KR20210117217A - Exosomes derived from umbilical cord blood plasma or their mimics and imunosuppression use of thereof - Google Patents

Abstract

The present invention relates to exosome derived from cord blood plasma or mimetibody thereof, and immunosuppressive uses thereof. More specifically, the present invention provides the use of exosomes derived from human cord blood plasma or exosomes mimicking the proteomics profile of the cord blood plasma-derived exosomes for the alleviation, prevention or treatment of various autoimmune diseases.

Description

Exosomes derived from umbilical cord blood plasma or their mimics and imunosuppression use of thereof

The present invention relates to cord blood plasma-derived exosomes or mimetics thereof and to their immunosuppressive use.

Human cord blood has been used as a source of hematopoietic stem cells for transplantation, but it contains a number of cells such as immune cells, mesenchymal stromal cells and endothelial precursor cells. Fetal immunity through the maternal immune system plays an important role in maintaining pregnancy. The immunological interaction between the fetus and the mother represents a two-way communication system that is regulated by the presentation of fetal antigens and/or recognition of these antigens by the maternal immune system and response thereto. Cells derived from human cord blood have specific immunomodulatory properties that contribute to the maintenance of pregnancy. Moreover, human umbilical cord blood is also a rich source of immunosuppressive cells such as regulatory T cells (Tregs) and monocyte-derived suppressor cells (MDSCs).

Exosomes are small cell membrane vesicles that mount a unique subset of functional proteins and reflect their cell lineage. Since human cord blood-derived cells are selected and expanded ex vivo, exosomes derived from these cells can be efficiently isolated from the cell culture supernatant. Exosomes released from cells derived from human umbilical cord blood may induce the differentiation of circulating immunosuppressive cells such as Tregs and MDSCs, and thus may have an immunosuppressive effect. However, despite advances in the study of cells and exosomes derived from human cord blood, studies on exosomes derived from human cord blood plasma (CBPexo) are poor. Proteomics studies on CBPexo showed that exosome proteins were involved in T-cell proliferation, differentiation and negative regulation; membrane permeability; wound-healing; arginase activity; and enzyme regulatory activity. In addition to cells derived from human cord blood, human cord blood plasma also contains various immunomodulatory factors, and thus plays a role in inhibiting T-cell proliferation by inhibiting IL-2 signaling. IL-2 and its receptor, IL-2R, are responsible for supporting the proliferation and survival of T cells and the differentiation of naive T cells. The IL-2R alpha chain (IL-2Rα/CD25) is an essential component of IL-2R. Moreover, the surface expression of IL-2Ra in CD4 + T cells and the level of IL-2 in activated T cells are significantly reduced by MMP-9. Inhibition of MMP activity by chemical inhibitors restores IL-2 production and partially restores T cell proliferation. MMP-9 as well as CBP immunomodulators are found in CBPexo, and these exosomes will exhibit concentrated immunosuppressive activity.

1. Ballen, K. K. et al., Blood, 2013. 122(4): p. 491-8. 2. Brunstein, C. G., et al., Blood, 2016. 127(8): p. 1044-51. 3. van Niel, G., et al., J Biochem, 2006. 140(1): p. 13-21. 4. Erices, A. et al., Br J Haematol, 2000. 109(1): p. 235-42. 5. Lin, S.J., et al., Immunol Res, 2014. 60(1): p. 105-11. 6. Zhang, B., et al., Stem Cells Dev, 2014. 23(11): p. 1233-44. 7. Jia, R., et al., Cell Physiol Biochem, 2015. 36(6): p. 2299-306. 8. Garanina, E.E., et al., Blood, 2017. 130. 9. Jia, R., et al., Cell Physiol Biochem. 36(6): p. 2299-306.

It is an object of the present invention to provide a pharmaceutical use thereof by identifying the proteomics profile of exosomes derived from human cord blood plasma compared to exosomes derived from adult plasma.

Another object of the present invention is to provide an exosome mimic that mimics the proteomics profile of exosomes derived from human cord blood plasma and pharmaceutical uses thereof.

In order to achieve the above object, the present invention is galectin-3 (galectin-3), Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7) ), Galectin-7 (GAL-7), Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B), Mannan-binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 ( ARG1) provides a composition for immunosuppression comprising, as an active ingredient, an exosome derived from umbilical cord blood plasma, wherein the expression of the gene group consisting of adult plasma is higher than that of adult plasma.

The present invention also provides galectin-3, Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7), Galectin-7 ( GAL-7), Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B) ), Mannan-binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 (ARG1). It provides a medium composition for inhibiting differentiation of Th1 and Th17 cells comprising exosomes derived from cord blood plasma as an active ingredient, the expression of which is higher than that of adult plasma-derived exosomes.

The present invention also provides galectin-3, Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7), Galectin-7 ( GAL-7), Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B) ), Mannan-binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 (ARG1). Provided is a method for inhibiting differentiation of Th1 and Th17 cells in vitro, comprising culturing in vitro exosomes derived from cord blood plasma and naive CD4 + T cells whose expression is higher than that of adult plasma-derived exosomes.

The present invention is also derived from HLA and MIC gene deficient cell lines, galectin-3, Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100 -A7 (S100A7), Galectin-7 (GAL-7), Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B), Mannan-binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Provided is an umbilical cord blood plasma exosome mimic expressing one or more selected from the gene group consisting of Arginase-1 (ARG1).

The present invention also provides a composition for immunosuppression comprising the umbilical cord blood plasma exosome mimic.

The present invention uses exosomes derived from human cord blood plasma for the improvement, prevention or treatment of various autoimmune diseases, and an exosome mimic that mimics the proteomics profile of the exosomes provides an effective immunosuppressive effect.

