JP2016069314A - Immunosuppressant - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunosuppressant represented by a graft-versus-host disease preventive/therapeutic agent.SOLUTION: An immunosuppressant comprises a caveolin 1 inhibitor as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to an immunosuppressive agent based on a new mechanism of action.

  Hematopoietic stem cell transplantation is the final treatment for blood immune diseases such as hematological malignancies and congenital immune diseases, but a decline in therapeutic results due to graft-versus-host disease (GVHD) due to immune response after transplantation is a major problem ing. In particular, due to the severe limitation of physical activity due to chronic respiratory disorders and skin sclerosis / joint disorders associated with chronic GVHD, there are many patients whose continuation of daily life is difficult even though the primary disease has been cured.

  Steroids are the standard treatment for chronic GVHD, but long-term immunosuppressive therapy is required for more than 2 years after onset, and there are many side effects associated with long-term administration. It is the limit of transplantation treatment.

On the other hand, CD26 is not only a marker for T cell activation, but is also associated with T cell signaling as a costimulatory molecule (Non-Patent Documents 1 to 4). Indeed, patients with autoimmune diseases such as multiple sclerosis (MS), Graves' disease (GD) and rheumatoid arthritis (RA) have an increased number of CD26 ⁺ CD4 T cells in both inflamed tissue and peripheral blood. (Non-Patent Documents 5 to 8), and in these autoimmune diseases, it is recognized that CD26 expression is enhanced in correlation with the severity of the disease (Non-Patent Document 9). In addition, the inventors have previously shown that T cells that migrate through endothelial cell monolayers in vitro express high levels of CD26 (Non-Patent Document 10). These findings suggest that CD26 ⁺ T cells means that play an important role in inflammatory processes and subsequent tissue damage in such diseases, CD26 ⁺ T cells can be from effector T cells under these conditions To suggest.

JP 2005-237234 A

J. Immunol. 1984; 133 (3): 1250-6 J. Immunol. 1989; 143 (11): 3430-6 J. Immunol. 1990; 144 (11): 4092-100 Trends Immunol. 2008; 29 (6): 295-301 N. Engl. J. Med. 1985; 312 (22): 1405-11 J. Immunol. 1989; 142 (12): 4233-40 Clin. Immunol. Immunopathol. 1996; 80 (1): 31-7 J. Rheumatol. 1996; 23 (12): 2022-6 Adv. Clin. Chem. 2011; 53: 51-84 J. Immunol. 1992; 148 (5): 1367-74

However, an effective therapeutic means for graft-versus-host disease, its complications, and various autoimmune diseases has not yet been found, and development of an immunosuppressive agent based on a new mechanism of action is desired.
Accordingly, an object of the present invention is to provide a new immunosuppressive agent.

  Therefore, the present inventor has been studying focusing on the CD26 molecule, which is a marker of T cell activity. The activation of donor T cells involved in the development of GVHD requires the signal of a costimulatory molecule at the same time as the recipient's antigen is presented, whereas CD26 molecules are T cells, particularly T cells that respond most strongly to memory antigens. A costimulatory molecule, the inventor has discovered that the costimulatory ligand for CD26 is caveolin 1 (Cav-1). To create a recombinant protein (Cav-Ig) in which the N-terminal region of human Cav-1 is fused to the constant region of human immunoglobulin G, and to suppress memory antigens or alloimmune reactions in in vitro experiments I found it. Furthermore, when recombinant Cav-Ig was administered to human immunized GVHD-onset mice, immune tolerance was induced without interfering with donor cell engraftment and GVHD was prevented. Moreover, the progress of GVHD was also suppressed by administration after the onset of GVHD. Furthermore, the present invention was completed by demonstrating in a tumor transplanted mouse model that an immunosuppressive effect is exhibited without interfering with the graft-versus-leukemia effect.

  That is, the present invention provides the following inventions [1] to [4].

[1] An immunosuppressant comprising a caveolin 1 inhibitor as an active ingredient.
[2] Selected from acute or chronic graft-versus-host disease, post-transplant rejection, autoimmune disease, idiopathic pulmonary hypertension, idiopathic pulmonary fibrosis, obstructive bronchiolitis, chronic glomerulonephritis and idiopathic nephrotic syndrome [1] The immunosuppressive agent according to [1], which is a prophylactic / therapeutic agent for diseases.
[3] Autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, polymyositis, dermatomyositis, scleroderma, Sjogren's syndrome, vasculitis syndrome, mixed connective tissue disease, autoimmune hepatitis, primary biliary cirrhosis, Primary sclerosing cholangitis, autoimmune pancreatitis, ulcerative colitis, Crohn's disease, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune neutropenia, type I diabetes, pemphigus [2] The immunosuppressive agent according to [2], which is a disease selected from pemphigoid, habitual abortion, causative disease and autoimmune optic neuritis.
[4] The caveolin 1 inhibitor is a peptide fragment of the N-terminal region of caveolin 1, a peptide fragment of the caveolin 1 scaffolding domain sequence, a peptide fragment lacking the scaffolding domain from the N-terminal region of caveolin 1, a caveolin of CD26 The immunity according to any one of [1] to [3], which is one or more selected from a peptide fragment of one binding region sequence and a fusion protein of any one of these peptide fragments and an immunoglobulin constant region Inhibitor.

  When the caveolin 1 inhibitor of the present invention is used, graft-versus-host disease, various autoimmune diseases and the like can be prevented or treated.

