion gene by inserting the gene into the medium of a gene encoding a protein, such as goat casein, which is produced only in milk. A DNA fragment comprising the inserted fusion of the antibody gene is injected into a goat embryo, and this embryo is introduced into a female goat. The desired antibody is obtained from the milk produced by the transgenic animal that is born from the goat that has received the embryo, or is produced by the progeny of this animal. To increase the amount of milk containing the desired antibody produced by the transgenic goat, hormones can be appropriately used in the transgenic goat (Ebers, K. M. et al., Bio / Technology (1994) 12, 699-702). Furthermore, when a silkworm is used, it is infected with baculoviruses into which the desired antibody gene has been inserted, and the desired antibody is obtained from the body fluid of this silkworm (Maeda, S. et al. ., Nature (1985) 315, 592-594). Moreover, when tobacco is used, the desired antibody gene is inserted into a plant expression vector (e.g., pMON530), and this vector is introduced into bacteria such as Agrobacterium tumefaciens. This bacterium is used to infect tobacco (for example, Nicotiana tabacum), in order to obtain the desired antibody from the leaves of this tobacco (Julián, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138). When an antibody is produced in in vitro or in vivo production systems as previously described, DNAs encoding the heavy chain (H chain) and the light chain (L chain) of the antibody can be inserted into separate expression vectors, and then co-transforms a host with the vectors. Alternatively, the DNAs can be inserted into a single expression vector to transform a host (see International Patent Application Publication No. WO 94/11523). The antibodies used in the present invention can be antibody fragments or modified products based thereon, so long as they can be used appropriately in the present invention. For example, fragments of antibodies include Fab, F (ab ') 2, Fv and single chain Fv (scFv), where Fv of the H and L chains bind via an appropriate linker.