1A-D are diagrams showing the results of characterization of exosome membrane vesicles purified from human umbilical cord blood plasma.
(a) Flow cytometry analysis of CBPexo bound to latex beads coated with various exosome-specific marker antibodies. For comparison, latex beads coated with a complete exosome preparation are included. Plots show intensities derived from exosome specific antibodies (CD9, CD63, CD82 and HSP72) against the corresponding bead-only controls.
(b) is a dot blot analysis result of CBPexo lysate. Corresponding dots were evaluated using the exosome antibody array kit. Exosome-specific antibody spots provide signals of varying intensities. The values presented represent the average of values obtained from three independent experiments. Error bars = standard error of the mean (SEM). “Blank” denotes negative control, denoted by φ, and GM130 denotes cell debris. Average intensity is analyzed using ImageJ software.
(c) is the result of morphological characterization of CBPexo. Using electron microscopy, the morphological characteristics of CBPexo with a lipid bilayer structure and a size (50-130 nm) were confirmed. Scale bar represents 100 nm.
(d) is the result of analysis of the size and number of particles of CBPexo. The size of the exosomes is 81 ± 1.4 nm and the number of particles is 1.64Х10 ¹² ± 2.37Х10 ¹⁰ particles/mL.
2A-C are diagrams showing molecular functions and biological processes associated with differentially expressed proteins.
(a) Proteomics analysis results of plasma-derived exosomes and various immunosuppressive molecules in exosomes screened by mass spectrometry (mass spectrometry data examination with ExoCarta and Mascot v2.3.01).
(b) Results of proteomics analysis of various biological functions of human CBPexo and ABPexo using the ExoCarta database and SBC analysis system. (c) Shows the results of proteomics analysis of human CBPexo and ABPexo screened using the ExoCarta database and SBC analysis system and various molecular functions of immunosuppressive molecules. Red boxes indicate higher associations of CBPexo as functional classification results.
3A-D depict the mechanism of CBPexo mediated immunosuppression in CD3/CD28 DYNABEAD-stimulated human CD4+ and CD8+ T cells.
(A) Representative experimental data demonstrating that CBPexo inhibits CD4+ and CD8+ T cell stimulation. The intensity of CFSE-labeled T cells is obtained by flow cytometry and further analyzed with ModFit LT 3.0 software.
(b) Concentration-dependent activation of human PBMCs using CD3/CD28 DYNABEAD under CBPexo or ABPexo. Rapamycin and cyclosporine are well known immunosuppressants and are used as standard positive controls. The intensity of CFSE-labeled T cells is obtained by flow cytometry and further analyzed with ModFit LT 3.0 software. All experiments were repeated at least 5 times using 5 different batches of CBPexo (1 batch of plasma exosomes=sum of 10 units of cord blood). Statistically significant difference (ANOVA test): *p <0.1 **p <0.05 ***p <0.01. Activated PBMCs represent positive control=φ.
(c) Representative experimental data of annexins V-FITC and 7AAD show that human PBMCs are activated with CD3/CD28 DYNABEAD under CBPexo or ABPexo.
(d) CBPexo induces apoptosis of CD4+ T cells activated by CD3/CD28 DYNABEAD. CD4+ and CD+8 T cells were harvested after 156 hours and stained with annexin V-FITC, 7AAD, anti-CD4-APC and anti-CD8-V450 antibodies. Cells gated on CD4 are shown. Analysis data is analyzed using FlowJo v10. This experiment is independently repeated 5 times. Statistically significant difference (ANOVA test): *p <0.1 **p <0.05 ***p <0.01.
4 shows the effect of G0/G1 cell cycle arrest in CD4 + T cells induced by CBPexo. Experiments were independently repeated 3 times and one-way ANOVA was used to calculate the significance between groups: * p <0.1 ** p <0.05 *** p <0.01.
5a-e show the effect of CBPexo on the induction of differentiation of Treg cells and the functional upregulation of T cell inhibition.
(a) Expression of CD4+CD25+ and FOXP3+ of PBMCs under CBPexo. This experiment was independently repeated 3 times, and the statistically significant difference (ANOVA test) was *p <0.1 **p <0.05 ***p <0.01.
(b) Regulatory T cell expansion or differentiation from umbilical cord blood CD25+ cells.
(c) Shows fold expansion of the same 1 unit cord blood CD25+ cells by cytokine, aAPC and antibody combination with and without CBPexo.
(d) Anti-CD3/CD28, expansion of cord blood CD25+ cells sorted according to the IL-2 cocktail and differentiation of Tregs with and without CBPexo. Cord blood mononuclear cells (CB-MNC) are used as negative controls.
(e) Comparison of the inhibitory functions of CBPexo-induced Tregs and nTregs in PBMCs after DYNABEAD stimulation. Inhibitory function is assessed by CFSE dilution. Flow cytometry data from three independent experiments are summarized in a straight line as the ratio of CBPexo-induced Treg and nTreg-mediated T cell proliferation in PBMCs. Data are presented as mean ± standard deviation. Statistically significant difference (two-way ANOVA test): *p <0.1 **p <0.05 ***p <0.01 (n=4).
6a-b show the effect of CBPexo on the induction of MDSC differentiation.
(A) Non-stimulated PBMCs are cultured in the absence (CTRL) or presence of CBPexo. Gating strategy: use physical parameters, namely forward scatter (FSC) and side scatter (SSC) to select monocytes (CD14+). MDSCs are differentiated by evaluating the expression of CD11b/CD33.
(b) In the PBMC population, CBPexos promotes the immunosuppressive phenotype of MDSCs and stimulates the production of arginase 1, NOS2 and IDO. Representative FACS histograms for intracellular staining of arginase 1, NOS2 and IDO and staining of CD14+/CD11b/CD33+ cells are shown.
7a-b show the effect of CBPexo on the induction of T cell immunosuppression by specific MMPs.
(a) Shows the results of MMP and TIMP antibody analysis of 300 μg of CBPexo or ABPexo lysate. Corresponding dots are assessed using the Exosome Antibody Assay Kit. MMP and TIMP antibody spots giving signals of varying intensities are calculated with ImageJ software. This experiment is independently repeated 3 times.
(b) CBPexo-mediated inhibition through MMP inhibition induces partial restoration of T cell proliferation. T cells were stimulated with DYNABEAD under CBPexo at 156 h and with or without GM6001 prior to comparison via CFSE assay. CFSE-labeled cells are obtained by FACSCanto and cells are gated at CD4+ events. The proportion of cells in each generation is calculated using ModFit LT4.0 software. This experiment is independently repeated 6 times. Statistically significant difference (ANOVA test): *p <0.1 **p <0.05 ***p <0.01.
8 shows the expression effect of the activation markers CD25 and CD69 in CD4 + T cells by the MMP inhibitor GM6001 treated CBPexo.
9A-E show mouse cross-reactivity of T cell inhibition and IL-2 downregulation by human CBPexo.
(A) The immunosuppressive effect of CBPexo is tested by DYNABEAD-stimulated mouse CD4+ T cell proliferation. This experiment is independently repeated 5 times. Statistically significant difference (ANOVA test): *p <0.1 **p <0.05 ***p <0.01.
(b) The immunosuppressive effect of CBPexo was tested by DYNABEAD-stimulated mouse CD8 + T cell proliferation. This experiment is independently repeated 5 times. Statistically significant difference (ANOVA test): *p <0.1 **p <0.05 ***p <0.01.
(c) ELISPOT assay is performed on day 6 to quantify the levels of IL-2, IFN-γ and IL-17 secreting T cells. Mouse splenocytes were stimulated with CBPexo or ABPexo, and IL-2, IFN-γ and IL-17 secreting T cell patterns were observed.
(d) CBPexo significantly reduces human T cell proliferation through the production of IL-2, IFN-γ and IL-6. CBPexo not only cleaves IL-2Ra in human T cells, but also downregulates IL-2, IFN-γ and IL-6 secretion associated with differentiation of Th17 and Th1 cells. These data suggest that cytokine production is inhibited by CBPexo, abrogating T cell differentiation capacity. Cytokine levels at 156 hours in harvested culture supernatants are analyzed using a human cytometric bead array. This experiment is independently repeated 3 times.
(E) Changes in cytokine levels are observed using a mouse cytometric bead array. As a result, CBPexo downregulates IL-2 secretion in human T cells as well as decreases IL-2 in mice. This experiment is independently repeated 3 times.
10A-C show the pattern of changes in Treg and IL-2 levels in exosome-treated EAE mice.
(A) EAE development is reduced in CBPexo treated EAE mice. EAE was induced in a total of 15 C57BL/6 mice treated with MOG/CFA and PTx for immunization. CBPexo and ABPexo were injected intravenously at a dose of 100 μg (black arrow) into 5 EAE mice twice on days 0 and 7, respectively. The clinical score of EAE was observed for 30 days according to the following criteria. Clinical score of diseased rats: 0, no signs of disease; 1, shriveled tail; 2, saggy tail and partial hindlimb weakness; 3, complete hindlimb paralysis; 4, complete hindlimb and partial forelimb paralysis; 5, death. Mice are assigned to different groups for analysis of clinical scores after EAE induction: EAE mice (n=5) vs. EAE mice injected with CBPexo or ABPexo (n=5). EAE represents positive control=φ.
(b) shows the detection result of MOG35-55 induced cytokine recall response in total splenocytes during acute EAE. C57BL/6 mice were immunized with MOG33-55 in CFA and tested 22 days after immunization. All mice tested exhibit clinical symptoms of EAE. The frequency of MOG peptide-specific IFN-γ, IL-2 and IL-17 secreting cells was measured using ELISPOT assay to compare with EAE control group, CBPexo-treated EAE group and ABPexo-treated EAE group. Analyze MOG peptide-specific T cells during restimulation with MOG peptide (25 μg/mL).
(c) Th1, Th2 and Th17 specific cytokine-gated CD4+ T cells in the presence or absence of MOG33-55 peptide measured using intracellular cytokine staining and EAE control, CBPexo injected EAE group and ABPexo injected EAE The results compared to the group are shown (n=4).
11 shows the detection results of MOG35-55-induced cytokine re-reaction in splenocytes of the EAE model.
12a-b show the pattern of Treg changes in the spleen ( FIG. 12a ) and the thymus ( FIG. 12b ) of exosome-treated EAE mice. The frequency of CD4+T gated MOG peptide-specific FOXP3+ cells is compared to EAE control, CBPexo-treated EAE and ABPexo-treated EAE groups using intracellular cytokine staining method.
13 shows the MDSC population of experimental autoimmune encephalomyelitis (EAE) mice treated with exosomes. The frequencies of CD11b + Gr1 + cells and CD11b + NOS2 + gated cells are determined by comparing the frequencies of the MDSC populations in healthy control, EAE control, CBPexo injected EAE and ABPexo injected EAE groups.
14 shows the ability of exosomes to migrate to the brain and spleen of experimental autoimmune encephalomyelitis (EAE) mice. Whether exosomes stained with PKH-67 fluorescence were delivered to DAPI-stained tissues were compared in the EAE control group, CBPexo-treated EAE and ABPexo-treated EAE groups.
15 compares the pathological treatment effect of CBPexo in the brain of experimental autoimmune encephalomyelitis (EAE) mice in the EAE control group, CBPexo-treated EAE and ABPexo-treated EAE groups.
16A-F are diagrams showing the CBPexo mimic and its immunosuppressive effect.
(A) Shows the establishment of an HLA class I/MIC null HEK 293T (H1ME-5) cell line using the multiplex CRISPR/Cas9 system. H1ME-5 cells show no expression of MICA/B and HLA class I.
(b) Shows the size distribution of exosomes released from H1ME-5. CBPexo has a size of 145.61 ± 75.89 nm.
(c) Expression of GAL-3, MMP-8, MMP-9, PIP, S100A7, GAL-7 and HSP72 contained in CBPexo transduced into H1ME-5 cells. On day 6 post-transduction, cells positive for each molecule are sorted using the MoFlo XDP Cell Sorter.
(d) CBPexo or CBPexo mimetics bound to latex beads coated with GAL-3, MMP-8, MMP-9, PIP, S100A7, GAL-7 and HSP72 antibodies are analyzed by flow cytometry. The intensities derived from the GAL-3, MMP-8, MMP-9, PIP, S100A7, GAL-7 and HSP72 antibodies are compared with the corresponding bead-only controls.
(E) Representative experimental data demonstrating that CBPexo and MMP-9-enriched CBPexo mimetics inhibit T cell stimulation. The intensity of CFSE-labeled T cells is obtained by flow cytometry and further analyzed using ModFit LT4.0 software. The concentrations of CBPexo, CBPexo mimic and H1ME-5 exosomes were 10 μg/well.
(f) Statistics of CBPexo, CBPexo mimetics and H1ME-5 exosomes are analyzed using the results obtained from the CFSE proliferation assay.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention is galectin-3, Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7), Galectin-7 (GAL) -7), Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B) , Mannan-binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 (ARG1). It relates to a composition for immunosuppression comprising, as an active ingredient, exosomes derived from cord blood plasma, whose expression is higher than that of adult plasma-derived exosomes.

Cord blood plasma-derived exosomes (CBPexo) of the present invention contain more immunosuppression-related proteins than adult plasma-derived exosomes (ABPexo). It is caused by the period arrest. In addition, CBPexo treatment increases the CD4+CD25+FOXP3+ Treg cell population and upregulates the function of polyclonal expanded Tregs. It is speculated that CBPexo arrests the cell cycle by cleaving CD25, which inhibits mTOR signaling downstream. Indeed, the secretion of IL-2 and the surface expression of IL-2Rα in CBPexo-treated activated T cells were significantly reduced in CD4+ T cells. Blockade of IL-2 signaling by CBPexo plays a pivotal role in the inhibition of proliferation of CD4+ and CD8+ T cells. Blockade of the IL-2 signaling pathway leads to suppression of the immune system and has therapeutic effects. IL-2/IL-2R interaction activates intracellular Ras/Raf/MARP, JAK/STAT and PI3K/AKT signaling pathways to promote growth, differentiation and survival of T cells.