A strategy for producing a fusion protein of a partial peptide of human caveolin 1 and an immunoglobulin constant region is shown. A schematic representation of the pEB6-CAG-MCS vector is shown. It is a figure which shows that chronic GVHD develops in the NOG mouse (huCB-NOG) which transplanted the human umbilical cord blood mononuclear cell by sublethal irradiation. NOG mice were irradiated at a sublethal dose (200 cGy) and the following day, 1 × 10 ⁵ T cell-depleted CD34 ⁺ cells (CD34 ⁺ transplanted) purified from human umbilical cord blood cells (huCB) or 1 × 10 ⁷ isolated from huCB Nuclear cells (all CB transplants) were transplanted. (A) Overall survival and (B) Clinical GVHD score (mean ± SEM). Data are cumulative results from three independent experiments (n = 10 each). (C) L ^& D staining of lungs, skin and liver of CD34 ⁺ transplants (panels ac) (8 weeks post-transplantation) and H & E staining or anti-human CD3 ⁺ of serial sections of lungs, skin and liver of all CB transplants Immunohistochemistry (panels df or gi) (8 weeks after transplantation). The arrowhead in panel f indicates perivascular invasion in the liver. Representative histology from three independent experiments (n = 10 each) is shown. The original magnification is 100x. The scale bar indicates 100 μm. (D) Pathological damage in lung, skin and liver of CD34 ⁺ transplant or all CB transplant groups was examined using a semi-quantitative scoring system. Each point represents an individual value and the horizontal bar represents the average value. ^* , ^** or ^*** represents P <0.0001. (E) Collagen deposition was measured in serial sections of lung tissue in panel a or d of (C) using an Azan-Mallory staining assay. The dark blue part indicating collagen deposition was clearly observed in all CB transplanted mice (panel b) compared to CD34 ⁺ transplanted mice (panel a). Representative histology from three independent experiments (n = 10 each) is shown. The original magnification is 100x. The scale bar indicates 100 μm. (F) Collagen deposition was quantified as the ratio of dark blue area to total tissue area. Quantification was performed using a Photoshop CS4 analysis tool. The average number of ratios per field (± SEM) was determined from 10 representative fields each. Data are cumulative results from three independent experiments (n = 10 each). ^{* Indicates} P <0.0001. It is a figure which shows the engraftment of the human CD45 ⁺ cell in the peripheral blood of a recipient mouse. NOG mice were irradiated at a sublethal dose (200 cGy) and the next day, 1 × 10 ⁵ CD34 ⁺ cells purified from human umbilical cord blood cells (huCB) (CD34 ⁺ transplanted) or 1 × 10 ⁷ mononuclear cells isolated from huCB ( All CB transplants) were transplanted. Recipient peripheral blood was collected at the indicated time points and analyzed for the population of human CD45 ⁺ cells using flow cytometry. Data are shown as the mean ± SEM of human CD45 ⁺ cells in whole peripheral blood leukocytes (PBL) (n = 10 in the CD34 ⁺ transplant group, respectively, and n = 0 for weeks 0-6 in the total CB transplant group). N = 8 in the 10th and 8th weeks and n = 3 in the 12th week). FIG. 2 shows significant infiltration of IL-26 ⁺ CD26 ⁺ CD4 T cells in cGVHD lung. HuCB-NOG mice were sacrificed at 4, 8 and 12 weeks after transplantation, lungs were excised, and single cell suspensions were then isolated. (A) The absolute number of human CD45 ⁺ , CD4 ⁺ or CD8 ⁺ cells per lung of huCB-NOG mice (mean ± SEM) was quantified by flow cytometry. CD4 ⁺ T cells infiltrated primarily into the lungs of mice with cGVHD. Data are cumulative results from three independent experiments (n = 10 at 4 weeks, n = 8 at 8 weeks and n = 3 at 12 weeks). ^* Indicates P <0.0001 for CD8 ⁺ cells. (B) Human cytokine or CD26 / DPP4 mRNA expression in human CD4 ⁺ cells isolated from the lungs of huCB-NOG mice was quantified by real-time RT-PCR. Each expression was normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT1). Week 0 data was obtained using freshly isolated huCB CD4 T cells from three different donors. Data are cumulative results from three independent experiments (n = 10 at 4 weeks, n = 8 at 8 weeks and n = 3 at 12 weeks). ^* Or ^** indicates P <0.0001 for the 0th week. (C) Serum from CD34 ⁺ transplanted or whole CB transplanted mice was collected at the indicated week after transplantation to quantify the serum levels of human IFN-γ, IL-17A, IL-26 and soluble CD26 / DPP4. . Data are from 3 independent experiments (n = 10 in each CD34 ⁺ transplant group and n = 10 at 0-6 weeks, n = 8 at 8 weeks and n = 3 at 12 weeks for all CB transplant groups, respectively. ). ^* Or ^** indicates P <0.0001 for the corresponding CD34 ⁺ transplant group. (D) Human IFN-γ ⁺ CD26 ⁺ CD4 (panel a), IL-26 ⁺ CD26 ⁺ CD4 (panel b), or IL-17A ⁺ CD26 ⁺ CD4 (panel c) in the lungs of CD34 ⁺ or all CB transplanted mice. ) The absolute cell number of the cells was quantified by flow cytometry. Data are from 3 independent experiments (n = 10 for the CD34 ⁺ transplant group, and n = 10 for the 4th week, n = 8 for the 8th week, and n = 3 for the 12th week for the all CB transplanted group). Is the cumulative result of ^* Indicates P <0.0001 for the corresponding CD34 ⁺ transplanted group, NS indicates not significant. A representative flow cytometry plot for panels a and b is shown in FIG. (E) Immunohistochemistry of anti-human CD26, IL-26 or IL-17A in serial sections of lungs from CD34 ⁺ transplanted or whole CB transplanted mice (8 weeks post-transplant). No human CD26, IL-26 and IL-17A positive cells were detected in the CD34 ⁺ transplanted group (panels a-c), whereas the lungs of all CB transplanted mice were clearly infiltrated with human CD26 or IL-26. (Panel d or e), IL-17A positive cells were not infiltrated (Panel f). Representative histology from three independent experiments (n = 10 each) is shown. The original magnification is 100x. The scale bar indicates 100 μm. FIG. 3 shows significant infiltration of human CD4 T cells in cGVHD lungs of huCB-NOG mice. Immunohistochemistry of anti-human CD4 (panel a) or CD8 (panel b) of serial sections of lung tissue from huCB-NOG mice (all CB transplantation) (8 weeks after transplantation). Representative histology from three independent experiments (n = 10 each) is shown. The original magnification is 100 × or 400 × (inset in each panel). It is a figure which shows the raise level of IL-26 ⁺ CD26 ⁺ CD4 T cell in the lung of huCB-NOG mouse | mouth. HuCB-NOG mice (all CB transplants) were sacrificed 8 and 12 weeks after transplantation, lungs were excised, and single cell suspensions were then isolated. Cells were then restimulated and then subjected to flow cytometry analysis. CB mononuclear cells (MNC) were analyzed as pre-transplant controls. (A) Human IFN-γ ⁺ CD26 ⁺ CD4 or (B) IL-26 ⁺ CD26 ⁺ CD4 cells in the lungs of all CB transplanted mice were analyzed using flow cytometry. From three independent experiments (n = 3 for pre-transplantation CB MNC, n = 10 for CD34 ⁺ transplantation group, and n = 8 for week 8 and n = 3 for week 12), respectively. A representative point plot is shown. Numbers indicate relative percentages per quadrant. It is a figure which shows infiltration of the human CD26 and IL-26 cell in the cGVHD skin of a huCB-NOG mouse | mouth. H & E staining (panel a), anti-human CD26 (pavel b), IL-26 (panel c) or IL-17A (panel d) of serial sections of skin tissue of all CB transplanted mice (8 weeks after transplantation) Histochemistry. Although infiltration by human CD26 and IL-26 positive cells was observed, no IL-17A positive cells were detected. Representative histology from three independent experiments (n = 10 each) is shown. Original magnification is 100x or 400x (inset in panels b and c). IL-26 stimulates fibroblasts and induces collagen deposition in cGVHD lungs in NOG mice that received bone marrow (BM) cells and splenocytes of IL-26 transgenic (Tg) mice. (A) Panel a, IL-20RA and IL-10RB expression in NIH3T3 cells, normal human lung fibroblasts (NHLF) and mouse fibroblasts, anti-IL-20RA recognizing both human and mouse antigens. Alternatively, it was measured by Western blotting using an anti-IL-10RB polyclonal antibody (pAb). Panel b, NHLF or NIH3T3 cells were treated with IL-26 (10 ng / ml in PBS) and harvested at each time point. Each lysate was separated by SDS-PAGE under reducing conditions, immunoblotted with anti-phosphorylated STAT3 (pY-STAT3), then stripped with anti-STAT3 pAb (total STAT3) and reprobed. STAT3 phosphorylation in NHLF and NIH3T3 was induced by the presence of exogenous IL-26. Representative data from three independent experiments with similar results are shown. (B) Collagen production of NHLF (panel a) or NIH3T3 (panel b) cells stimulated with exogenous IL-26 in the presence or absence of neutralizing anti-IL-20RA pAb or rabbit Ig (control pAb). The amount of soluble collagen secreted increases in a dose-dependent manner with increasing levels of exogenous IL-26 ( ^* P <0.0001 versus the corresponding control solvent), and collagen production is neutralizing anti-IL-20RA. Inhibited by the presence of pAb ( ^** P <0.0001 vs. corresponding control pAb). Data are presented as mean ± SEM resulting from three independent experiments performed in triplicate. (C) Post-transplantation of BM and splenocytes isolated from parent B6 (WT) (panels a-c), 190-IFNG BAC Tg (panels df) or ΔCNS-77 Tg (panels g-i) mice H & E, Azan-Mallory staining and anti-IL-26 immunohistochemistry of serial sections of lungs from weekly NOG mice. The recipient lungs of 190-IFNG BAC Tg mice showed areas of collagen deposition and IL-26 ⁺ cell infiltration, whereas recipients of WT or ΔCNS-77 Tg mice received collagen deposition or IL-26 ⁺ cells. Showed BO and septal infiltration without it. Representative histology from 3 independent experiments (n = 6 each) is shown. The original magnification is 100x. The scale bar indicates 100 μm. (D) Collagen deposition was quantified as a ratio of dark blue area to total tissue area, as shown in panel b, e or h of (C). Quantification was performed using a Photoshop CS4 analysis tool. The average number of ratios per field (± SEM) was determined from 10 representative fields each. Data are cumulative results from three independent experiments (n = 6 each). ^* Indicates P <0.0001 relative to that in recipients of WT or ΔCNS-77 Tg mice. FIG. 6 shows that CD26 costimulation enhances IL-26 production in human umbilical cord blood (huCB) CD4 T cells, and blockade of the CD26 ligand caveolin-1 reduces IL-26 production. (A) huCB CD4 T cells were stimulated with immobilized anti-CD3 (0.05 μg / mL) plus anti-CD28 (10 μg / mL) or anti-CD26 (10 μg / mL) mAb. Seven days after stimulation, human IL2, IFNG, IL17A, IL26 and DPP4 mRNA levels were quantified by real-time RT-PCR. Data are presented as mean ± SEM resulting from three independent experiments performed in triplicate. ^{* Indicates} P <0.05; ^** or ^*** indicates P <0.0001; NS indicates not significant. (B) huCB CD4 T cells were stimulated with anti-CD3, anti-CD28, anti-CD26 mAb, or immobilized Cav-Ig bound to the indicated concentration of plate. Supernatants were collected 7 or 14 days after stimulation to quantify human IL-26 levels. Data are presented as mean ± SEM resulting from three independent experiments performed in triplicate. ^* Or ^** indicates P <0.0001 for the corresponding sample on day 0, or ^*** indicates P <0.05. (C) huCB CD4 T cells were treated with the indicated concentrations of blocking Cav-Ig (solid line) or control Ig (dotted line). After 1 hour incubation with blocking Cav-Ig or control Ig, cells were immobilized with anti-CD3 (0.05 μg / mL) plus Cav-Ig (20 μg / mL), anti-CD28 (20 μg / mL) or anti-CD26 (20 μg / mL). mL) mAb stimulated. Seven days