Specifically, antibody fragments are produced by treating an antibody with an enzyme, for example, papain or pepsin, or alternatively, genes encoding these fragments are constructed, introduced into expression vectors and expressed in appropriate host cells ( see, for example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Bete M. &Horwitz, AH, Methods in Enzymology (1989) 178, 476-496; Plueckthun, A. &Skerra, A., Methods in Enzymology (1989) 178, 497-515; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-666; Bird, RE et al., TIBTECH (1991) 9, 132-137). It is possible to obtain a scFv by joining the V region of the H chain and the V region of the L chain of an antibody. In the scFv, the V region of the H chain and the V region of the L chain are linked by a linker, preferably via a peptide linker (Huston, JS et al., Proc. Nati. Acad. Sci. USA ( 1988) 85, 5879-5883). The V regions of the H and L chains in a scFv can be derived from any of the antibodies previously described. Peptide linkers for joining the V regions include, for example, a single arbitrary peptide chain consisting of between 12 and 19 amino acid residues. It is possible to obtain a DNA encoding a scFv using as a template the DNA encoding the H chain, or its V region, and the DNA encoding the L chain, or its V region, of the aforementioned antibodies, performing the PCR amplification of the DNA portion encoding the desired amino acid sequence in the template sequence, using primers that define the ends of the portion, and then further amplifying the amplified DNA portion with a DNA encoding a peptide linker moiety, with a pair of primers that binds both ends of the linker to the H chain and the L chain. In addition, once a DNA encoding a scFv has been obtained, an expression vector comprising the DNA and a host transformed with the vector of according to conventional methods. In addition, the scFv can be obtained according to conventional methods using the host. In a manner similar to that described above, it is possible to produce these antibody fragments from the host by obtaining and expressing their genes. In the present documentation, an "antibody" includes these antibody fragments. As the modified antibody, an antibody bound to various molecules, such as polyethylene glycol (PEG), can also be used. In the present documentation, an "antibody" encompasses these modified antibodies. These modified antibodies can be obtained by modifying the antibodies obtained by chemical means. These methods are already established in the art. The antibodies produced and expressed as described above can be isolated from the interior or exterior of the cell or the host, and can be purified until they are homogeneous. The isolation and / or purification of the antibodies used in the present invention can be carried out by affinity chromatography. Columns that can be used for affinity chromatography include, for example, the protein A column and the G protein column. The vehicles used for the protein A column include, for example, HiperD, POROS, SepharoseF.F. and similar. In addition to the above, other methods can be used to isolate and / or purify common proteins, and these are not limited in any way. For example, the antibodies used in the present invention can be isolated and / or purified by selecting and appropriately combining chromatographies, beyond affinity chromatography, filters, ultrafiltration, desalting, dialysis and the like. Chromatographies include, for example, ion exchange chromatography, hydrophobic chromatography, gel filtration and the like. These chromatographies can be applied to a high performance liquid chromatography (HPLC). As an alternative, reverse phase HPLC can be used. The concentration of the antibodies obtained as described above can be determined with absorbance, ELISA or similar measurements. Specifically, the absorbance is determined by appropriately diluting the antibody solution with PBS (-), measuring the absorbance at 280 nm and calculating the concentration (1.35 OD = 1 mg / ml). Alternatively, when using an ELISA, the measurement can be performed as indicated below. Specifically, 100 μ? of goat anti-human IgG (TAG), diluted to 1 pg / ml with 0.1 M bicarbonate buffer (pH 9.6), to a 96-well plate (Nunc), and incubated overnight at 4 ° C to immobilize the antibody. After blocking, 100 μ? of an antibody of the present invention properly diluted, or a sample comprising the appropriately diluted antibody, and human IgG (CAPPEL) as a reference, and incubated one hour at room temperature. After washing, 100 μ? of anti-human IgG labeled with alkaline phosphatase (BIO SOURCE), diluted 5000 x, and incubated one hour at room temperature. After performing another wash, the substrate solution is added and incubated, and the absorbance at 405 nm is measured using the model 3550 microplate reader (Bio-Rad), to calculate the concentration of the antibody of interest. The IL-6 variants used in the present invention are substances that exhibit IL-6 receptor binding activity, and that do not transmit the biological activity of IL-6. That is, IL-6 variants compete with IL-6 for binding to IL-6 receptors, but they can not transmit the biological activity of IL-6, so they block IL-6-mediated signal transduction. . IL-6 variants are produced by introducing one or more mutations, by substituting amino acid residues in the amino acid sequence of IL-6. The origin of IL-6 used as a base for IL-6 variants is not limited; however, preferably it is human IL-6, when its antigenicity is considered, and similar factors. More specifically, amino acid substitution is performed by predicting the secondary structure of the amino acid sequence of IL-6 with known molecular modeling programs (eg, 7 WHATIF; Vriend et al., J. Mol. Graphics (1990) 8, 52-56), and further evaluating the influence of the one or more substituted amino acid residues on the entire molecule. Once the appropriate amino acid residue to be replaced is determined, PCR methods are commonly practiced, using the nucleotide sequence encoding the human IL-6 gene as a template