In addition, MMP-9 of CBPexo is involved in the cleavage of CD25 expressed in T cells when it encounters cancer cells, and MMP-8 also induces the disruption of the extracellular domain, resulting in cleavage of surface molecules related to immune responses, resulting in immunomodulatory effects. indicates Cleavage of IL-2Ra is dependent on MMP-9.

When human CBPexo was applied to a mouse model, an immunosuppressive effect was observed in mouse T cells in vitro, confirming the mouse cross-reactivity of human CBPexo.

In addition, the EAE mouse model is used comprehensively to investigate the role of Th cells in disease development. For example, we show that transplantation of myelin-specific CD4+Th1 cells into immature recipient mice promotes the induction of EAE. Similarly, IL-17 secreting T cells known as Th17 cells have also been reported to be a driving force in EAE development. In addition, EAE promotes disease initiation by lowering the frequency of Tregs and impairing their inhibitory function. The CBPexo of the present invention can change the EAE development process and regulate the differentiation of antigen-specific CD4+ T cells by increasing Treg differentiation and decreasing Th1 and Th17 differentiation in vivo.

As described above, the exosome of the present invention inhibits T cell proliferation by 1) reducing IL-2 production by 1) MMP-9 expressed in the exosome degrades IL-2 receptor α (CD25) on the activated T cell surface. and 2) induce regulatory T cell (Treg cell) and monocyte-derived suppressor cell (MDSC) differentiation, and 3) inhibit the differentiation of Th1 and Th17 cells.

As used herein, the term "cord blood plasma-derived exosome" or "CBPexo" is a cell membrane particle derived from cord blood plasma. The exosome (exosome) has the same meaning as microvesicles, circulating microvesicles or microvesicles in the art, and refers to a fragment having a plasma membrane of 50 nm to 100 nm removed from the cell. Microvesicles mediate the transport of mRNA, miRNA and proteins between cells and play an important role in intracellular interactions. Microvesicles represent a heterogeneous population depending on the cell from which it is derived, the number of cells, the size of the cells and the composition of the antigen. Preferably, the exosome of the present invention is galectin-3 (galectin-3), Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7) ), Galectin-7 (GAL-7), Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B), Mannan-binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 ( It is characterized in that the expression of the gene group consisting of ARG1) is higher than that of exosomes derived from adult plasma.

As used herein, the term "active ingredient" refers to a component capable of exhibiting the desired activity by itself or in combination with a carrier having no activity by itself.

In addition, as used herein, the term "immunosuppression" is meant to include improvement (relief of symptoms), treatment, prevention, suppression or delay of the onset of immune diseases caused by hyperimmune or abnormal immune responses. The "immune disease" means graft-versus-host disease, autoimmune disease (rheumatoid arthritis, etc.), hyperproliferative skin disease (atopic dermatitis, contact dermatitis, etc.), chronic obstructive pulmonary disease (COPD), allergy It includes, but is not limited to, sexual asthma, asthma, bronchitis, allergic rhinitis, and autoimmune hepatitis.

The present invention also provides galectin-3, Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7), Galectin-7 ( GAL-7), Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B) ), Mannan-binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 (ARG1). To a medium composition for inhibiting differentiation of Th1 and Th17 cells comprising exosomes derived from cord blood plasma as an active ingredient, the expression of which is higher than that of adult plasma-derived exosomes.

In addition, the present invention is galectin-3 (galectin-3), Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7), Galectin-7 (GAL-7), Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B ( C4B), Mannan-binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 (ARG1) Provided is a method for inhibiting differentiation of Th1 and Th17 cells in vitro, comprising culturing in vitro exosomes derived from cord blood plasma and naive CD4 + T cells, wherein the group expression is higher than that of adult plasma.

As used herein, the term "medium" refers to a medium capable of inhibiting the differentiation of naive CD4 + T cells into Th1 and Th17 cells in vitro, and includes any medium used in the art suitable for cell culture. Depending on the type of cell, the medium and culture conditions can be selected. The medium used for culture generally contains a carbon source, a nitrogen source, and a trace element component. Such cell culture medium includes, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (α Minimal essential Medium), GMEM ( Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium, etc., but are not limited thereto.

In addition, the medium may contain antibiotics such as penicillin, streptomycin, and gentamicin.

The medium composition of the present invention can be used alone or in combination with other substances to form a microenvironment for culturing and inhibiting differentiation of Th1 and Th17 cells.

In the medium composition of the present invention, a differentiation inhibitory material may be additionally added as the other material, and any differentiation inhibitory material known in the art may be used as the differentiation inhibitory material. It may be added alone or in combination in various ways depending on the state.

Culturing conditions may be carried out at 35 to 37° C. with an aeration of 5 to 15% carbon dioxide in a _{CO 2 incubator, but is not particularly limited thereto.}

The composition of the present invention is galectin-3 (galectin-3), Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7), Galectin-7 (GAL-7), Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B ( C4B), Mannan-binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 (ARG1) overexpressing Since it includes exosomes derived from cord blood plasma, the overlapping content will be omitted to avoid excessive complexity of the present specification.

The present invention is also derived from HLA and MIC gene deficient cell lines, galectin-3, Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100 -A7 (S100A7), Galectin-7 (GAL-7), Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B), Mannan-binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and It relates to an umbilical cord blood plasma exosome mimic expressing one or more selected from the gene group consisting of Arginase-1 (ARG1).

The present inventors have identified GAL-3, MMP-9, HSP72, PIP, S100A7, GAL-7, LAMP1, SERPINB12, LTF, ORM1, CD5L, C4B associated with pharmaceutical uses in the proteomics profile of CBPexo, such as immunosuppression, wound healing, etc. , MASP1, PSMA6, PRDX1, DEFA3, CD44 antigen, ARG1, etc. were confirmed to be included. However, since it is difficult to isolate sufficient exosomes from umbilical cord blood plasma, the nucleic acid encoding the immunosuppression-related protein alone or two or more types was added to the H1ME-5 (HLA class I and MIC-edited clone-5 cell line) cell line, which has no immunosuppressive effect. introduced, and the exosomes were separated and purified therefrom. According to one embodiment of the present invention, by selecting GAL-3, GAL-7, PIP, HSP72 and S100-A7 as such proteins, exosome mimetics, such as GAL-3 exo, GAL-7 exo, PIP exo, HSP72 exo, S100-A7 exo, HSP72 & PIP dual exo were separated and purified.

Accordingly, as used herein, the term "umbilical cord blood plasma exosome mimic" refers to an exosome that mimics the proteomics profile of an exosome isolated from cord blood plasma, and in particular, a single or a plurality of proteins overexpressed in the exosome for a specific purpose. refers to an exosome derived from a cell line without an immunosuppressive effect prepared to be overexpressed using a genetic recombination technique. Therefore, the umbilical cord blood plasma exosome mimic can be prepared by introducing a nucleic acid encoding an exosome protein into a cell line having no immunosuppressive effect and separating it therefrom. The nucleic acid is used in the broadest sense, and includes single-stranded (ss) DNA, double-stranded (ds) DNA, cDNA, (-)-RNA, (+)-RNA, dsRNA, and the like.

The size of the exosome mimic may have a diameter of 60 nm to 250 nm, or 70 nm to 230 nm.

As the cell line without the immunosuppressive effect, a cell line deficient in HLA and MIC genes is used. For example, by using the CRISPR-Cas9 system to delete between exons 2 and 3 of each of HLAA-A, HLA-B, HLA-C and MICA/B, the HLAA-A, HLA-B, HLA-C and MICA/B genes H1ME-5 (Accession No.: KCTC 13602BP), which is a 293T cell line deficient in HLA and MICA/B completely removed from the pseudogenome, can be used.

GAL-3, MMP-9, HSP72, PIP, S100A7, GAL-7, LAMP1, SERPINB12, LTF, ORM1, CD5L, C4B, MASP1, PSMA6, PRDX1, DEFA3, CD44 antigen and ARG1 genes were added to the H1ME-5 cell line. After transfection with an expressing vector, and selecting an H1ME-5 cell line expressing GAL-3, MMP-9, HSP72, S100A7, GAL-7 and PIP genes alone or two or more types, exosomes (CBPexo mimic) separate

Therefore, preferably, the umbilical cord blood plasma exosome mimic of the present invention may express LAMP1, SERPINB12, LTF, ORM1, CD5L, C4B, MASP1, PSMA6, PRDX1, DEFA3, CD44 antigen and ARG1.

Alternatively, the umbilical cord blood plasma exosome mimetic of the present invention may express HSP72 and PIP alone or both.

The transfection includes microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, protein transduction domain mediated introduction, virus It can be delivered to cells by various methods in the art, such as, but not limited to -mediated gene delivery, and PEG-mediated transfection in protozoa.

As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector that can ligate additional DNA segments into the viral genome. Some vectors are capable of self-replication within a host cell when introduced into the host cell (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell and replicated along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful for recombinant DNA IVT mRNA techniques are usually in the form of plasmids, since plasmids are the most commonly used type of vector, so "plasmid" and "vector" can be used interchangeably. However, the present invention relates to viral vectors (eg, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors) and IVT mRNAs that provide equivalent functions Other types of expression vectors are also included. Preferably, a lentiviral vector can be used. Transformation includes any method of introducing a nucleic acid into an organism, cell, tissue or organ, and as is known in the art, it can be carried out by selecting the above-mentioned standard technique suitable for the host cell.