after stimulation, supernatants were collected and human IL-26 levels were quantified. Data are presented as mean ± SEM resulting from three independent experiments performed in triplicate. ^* Indicates P <0.0001 relative to the corresponding control Ig. The alignment of the N-terminal amino acid sequence of caveolin-1 is shown. The N-terminal amino acid (AA) sequence of human and mouse caveolin-1 suggests that the scaffold domain required for CD26 binding is conserved between species. The dot in mouse caveolin-1 indicates the same amino acid residue (red box) as the corresponding human caveolin-1. ^* Or ^** refers to a reference in the text. Figure 2 shows caveolin-1 expression in cGVHD lungs of huCB-NOG mice. Immunohistochemistry of lung anti-caveolin-1 in all CB-transplanted mice (8 weeks after transplantation). Caveolin-1 is a macrophage-like large cell (arrowhead in panel B) around the inflammatory vessel (panel C) in the epithelial cell surface (yellow arrow in panel A) and bronchioloocclusive lesion (BO in panel A). ). Original magnification: 100 × (A) or 400 × (B, C). FIG. 4 shows that administration of Cav-Ig prevents cGVHD by reducing the level of IL-26 ⁺ CD26 ⁺ CD4 cells. NOG mice irradiated sub-lethally were transplanted with 1 × 10 ⁷ mononuclear cells isolated from human umbilical cord blood. Cav-Ig or control Ig (100 μg / dose each) was administered intraperitoneally three times a week, starting on day +1 after transplantation until day +28. (A) Overall survival and (B) Clinical GVHD score (mean ± SEM). Data are cumulative results from three independent experiments (n = 10 each). (C) Lung, skin and liver H & E staining or anti-human CD3 ⁺ immunohistochemistry (panels ac or df) for control Ig group (6 weeks post-transplant), and Cav-Ig group (transplant Lung, skin and liver H & E staining (Panel g to i) for 6 weeks after). The arrowhead in panel c indicates perivascular invasion in the liver. Representative histology from three independent experiments (n = 10 each) is shown. The original magnification is 100x. The scale bar indicates 100 μm. (D) Pathological damage in the lung, skin and liver of recipients receiving Cav-Ig or control Ig was examined 6 weeks after transplantation using a semi-quantitative scoring system. Each point represents an individual value and the horizontal bar represents the average value. ^* , ^** or ^*** represents P <0.0001. (E) Cav-Ig or control Ig group recipient sera were collected at the indicated weeks after transplantation to quantify serum levels of human IL-26 and soluble CD26 / DPP4. Data are from 3 independent experiments (n = 10 in the Cav-Ig group, and n = 10 in the 0 to 6 weeks, n = 9 in the 8th week, and n = 2 in the 12th week in the control Ig group). Is the cumulative result from ^* Indicates P <0.0001 relative to the corresponding control Ig group. (F) Absolute cell number of human IL-26 ⁺ CD26 ⁺ CD4 cells in the lungs of recipients of Cav-Ig or control Ig groups. Data are from 3 independent experiments (n = 10 in the Cav-Ig group, and n = 10 in the 0 to 6 weeks, n = 9 in the 8th week, and n = 2 in the 12th week in the control Ig group). Is the cumulative result from ^* Indicates P <0.0001 for the corresponding CD34 ⁺ transplanted group. ^* Indicates P <0.0001 relative to the corresponding control Ig group. (G) Lung anti-human CD26 or IL-26 immunohistochemistry of recipients of control Ig group (panels a, b) or Cav-Ig group (panels c, d) (8 weeks post-transplant). Representative histology from 3 independent experiments (n = 6 each) is shown. The original magnification is 100x. The scale bar indicates 100 μm. (H) Collagen deposition was measured in the lung tissue of recipients of Cav-Ig or control Ig groups (8 weeks post-transplant) using an Azan-Mallory staining assay. Representative histology from 3 independent experiments (n = 6 each) is shown. The original magnification is 100x. The scale bar indicates 100 μm. (I) Collagen deposition was quantified as the ratio of dark blue area to total tissue area. Quantification was performed using a Photoshop CS4 analysis tool. The average number of ratios per field (± SEM) was determined from 10 representative fields each. Data are cumulative results from three independent experiments (n = 6 each). ^{* Indicates} P <0.0001. It is a figure which shows that GVHD is suppressed in the recipient of the huCB-NOG mouse | mouth treated with Cav-Ig. Sublethal irradiated NOG mice were transplanted with 1 × 10 ⁷ mononuclear cells isolated from human umbilical cord blood and administered with Cav-Ig or control Ig by the same method as described in FIG. BM cells and splenocytes collected 4 weeks after transplantation were transferred to another NOG mouse (New NOG) 1 day after sublethal irradiation. (A) Overall survival and (B) Clinical GVHD score (mean ± SEM). Data are cumulative results from three independent experiments (n = 10 each). (C) H & E staining or anti-human CD3 ⁺ immunohistochemistry of lungs (panel a or c) or liver (panel b or d) or anti-human CD3 ⁺ in control Ig group (2 weeks after transplantation), and Cav-Ig group (post-transplant) H & E staining of lung (panel e) or liver (panel f) at 12 weeks). The arrowhead in panel b indicates perivascular invasion in the liver. Representative histology from three independent experiments (n = 10 each) is shown. The original magnification is 100x. The scale bar indicates 100 μm. (D) Absolute cell number of human IFN-γ ⁺ CD26 ⁺ CD4 or IL-26 ⁺ CD26 ⁺ CD4 cells in the lungs of recipients of Cav-Ig or control Ig groups. Data are from 3 independent experiments (n = 10 in the Cav-Ig group, respectively, and n = 10 in the 2nd week, n = 4 in the 4th week, and n = 4 in the 8th and 12th weeks in the control Ig group). Cumulative results from 0 (shown as †) ^* indicates P <0.0001 relative to the corresponding control Ig group. It is a figure which shows the engraftment of the human CD45 ⁺ cell in the peripheral blood of a recipient mouse. NOG mice irradiated sub-lethally were transplanted with 1 × 10 ⁷ mononuclear cells isolated from human umbilical cord blood. Cav-Ig or control Ig (100 μg / dose each) was administered intraperitoneally three times a week, starting on day +1 after transplantation until day +28. Recipient peripheral blood was collected at the indicated time points and the population of human CD45 ⁺ cells was analyzed by flow cytometry. Data are shown as the mean ± SEM of human CD45 ⁺ cells in whole peripheral blood leukocytes (PBL) (n = 10 in the Cav-Ig group and n = 10 in the control Ig group at weeks 0-6, respectively). , Week 8 n = 9 and week 12 n = 2). FIG. 5 shows that administration of Cav-Ig during the onset of early GVHD prevents lethal GVHD. 1 × 10 ⁷ mononuclear cells isolated from human umbilical cord blood were transplanted into sublethal irradiated NOG mice. Cav-Ig or control Ig (100 μg / dose each) was administered intraperitoneally three times a week, starting on day +29 after transplantation until day +56. (A) Overall survival and (B) Clinical GVHD score (mean ± SEM). Data are cumulative results from three independent experiments (n = 10 each). (C) H & E staining of lung, skin and liver of control Ig group (panels af) and Cav-Ig group (panels g-1) at 5 or 10 weeks after transplantation. Representative histology from three independent experiments (n = 10 in the Cav-Ig group and n = 10 at 5 weeks and n = 5 at 10 weeks in the control Ig group, respectively) is shown. The arrowheads in panels e, f, and k indicate perivascular invasion in the liver, and the arrowheads in panel g indicate slight peribronchial cuffing in the lungs. The original magnification is 100x. The scale bar indicates 100 μm. (D) Pathological damage in lung, skin and liver of recipients receiving Cav-Ig or control Ig was examined at 5 and 10 weeks post-transplant using a semi-quantitative scoring system. Control Ig recipients developed progression of GVHD lesions ( ^* , P <0.0001 in lung and skin, ^* , P <0.05 in liver). In contrast, Cav-Ig recipients compared to recipients in the control Ig group at 10 weeks ( ^** , P <0.0001) and at 10 weeks rather than 5 weeks after transplantation. ( ^*** , P <0.0001), showed significant reduction of GVHD lesions. Each point represents an individual value and the horizontal bar represents the average value. Data are cumulative results from three independent experiments, each with n = 10 in the Cav-Ig group and n = 10 at 5 weeks and n = 5 at 10 weeks in the control Ig group. (E) Absolute cell number of human IFN-γ ⁺ or IL-26 ⁺ CD26 ⁺ CD4 cells in the lungs of recipients of Cav-Ig or control Ig groups. Data are cumulative results from three independent experiments, each with n = 10 in the Cav-Ig group and n = 10 at 5 weeks and n = 5 at 10 weeks in the control Ig group. ^* , ^** or ^*** represents P <0.0001. It is a figure which shows that Cav-Ig preserve | saves a GVL effect. (A) NOG mice were irradiated at a sublethal dose (200 cGy) and the following day, 1 × 10 ⁴ A20-luc cells were inoculated via the tail vein and then the next day, 1 × isolated from human umbilical cord blood 10 ⁷ mononuclear cells were transplanted. Cav-Ig or control Ig (100 μg / dose each) was administered intraperitoneally three times a week, starting on day +1 after transplantation until day +28. Overall survival is plotted from cumulative results from three independent experiments (n = 10 each). P <0.0001 vs. control Ig recipients by log rank test. (B) In vivo bioluminescence imaging (BLI) was performed at the indicated time points after the treatment described in Panel A. Representative mice are shown. Crosses (†) indicate death of all mice in the tumor alone group or the control Ig group. Hair perturbations consistent with cutaneous GVHD are shown in the control Ig group recipients 10 weeks after CB transplantation (middle panel). (C) MNCs isolated from huCB were transplanted into sublethal irradiated NOG mice. Cav-Ig or control Ig was administered intraperitoneally three times a week starting on day +1 after transplantation until day +28. On day +28 after transplantation, 1 × 10 ⁵ A20-luc cells were inoculated via the tail vein. Overall survival is plotted from cumulative results from three independent experiments (n = 10 each). P <0.0001 vs. control Ig recipients by log rank test. (D) In vivo BLI was performed at the indicated time points after the treatment described in Panel C. Representative mice are shown. Crosses (†) indicate death of all mice in the tumor alone group or the control Ig group. Slight coat disturbance consistent with cutaneous GVHD is shown in the control Ig group recipients 6 weeks after CB implantation (middle panel). It is a figure which shows the engraftment of the human CD45 ⁺ cell in the peripheral blood of a secondary recipient mouse. Sublethal irradiated NOG mice were transplanted with 1 × 10 ⁷ mononuclear cells isolated from human umbilical cord blood and administered with Cav-Ig or control Ig by the same method as described in FIG. Bone marrow (BM) cells and splenocytes were harvested 4 weeks after transplantation, and 1 × 10 ⁶ BM cells and 1 × 10 ⁷ splenocytes were transferred to another NOG mouse (New NOG) one day after sublethal irradiation. did. Recipient peripheral blood was collected at the indicated time points and the population of human CD45 ⁺ cells was analyzed by flow cytometry. Data are shown as mean ± SEM of human CD45 ⁺ cells in whole peripheral blood leukocytes (PBL) (n = 10 in the Cav-Ig group, respectively, and n = 10, 4 in the second week in the control Ig group). N = 4 for week and n = 1 for week 6). It is a figure which shows the lymphocyte proliferation inhibitory effect of each fusion protein. The graph shows the mean value (cpm) and standard error of triplicate. -P <0.0001 v. s. Control Fcg1 (two-tailed student t test).