for introducing mutations, in order to replace the amino acids, which results in a gene that codes for a variant of IL-6. If necessary, this gene is inserted into an appropriate expression vector, and the IL-6 variant can be obtained by applying the previously mentioned methods to express, produce and purify the recombinant antibodies. Specific examples of IL-6 variants are described in Brakenhoff et al., J. Biol. Chem. (1994) 269, 86-93, Savino et al., EMBO J. (1994) 13,1357-1367, WO 96/18648 and WO 96/17869. The partial peptides of IL-6 and the partial peptides of IL-6 receptors to be used in the present invention are substances that exhibit binding activity to IL-6 and IL-6 receptors, respectively, and that do not transmit the activity biological activity of IL-6. To know, when binding and capturing an IL-6 or an IL-6 receptor, the partial peptide of IL-6 or the partial peptide of the IL-6 receptor specifically inhibits the binding of IL-6 to the IL-6 receptor. 6 as a result, the biological activity of IL-6 is not transmitted, so signal transduction mediated by IL-6 is blocked. The partial peptides of IL-6 or the IL-6 receptor are peptides that comprise part or all of the amino acid sequence of the region of the amino acid sequence of IL-6 or the participating IL-6 receptor. in the binding of IL-6 and the IL-6 receptor. These peptides commonly comprise between 10 and 80, preferably between 20 and 50, more preferably between 20 and 40 amino acid residues. The partial peptides of IL-6 or the partial peptides of the IL-6 receptor can be produced according to generally known methods, for example, genetic engineering techniques or peptide synthesis methods, by specifying the region of the amino acid sequence of the peptide. IL-6 or the IL-6 receptor that participates in the binding of IL-6 and the IL-6 receptor, and using a portion or all of the amino acid sequence of the specified region. When a partial peptide of IL-6 or a partial peptide of an IL-6 receptor is prepared with a genetic engineering method, a DNA sequence encoding the desired peptide is inserted into an expression vector, and then the peptide by applying the aforementioned methods to express, produce and purify recombinant antibodies. To produce a partial peptide of IL-6 or a partial peptide of an IL-6 receptor through peptide synthesis methods, general-purpose peptide synthesis methods can be used, for example, solid-phase synthesis methods or synthesis methods in liquid phase. Specifically, the synthesis can be performed according to the method described in "Continuation of Development of Pharmaceuticals, Vol 14, Peptide Synthesis (in Japanese) (edited by Hauaki Yajima, 1991, Hirokawa Shoten)". As a solid-phase synthesis method, for example, the following method can be employed: the amino acid corresponding to the C-terminus of the peptide to be synthesized is attached to a support that is insoluble in organic solvents, then the peptide chain is elongated by repeating ( 1) the condensation reaction of the amino acids whose a-amino groups and their functional branching chain groups are protected with appropriate protecting groups, one at a time, in a direction from the C-terminus to the N-terminus; and (2) the removal reaction of the protecting groups of the a-amino groups of the amino acid or the peptide bound to the resin. Peptide synthesis in solid phase is broadly classified into the Boc method and the Fmoc method, based on the type of protecting group used. Once the protein of interest has been synthesized as previously described, the deprotection reaction and the reaction to separate the peptide chain from the support are carried out. For the separation reaction of the peptide chain, in general hydrogen fluoride or trifluoromethane sulphonic acid is used for the Boc method, and TFA for the Fmoc method. According to the Boc method, for example, the resin with the protected peptide mentioned above is treated in hydrogen fluoride in the presence of anisole. The peptide is then recovered by removing the protecting group and separating the peptide from the support. Upon freeze drying of the recovered peptide, a crude peptide can be obtained. On the other hand, in the Fmoc method, for example, it is possible to carry out the deprotection reaction and the reaction to separate the peptide chain from the support in TFA, according to a method similar to that previously described. The crude peptide obtained can be separated and / or purified by performing an HPLC. Elution can be carried out under optimal conditions, using a water-acetonitrile solvent system, which is generally used to purify proteins. The fractions corresponding to the peaks of the obtained chromatographic profile are collected and dried by freezing. Accordingly, fractions of purified peptides are identified through molecular weight analysis, mass spectrum analysis, amino acid composition analysis, amino acid sequence analysis, or the like. Specific examples of partial peptides of IL-6 and partial peptides of IL-6 receptors are described in JP-A (Kokai) H02-188600, JP-A (Kokai) H07-324097, JP-A (Kokai) H08-311098 , and in U.S. Patent Publication No. US 5210075. The antibodies used in the present invention can also be conjugated antibodies, which are attached to various molecules, such as polyethylene glycol (PEG), radioactive substances and toxins. These conjugated antibodies can be obtained by chemically modifying the antibodies obtained. Methods for modifying antibodies are already established in the art. The "antibodies" of the present invention encompass these conjugated antibodies. The agents of the present invention for suppressing damage to transplanted islets after islet transplantation can be used to treat diabetes. Diabetes includes type 1 diabetes, type 2 diabetes, pancreatic diabetes, gestational diabetes, and the like. The types of diabetes described previously also include secondary diabetes caused by pancreatitis or induced by the use of steroidal drugs, and diabetes due to specific causes, such as diabetes caused by abnormalities in particular genes.