The present invention also relates to a composition for immunosuppression comprising the umbilical cord blood plasma exosome mimic.

According to one embodiment of the present invention, it is shown that MMP-9-enhanced exosomes among CBPexo mimics are promising therapeutic molecules for T cell immunosuppression. These results indicate that several complex effects such as cleavage of CD25 by MMP, inhibition of IL-2 production, changes in cytokine secretion pattern, and proliferation of Treg cells are simultaneously associated with T cell differentiation and proliferation. In addition, it is known that exosomes expressing HSP72 inhibit T cell proliferation, and HSP72 upregulates MMP-9.

Therefore, the umbilical cord blood plasma exosome mimic of the present invention can be used as an active ingredient of the composition for immunosuppression.

The immunosuppression is for the improvement, prevention, or treatment of the above-mentioned immune diseases, and the overlapping contents related to the immune diseases are omitted in order to avoid excessive complexity of the present specification.

On the other hand, the immunosuppressive composition of the present invention may include an active ingredient and an active or inactive pharmaceutically acceptable carrier constituting a composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

The pharmaceutically acceptable carrier includes exosome mimetics and protein excipients including phosphate buffered saline solution, serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. any pharmaceutical carrier that is compatible with it. Examples of carriers, stabilizers and adjuvants can be found in Martin REMINGTON'S PHARM. ^{SCI, 18 th Ed. (Mack} Publ. Co., Easton (1995)) and the "PHYSICIAN'S DESK REFERENCE", 58nd Ed., Medical Economics, Montvale, NJ. (2004). The term "carrier" may include buffers or pH adjusting agents, typically buffers are salts prepared from organic acids or bases. Representative buffers include organic acid salts such as citric acid salt, ascorbic acid salt, gluconic acid salt, carbonic acid salt, tartaric acid salt, succinic acid salt, acetic acid salt or phthalic acid salt; tris, tromethamine hydrochloride or phosphate buffers. As additional carriers, polyvinylpyrrolidone, ficol (polymer sugar), dextrates (eg, cyclodextrins such as 2-hydroxypropyl-quadrature, -cyclodextrin), polyethylene glycol, antioxidants, anti- Polymers such as antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g. phospholipids, fatty acids), steroids (e.g. cholesterol) and chelating agents (e.g. EDTA) Contains excipients/additives. An anti-freeze or de-icing agent may also be included.

The composition for immunosuppression of the present invention can be prepared in various suitable formulations. Formulations and carriers suitable for parenteral administration such as, for example, intratumoral, intraarterial (in the joint), intravenous, intramuscular, intradermal, intraperitoneal, intranodal and subcutaneous routes include antioxidants, buffers, bacteriostatic agents. , and aqueous and non-aqueous sterile suspensions which may contain solutes which will render the formulation isotonic with the blood of the intended recipient, and suspending, solubilizing, thickening, stabilizing, and preservative agents. Intravenous or intraperitoneal administration is the preferred method. The dosage of cells administered to a subject is an amount effective to achieve a desired beneficial therapeutic response in the subject over time, or an amount effective to inhibit the growth of cells or an amount effective to inhibit infection. For example, it can be carried out in such a way that a blood sample is obtained from the subject prior to injection and stored for use in subsequent analysis and comparison. In general, at least about 10 ⁴ to 10 ⁶ and typically 1×10 ⁸ to 1×10 ¹⁰ cells may be infused intravenously or intraperitoneally into a 70 kg patient over approximately 60 to 120 minutes. In the case of administration, taking into account the overall health and weight of the individual, the exosomes of the present invention can be administered according to the LD-50 (or other toxicity measurement method) according to the type of cell and the side effect determined by the type of cell at various concentrations. administered in proportion. Administration may be administered at one time or in several divided doses. The exosomes of the present invention can supplement treatment for other specific conditions using known conventional therapies including cytotoxic agents, nucleotide analogs and biological response modifiers. Similarly, biological response modifiers may optionally be added to treatment with the exosomes of the present invention.

Hereinafter, the present invention will be described in more detail through Examples according to the present invention, but the scope of the present invention is not limited by the Examples presented below.

<Example 1> Preparation and characterization of exosomes derived from cord blood plasma

1. Sample Preparation

Human peripheral blood mononuclear cells and human umbilical cord blood (UCB) were provided by the Catholic Hematopoietic Stem Cell Bank after informed consent was obtained from a healthy donor or from a normal full-term pregnant woman. All experiments on human subjects were performed in accordance with the recommendations of the Declaration of Helsinki. The protocol was approved by the Animal Experimental Ethics Committee (IACUC) of the Catholic University of Korea. All subjects received written consent to donate samples in accordance with the Declaration of Helsinki.

2. Mouse

C57BL/6 mice were purchased from OrientBio, Inc. (Seoul, Korea) and maintained free of specific pathogens according to the guidelines of the Institute of Laboratory Animal Resources, Catholic University of Korea, Korea. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Catholic University of Korea, Korea. All animal experiments were performed according to the researcher's protocol previously approved by the Institutional Animal Care and Use Committee (Permission No. CUMC-2017-0273-05), Catholic University of Korea Medical School.

3. Isolation of exosomes

Human cord blood plasma was sequentially centrifuged at 400 g for 5 minutes and at 2,000 g for 10 minutes to remove cells and cell debris. Cord blood plasma was then filtered through a 0.45-μm polyvinylidene fluoride (PVDF) membrane (Nalgene™ Rochester, NY). Protein elution was checked by reading the absorbance of each fraction at 280 nm using a nanodrop spectrometer (Thermo Scientific, San Diego, CA). CBP was ultracentrifuged at 100,000 g for 2 hours, and cord blood plasma pellets were used for comparative experiments. As a control, plasma from adult blood was isolated and the same exosome isolation procedure was followed. All fractions were stored at 4°C and used within 24 hours for in vitro experiments or frozen at -80°C. CBPexos was obtained by continuously collecting CBP samples from a total of 10 healthy donors per batch. Exo-Check Exosome Antibody Array (System Biosciences, Palo Alto, CA) or PE-conjugated anti-human CD9 (e-Bioscience, San Diego, CA), anti-human CD63 (BD Biosciences, San Jowe, CA), or anti-human CD82 (Biolegend, San Diego, CA), or anti-human HSP70/HSP72 (Enzo Life Sciences, Farmingdale, NY) FACS antibodies were used to characterize the exosomes on the false batches. There was no difference in the properties of exosomes between batches.

4. Human Cord Blood Treg Expansion

Tregs were purified from 1 UCB unit (The Catholic Hematopoietic Stem Cell Bank) through positive selection using magnetic microbeads directly conjugated with anti-CD25 and a manual column (Miltenyi, Bergisch-Gladbach, Germany). CD25+ cells were cultured with a lentivirus-transduced K562 cell line (aAPC) expressing CD80, CD83, 4-1BBL and CD32 at a ratio of 2:1. aAPCs were irradiated with 10000 cGY and incubated with 0.5 μg/mL of anti-CD3 (OKT3) and anti-CD28 (CD28.2; both BD Pharmingen, San Diego, CA). Recombinant IL-2 (300 IU/mL; Chiron, Emeryville, CA) was added every 3 days and maintained for the incubation period. Cells were cultured for 14 days and split every 3-4 days.

5. LC-MS/MS analysis

Half of the digested samples were analyzed by nano LC-MS/MS using a Waters NanoAcquity HPLC system connected to a ThermoFisher Q Exactive HF mass spectrometer. The peptide was loaded onto the trapping column and eluted at 350 nL/min using a 2-h reversed-phase gradient on a 75 μm analytical column: both columns were packed with Luna C18 resin (Phenomenex). The mass spectrometer was operated in a data-dependent manner using Orbitrap operating at 60,000 FWHM and 17,500 FWHM for MS and MS/MS, respectively. The 15 most abundant ions were selected for MS/MS. Mascot DAT files were analyzed with Scaffold (Proteome Software) to validate, filter and generate an unknown list per sample. Data were filtered at 1% protein and peptide FDR, requiring at least 2 specific proteins per protein.

6. CBPexo mimic preparation

H1ME-5 cells (Null-293T (H1ME-5, accession number: KCTC 13602BP) were cultured in DMEM medium (Lonza) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin-streptomycin. T2 cells were cultured in RPMI-1640 medium (Lonza) supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin.

^{H1ME-5 cells were seeded at 2×10 6} cells/10 mL in DMEM without antibiotics (Lonza). After 24 hours, four targets GAL-3 (SEQ ID NO: 5), MMP-9 (SEQ ID NO: 1), HSP72 (SEQ ID NO: 4) S100A7 (SEQ ID NO: 6), GAL-7 ( A mixture of six individual all-in-one plasmids specific for each of SEQ ID NO: 2), and PIP (SEQ ID NO: 3) (NCBI reference sequence; self-made using pCDH vector) was combined with Lipofectamine reagent ( Invitrogen, Carlsbad, CA) was used to transfect 293T cells. 48 hours after transfection, cells were analyzed by flow cytometry. After 6 days of transfection, cells positive for GAL-3, MMP-9, HSP72, S100A7, GAL-7 and PIP were sorted using a Moflo XDP Cell Sorter (Beckman) and clones were established.