  The active ingredient of the immunosuppressive agent of the present invention is a caveolin 1 inhibitor.

  Caveolin 1 is one of caveolae, which is the most important structural protein of caveola, which is a small depression on the cell surface. Caveolin 1 is known to be a symptom marker of human prostate cancer, but its relationship with immunosuppressive action is not known (Patent Document 1).

  The amino acid sequence of caveolin 1 is known and consists of a 178 amino acid sequence (SEQ ID NO: 1), the 1-101th is the N-terminal region, and the 102-134th is the hydrophobic membrane penetration part. . The 82nd to 101st is a scaffolding domain.

  As caveolin-1 inhibitors, caveolin-1 is a costimulatory molecule for the activation of caveolin-1 T cells, since it is a CD26 costimulatory ligand for T cell activation, particularly T cell activation that reacts most strongly to memory antigens. What is necessary is just to inhibit the function. For example, (1) peptide fragment of N-terminal region of caveolin 1 (SEQ ID NO: 2); (2) peptide fragment of caveolin 1 scaffolding domain sequence (SEQ ID NO: 3); 3) Peptide fragment lacking the scaffolding domain from the N-terminal region of caveolin 1 (SEQ ID NO: 4); (4) Peptide fragment of caveolin 1 binding region sequence of CD26 (SEQ ID NO: 5); and (5) any of these And a fusion protein of such a peptide fragment and an immunoglobulin constant region.

  Since these caveolin-1 inhibitors have known amino acid sequences, they can be produced by various methods such as gene recombination and peptide synthesis. Moreover, the said fusion protein can be manufactured, for example by a gene recombination method.

Administration of caveolin 1 inhibitor induces immune tolerance and prevents GVHD without interfering with donor cell engraftment in mice immunized with human immunized GVHD, as shown in the Examples below. In addition, administration of a caveolin 1 inhibitor after the onset of GVHD also suppresses the progression of GVHD. Furthermore, when administered to a tumor-transplanted mouse model, it exerts an immunosuppressive effect without interfering with the graft-versus-host disease effect.
Therefore, caveolin 1 inhibitors are useful as immunosuppressants in animals including humans. Specific examples of the immunosuppressive agent of the present invention include acute or chronic graft-versus-host disease, post-transplant rejection, autoimmune disease, idiopathic pulmonary hypertension, idiopathic pulmonary fibrosis, obstructive bronchiolitis, chronic thread Examples include preventive and therapeutic agents for diseases selected from spherical nephritis and idiopathic nephrotic syndrome. The autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, polymyositis, dermatomyositis, scleroderma, Sjogren's syndrome, vasculitis syndrome, mixed connective tissue disease, autoimmune hepatitis, primary biliary cirrhosis , Primary sclerosing cholangitis, autoimmune pancreatitis, ulcerative colitis, Crohn's disease, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune neutropenia, type I diabetes, celestial Includes pemphigus, pemphigoid, habitual abortion, causative disease and autoimmune optic neuritis.

  The immunosuppressive agent of the present invention can be formulated for oral or parenteral use by mixing, dissolving, emulsifying, encapsulating, lyophilizing, etc. with a pharmaceutically acceptable carrier well known in the art. .

  Suitable preparations for oral administration include a solution in which caveolin 1 inhibitor is dissolved in an effective amount in a diluent such as water or physiological saline, and a capsule, granule or powder containing the effective amount as a solid or granule Or a tablet, a suspension in which an effective amount is suspended in an appropriate dispersion medium, an emulsion in which a solution in which an effective amount is dissolved is dispersed in an appropriate dispersion medium and emulsified.

  For parenteral administration, the caveolin 1 inhibitor is combined with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc., together with injection solutions, suspensions, emulsions, creams, It can be formulated into dosage forms such as ointments, inhalants, suppositories and the like. In an injectable formulation, the caveolin 1 inhibitor can be dissolved in an aqueous solution, preferably a physiologically compatible buffer such as Hanks's solution, Ringer's solution, or physiological saline buffer. Furthermore, the medicament of the present invention can be in the form of a suspension, solution, emulsion or the like in an oily or aqueous vehicle. Alternatively, the caveolin 1 inhibitor may be produced in the form of a powder, and an aqueous solution or suspension may be prepared using sterilized water or the like before use. For administration by inhalation, the caveolin 1 inhibitor can be powdered and made into a powder mixture with a suitable base such as lactose or starch. Suppository formulations can be prepared by mixing the caveolin 1 inhibitor with a conventional suppository base such as cocoa butter. Furthermore, the medicament of the present invention can be encapsulated in a polymer matrix or the like and formulated as a sustained release preparation.

  The dose of caveolin 1 inhibitor varies depending on the patient's symptoms, administration route, body weight, age, etc., but is preferably 1 μg to 500 mg per day for adults, for example.

  EXAMPLES Next, an Example is given and this invention is demonstrated in detail.

1. Test method (1) antibodies, reagents and human _CD3, OKT3 (IgG 2b) for cell costimulation experiments, murine mAb to CD26,1F7 (IgG ₁₎ and CD28,4B10 (IgG ₁₎ we developed (Non Patent Document 2). Human recombinant IL-26 was purchased from R & D Systems and stored at 1 μg / mL in phosphate buffered saline (PBS). Cav-Ig and its control Fc protein were generated by J. Biol. Chem. 2007; 282 (13): 10117-31. Normal human lung fibroblasts (NHLF) and medium FGF-2 were purchased from LONZA. Mouse fibroblast cell line NIH3T3 and MNC isolated from human umbilical cord blood by Ficoll density gradient method were purchased from RIKEN BioResource Center (Tsukuba, Japan). huCB-derived CD34 ⁺ cells were purified from huCB MNC using CD34 MicroBead Kit (Miltenyi Biotec).

(2) Preparation of caveolin 1 inhibitor i) Fusion protein (Cav-1-NT-Fcγ1 (NT-Fc)) of peptide fragment of N-terminal region of caveolin 1 and immunoglobulin constant region by the strategy shown in FIG. A fusion protein (Cav-1-NTΔSCD-Fcγ1 (NTΔSCD-Fc)) of a peptide fragment lacking the scaffolding domain from the caveolin 1 N-terminal region and an immunoglobulin constant region, and a peptide fragment of the caveolin-1 scaffolding domain sequence And an immunoglobulin constant region fusion protein (Cav-1-SCD-Fcγ1 (SCD-Fc)).
That is, among the N-terminal region of human caveolin 1, the 1st to 101st amino acid residue part (NT), the 1st to 82nd amino acid residue part (NTΔSCD), the 82nd to 101st amino acid residue part ( SCD) was prepared by PCR and incorporated into the fusion protein expression vector of FIG.
pEB6-CAG-huECDSP-Fcγ ₁ vector Hind III site, 1-101 amino acid portion of caveolin-1 (NT), 1-82 amino acid moiety (NTΔSCD), 82-101 amino moiety (SCD) in In-framde After cloning, a Caveolin-1-Fc fusion protein expression vector was constructed. P CAG, CAG promoter; huECDSP, human E-cadherin signal peptide; huFcγ1, Fc portion of human IgG1 ( human IgG-H chain _{hinge + C H 2 + C H} 3 portion (SEQ ID NO: 28)).
(3) Mouse NOD / SCID / γ _c ^{− / −} NOD. Cg-Prkdcscid Il2rgtm1Sug / Jic mouse (NOG mouse) ^{(H-2 d)} were purchased from Central Institute for Experimental Animals (Kawasaki, Japan), B6 mice ^{(H-2 b)} is Charles River Laboratories Japan, Inc. (Yokohama, Japan). A 190 kb bacterial artificial chromosome (BAC) transgene with human IFNG and IL26 genes (190-IFNG Tg) and a BAC Tg lacking a conserved coding sequence (CNS) located upstream of the IFNG transcription start site ( B6 mice carrying (ΔCNS-77 Tg) were developed in the laboratory of Thomas Ane (J. Immunol. 2010; 185 (3): 1492-501). All mice were housed in microisolator cages at specific pathogen removal facilities. Mice were used at 8-12 weeks. The animal protocol was approved by the Institutional Animal Care and Use Committee.

(4) Transplantation and evaluation of GVHD On the first day, NOG mice were irradiated with 200 cGy. On day 0, mice were injected intraperitoneally with MNC (1 × 10 ⁷ ) or 1 × 10 ⁵ CD34 ⁺ cells purified from huCB. Mice were evaluated daily for survival and body weights were measured three times a week. To record human lymphocyte-related peripheral T cell proliferation, blood was collected from mice (50-100 μL), erythrocytes were lysed, and peripheral blood mononuclear cell (PBMC) subsets were counted by flow cytometry. In the cGVHD prevention experiment, mice were intraperitoneally administered 100 μg human Cav-Ig or control human Fc three times a week from day +1 to day +28 (total 12 doses). In the cGVDH treatment experiment, mice were treated 3 times a week from day 29 to day +56 after confirmation of engraftment (total 12 doses) when the first clinical signs of disease developed on day 28 after huCB transplantation. 100 μg human Cav-Ig or control human Fc was administered intraperitoneally for each dose. HuCB engraftment was confirmed by quantification of human CD45 ⁺ leukocytes in mouse peripheral blood using flow cytometry. Clinical GVHD scores were assessed at least once a week according to Blood. 1996; 88 (8): 3230-9.