The agents of the present invention for suppressing damage to transplanted islets can be used in islet transplantation. Islet transplantation includes the transplantation of islets from brain-dead donors, from donors with cardiac arrest and from living donors. Islet transplantation according to the present invention may include isografts and allografts. Isografts are transplants between animals of a homogenous system, which in humans are transplants between identical twins. Allografts are transplants between individuals of the same species that present genetic differences, which in humans are transplants between individuals without any relationship or between diovicular twins. In the present invention, the activity of IL-6 inhibitors in the inhibition of IL-6 signal transduction can be evaluated with conventional methods. Specifically, IL-6 is added to cultures of IL-6 dependent human myeloma cell lines (S6B45 and KPMM2), the human Lennert lymphoid T cell line KT3 or the IL-6 dependent MH60 cell line. BSF2; and assimilation of 3H-thymidine by the IL-6 dependent cells in the presence of an inhibitor of IL-6 is measured. Alternatively, U266 cells expressing the IL-6 receptor are cultured, 25 L-labeled IL-6 and an IL-6 inhibitor are added to the culture at the same time; and then the 125 I-labeled IL-6 bound to the cells expressing the IL-6 receptor is quantified. In addition to the IL-6 inhibitor group, a negative control is included that does not contain the IL-6 inhibitor in the test system previously described. The activity of the inhibitor of IL-6 in the inhibition of IL-6 can be evaluated by comparing the results of both groups. As will be indicated below in the Examples, it was found that administration of an anti-IL-6 receptor antibody suppressed damage to the transplanted islets after islet transplantation. This discovery suggests that IL-6 inhibitors, such as anti-IL-6 receptor antibodies, are useful as agents to suppress damage to transplanted islets after islet transplantation. The subjects who will receive the administration of the agents of the present invention to suppress damage to the transplanted islets are mammals. Preferably, mammals are human beings. The agents of the present invention for suppressing damage to the transplanted islets can be administered as pharmaceutical substances, and can be administered systemically or locally, by oral or parenteral administration. For example, an intravenous injection, such as a drip infusion, an intramuscular injection, an intraperitoneal injection, a subcutaneous injection, a suppository, an enema, oral enteric tablets or the like, may be selected. It is possible to select an appropriate method of administration depending on the age and symptoms of the patient. The effective dose per administration is selected from the range of between 0.01 and 100 mg / kg of body weight. Alternatively, the dose may be selected within the range of 1 to 1000 mg / patient, preferably within the range of 5 to 50 mg / patient. A preferred dose and method of administration are as follows: for example, when an anti-IL-6 receptor antibody is used, the effective dose is such an amount that the antibody libe is present in the blood. Specifically, a dose of between 0.5 and 40 mg / kg body weight / month (four weeks), preferably between 1 and 20 mg / kg body weight / month is administered by intravenous injection, such as a drip infusion, a subcutaneous injection or similar, between one and several times per month, for example, twice a week, once a week, once every two weeks or once every four weeks. The administration schedule can be adjusted, for example, by extending the administration interval from twice a week or once a week to once every two weeks, once every three weeks or once every four weeks, while monitoring the condition after the transplant and changes in blood test values. In the present invention, the agents for suppressing damage to the transplanted islets may contain vehicles acceptable for pharmaceutical use, such as preservatives and stabilizers. "Acceptable vehicles for pharmaceutical use" refer to materials that can be co-administered with an agent described above, and they themselves may or may not produce the previously described effect of suppressing damage to the transplanted islets. Alternatively, vehicles can be materials that do not have the effect of suppressing damage to the transplanted islets, but produce an additive or synergistic stabilizing effect when used in combination with an IL-6 inhibitor. These materials acceptable for pharmaceutical use include, for example, sterile water, physiological saline, stabilizers, excipients, buffers, preservatives, detergents, chelating agents (EDTA and the like) and binders. In the present invention, detergents include non-ionic detergents, and typical examples thereof include sorbitan esters of fatty acids, such as sorbitan monocaprylate, sorbitan monolaurate and sorbitan monopalmitate.; glycerin esters of fatty acids, such as glycerin monocaprylate, glycerin monomiristate and glycerin monostearate; acid polyglycerol esters, such as decaglyceryl monostearate, decaglyceryl distearate and decaglyceryl monolinoleate; polyoxyethylene sorbitan esters of fatty acids, such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate and polyoxyethylene sorbitan distearate; polyoxyethylene sorbit fatty acid esters, such as polyoxyethylene sorbit tetrastearate and polyoxyethylene sorbit tetraoleate; polyoxyethylene glycerin esters of fatty acids, such as polyoxyethylene glyceryl monostearate; polyethylene glycol esters of fatty acids, such as polyethylene glycol distearate; polyoxyethylene alkyl esters, such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene alkyl esters, such as polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether and polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylene alkyl phenyl esters, such as polyoxyethylene nonylphenyl ether; castor oils hardened with polyoxyethylene, such as polyoxyethylene castor oil and castor oil hardened with polyoxyethylene (castor oil hardened with polyoxyethylene); polyoxyethylene beeswax derivatives, such as polyoxyethylene sorbit beeswax; polyoxyethylene lanolin derivatives, such as polyoxyethylene lanolin; and polyoxyethylene amides of fatty acids and the like, with a HLB value of between 6 and 18, such as polyoxyethylene amide stearic acid. Detergents also include anionic detergents, and typical examples thereof include, for example, alkyl sulfates having an alkyl group with 10 to 18 carbon atoms, such as sodium cetyl sulfate, sodium lauryl sulfate and sodium oleyl sulfate; the polyoxyethylene alkyl ether sulfates, wherein the alkyl group has between 10 and 18 carbon atoms, and wherein the average molar amount of ethylene oxide is between 2 and 4, such as sodium polyoxyethylene lauryl sulfate; salts of alkyl sufosuccinate esters having an alkyl group with 8 to 18 carbon atoms, such as the sodium lauryl sulfosuccinate ester; natural detergents, for example, lecithin; the glycerophospholipids; sphingospholipids, such as sphingomyelin; and the esters of sucrose and fatty acids, in which the fatty acids have between 12 and 18 carbon atoms. One, two or more of the detergents described above may be combined, and may be added to the agents of the present invention. Detergents which are preferably used in the preparations of the present invention include the esters of polyoxyethylene sorbitan fatty acids, such as polysorbates 20, 40, 60 and 80. Polysorbates 20 and 80 are particularly preferred. Also preferred are polyoxyethylene polyoxypropylene glycols, such as poloxamer (Pluronic F-68® and the like). The amount of detergent added varies depending on the type of detergent used. When polysorbate 20 or 80 is used, the amount in general is in the range of 0.001 to 100 mg / ml, preferably in the range of 0.003 to 50 mg / ml, more preferably, in the range of 0.005 to 2 mg / ml. In the present invention, the buffers include phosphate, citrate buffer, acetic acid, malic acid, tartaric acid, succinic acid, lactic acid, potassium phosphate, gluconic acid, capric acid, deoxycholic acid, salicylic acid, triethanolamine, fumaric acid and other organic acids, and carbonic acid buffer, Tris buffer, histidine buffer and imidazole buffer. Liquid preparations can be formulated by dissolving the agents in aqueous buffers known in the field of liquid preparations. The concentration of the buffer is generally in the range of 1 to 500 mM, preferably in the range of 5 to 100 mM, more preferably in the range of 10 to 20 mm. The agents of the present invention may also comprise other low molecular weight polypeptides; proteins such as serum albumin, gelatin and immunoglobulin; amino acids; sugars and carbohydrates, such as polysaccharides and monosaccharides, sugar alcohols and the like. In the present documentation, the amino acids include basic amino acids, for example, arginine, lysine, histidine and ornithine, and inorganic salts of these amino acids (preferably hydrochloride salts and phosphate salts, known, phosphate amino acids). When free amino acids are used, the pH is adjusted to a preferred value by adding buffer substances suitable for physiological use, for example, inorganic acids, in particular, hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid and formic acid, and salts of these. In this case, the use of phosphate is particularly beneficial, one that provides fairly stable products by drying them by freezing. The phosphate is particularly advantageous when preparations are made which substantially do not contain organic acids, such as malic acid, tartaric acid, citric acid, succinic acid and fumaric acid, or which do not contain the corresponding anions (malate ion, tartrate ion, citrate ion, succinate ion, fumarate ion and the like). The preferred amino acids are arginine, Usin, histidine and ornithine. In addition, it is possible to use acidic amino acids, for example, glutamic acid and aspartic acid, and salts thereof (preferably salts of amino acids); neutral amino acids, for example, isoleucine, leucine, glycine, serine, threonine, valine, methionine, cysteine and alanine; and aromatic amino acids, for example, phenylalanine, tyrosine, tryptophan and its derivative, N-acetyl tryptophan. In the present documentation, sugars and carbohydrates, such as polysaccharides and monosaccharides, include, for example, detritus, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose and raffinose. In the present documentation, the sugar alcohols include, for example, mannitol, sorbitol and inositol. When the agents of the present invention are prepared as aqueous solutions for injection, the agents can be mixed, for example, with physiological saline solution and / or with an isotonic solution containing glucose or other auxiliary agents (such as D-sorbitol, D). -manose, D-mannitol and sodium chloride). The aqueous solutions can be used in combination with suitable solubilizing agents, such as alcohols (ethanol and the like), polyalcohols (propylene glycol, PEG and the like), or non-ionic detergents (polysorbate 80 and HCO-50).