7. Proliferation Assay

Peripheral blood mononuclear cells (PBMC) or splenocytes (5Х10 ⁵ ) in culture medium were seeded in 96-well tissue culture plates supplemented with anti-CD3/CD28 DYNABEAD (Invitrogen, Oslo, Norway). CBPexo or ABPexo was added simultaneously to the appropriate wells. Cell proliferation was measured by 5,6-carboxyfluorescein diacetate-succinimidyl ester (CFSE) staining and flow cytometry. Briefly, 5Х10 ⁵ splenocytes or PBMCs were incubated with 5 μM CFSE (Molecular Probes, Eugene, OR, USA) in 1 mL of PBS at 37 °C for 10 min, followed by washing twice with ice-cold culture medium containing 10% FBS. did. Stained cells were stimulated with DYNABEAD as described above in the presence of CBPexo or ABPexo. After incubation for the indicated time, cells were acquired with a FACSCanto (Becton Dickinson Biosciences, Heidelberg, Germany) and data were analyzed with Modfit LT 3.0 software (Verity Software House, Topsham, ME). At least three independent experiments were performed to verify the immunosuppressive effect of CBPexo on activated PBMCs or splenocytes from 10 healthy donors.

8. Apoptosis Assay and Cell Cycle Assay

Apoptotic cells were stained with Annexin V and analyzed according to the manufacturer's instructions. Briefly, 1Х10 ⁶ cells were resuspended in 1 mL of annexin binding buffer. 5 μl of a working solution of APC-Annexin V or FITC-Annexin V (BD Biosciences, San Jose, CA, USA) and 7AAD (BD Biosciences, Catalog # 551076, San Jose, CA) was added to 5Х10 ⁵ cells in 100 μl. and incubated at 37° C., 5% CO ₂ for 15 minutes. Analysis data were analyzed using FlowJo v10.

PBMCs were stimulated with DNYNABEAD and incubated for 12-48 hours in 96-well flat bottom plates. Cells were harvested, fixed in 70% ethanol, and the cell pellet was stained with a solution of propidium iodide (PI; Sigma) supplemented with RNase A in PBS. DNA content was measured by FACS Canto (Becton Dickinson) and analyzed using ModFITLT 4.0 software to measure sub-G1, G1, S and G2 phases of the cell cycle.

9. protease control

To inhibit protease activity, 10 μg/mL of the MMP inhibitor GM6001 (Calbiochem, San Diego, USA) was added to CBPexo and incubated at 21° C. for 2 hours. GM6001 pretreated CBPexo was used for further experiments as indicated.

10. ELISPOT Analysis

ELISPOT analysis was performed according to the manufacturer's protocol. Briefly, monoclonal antibodies specific for human IL-2 (Catalog #551076, BD Biosciences), IFN-γ (Catalog #551083, BD Biosciences), IL-17 (Catalog #SEL421, R&D Systems) were microplate (Millipore, Billerica, MA). For in vitro experiments, cells cultured for 6 days were washed 3 times with medium, resuspended in 1 mL of medium before proceeding with ELISPOT analysis, and incubated overnight. For ex vivo experiments, MOG33-55 was added to 96-well microplates at a concentration ^{of 1Х10 6 cells/well in medium.} Microplates were incubated at 37° C., in a CO ₂ incubator for 20 hours. The microplate was then washed 4 times with wash buffer. The wells were then filled with 100 μl/well of biotinylated polyclonal anti-mouse IL-2, IFN-γ and IL-17. Plates were incubated at 21° C. for 2 hours, washed with buffer, and then incubated for 1 hour at ambient temperature in the presence of 100 μl/well of streptavidin-horseradish peroxidase (HRP). Unbound enzyme was then removed by washing and enzyme substrate solution was added. After the spot developed, distilled water was added to stop the reaction. The plate was inverted and dried overnight protected from light. The number of spots corresponding to IL-2, IFN-γ, IL-17 secreting cells was determined using an automatic AID-ELISPOT reader (AID Diagnostika GmbH, Strassberg, Germany).

11. Cytometric Bead Array (CBA) Human and Mouse Th1/Th2/Th17 cytokine kit Evaluation of Th1, Th2 and Th17 cytokines using

Human and Mouse Cytometric Bead Array (CBA) Th1/Th2/Th17 Cytokine Kits were purchased from BD Biosciences (Catalog #560484 and #560485). 50 μl of assay beads, 50 μl of detection reagent and 50 μl of study sample or standard were successively added to each sample tube and incubated at 21° C. for 3 hours in the dark. Next, the samples were washed with 1 mL of wash buffer and centrifuged at 500 Хg for 5 minutes. After discarding the supernatant, the pellet was resuspended in 300 μl of buffer and analyzed by flow cytometry on the same day.

12. EAE induction and exosome administration

Meningitis was treated in mice using MOG35-55 peptide in complete Freund's adjuvant (CFA) with pertussis toxin (PTx) as previously described (McCarthy, DP et al. Methods Mol Biol, 2012. 900: p.381-401). induced (n=5 per condition). After the onset of EAE symptoms, mice were scored daily, stratified, and assigned to separate test groups by two independent investigators to obtain an equally weighted mean disease score prior to experimental intervention (0, no signs of disease). ; 1, saggy tail; 2, saggy tail and partial hindlimb weakness; 3, complete hindlimb paralysis; 4,complete hindlimb and partial forelimb paralysis; 5, death). Exosomes were injected at weekly intervals in the “challenge state” at 100 μg/100 μl per mouse.

13 . statistical analysis

All experiments were performed at least 3 replicates per group, and in vitro experiments were repeated at least 3 times. Data are representative of these experiments and are plotted as mean ± standard deviation (SEM). Data were analyzed for statistical significance using Prism version 7.0 (GraphPad, San Diego, CA). Means of multiple groups were compared by one-way analysis of variance (ANOVA). An independent-sample t-test was used to compare the means of two different groups. Statistical analysis was performed using GraphPad Prism software, and P < 0.05 was considered statistically significant.

(1) Molecular properties of umbilical cord blood plasma-derived exosomes

Using flow cytometry, it was confirmed that CBPexo contains the typical exosome marker proteins CD9, CD63, CD82 and HSP72. These markers were absent in the bead-only control ( FIG. 1A ). In addition, the purity of these exosomes was assayed for exosome antibodies, including the well-characterized exosomal protein markers CD81, ICAM, CD63, ALIX, TSG101, EpCAM and FLOT-1, as well as the cis-Golgi marker GM130 for monitoring cellular contamination. confirmed using the kit. The Exo Anibody Array Kit consists of a positive (+) control, a standard exosomal protein, and a negative (-) control, a blank. The results obtained from densitometry analysis using the Exo Anibody Array Kit with ImageJ software were graphically expressed to indicate the positive expression of specific exosome markers. CBPexo was highly positive for FLOT1, ICAM, ALIX, CD81, TSG101, EpCAM and ANXA5, but the expression of CD63 was low. The isolated exosomes were free of cellular debris and other contaminants as evidenced by negative staining for GM130 (Fig. 1b).

(2) Comparison of proteomics profiles of human CBPexo and ABPexo

Liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis identified 150 proteins in CBPexo and 214 proteins in ABPexo, each with at least two mapping peptides per sequence. Of the 150 proteins of CBPexo, 11 proteins were divided into two subgroups (4 immunomodulatory proteins or 7 unrelated immunomodulatory proteins), whereas 19 immunomodulatory proteins were differentially expressed (Fig. 2a). Functional classification of ABPexo and CBPexo was performed using the ExoCarta databse (table on the right of Fig. 2a). The functional classification of ABPexo and CBPexo and further comparisons between them highlighted five different biological processes and molecular functions. Some of them are found exclusively within certain pathways, such as negative regulation of IL-6, cell differentiation, negative regulation of cell proliferation, transmembrane transport and wound healing (Fig. 2b). Focusing on the molecular functions of ABPexo and CBPexo, we discovered interesting functions related to metalloendopeptidase activity. CBPexo appears to be associated with arginase activity, heat shock protein binding, S100 protein binding and hyaluronic acid binding to a higher degree than ABPexo (Fig. 2c). Thus, proteomics analysis of CBPexo indicates that exosomal proteins are associated with immunosuppression and wound healing. Table 1 lists the immunomodulation-related proteins in CBPexo.

(3) Inhibitory effect of CBPexo on human T-cell proliferative response

As positive controls, rapamycin and cyclosporine inhibited immunosuppression in a concentration-dependent manner ( FIGS. 3A and 3B ). In this experimental example, the focus was mainly on the immunosuppression mechanism mediated by direct contact between CBPexo and immune cells. To address this, DYNABEAD anti-CD3/CD28 activated PBMCs were cultured with CBPexo or ABPexo to determine whether CBPexo had a potent immunosuppressive effect on activated immune cells. The immunosuppressive effect of CBPexo was investigated by CD4+ and CD8+ T cell proliferation.

As analyzed by CFSE dilution analysis, ABPexo did not, but CBPexo did not inhibit CD4+ and CD8+ T cell proliferation ( FIGS. 3A and 3B ).

To elucidate the mechanism underlying the inhibitory effect, the proportion of apoptotic cells and flow cytometry analysis were investigated in CBPexo or ABPexo-treated groups with Annexin V and 7AAD. In addition, it was confirmed whether CBPexo can affect the apoptosis of activated T cells.

As a result, PBMCs were activated by DYNABEAD in the presence of exosomes. Apoptosis of T cells was measured at 156 hours by flow cytometry. Annexin V and 7AAD double negative staining indicates live cells, and 7AAD and Annexin V positive staining indicates dead and apoptotic cells, respectively. As a result, the apoptosis rate in the CBPexo treatment group was increased compared to the ABPexo treatment group. However, compared to the positive control group, the exosome-treated group did not show any difference in the initial apoptosis period. Moreover, when comparing CD8+ T cell apoptosis, there was no difference between the CBPexo-treated group and the control group. These results show that apoptosis of CD4+ T cells is increased in CBPexo-treated PBMCs, but there is no significant effect on CD8+ T cells ( FIGS. 3c and 3d ).