For continuous transplantation experiments using Cav-Ig or control Ig recipients, BM from recipients of huCB-NOG mice administered Cav-Ig or control Ig from day +1 to +28 days after transplantation. Cells and splenocytes were harvested. BM was washed from the donor femur and tibia with Dulbecco's modified Eagle medium (Invitrogen) and passed through sterile mesh fibers to obtain a single cell suspension. Spleen cells were isolated from donor spleens using the Spleen Dissociation Kit (Miltenyi Biotec) and human CD45 ⁺ cells were purified using the human CD45 MicroBeads Kit (Miltenyi Biotec). Next, 1 × 10 ⁶ BM cells and 1 × 10 ⁷ splenocytes were transferred to another NOG mouse (New NOG) one day after sublethal irradiation (200 cGy).

For allogeneic BM transplantation experiments using Tg mice, sublethal irradiated (200 cGy) NOG mice were isolated from 190-IFNG BAC, ΔCNS-77 Tg or parental B6 (B6 WT) mice 1 × 10 ^7. BM and 1 × 10 ⁶ splenocytes were transplanted. Recipients were sacrificed 4 weeks after transplantation and lungs were excised for histological evaluation.

(5) Tissue histopathology and immunohistochemistry Skin, left lung, liver and colon specimens from the upper back of the recipient were fixed in 10% formalin, embedded in paraffin, sectioned and placed on a glass slide. The lesions were measured by staining with hematoxylin and eosin (H & E). Images were captured with an Olympus digital camera DP21 attached to an Olympus BX43 microscope using CellSens software (Olympus Corporation). The slides were scored by a pathologist who was not informed of the experimental group. Skin slides were scored based on skin fibrosis, fat loss, inflammation, changes in the epidermal interface, and vesicle shedding (0-2 for each category; maximum score was 10) (Blood. 2004; 104 (5): 1565-73). Lung slides were scored by periluminal infiltrate, pneumonia, and extent of injury (0-3 for each category; maximum score was 9) (Blood. 1996; 88 (8): 3230-9 ). Liver slides were scored by bile duct injury and inflammation (0-4 for each category; maximum score was 8) (J. Immunol. 2004; 173 (9): 5467-75). For detection of collagen, slides were stained with Azan-Mallory staining, and collagen deposition was determined by using the Photoshop CS4 analysis tool (Adobe Systems Inc.) as the ratio of the area of blue staining to the total stained area as an Azan-Malory stained section. Quantified in terms of The antibodies used in immunohistochemistry were anti-human CD3 mAb from Dako (F7.2.38, mouse IgG ₁ ), anti-human CD26 rabbit pAb from R & D Systems (AF1180), Bioss Inc. Anti-IL-26 rabbit pAb (bs-2626R), anti-IL-17A rabbit pAb (H-132) from Santa Cruz Biotechnology and anti-caveolin-1 rabbit pAb (D46G3) from Cell Signaling Technology. Immunohistochemical staining for CD3, CD26 or IL-26 was performed as described previously (Br J. Haematol. 2013; 162 (2): 263-77) Department of Pathology, Keio University School of Medicine ( (Tokyo, Japan). Immunohistochemical staining for IL-17A or caveolin-1 was performed in the laboratory of MorphoTechnology (Sapporo, Japan).

(6) Real-time quantitative RT-PCR
In experiments to evaluate the expression of effector molecule mRNA, single cell suspensions were isolated from the lungs of huCB-NOG mice using the Lung Dissociation Kit (Miltenyi Biotech) and then human using the human CD4 MicroBead Kit (Miltenyi Biotech). CD4 T cells were purified. mRNA isolation and quantification was performed as previously described (Immunology 2013; 138 (2): 165-72). The expression level of mRNA was calculated based on a standard curve generated for each gene, and hypoxanthine phosphoribosyltransferase 1 (HPRT1) mRNA was used as an invariant control. The primer sequences used in the real-time RT-PCR analysis are shown in (Table 1).

(7) Flow cytometry The following antibodies were from BD Biosciences: anti-human CD3 (UCHT1, mouse IgG ₁ ), anti-human CD4 (RPA-Ta, mouse IgG ₁ ), anti-human CD8 (HIT8a, mouse IgG _1). ), anti-human CD26 (M-A261, mouse IgG _1), anti-human CD45 (HI30, mouse IgG _1), anti-human IFN-γ (B27, mouse IgG _1), anti-human IL-17A (N49-653, mouse IgG ₁ ) antibody, and related control mAb of the same Ig isotype. The fluorescent complexes were FITC, phycoerythrin (PE), peridinin-chlorophyll-Cy5.5 (PerCP-Cy5.5), and allophycocyanin (APC). Intracellular IL-26 antibody staining (clone 510414, mouse IgG ₁ ) was purchased from R & D Systems and conjugated with Alexa Fluor 647 using the Antibody Labeling Kit (Invitrogen). Red blood cells in samples of mouse peripheral blood were lysed using BD FACS Lysing Solution (BD Biosciences). To analyze lymphocytes infiltrating the lungs, single cell suspensions were prepared using the Lung Dissociation Kit and then purified with a human CD45 MicroBeads Kit. After blocking non-specific binding with rabbit / mouse IgG (BD Biosciences), cells were stained with PBS plus 1% BSA. For intracellular cytokine analysis, purified human CD45 ⁺ cells were treated with 25 ng / mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) plus 1 μg / mL in the presence of BD GolgiPlug (BD Biosciences). Restimulation with ionomycin (Sigma-Aldrich) for 4 hours. Intracellular flow cytometry was performed using a Human Intracellular Cytokine Staining Kit (BD Biosciences) according to the manufacturer's instructions. Analysis was performed with FACSCalibur (BD Biosciences) with two lasers, and files were analyzed with FlowJo (Tree Star).

(8) Measurement of Cytokine and Soluble CD26 / DPP4 Collected serum was collected from inflammatory cytokines (human IL-1β, IL-2, IL-) using Bio-Plex Pro Human Cytokine 27-Plex Assay (Bio-Rad Laboratories). 4, IL-6, IL-10, IL-17A, TNF-α and IFN-γ). IL-26 was assayed using a Human IL-26 ELISA Kit (CUSABIO). An assay for soluble CD26 / DPP4 was developed in our laboratory (Br J. Haematol. 2013; 162 (2): 263-77). All samples were assayed in triplicate.

(9) Western Blotting To analyze the expression of IL-20RA or IL-10RB, 10 μg of NHLF and NIH3T3 lysate was prepared using RIPA buffer, separated by SDS-PAGE under reducing conditions, Immunoblot was performed using anti-IL20RA (ab25922, Abcam) or anti-IL10RB (bs-2602R, Bioss Inc.) rabbit pAbs that recognize both human and mouse antigens. For analysis of phosphorylated STAT3 induced by exogenous IL-26, 1 × 10 ⁵ cells were seeded in 96-well flat bottom plates and IL-26 (10 ng / mL in PBS) was added to each well the next day. Cells were harvested at each time point and cell lysates were prepared in RIPA buffer containing Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). 10 μg of each lysate was separated by SDS-PAGE under reducing conditions, immunoblotted with anti-phosphorylated STAT3 (pY-STAT3) (No. 11045, SAB) that recognizes both human and mouse antigens, and then human And anti-STAT3 pAb (total STAT3) (GTX15523, GeneTex) that recognizes both antigen and mouse antigen and reprobed.

(10) In Vitro Collagen Production Assay NHLF or NIH3T3 cells are seeded in 96 well flat bottom plates (1 × 10 ⁵ / well) and the next day IL-26 (0.1, 2.0, 5.0 or 10 ng / mL in PBS) ) Or PBS alone was added to each well. For neutralization experiments, neutralizing anti-IL20RA pAb (ab25922, Abcam) or control rabbit Ig (Abcam) (each 10 μg / mL) was added to each well 1 hour prior to the addition of exogenous IL-26. The supernatant was collected 48 hours after the addition of IL-26, and then soluble collagen was measured using a Sircol Collagen Assay Kit (Biocolor). All samples were examined in triplicate.

(11) In vitro costimulation assay For analysis of mRNA expression, CD4 T cells were purified and isolated from huCB MNC and 1 × 10 ⁵ cells / well were added to anti-CD3 (0.05 μg / mL) plus anti-CD28 (10 μg / ML) or anti-CD26 (10 μg / mL) mAb was inoculated into 96 well flat bottom plates. Seven days after stimulation, cells were harvested, mRNA was prepared, and transcript levels of human IL2, IFNG, IL17A, IL26 and DPP4 were quantified by real-time RT-PCR. For analysis of IL-26 production by costimulation, 1 × 10 ⁵ huCB CD4 T cells were stimulated with anti-CD3, anti-CD28, anti-CD26 mAb or immobilized Cav-Ig bound to the indicated concentration of plate. did. Supernatants were collected 7 or 14 days after stimulation. For inhibition assays, 1 × 10 ⁵ cells / well of huCB CD4 T cells were injected with various concentrations (0, 1, 5, 10, 20 or 50 μg / mL) of blocking Cav-Ig or control Ig prior to stimulation. Was processed. After a 1 hour blocking period, cells were stimulated with immobilized anti-CD3 (0.05 μg / mL) plus Cav-Ig (20 μg / mL), anti-CD28 (20 μg / mL) or anti-CD26 (20 μg / mL) mAb. Supernatants were collected 7 days after stimulation. HuCB CD4 T cells were cultured in AIM V serum-free medium (Invitrogen).