In addition, if necessary, the agents may comprise diluents, solubilizers, substances for adjusting the pH, softening agents, reducing agents containing sulfur, antioxidants and the like. In the present documentation, reducing agents containing sulfur include, for example, compounds comprising sulfhydryl groups, such as N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and thioalkanoic acids having between 1 and 7 carbon atoms. Moreover, the antioxidants in the present invention include, for example, erythorbic acid, dibutylhydroxy toluene, butylhydroxy anisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbic acid palmitate, acid stearate. L-ascorbic, sodium bisulfite, sodium sulfite, thiamyl gallate, propyl gallate, and chelating agents, such as disodium ethylenediamine tetraacetate (EDTA), sodium pyrophosphate and sodium metaphosphate. If necessary, the agents can be encapsulated in microcapsules (microcapsules of hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid] or the like), or they can be prepared as colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules and the like (see Remington's Pharmaceutical Science, 16th edition, edited by Oslo, 1980, and the like.) In addition, methods for preparing agents as sustained release agents are also known, and can be applied to the present invention (Langer et al., J. Biomed, Mater. Res. 1981, 15: 167-277; Langer, Chem Tech. 1992, 12: 98-105; US Patent No. 3773919; European Patent Application No. (EP) 58481; Sidman et al. , Biopolimers 1983, 22: 547-556, and EP 133988.) The acceptable pharmaceutical vehicles for use are appropriately selected from those previously described, or are combined depending on the type of dosage form. dosage, but not limited to those. The present invention relates to methods for suppressing damage to transplanted islets, comprising the step of administering an IL-6 inhibitor to subjects who have received an islet transplant. In addition, the present invention relates to methods for improving the survival of transplanted islets in the subject, comprising the step of administering an IL-6 inhibitor to subjects who have received an islet transplant.