From the above results, it was hypothesized that the decrease in the proliferative response could be due to the induction of cell cycle arrest. Therefore, cell cycle analysis was performed by measuring the DNA content of each cell group by flow cytometry after PI staining. PBMCs were stimulated with DYNABEAD in the presence of CBPexo or ABPexo, incubated for 156 h, and then stained with PI.

The ratio of cells in the G0/G1 phase showing apoptosis was higher in the CBPexo-treated group than in the control group, but this difference was not significant. Moreover, the frequency of cells in sub-G2/M and S phases was higher in the CBPexo-treated group than in the control group at 156 h ( FIG. 4 ). These results suggest that CBPexo reduces T cell proliferation by inducing G0/G1 cell cycle arrest along with apoptosis.

(4) Induction effect of immunosuppressive cell differentiation in vitro by CBPexo

From the above results, it was hypothesized that the inhibitory effect of CBPexo observed on T cells in the entire PBMC population may be due to the induction of immunosuppressive cells. As a result of the experiment, incubation with CBPexo increased the ratio of CD4+/CD25+/FoxP3+ cells in stimulated PBMCs on day 2 (Fig. 5a). However, CD25+ cells gated based on the proportion of CD4+ T cells were significantly downregulated in the presence of CBPexo at day 6, indicating that the IL-2 receptor alpha chain, a factor that downregulates CD25, is present in CBPexo; The present inventors have shown through previous references that MMP9 or MMP2 plays this role.

To further test the effect of CBPexo associated with regulatory T cells, CBPexo was co-cultured with cord blood-derived CD25+ cells, and the cells were incubated for 21 days prior to detection ^{of CD4+/CD25+/FoxP3+ and CD127 low cells.} Cells were effectively expanded by the combination of artificial antigen presenting cells and anti-CD3/CD28 and IL-2 (Fig. 5b). Treg and CBPexo-Treg cells were generated from the same 1 unit of umbilical cord blood, and increased 16.32-fold in CBPexo-Treg on day 17 after culture ( FIG. 5c ). Foxp3 expression increased expanded cord blood CD25+ cells in CBPexo-induced Tregs compared to their expression in nTregs. However, there was no difference in the expression of CD25 and CD127 on day 21 after CBPexo and Treg culture. Thus, CBPexo induces Foxp3 upregulation 21 days after Treg differentiation (Fig. 5d). To investigate whether signaling through CBPexo enhances the inhibitory properties of nTregs, we tested the immunosuppressive differences between nTregs and CBPexo-induced Tregs using CFSE staining. Moreover, CBPexo-derived Tregs were potent in almost all Treg:PBMC ratios tested. Inhibition of >50% was observed at a Tregs:PBMC ratio of 1:4, demonstrating the strong inhibitory ability of CBPexo-induced Tregs ( FIG. 5E ). Therefore, CBPexo can directly suppress the immunity of T cells, but it can also induce the differentiation of Tregs at an early stage and participate in the improvement of the function of cord blood Treg cells.

For characterization of the phenotype of these inhibitory monocytes in the PBMC population, cells treated with CBPexo for 24 h were stained to evaluate CD33, CD11b and CD14 (Fig. 6a). As a result, CBPexo promoted the expression of arginase-1, NOS2 and IDO in CD33+CD11b+CD14+ cells, a population of MDSCs ( FIG. 6b ). These results indicate that CBPexo not only directly participates in immunosuppression but also induces immunosuppressive cells.

(5) Restoration of T cell proliferation by inhibition of MMP expressed in CBPexo

We tried to identify factors in CBPexo that can inhibit T cell proliferation. CD25 is proteolytically cleaved by MMP-9 and shows an immunosuppressive effect on T cells. These zinc-dependent endopeptidases are essential factors for cell invasion through the extracellular matrix. To determine the presence of MMPs in CBPexo, various MMPs and TIMPs were assayed in CBPexo using a human MMP antibody assay.

As shown in FIG. 7a , despite the expression of the MMP inhibitory molecules TIMP1 and TIMP2, CBPexo significantly contained high concentrations of MMP9 and MMP8. Thus, it was speculated that CBPexo cleaves membrane-bound IL-2Rα (CD25) by MMP9 and inhibits the proliferation of activated T lymphocytes. To further confirm whether MMPs are involved in immunosuppression, CBPexo and GM6001, inhibitors of various MMPs, including MMP-1, -2, -3, -8 and -9, were incubated for 2 h prior to incubation with human T cells. did. Human CD4 + T cells cultured with GM6001-treated CBPexo restored cell proliferation (Fig. 7b).

In addition, CD4+ and CD8+ T cells cultured with the MMP inhibitor GM6001 (10 μg/mL)-treated CBPexo showed recovery of CD25 expression, but there was no difference in the activation marker CD69 ( FIG. 8 ). These results indicate that MMP-9 is mainly associated with the immunosuppression of CBPexo induced by inhibition of CD25 by MMP-9.

(6) Confirmation of immunosuppressive function of CBPexo in mouse system

CBPexo showed a strong immunosuppressive effect on mouse splenocytes and human PBMC. The immunosuppressive effect of CBPexo was tested by DYNABEAD-stimulated mouse CD4+ and CD8+ T cell proliferation. CBPexo inhibited CD4+ and CD8+ T cell proliferation, but not ABPexo, as analyzed by CFSE dilution assay ( FIGS. 9A-B ). Human CBPexo exhibited mouse cross-reactivity of proliferative T cell inhibition. Therefore, these results indicate that CBPexo is applicable to EAE, an animal model of autoimmune disease.

(7) Effect of CBPexo on IL-2 signaling and regulation of Th1 and Th2 cell-associated cytokines

^{Cells (1Х10 5} cells/well in 96-well culture plates) were cultured for 6 days with anti-CD3/CD28 to allow T cells to expand and respond to CBPexo. Cells in each well were washed and left to be used for ELISPOT analysis.

As a result, when co-cultured with CBPexo, the population of IL-2 and IFN-γ-secreting cells was significantly reduced, but there was no difference in L-17-secreting cells between the control group, CBPexo-treated group and ABPexo-treated group ( FIG. 9c ). . CBPexo showed immunosuppressive function by inhibiting IL-2 and IFN-γ secreting cells.

To assess cytokine secretion, levels of IL-2, IFN-γ and IL-6 were assessed using a cytometric bead assay. As a result, CBPexo down-regulated human IL-2, IFN-γ and IL-6 associated with the differentiation of Th1 and Th17 cells and the development of autoimmune diseases ( FIG. 9D ). However, as a result of evaluation of the culture supernatant, only the concentration of IL-2 was significantly reduced when incubated with CBPexo in mouse T cells (FIG. 9e). These data indicate that inhibition of IL-2 production by CBPexo is an important mechanism of T cell inhibition in both humans and mice.

(8) Effect of CBPexo on IL-2 cytokine level regulation and induction of mutual regulation of Th1 and Th17 cell differentiation to alleviate EAE symptoms

As shown in FIG. 10A , the clinical score of the CBPexo-treated EAE group was significantly lower than that of the ABPexo-treated EAE group and the EAE control group. These results show that CBPexo can alleviate autoimmune symptoms. To determine whether CBPexo has an immunomodulatory function in helper T cells, the expression level of cytokines in splenocytes was measured by ELISPOT analysis. The frequency of cells secreting MOG peptide-specific IFN-γ, IL-2 and IL-17 was determined by ELISPOT assay compared among the EAE control group, CBPexo-treated EAE group and ABPexo-treated EAE group.

As a result, MOG peptide-specific IL-2 cells were significantly reduced in the CBPeox-treated EAE group, but there was no difference in the cells secreting IFN-γ and IL-17, similar to the in vitro analysis results (Fig. 10b).

Donor CD4+ T cells differentiate from each other into Th1, Th2 and Th17 cells, which mediate the balance of EAE and Th subsets, playing an important role in regulating the T cell immune response. Therefore, their ability to reduce Th1, Th2 and Th17 cell-associated cytokines by CBPexo was further investigated. To confirm this, cytokine levels were evaluated using Th1, Th2 and Th17 cytometric bead assays. As a result of evaluating the concentration of the secreted protein, CBPexo administration induced a decrease in the levels of IFN-γ, IL-2, IL-6, IL-17 and IL-4 compared to the EAE group ( FIG. 10c ).

Intracellular staining was performed to compare the cytokine profiles of Th1, Th2, Th17 and IL-2 in the EAE model. As a result, the ratio of IL-2 was reduced two-fold, and IFN-γ and IL-17 were decreased by 1-4% in the CBPexo-treated group compared to the ABPexo-treated group ( FIG. 11 ). These results demonstrate that CBPexo administration modulates IL-2 signaling and induces mutual regulation of Th1 and Th17 cell differentiation to have a protective effect against EAE.

(9) Effect of CBPexo on induction of immune regulatory cell differentiation in EAE model

The frequency of CD4+CD25+FOXP3+ Treg cells was measured, and the untreated control (negative control), EAE control (positive control), CBPexo-treated EAE group and ABPexo-treated EAE group were compared.

As a result, the Treg population was increased in the CBPexo-treated EAE group compared to the control group (FIGS. 12a-b). These results demonstrate that CBPexo not only inhibits IL-2 signaling, but also increases the CD4+CD25+FOXP3+ Treg population in vivo, demonstrating their immunosuppressive effect. The phenotype of CD11b+Gr-1+ cells (mouse MDSCs) expanded during EAE disease progression in CBPexo-injected mice ( FIG. 13 ). Enriched and accumulated MDSCs in splenocytes of CBPexo-injected EAE mice play a regulatory role.