(12) GVL and bioluminescence imaging (BLI)
A20 mouse lymphoma cells (A20-luc) transfected with a firefly luciferase of Balb / c background (H-2 ^d ) were obtained from Dr. Courtesy of Xiao Chen (Medical College of Wisconsin, Milwaukee, Wis., USA) (70). NOG mice were irradiated at a sublethal dose (200 cGy) and the next day, 1 × 10 ⁴ A20-luc cells were inoculated via the tail vein, and the next day, transplanted with 1 × 10 ⁷ MNC isolated from huCB did. Cav-Ig or control Ig (100 μg / dose) was administered intraperitoneally three times a week (total of 12 doses) starting on day +1 after transplantation until day +28. For other GVL experiments, NOG mice were irradiated with a sublethal dose (200 cGy) and the following day, 1 × 10 ⁷ MNCs isolated from huCB were transplanted. Cav-Ig or control Ig (100 μg / dose) was administered intraperitoneally three times a week (total of 12 doses) starting on day +1 after transplantation until day +28. 1 × 10 ⁵ A20-luc cells were inoculated via the tail vein +28 days after transplantation. Tumor dissemination was monitored weekly using in vivo BLI as previously described in the literature (Br J. Cancer. 2014; 110 (9): 2232-45).

(13) Statistics Data were analyzed by two-tailed Student t-test for two-group comparisons or by ANOVA test followed by Tukey-Kramer post-hoc test for multiple comparisons. A P value ≦ 0.05 was considered statistically significant. Survival was analyzed by log rank test using the Kaplan-Meier method. Calculations were performed using GraphPad Prism 6 (GraphPad Software Inc.) to produce a graph.

2. Results (1) Pulmonary fibrosis in all huCB recipients experiencing cGVHD To evaluate the exact mechanism of cGVHD induced by human immune cells in a mouse model, NOG mice are used as recipients and huCB as donor cells did. Chronic NOG mice are pre-treated with sublethal dose irradiation and then given either T cell depleted CD34 positive huCB (CD34 ⁺ transplant) or total huCB containing immature T cells and do not lead to early post-transplant mortality Heterogeneous responses were induced. CD34 ⁺ transplanted mice survived for 5 months without any signs or symptoms of GVHD (solid lines in FIGS. 3A and 3B). On the other hand, all CB transplanted mice (huCB-NOG) showed GVHD clinical signs / symptoms such as weight loss or hair disturbance as early as 4 weeks after transplantation (dotted line in FIG. 3B) and significantly lower survival. The rate was clarified (dotted line in FIG. 3A). Human cells engrafted similarly in both groups as shown in FIG.

To examine cGVHD histopathology in huCB-NOG mice, we next performed a histological analysis of GVHD target organs. The CD34 ⁺ transplant control group showed a normal appearance of GVHD target organs such as lung, skin and liver (panels a-c in FIG. 3C) and none had a positive lesion score (FIG. 3D). On the other hand, histological examination of the lungs of huCB-NOG mice showed peribronchial infiltration and cuffing with human CD3 ⁺ cells along with subepithelial enlargement of fibrous tissue, suggesting the onset of bronchiole occlusion (FIG. 3C). Panels d and g). The lung lesion score was significantly higher in the huCB-NOG group than in the CD34 ⁺ transplant control group ( ^{* in} FIG. 3D). Skin histology of huCB-NOG mice showed skin atrophy, fat loss and vesicle shedding associated with human CD3 ⁺ cell infiltration (panels e and h in FIG. 3C). In the liver, perivascular infiltration by human CD3 ⁺ cells was observed in tissues surrounding the bile duct (panels f and i in FIG. 3C), but the degree was milder than that seen in the lung or skin. Both skin and liver findings resulted in a significant increase in lesion score in huCB-NOG mice compared to CD34 ⁺ transplanted control mice ( ^** or ^{*** in} FIG. 3D, respectively). On the other hand, no lesion findings were observed in other GVHD target organs such as the small intestine and colon of huCB-NOG mice and CD34 ⁺ transplant control group. These findings suggest that human CD3 ⁺ lymphocytes in huCB grafts induced cGVHD, including life-threatening organ damage in the lungs of huCB-NOG mice.

Since cGVHD can be characterized as a fibroproliferative disease (Biol Blood Marrow Transplant. 2010; 9 (10): 657-66), we also have a Mallory staining assay that quantifies collagen levels as a measure of the extent of the disease. Carried out. The lungs of the cGVHD group show a significant increase in peribronchial and perivascular collagen deposition (dark blue part in panel b of FIG. 3E and ^{* in} FIG. 3F). In particular, FIG. 3E clearly shows the organization of collagen surrounding the bronchioles and blood vessels, revealing that elevated collagen levels are localized to specific structures and are not evenly distributed throughout the lungs. , Suggesting that huCB NOG mice developed lung cGVHD. Overall, sublethal irradiation of NOG mice followed by whole huCB transplantation reproduced the pathology of cGVHD, especially in the lungs and skin. The pathology of cGVHD is due to the absence of early significant weight loss (J. Clin. Invest. 2000; 105 (9): 1289-98) and late overall post-transplant weight loss, BO and lung fibers It was distinguished from aGVHD by the disease (Best Pract Res Clin Haematol. 2008; 21 (2): 251-7). The mouse cGVHD model has been reported previously (Am. J Respir Crit Care Med 2007; 176 (7): 713-23), but no documented document of lung lesions in cGVHD mice induced by human lymphocytes has been described . According to the National Institutes of Health accreditation standards, BO is the only characteristic manifestation of pulmonary cGVHD (Biol Blood Marrow Transplant 2005; 11 (12): 945-56, Biol Blood Marrow Transplant 2006; 12 (1): 31-47), therefore, our findings indicate a genuine humanized mouse model of lung cGVHD.

(2) Infiltration of IL-26 producing CD4 ⁺ T cells in the lungs of cGVHD mice To elucidate potential pathogenic pathogenic mechanisms associated with cGVHD in this mouse model, we demonstrated the existence of BO and fibrosis, Subsequent studies were focused on the lung as the most severely affected cGVHD organ. Since there was perivascular and peribronchial infiltration of human CD3 ⁺ cells in the lungs of huCB-NOG mice (FIG. 3C), we next quantified the percentage of human lymphocytes in the cGVHD lung. Unlike the reported results (Int. Immunol. 2009; 21 (7): 843-58) where human B cells are found mainly in the peripheral blood and spleen of humanized NOG mice, the presence of human CD4 T cells is mainly cGVHD. Observed in the lung (FIG. 5A). Furthermore, the remainder of the human CD45 ⁺ blood cells found in the cGVHD lung appears to consist of CD8 T cells (FIG. 5A). These findings were also confirmed by immunohistochemical examination of lung specimens (FIG. 6). Our findings suggest that the pulmonary pathophysiology of cGVHD mice includes human CD3 lymphocytes, particularly CD4 T cells.

To identify inflammatory cytokines that have a role in the pathophysiology of cGVHD lung, the expression profiles of various inflammatory cytokine mRNAs in human CD4 T cells isolated from cGVHD lung were analyzed. As shown in FIG. 5B, IFNG, IL17A and IL26 increase significantly during the development of cGVHD after all CB transplantation ( ^** ), while IL1B, IL2, TNF (TNF-α), IL4, IL6 and IL10 are Decreased ( ^* ). IFN-γ, IL-17A and IL-26 are produced by Th17 cells (Citokine Growth Factor Rev. 2010; 21 (5): 393-401), and IFN-γ and IL-26 are produced by Th1 cells ( Genes Immun 2012; 13 (6): 481-8). Since Th1 and Th17 cells both strongly express CD26 (Immunol Rev. 1998; 161: 55-70, Immunol 2012; 188 (11): 5438-47), we next analyzed the expression level of CD26 / DPP4 did. DPP4 mRNA expression in human CD4 T cells infiltrating the cGVHD lung was significantly increased (FIG. 5B), but mRNA expression of the costimulatory molecule CD28 was not enhanced. These data suggest that human CD26 ⁺ CD4 T cells play an important role in the pathophysiology of cGVHD lung.

To further confirm the above findings, we next examined the protein levels of the relevant cytokines. In addition, since soluble CD26 / DPP4 levels correlate with T cell CD26 protein (Clin. Chem. Lab Med 1993; 37 (8): 839-40, Scand J Immunol 2001; 54 (3): 249-64), The protein level of human soluble CD26 / DPP4 in mouse serum was also examined. As shown in FIG. 5C, serum levels of human IFN-γ, IL-17A, IL-26, and human soluble CD26 / DPP4 were significantly elevated in cGVHD mice compared to CD34 ⁺ transplanted control mice. To determine whether these cytokines were produced by infiltrating human CD26 ⁺ CD4 T cells, lymphocytes isolated from the lungs of cGVHD mice and CD34 ⁺ transplanted control mice were analyzed using flow cytometry. As shown in FIG. 5D, human IFN-γ ⁺ or IL-26 ⁺ CD26 ⁺ CD4 T cell levels were significantly elevated in cGVHD mice compared to CD34 ⁺ transplanted control mice (panel a or b) IL-17A ⁺ CD26 ⁺ CD4 T cell levels were similar between cGVHD and CD34 ⁺ control groups (panel c). These findings were also confirmed by immunohistochemical examination of lung specimens (FIG. 5E). In addition, CD26 ⁺ CD4 T cells in cGVHD lung mainly produced IL-26 but not IFN-γ (FIG. 7). Furthermore, dermatological histology of huCB-NOG mice showed atrophy, fat loss and vesicle shedding associated with CD26 ⁺ and IL-26 ⁺ cell infiltration (FIG. 8, panels b and c) (FIG. 3C). Panel e and FIG. 8 panel a), IL-17A ⁺ cells were not detected (FIG. 8, panel d). Taken together, these findings strongly suggest that IFN-γ ⁺ and / or IL-26 ⁺ CD26 ⁺ CD4 T cells play an important role in the pathophysiology of lung and cutaneous cGVHD.