In the present documentation, the "subject" refers to organisms, body parts of the organisms, or a separate or proportionate part of the organisms in which it is desired to administer an agent of the present invention to suppress damage to the transplanted islets. Organisms include animals (eg, humans, species of domestic animals and wild animals), but are not particularly limited. The "body parts of the organisms" are not particularly limited, but preferably include organs in which it is desired to transplant the islets, omentum major, subrenal capsule and mesentery. In the present invention, the organs in which it is desired to transplant the islets include the liver, and more specifically, the blood vessels of the liver.

In the present documentation, "administration" includes oral and parenteral administrations. Oral administration includes, for example, the administration of oral agents. These oral agents include, for example, granules, powders, tablets, capsules, solutions, emulsions and suspensions. Parenteral administration includes, for example, the administration of injections. These injections include, for example, subcutaneous injections, intramuscular injections and intraperitoneal injections. Meanwhile, the effects of the methods of the present invention can be achieved by introducing genes comprising the oligonucleotides that it is desired to administer in living bodies., using gene therapy techniques. Alternatively, the agents of the present invention can be administered locally in the desired treatment areas. For example, agents can be administered through a local injection during surgery, using catheters, or by direct administration of genes with DNA encoding a peptide of the present invention. The suppressive agents of the present invention can be administered to subjects prior to organ transplantation, at the time of organ transplantation or after organ transplantation. In addition, suppressive agents can be administered once or repeatedly. Alternatively, when administered to a separate or proportionate part of an organism, the suppressive agents of the present invention can "contact" the organism part. In the present invention, the "contacting" is carried out according to the condition of the organism. Examples include, without limitation, the spraying of the suppressive agents of the present invention on the parts of the organism, and the addition of the suppressive agents of the present invention to compressed parts of the organism. When the part of the organism is composed of cultured cells, the "contact" mentioned previously can be achieved by adding the suppressive agents of the present invention to the culture medium of these cells, or introducing DNA comprising the oligonucleotides of the present invention into the cells that they constitute the part of the organism. When the methods of the present invention are put into practice, the suppressive agents of the present invention can be administered as parts of pharmaceutical compositions, in combination with at least one known chemotherapeutic agent. Alternatively, the suppressive agents of the present invention can be administered simultaneously with at least one known immunosuppressant. In one embodiment, the known chemotherapeutic agents and suppressive agents of the present invention can be administered in a virtually simultaneous manner. The suppressive agents of the present invention can be administered at the sites where the islet transplantation has been performed, once the islets have been transplanted, or they can be administered on the targets at the same time as the islets. Alternatively, agents can be added to the islets in vitro, before transplantation. In the present documentation, the phrase "suppression of damage to transplanted islets" denotes that the damage to the transplanted islets is suppressed and viability is improved. The phrase "suppression of damage to transplanted islets" also includes the suppression of natural immune responses that accompany transplantation.