(10) Effects of CBPexo acting locally and systemically in the EAE model

To confirm the effect of CBPexo acting locally or systemically, CBPexo or ABPexo labeled with PKH67 was injected into the tail vein of EAE mice. The EAE model can be localized to the brain or co-occurring throughout the body. Therefore, the cell nucleus of the brain, which is the most important organ in disease development, and the spleen tissue, which is the most important lymph organ acting on the whole body, were stained with DAPI, and CBPexo labeled with PKH67 was detected in both the spleen and brain tissue (FIG. 14).

As a result of comparing the number of cell infiltration and the area of inflammatory cells in the brains of EAE mice, EAE mice, and normal mice treated with CBPexo or ABPexo through histopathological examination, the cell infiltration and area of inflammatory cells were reduced in the CBPexo-administered group. (Fig. 15).

<Example 2> Preparation of artificial exosomes (exosome mimics) and immunosuppressive effect

By focusing on the molecular function of CBPexo, we found interesting functions associated with MMP-9, GAL-3, HSP72, S100A7, GAL-7 and PIP associated with immune regulation ( FIGS. 2 and 7 ). MICA induces natural killer cell proliferation, and MHC-I deficiency jeopardizes immune activation. Therefore, H1ME-5 cells can be silenced using the CRISPR/cas9 system to create cells without immune-activating effects. HEK 293T cells have several advantages, including high transfection efficiency and homologous haploids of HLA class I (A*02:01, B*07:02 and C*07:02) and MICA/B. For complete removal of HLA class I and MICA/B genes, seven plasmids encoding Cas9 proteins and gRNAs targeting exons 2 and 3 of HLAA-A, HLA-B, HLA-C and MICA/B genes was used (see Hong, CH, et al., J Immunother, 2017. 40(6): p. 201-210). These H1ME-5 cells exhibiting a lack of expression of HLA class I and MICA/B were analyzed by flow cytometry ( FIG. 16A ), encoding the GAL-3, MMP-9, HS672, S100A7, GAL-7 and PIP genes. was transduced with a lentivirus. After 6 days of transduction, GAL-3, MMP-9, HSP72, S100A7, GAL-7 and PIP-positive cells were sorted.

The size distribution of the exosomes released from H1ME-5 was characterized using DLS, and the observed particle size of the exosomes was 145.61±75.89 nm ( FIG. 16b ).

As a result of flow cytometry analysis, more than 90% of H1ME-5 cells expressed GAL-3, MMP-9, HSP72, S100A7, GAL-7 and PIP (FIG. 16c). In addition, exosomes derived from this cell line expressed target molecules GAL-3, MMP-9, HSP72, S100A7, GAL-7 and PIP (FIG. 16d).