(3) IL-26 contributes to collagen deposition in cGVHD lung Since significant collagen deposition was observed in the peribronchial blood vessel of cGVDH lung (FIG. 3E) (Biol Blood Marrow Transplant 2010; 16 (1Suppl); 106- 14, Biol Blood Marrow Transplant 2006; 12 (1): 31-47), we determined whether collagen production was induced by cGVHD lung infiltration of IFN-γ ⁺ and / or IL-26 ⁺ CD26 ⁺ CD4 T cells. I tried to judge. Although IFN-γ is a key regulator of cellular immunity, cGVHD lung development has been reported to be independent of IFN-γ (Blood 2009; 114 (14): 3101-12). On the other hand, the effect of IL-26 on the pathophysiology of cGVHD has not been elucidated. We therefore focused on IL-26 as a potential effector cytokine for lung cGVHD. The human IL-26 gene IL26 is located adjacent to IFNG on chromosome 12q15, and there is no IL-26 gene in rodents (Genes Immun 2012; 13 (6): 481-8, Int Immunopharmacol. 2004; 4 (5): 609-13), but we cannot formally exclude the possibility that human IL-26 activates mouse cells. Therefore, an in vitro assay was performed that measures the effect of human IL-26 on mouse cells. In human cells, IL-26 first binds to IL-20RA and forms the binary complex IL-26 plus IL-20RA, and then recruits the IL-10RB chain, leading to phosphorylation of STAT3 (J Biol Chem 2004; 279 (32): 33343-51). As shown in FIG. 9A, both IL-20RA and IL-10RB are expressed in the mouse fibroblast cell line NIH3T3 and normal human lung fibroblasts (NHLF) (panel a), and exogenous recombinant human IL-26 is Both NHLF and NIH3T3 induced phosphorylation of STAT3 (panel b), suggesting that human IL-26 is active not only in NHLF but also in mouse fibroblasts. To examine the functional effects of IL-26 on mouse fibroblasts, we next performed an in vitro assay for collagen synthesis. As shown in FIG. 9B, increased collagen production in NIH3T3 and NHLF was observed in a dose-dependent manner after addition of exogenous IL-26 ( ^{* in} panels a and b) and anti-IL-20RA polyclonal antibody (pAb) was Neutralization inhibited collagen production ( ^{** in} panels a and b). These results strongly suggest that human IL-26 activates both human and mouse fibroblasts via IL-20RA, leading to increased collagen production.

To further extend the in vitro results to an in vivo system, human allo-reactive GVHD lungs were analyzed using human IL26 Tg mice. To this end, mice carrying human IFNG and IL26 transgene (190-IFNG Tg mice) or mice carrying human IFNG transgene and lacking IL26 transcription (ΔCNS-77 Tg mice) were used. 190-IFNG Tg mice showed IL-26 production by CD4 T cells under Th1 or Th17 polarized conditions, whereas IL-26 expression was completely eliminated in ΔCNS-77 Tg mice (Genes Immun 2012; 13 (6): 481-8). In addition, IFN-γ production by T cells or NK cells was comparable in both 190-IFNG Tg mice and ΔCNS-77 Tg mice (J Immunol. 2010; 185 (3): 1492-501). As shown in FIG. 9C, pulmonary histology of recipient NOG mice or ΔCNS-77 Tg mice derived from parental C57BL / 6 (B6 WT) mice showed peribronchial infiltration and cuffing suggesting GVHD ( Panel a or g), collagen deposition was not detected by Mallory staining (panel b or h), and IL-26 ⁺ cells were not detected (panel c or i). On the other hand, histology of recipient NOG mice derived from 190-IFNG Tg mice showed collagen deposition and IL-26 ⁺ cell infiltration (panels e and f) with peribronchial infiltration and cuffing (panel d) suggesting GVHD. )showed that. A significant increase in collagen deposition in the lungs of recipient NOG mice derived from 190-IFNG Tg mice was quantified by Mallory staining and is shown in FIG. 9D. These results suggest that human IL-26 plays a crucial role in pulmonary fibrosis associated with pulmonary cGVHD, whereas human IFN-γ does not play an important role.

(4) IL-26 production by CD26-mediated T cell costimulation IL-26 is co-expressed with IFN-γ by Th1 cells (Cytokine Growth Factor Rev. 2010; 21 (5): 393-401), And CD26 / DPP4 has been reported to be selectively expressed on Th1 cells activated by CD26-mediated costimulation (Immunol Rev. 1998; 161: 55-70). In addition, CD26 ⁺ CD4 T cells in cGVHD lung mainly produced IL-26 but not IFN-γ (FIG. 7). We therefore hypothesize that human CD4 T cells produce IL-26 after CD26 costimulation. To test this hypothesis, in vitro costimulation experiments using huCB CD4 T cells were performed to analyze the expression of various inflammatory cytokines. As shown in FIG. 10A, IL26 and DPP4 expression levels were enhanced by CD28 or CD26 costimulation ( ^* or ^** , respectively), but significantly greater levels of IL26 and DPP4 after CD26 costimulation than CD28 costimulation. An increase was observed ( ^*** ). On the other hand, the expression levels of IL2, IFNG and IL17A, as previously reported (Blood 2004; 103 (3): 1002-10, Hum. Immunol. 2006; 67 (11): 874-83), huCB Due to T cell immaturity, it did not increase after CD26 costimulation or CD28 costimulation (NS in each panel in FIG. 10A). We next performed costimulation experiments that assessed dose and temporal kinetics using the CD26 costimulatory ligand Cav-Ig and anti-CD26 or anti-CD28 monoclonal antibody (mAb) and were secreted using an ELISA. IL-26 was assayed. As shown in FIG. 10B, IL-26 production increased in a dose- and time-dependent manner following CD26 costimulation with Cav-Ig or anti-CD26 mAb ( ^* or ^** , respectively), but higher doses of mAb. And only for longer stimulation periods, a slight increase in IL-26 levels was observed after CD28 costimulation ( ^*** ). Blocking experiments were then performed for further confirmation, and IL-26 production induced by Cav-Ig or anti-CD26 mAb was clearly inhibited in a dose-dependent manner by treatment with soluble Cav-Ig (respectively in the figure). 10C left or middle panel), showing no change with CD28 costimulation (right panel in FIG. 10C). These findings strongly suggest that IL-26 production by huCB CD4 T cells is regulated by CD26-mediated costimulation. In addition, the functional sequence at the N-terminus of caveolin-1 is highly conserved between humans and mice (FIG. 11) and can bind to human CD26 as a costimulatory ligand and is transferred to mice. It is possible that the donor huCB T cells were activated by CD26 costimulation brought about by mouse caveolin-1. Indeed, expression of caveolin-1 was detected in endothelial cells and macrophage-like cells of BO-like lesions in cGVHD lungs using a pAb that recognizes the N-terminus of both human and mouse caveolin-1 (FIG. 12). . Considered in conjunction with the results of FIG. 10, CD26-mediated IL-2 production caused by caveolin-1 is identified as a potential therapeutic target in cGVHD using huCB NOG mice.

(5) CD26 / DPP4 blockade is sufficient to prevent cGVHD and associated pulmonary fibrosis The above data show that IL-26 ⁺ CD26 ⁺ CD4 T cells play an effector role in cGVHD lung and Cav -Ig blockade of CD26 costimulation suggests that T cell activation is inhibited. In view of the role of CD26 costimulation in IL-26 production and IL-26 regulation of collagen production in pulmonary cGVHD, we therefore blocked blocking CD26 costimulation by the blocking reagent Cav-Ig reduces the incidence of cGVHD And attempted to determine whether to extend the survival of the recipient mice. Recipient mice were treated with Cav-Ig or control Ig three times a week from day +1 to day 28 after transplantation. Recipients treated with Cav-Ig survived for 7 months without clinical findings of GVHD (solid lines in FIGS. 13A and 13B). On the other hand, the survival rate of recipient mice treated with control Ig decreased significantly (dotted line in FIG. 13A), and clinical signs / symptoms of GVHD such as weight loss or hair disturbance were observed as early as 4 weeks after transplantation. (Dotted line in FIG. 13B). Human cells were similarly engrafted in both groups as shown in FIG. In recipients treated with control Ig, lung, skin and liver histology showed human CD3 ⁺ cell infiltration associated with the development of cGVHD (panels af of FIG. 13C). On the other hand, recipients treated with Cav-Ig show a normal appearance of GVHD target organs such as lung, skin and liver compared to mice treated with control Ig (panels g-i in FIG. 13C), None had a positive lesion score (FIG. 13D). Furthermore, human IL-26 and human soluble CD26 / DPP4 serum levels were significantly lower in recipients treated with Cav-Ig than in recipients treated with control Ig (FIG. 13E). Similarly, IL-26 ⁺ CD26 ⁺ CD4 T cells in the lung were decreased in Cav-Ig treated recipients than in control Ig treated mice (FIG. 13F). These findings were also confirmed by immunohistochemistry of lung specimens (FIG. 13G), and the lungs of control Ig treated mice showed a significant increase in bronchiole and perivascular collagen deposition (in panel a of FIG. 13H). Of dark blue and ^{* in} FIG. 13I). Taken together, the above results indicate that administration of Cav-Ig prevents the development of cGVHD after huCB transplantation with sublethal irradiation by reducing the number of IL-26 ⁺ CD26 ⁺ CD4 T cells. Support the concept.