The suppression of damage to the transplanted islets can be confirmed by measuring blood sugar levels in living bodies, according to the methods described in the Examples. Blood sugar levels can be measured, for example, by collecting blood from the orbital sinus, and by determining blood sugar levels with a Beckman glucose analyzer after separating the plasma. When the administration of the agents of the present invention to suppress damage to the transplanted islets causes a sustained decrease in the blood sugar level, it is considered that the damage to the transplanted islets has been suppressed. Alternatively, when the survival of the islets improves as a result of this administration, it is also possible to conclude that the agents "suppress the damage to the transplanted islets" after the islet transplantation. The survival of the islets can be assessed by determining whether normal blood sugar levels are restored after a transplant in diabetic recipients. For example, when 200 islets, which can be isolated from a single donor, are transplanted to the control group described in the Examples of the present documentation, the hyperglycemic state is maintained, so the viability can be considered as 0%. However, when the same number of islets used for the control group is transplanted into the group that has received the administration of anti-IL-6 receptor antibodies described in the Examples herein, normal levels of blood sugar are restored. in all the receivers, so it is considered that the viability is 100%. All prior art documents cited in this documentation are incorporated herein by way of reference. EXAMPLES Next, the present invention will be specifically described with reference to the Examples, but should not be construed as being limited thereto. Example 1 Investigation of deletion of damage to transplanted islets after isogenic islet transplantation, where the effect is due to the administration of anti-IL-6 receptor antibodies. Diabetic recipient mice were prepared by administering streptozotocin (180 mg / kg) to male C57BL / 6 mice intravenously. Streptozotocin is an agent that selectively destroys the islets of Langerhans in the pancreas. A single administration of streptozotocin removes most B cells, thus inducing type 1 diabetes. Without islet transplantation, a hyperglycaemic state remained in all mice (Figure 1). Between three and five days after the administration of streptozotocin, isogenic islets of mice were transplanted to the livers of diabetic mice through the portal vein. The islets were isolated from islet donors using collagenase-based methods. From the pancreas of a single mouse, 200 islets can be isolated. In the case of the transplantation of 400 islets from two donor mice, normal blood sugar levels were restored after transplantation to diabetic recipients, and it was possible to treat diabetes (Figure 2). In this Example, blood was collected from the orbital sinus and the blood sugar levels were determined using a Beckman glucose analyzer after separating the plasma. However, the transplantation of 200 islets of a mouse did not restore normal blood sugar levels, and hyperglycaemia remained in recipients (Figure 3). When three administrations of 500 pg of anti-IL-6 receptor antibody (R16-1) were administered intraperitoneally after transplantation of 200 islets (days 0, 2, and 4), normal blood sugar levels were restored in all recipients after transplantation (Figure 4). When a single administration of an equal dose of anti-IL-6 receptor antibody was performed, normal blood sugar levels were restored in three-quarters of the recipients (Figure 5). As an alternativer. , when a single administration of 200 g of anti-IL-6 receptor antibody was performed, normal blood sugar levels were restored in one third of the receptors (Figure 6). The above results demonstrate that anti-IL-6 receptor antibodies reduced damage to the transplanted islets, improved islet survival and corrected hyperglycemia at the receptors. Specifically, it was discovered that anti-IL-6 receptor antibodies can be used as agents to suppress damage to transplanted islets after an isogenic islet transplant (isograft). Example 2 Investigation of the suppression of inflammatory cytokine production, where the effect is due to the administration of anti-IL-6 receptor antibodies. Flow cytometry methods were used to investigate whether the administration of IL-6 receptor antibodies. -6 of the present invention suppressed the post-transplant production of inflammatory cytokines by infiltrating cells. Two hundred isogenic islets were transplanted into the livers of mice with diabetes caused by streptozotocin, and these animals were sacrificed six hours later, their livers were removed and their hepatic mononuclear cells were isolated and analyzed by flow cytometry (Figure 7). No production of IFN-? and TNF-a in hepatic mononuclear cells of untreated mice (virgins). The production of IFN-? and TNF-α was increased in the hepatic mononuclear cells of the control group, in which no anti-IL-6 receptor antibodies had been administered. The production of IFN-α was not observed and TNF-a in hepatic mononuclear cells from mice given a single administration of anti-IL-6 receptor antibodies (ip; 200 g / injection / mouse) simultaneously with the transplant. These findings suggest that administration of anti-IL-6 receptor antibodies prevents damage to transplanted islets by suppressing the production of inflammatory cytokines. Example 3 Investigation of damage suppression to transplanted islets in allogeneic islet transplantation, where the effect is due to the administration of IL-6 anti-receptor antibodies. Diabetic receptor mice were prepared by administering streptozotocin (180 mg / kg) to C57BL mice / 6 male intravenously. Between three and five days after the administration of streptozotocin, allogenic mouse islets were transplanted into the livers of the diabetic mice (BALB / c) through the portal vein. The islets were isolated from donor pancreas using collagenase-based methods. Blood sugar levels were determined with the same method described in Example 1. After transplanting 200 islets, three administrations of 500 pg of an anti-IL-6 receptor antibody (MR16-1) were performed intraperitoneally ( days 0, 2 and 4), and a single administration of 200 pg of anti-CD4 antibody intraperitoneally, simultaneously with the first administration of the anti-IL-6 receptor antibody, and normal sugar levels were restored in blood in all recipients after transplantation (FiguralO). On the other hand, when the same number of administrations of 500 pg of a control antibody (rat IgG) (Figure 8) or 200 of anti-CD4 antibody alone (Figure 9) was performed, in all the mice the state of hyperglycemia prevailed . The above results demonstrate that in recipients receiving allografts, anti-IL-6 receptor antibodies also reduced damage to the transplanted islets, improved islet survival and corrected hyperglycemia. Specifically, it was discovered that anti-IL-6 receptor antibodies can be used as agents to suppress damage to transplanted islets after an allogeneic islet transplant. Industrial Applicability The methods and agents of the present invention for suppressing damage to transplanted islets improve islet survival after islet transplantation, and are expected to allow efficient treatment of diabetes using fewer islet donors. Previously, when isolated islets were transplanted from the pancreas of brain-dead donors or donors with cardiac arrest, a single receptor required isolated islets from two or three donor pancreas, since functional failure of the islets was induced. reduced the viability of the grafts. By using the agents of the present invention to suppress damage to the transplanted islets, the amount of islets required for a single receptor is reduced, and as a result, it is expected that more receptors can receive islet transplants.

Recent reports describe successful cases of islet transplants from living donors, in which islets were isolated and purified from a portion of pancreas extracted from healthy donors and transplanted to diabetic patients; however, it is believed that the use of the agents of the present invention to suppress islet damage allows the establishment of therapeutic methods that are less invasive for donors.

Claims (23)

1. An agent for suppressing damage to the transplanted islets after islet transplantation, comprising an inhibitor of IL-6 as an active ingredient.

2. The agent for suppressing damage to the transplanted islets of claim 1, wherein the IL-6 inhibitor is an antibody that recognizes an IL-6.

3. The agent for suppressing damage to the transplanted islets of claim 1, wherein the IL-6 inhibitor is an antibody that recognizes an IL-6 receptor.

4. ?? agent for suppressing damage to the transplanted islets of claim 2 or 3, wherein the antibody is a monoclonal antibody.

5. The agent for suppressing damage to the transplanted islets of any of claims 2 to 4, wherein the antibody is a human anti-IL-6 antibody or an anti-human IL-6 receptor antibody.

6. The agent for suppressing damage to the transplanted islets of any of claims 2 to 5, wherein the antibody is a recombinant antibody.

7. The agent for suppressing damage to the transplanted islets of claim 6, wherein the antibody is a chimeric, humanized or human antibody.

8. The agent for suppressing damage to the transplanted islets of any of claims 1 to 7, which is used to treat diabetes.

9. A method for suppressing damage to transplanted islets in a subject undergoing an islet transplant, comprising the step of administering an IL-6 inhibitor to the subject.

10. A method for improving the viability of an islet transplant in a subject, comprising the step of administering an IL-6 inhibitor to the subject. The method of claim 9 or 10, wherein the inhibitor of IL-6 is an antibody that recognizes an IL-6. The method of claim 9 or 10, wherein the inhibitor of IL-6 is an antibody that recognizes an IL-6 receptor. The method of claim 11 or 12, wherein the antibody is a monoclonal antibody. 14. The method of any of claims 11 to 13, wherein the antibody is a human anti-IL-6 antibody or an anti-human IL-6 receptor antibody. 15. The method of any of claims 11 to 14, wherein the antibody is a recombinant antibody. 16. The method of claim 15, wherein the antibody is a chimeric, humanized or human antibody. 17. The use of an IL-6 inhibitor to produce agents to suppress damage to the transplanted islets after islet transplantation. 18. The use of claim 17, wherein the IL-6 inhibitor is an antibody that recognizes an IL-6. The use of claim 17, wherein the IL-6 inhibitor is an antibody that recognizes an IL-6 receptor. The use of claim 18 or 19, wherein the antibody is a monoclonal antibody. The use of any of claims 18 to 20, wherein the antibody is a human anti-IL-6 antibody or an anti-human IL-6 receptor antibody. 22. The use of any of claims 18 to 21, wherein the antibody is a recombinant antibody. 23. The use of claim 22, wherein the antibody is a chimeric, humanized or human antibody.