(6) In vivo immunosuppression induced by Cav-Ig We previously reported that Cav-Ig induces hyporesponsiveness to recall and alloantigens in in vitro experiments using adult peripheral T cells (Biochem Biophys. Res. Commun. 2009; 386 (2): 327-32). In order to further expand these in vitro findings to in vivo systems, we then performed serial transplantation experiments after treatment with Cav-Ig or control Ig. Mice that received bone marrow (BM) cells and splenocytes isolated from huCB-NOG mice treated with Cav-Ig survived for 5 months without clinical findings of GVHD (solid lines in FIGS. 14A and 14B). . On the other hand, the survival rate of recipient mice transplanted with BM cells and spleen cells isolated from huCB-NOG mice treated with control Ig was significantly reduced (dotted line in FIG. 14A), and as early as 2 weeks after transplantation. Clinical signs / symptoms of GVHD such as weight loss were shown (dotted line in FIG. 14B). Human cells engrafted in both groups as shown in FIG. In recipients of BM cells and splenocytes isolated from huCB-NOG mice treated with Cav-Ig, lung and liver histology findings showed intact tissue (panels e and f in FIG. 14C). On the other hand, in recipient mice that received BM cells and splenocytes isolated from huCB-NOG mice treated with control Ig, lung histology was similar to findings in xenogeneic aGVHD (Blood 2009; 114 (14) : 3101-12), revealing massive infiltration of human CD3 ⁺ cells around bronchioles and blood vessels and in the alveolar septum (FIGS. 14C, panels a and c). In the liver, perivascular infiltration by human CD3 ⁺ cells was observed in the tissue surrounding the bile duct (panels b and d in FIG. 14C). Importantly, the levels of human IFN-γ ⁺ or IL-26 ⁺ CD26 ⁺ CD4 T cells in the lung were determined by recipients transplanted with BM cells and splenocytes isolated from huCB-NOG mice treated with control Ig. Was significantly lower in recipients of BM cells and splenocytes isolated from huCB-NOG mice treated with Cav-Ig (FIG. 14D). These findings suggest that in vivo immunosuppression is achieved in huCB-NOG mice treated with Cav-Ig.

(7) Delayed administration of Cav-Ig suppresses the onset of cGVHD Since cGVHD progresses painlessly over weeks to months, patients often suffer from clinical findings before being diagnosed with cGVHD (Bone Marrow Transplant. 2008; 42 Suppl 1: S66-S9). We therefore sought to determine whether blockade of the caveolin-1 / CD26 interaction effectively suppressed the development of cGVHD after the appearance of clinical signs / symptoms. For this, recipient mice were treated with Cav-Ig or control Ig from day +29 to day +56 after transplantation. Treatment started at +29 days after the appearance of weight loss and an increase in GVHD score, suggesting an early stage of cGVHD development. Recipients treated with Cav-Ig survived for 7 months with remission of GVHD symptoms (solid lines in FIGS. 16A and 16B). On the other hand, the survival rate of recipients treated with control Ig was significantly reduced (dotted line in FIG. 16A) and clinical signs / symptoms of GVHD progressed (dotted line in FIG. 16B). Human cells were similarly engrafted in both groups as shown in FIG. In control Ig-treated recipients, lung histology showed BO at 5 weeks after transplantation and diffuse alveolar damage at 10 weeks after transplantation (panels a and b in FIG. 16C). In skin and liver, similar GVHD findings as shown in FIG. 3C were observed (panels cf in FIG. 16C). On the other hand, in Cav-Ig treated recipients, slight infiltration of mononuclear cells surrounding bronchioles, vessels or bile ducts was observed 5 weeks after transplantation (panels g and k in FIG. 16C). Ten weeks later, intact histological findings were observed (panels h and l in FIG. 16C). In addition, the cutaneous GVHD findings shown in FIG. 3C were not observed both at 5 and 10 weeks after transplantation (panels i and j in FIG. 16C). Correspondingly, the lesion score of control Ig-treated recipients was significantly elevated at 10 weeks compared to that at 5 weeks ( ^{* in} FIG. 16D). On the other hand, the lesion score of Cav-Ig treated recipients was significantly reduced at 10 weeks compared to that at 5 weeks ( ^{** in} FIG. 16D) and compared to that of control Ig treated recipients. It was clearly low ( ^{*** in} FIG. 16D). Furthermore, the levels of human IFN-γ ⁺ and IL-26 ⁺ CD26 ⁺ CD4 T cells in the lungs of control Ig-treated recipients were significantly elevated at 10 weeks compared to those at 5 weeks (FIG. 16E). ^* ). On the other hand, the levels of human IFN-γ ⁺ and IL-26 ⁺ CD26 ⁺ CD4 T cells in the lungs of Cav-Ig treated recipients are significantly reduced at 10 weeks compared to those at 5 weeks ( ^** ) in FIG. 16E, clearly lower compared to that of the control Ig-treated recipient ( ^{*** in} FIG. 16E). Taken together, these data indicate that administration of Cav-Ig not only prevents the development of cGVHD, but also a novel therapeutic approach for early cGVDH by modulating the levels of IL-26 ⁺ CD26 ⁺ CD4 T cells Suggest that also provide.

(8) Treatment with Cav-Ig preserves GVL ability in recipient mice Since GVHD and GVL effects are highly related immune responses (Clin. Cancer Res. 2009; 15 (14): 4515-17) We evaluated the potential impact of Cav-Ig treatment on the GVL effect. To this end, a cohort of huCB-NOG mice treated with Cav-Ig or control Ig was irradiated at a sublethal dose and then transfected with luciferase one day prior to total CB transplantation to allow tumor cell seeding. A20 (A20-luc) cells were injected intravenously. The day after transplantation, treatment with Cav-Ig or control Ig three times a week started on day +1 and continued until day +28. All mice inoculated with A20 cells alone died from tumor progression within 6 weeks (blue line in FIG. 17A and left panel in FIG. 17B). Recipients treated with control Ig showed clinical evidence of GVHD, such as weight loss and hair turbulence, and died of GVHD without tumor progression at 13 weeks (dotted line in FIG. 17A and FIG. 17B). Middle panel). In contrast, recipient mice treated with Cav-Ig showed significantly longer survival (black line in FIG. 17A) without A20-luc cells involved (right panel in FIG. 17B). In order to characterize the efficacy of the GVL effect in more detail, these studies were repeated by injection of A20-luc cells +28 days after the whole CB transplantation to allow acquisition of immunosuppression by Cav-Ig treatment. All mice inoculated with A20 cells alone died from tumor progression within 2 weeks after tumor inoculation (blue line in FIG. 17C and left panel in FIG. 17D). Recipient mice treated with control Ig revealed clinical evidence of GVHD, such as weight loss and coat disturbance, and died of GVHD without tumor progression within 13 weeks after transplantation (FIG. 17C). (Dotted line and middle panel of FIG. 17D). In contrast, recipients treated with Cav-Ig showed significantly longer survival without the involvement of A20-luc cells (right panel in FIG. 17D) (black line in FIG. 17C). Taken together, these results demonstrate that Cav-Ig treatment of huCB-NOG mice was effective in reducing the symptoms of cGVHD without a concomitant loss of GVL effect.

(9) Lymphocyte growth inhibitory effect of fusion protein of caveolin 1 partial peptide and immunoglobulin constant region The inhibitory effect of Fc fusion protein treatment on T cell proliferation response by tetanus toxoid stimulation was examined.
Lymphocytes were isolated from peripheral blood of healthy adults immunized with tetanus toxoid, blocked with 10 μg / mL Fc fusion protein or control immunoglobulin, tetanus toxoid was added, and proliferation reaction was measured by ³ H-thymidine incorporation. The results are shown in FIG.

Claims (4)

  An immunosuppressant comprising a caveolin 1 inhibitor as an active ingredient.   A disease selected from acute or chronic graft-versus-host disease, post-transplant rejection, autoimmune disease, idiopathic pulmonary hypertension, idiopathic pulmonary fibrosis, obstructive bronchiolitis, chronic glomerulonephritis and idiopathic nephrotic syndrome The immunosuppressive agent according to claim 1, which is a prophylactic and therapeutic agent.   Autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, polymyositis, dermatomyositis, scleroderma, Sjogren's syndrome, vasculitis syndrome, mixed connective tissue disease, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosis Cholangitis, autoimmune pancreatitis, ulcerative colitis, Crohn's disease, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, autoimmune neutropenia, type I diabetes, pemphigus, celestial The immunosuppressive agent according to claim 2, wherein the immunosuppressive agent is a disease selected from pemphigus, habitual miscarriage, causative disease and autoimmune optic neuritis.   The caveolin 1 inhibitor is a peptide fragment of the caveolin 1 N-terminal region, a peptide fragment of the caveolin 1 scaffolding domain sequence, a peptide fragment lacking the scaffolding domain from the caveolin 1 N-terminal region, the caveolin 1 binding region of CD26 The immunosuppressive agent according to any one of claims 1 to 3, wherein the immunosuppressive agent is one or more selected from peptide fragments of sequences and fusion proteins of any one of these peptide fragments and an immunoglobulin constant region.