Among the various CBPexo mimetics, the exosomes expressing MMP-9 and HSP72 showed a significant immunosuppressive effect. was shown (FIGS. 16e-f). HSP72 increases the production of MMP-9. Thus, these data indicate that MMP-9 plays an important role among various immunosuppressive molecules present in CBPexo.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Exosomes derived from umbilical cord blood plasma or their mimics and imunosuppression use of them <130> P21U11C0247 <150> KR 10-2020-0033254 <151> 2020-03-18 <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 2387 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens matrix metallopeptidase 9 (MMP9) <400> 1 agacacctct gccctcacca tgagcctctg gcagcccctg gtcctggtgc tcctggtgct 60 gggctgctgc tttgctgccc ccagacagcg ccagtccacc cttgtgctct tccctggaga 120 cctgagaacc aatctcaccg acaggcagct ggcagaggaa tacctgtacc gctatggtta 180 cactcgggtg gcagagatgc gtggagagtc gaaatctctg gggcctgcgc tgctgcttct 240 ccagaagcaa ctgtccctgc ccgagaccgg tgagctggat agcgccacgc tgaaggccat 300 gcgaacccca cggtgcgggg tcccagacct gggcagattc caaacctttg agggcgacct 360 caagtggcac caccacaaca tcacctattg gatccaaaac tactcggaag acttgccgcg 420 ggcggtgatt gacgacgcct ttgcccgcgc cttcgcactg tggagcgcgg tgacgccgct 480 caccttcact cgcgtgtaca gccgggacgc agacatcgtc atccagtttg gtgtcgcgga 540 gcacggagac gggtatccct tcgacgggaa ggacgggctc ctggcacacg cctttcctcc 600 tggccccggc attcagggag acgcccattt cgacgatgac gagttgtggt ccctgggcaa 660 gggcgtcgtg gttccaactc ggtttggaaa cgcagatggc gcggcctgcc acttcccctt 720 catcttcgag ggccgctcct actctgcctg caccaccgac ggtcgctccg acggcttgcc 780 ctggtgcagt accacggcca actacgacac cgacgaccgg tttggcttct gccccagcga 840 gagactctac acccaggacg gcaatgctga tgggaaaccc tgccagtttc cattcatctt 900 ccaaggccaa tcctactccg cctgcaccac ggacggtcgc tccgacggct accgctggtg 960 cgccaccacc gccaactacg accgggacaa gctcttcggc ttctgcccga cccgagctga 1020 ctcgacggtg atggggggca actcggcggg ggagctgtgc gtcttcccct tcactttcct 1080 gggtaaggag tactcgacct gtaccagcga gggccgcgga gatgggcgcc tctggtgcgc 1140 taccacctcg aactttgaca gcgacaagaa gtggggcttc tgcccggacc aaggatacag 1200 tttgttcctc gtggcggcgc atgagttcgg ccacgcgctg ggcttagatc attcctcagt 1260 gccggaggcg ctcatgtacc ctatgtaccg cttcactgag gggcccccct tgcataagga 1320 cgacgtgaat ggcatccggc acctctatgg tcctcgccct gaacctgagc cacggcctcc 1380 aaccaccacc acaccgcagc ccacggctcc cccgacggtc tgccccaccg gaccccccac 1440 tgtccacccc tcagagcgcc ccacagctgg ccccacaggt cccccctcag ctggccccac 1500 aggtcccccc actgctggcc cttctacggc cactactgtg cctttgagtc cggtggacga 1560 tgcctgcaac gtgaacatct tcgacgccat cgcggagatt gggaaccagc tgtatttgtt 1620 caaggatggg aagtactggc gattctctga gggcaggggg agccggccgc agggcccctt 1680 ccttatcgcc gacaagtggc ccgcgctgcc ccgcaagctg gactcggtct ttgaggagcg 1740 gctctccaag aagcttttct tcttctctgg gcgccaggtg tgggtgtaca caggcgcgtc 1800 ggtgctgggc ccgaggcgtc tggacaagct gggcctggga gccgacgtgg cccaggtgac 1860 cggggccctc cggagtggca gggggaagat gctgctgttc agcgggcggc gcctctggag 1920 gttcgacgtg aaggcgcaga tggtggatcc ccggagcgcc agcgaggtgg accggatgtt 1980 ccccggggtg cctttggaca cgcacgacgt cttccagtac cgagagaaag cctatttctg 2040 ccaggaccgc ttctactggc gcgtgagttc ccggagtgag ttgaaccagg tggaccaagt 2100 gggctacgtg acctatgaca tcctgcagtg ccctgaggac tagggctccc gtcctgcttt 2160 ggcagtgcca tgtaaatccc cactgggacc aaccctgggg aaggagccag tttgccggat 2220 acaaactggt attctgttct ggaggaaagg gaggagtgga ggtgggctgg gccctctctt 2280 ctcacctttg ttttttgttg gagtgtttct aataaacttg gattctctaa cctttaaaaa 2340 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 2387 <210> 2 <211> 515 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens galectin 7 (LGALS7) <400> 2 acggctgccc aacccggtcc cagccatgtc caacgtcccc cacaagtcct cactgcccga 60 gggcatccgc cctggcacgg tgctgagaat tcgcggcttg gttcctccca atgccagcag 120 gttccatgta aacctgctgt gcggggagga gcagggctcc gatgccgcgc tgcatttcaa 180 cccccggctg gacacgtcgg aggtggtctt caacagcaag gagcaaggct cctggggccg 240 cgaggagcgc gggccgggcg ttcctttcca gcgcgggcag cccttcgagg tgctcatcat 300 cgcgtcagac gacggcttca aggccgtggt tggggacgcc cagtaccacc acttccgcca 360 ccgcctgccg ctggcgcgcg tgcgcctggt ggaggtgggc ggggacgtgc agctggactc 420 cgtgaggatc ttctgagcag aagcccaggc gggcccgggg ccttggctgg caaataaagc 480 gttagcccgc agcgaaaaaa aaaaaaaaaa aaaaa 515 <210> 3 <211> 618 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens prolactin-induced protein (PIP) <400> 3 ggggaccact tctctgggac acattgcctt ctgttttctc cagcatgcgc ttgctccagc 60 tcctgttcag ggccagccct gccaccctgc tcctggttct ctgcctgcag ttgggggcca 120 acaaagctca ggacaacact cggaagatca taataaagaa ttttgacatt cccaagtcag 180 tacgtccaaa tgacgaagtc actgcagtgc ttgcagttca aacagaattg aaagaatgca 240 tggtggttaa aacttacctc attagcagca tccctctaca aggtgcattt aactataagt 300 atactgcctg cctatgtgac gacaatccaa aaaccttcta ctgggacttt tacaccaaca 360 gaactgtgca aattgcagcc gtcgttgatg ttattcggga attaggcatc tgccctgatg 420 atgctgctgt aatccccatc aaaaacaacc ggttttatac tattgaaatc ctaaaggtag 480 aataatggaa gccctgtctg tttgccacac ccaggtgatt tcctctaaag aaacttggct 540 ggaatttctg ctgtggtcta taaaataaac ttcttaacat gcttctacaa aaaaaaaaaa 600 aaaaaaaaaa aaaaaaaa 618 <210> 4 <211> 2802 <212> DNA <213> Artificial Sequence <220> <223> Homo sapiens heat shock protein family A <400> 4 ccagcagcag gaggcgcgcg aggcaccacg gcctggcggc cgagagtcag ggaggaacct 60 catttacata acggccgccc ctctgtctcc tggcgggggc cggagtcccg cccctcgtcc 120 aacttgaaat ctgttgggtc acgggccagt cactccgacc taggcaagcc tgtggtggag 180 ctggaagagt ttgtgagggc ggtcccggga gcggattggg tctgggagtt cccagaggcg 240 gctataagaa ccgggaactg ggcgcgggga gctgagttgc tggtagtgcc cgtggtgctt 300 ggttcgaggt ggccgttagt tgactccgcg gagttcatct ccctggtttt cccgtcctaa 360 cgtcgctcgc ctttcagtca ggatgtctgc ccgtggcccg gctatcggca tcgacctggg 420 caccacctat tcgtgcgtcg gggtcttcca acatggcaag gtggagatca tcgccaacga 480 ccagggcaat cgcaccaccc ccagctacgt ggccttcacg gacaccgagc gcctcatcgg 540 cgacgccgcc aagaaccagg tggccatgaa ccccaccaac accatcttcg acgccaagag 600 gctgattgga cggaaattcg aggatgccac agtgcagtcg gatatgaaac actggccgtt 660 ccgggtggtg agcgagggag gcaagcccaa agtgcaagta gagtacaagg gggagaccaa 720 gaccttcttc ccagaggaga tatcctccat ggtcctcacg aagatgaagg agatcgcgga 780 agcctacctg gggggcaagg tgcacagcgc ggtcataacg gtcccggcct atttcaacga 840 ctcgcagcgc caggccacca aggacgcagg caccatcacg gggctcaatg tgctgcgcat 900 catcaacgag cccacggcgg cggccatcgc ctacggcctg gacaagaagg gctgcgcggg 960 cggcgagaag aacgtgctca tctttgacct gggcggtggc actttcgacg tgtccatcct 1020 gaccatcgag gatggcatct tcgaggtgaa gtccacggcc ggcgacaccc acctgggcgg 1080 tgaggacttc gacaaccgca tggtgagcca cctggcggag gagttcaagc gcaagcacaa 1140 gaaggacatt gggcccaaca agcgcgccgt gaggcggctg cgcaccgctt gcgagcgcgc 1200 caagcgcacc ctgagctcgt ccacgcaggc gagcatcgag atcgactcgc tctacgaggg 1260 cgtggacttc tatacgtcca tcacgcgcgc ccgcttcgag gagctcaatg ccgacctctt 1320 tcgcgggacc ctggagccgg tggagaaggc gctgcgcgac gccaagctgg acaagggcca 1380 gatccaggag atcgtgctgg tgggcggctc cactcgtatc cccaagatcc agaagctgct 1440 gcaggatttc ttcaacggca aggagctgaa caagagcatc aaccccgacg aggcggtggc 1500 ctatggcgcc gcggtgcagg cggccatcct catcggcgac aaatcagaga atgtgcagga 1560 cctgctgcta ctcgacgtga ccccgttgtc gctgggcatc gagacagctg gcggtgtcat 1620 gaccccactc atcaagagga acaccacgat ccccaccaag cagacgcaga ccttcaccac 1680 ctactcggac aaccagagca gcgtactggt gcaggtatac gagggcgaac gggccatgac 1740 caaggacaat aacctgctgg gcaagttcga cctgaccggg attccccctg cgcctcgcgg 1800 ggtcccccaa atcgaggtta ccttcgacat tgacgccaat ggcatcctta acgttaccgc 1860 cgccgacaag agcaccggta aggaaaacaa aatcaccatc accaatgaca aaggtcgtct 1920 gagcaaggac gacattgacc ggatggtgca ggaggcggag cggtacaaat cggaagatga 1980 ggcgaatcgc gaccgagtcg cggccaaaaa cgccctggag tcctatacct acaacatcaa 2040 gcagacggtg gaagacgaga aactgagggg caagattagc gagcaggaca aaaacaagat 2100 cctcgacaag tgtcaggagg tgatcaactg gctcgaccga aaccagatgg cagagaaaga 2160 tgagtatgaa cacaagcaga aagagctcga aagagtttgc aaccccatca tcagcaaact 2220 ttaccaaggt ggtcctggcg gcggcagcgg cggcggcggt tcaggagcct ccggggggacc 2280 caccatcgaa gaagtggact aagcttgcac tcaagtcagc gtaaacctct ttgcctttct 2340 ctctctctct ttttttttgt ttgtttcttt gaaatgtcct tgtgccaagt acgagatcta 2400 ttgttggaag tctttggtat atgcaaatga aaggagaggt gcaacaactt agtttaatta 2460 taaaagttcc aaagtttgtt ttttaaaaac attattcgag gtttctcttt aatgcatttt 2520 gcgtgtttgc tgacttgagc atttttgatt agttcgtgca tggagatttg tttgagatga 2580 gaaaccttaa gtttgcacac ctgttctgta gaagcttgga aacagtaaaa tatataggag 2640 cttaaattgt ttatttttat gtactacttt aaaactaaac tgaacattgc agtaatgtta 2700 aggacaggta tactttttgc aaacaaatgc ataaatgcaa atgtaaagta aagctgaaat 2760 tgatctcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 2802 <210> 5 <211> 1017 <212> DNA <213> Artificial Sequence <220> <223> galectin 3 (LGALS3) <400> 5 gagtatttga ggctcggagc caccgccccg ccggcgcccg cagcacctcc tcgccagcag 60 ccgtccggag ccagccaacg agcggaaaat ggcagacaat ttttcgctcc atgatgcgtt 120 atctgggtct ggaaacccaa accctcaagg atggcctggc gcatggggga accagcctgc 180 tggggcaggg ggctacccag gggcttccta tcctggggcc taccccgggc aggcaccccc 240 aggggcttat cctggacagg cacctccagg cgcctaccct ggagcacctg gagcttatcc 300 cggagcacct gcacctggag tctacccagg gccacccagc ggccctgggg cctacccatc 360 ttctggacag ccaagtgcca ccggagccta ccctgccact ggcccctatg gcgcccctgc 420 tgggccactg attgtgcctt ataacctgcc tttgcctggg ggagtggtgc ctcgcatgct 480 gataacaatt ctgggcacgg tgaagcccaa tgcaaacaga attgctttag atttccaaag 540 agggaatgat gttgccttcc actttaaccc acgcttcaat gagaacaaca ggagagtcat 600 tgtttgcaat acaaagctgg ataataactg gggaagggaa gaaagacagt cggttttccc 660 atttgaaagt gggaaaccat tcaaaataca agtactggtt gaacctgacc acttcaaggt 720 tgcagtgaat gatgctcact tgttgcagta caatcatcgg gttaaaaaac tcaatgaaat 780 cagcaaactg ggaatttctg gtgacataga cctcaccagt gcttcatata ccatgatata 840 atctgaaagg ggcagattaa aaaaaaaaaa agaatctaaa ccttacatgt gtaaaggttt 900 catgttcact gtgagtgaaa atttttacat tcatcaatat ccctcttgta agtcatctac 960 ttaataaata ttacagtgaa ttacctgtct caatatgtca aaaaaaaaaa aaaaaaa 1017 <210> 6 <211> 450 <212> DNA <213> Artificial Sequence <220> <223> S100 calcium binding protein A <400> 6 gtccaaacac acacatctca ctcatccttc tactcgtgac gcttcccagc tctggctttt 60 tgaaagcaaa gatgagcaac actcaagctg agaggtccat aataggcatg atcgacatgt 120 ttcacaaata caccagacgt gatgacaaga ttgagaagcc aagcctgctg acgatgatga 180 aggagaactt ccccaacttc cttagtgcct gtgacaaaaa gggcacaaat tacctcgccg 240 atgtctttga gaaaaaggac aagaatgagg ataagaagat tgatttttct gagtttctgt 300 ccttgctggg agacatagcc acagactacc acaagcagag ccatggagca gcgccctgtt 360 ccgggggcag ccagtgaccc agccccacca atgggcctcc agagacccca ggaacaataa 420 aatgtcttct cccaccagaa aaaaaaaaaa 450 <210> 7 <211> 1254 <212> DNA <213> Artificial Sequence <220> <223> Lysosome-associated membrane glycoprotein 1 <400> 7 atggcggccc ccggcagcgc ccggcgaccc ctgctgctgc tactgctgtt gctgctgctc 60 ggcctcatgc attgtgcgtc agcagcaatg tttatggtga aaaatggcaa cgggaccgcg 120 tgcataatgg ccaacttctc tgctgccttc tcagtgaact acgacaccaa gagtggccct 180 aagaacatga cctttgacct gccatcagat gccacagtgg tgctcaaccg cagctcctgt 240 ggaaaagaga acacttctga ccccagtctc gtgattgctt ttggaagagg acatacactc 300 actctcaatt tcacgagaaa tgcaacacgt tacagcgtcc agctcatgag ttttgtttat 360 aacttgtcag acacacacct tttccccaat gcgagctcca aagaaatcaa gactgtggaa 420 tctataactg acatcagggc agatatagat aaaaaataca gatgtgttag tggcacccag 480 gtccacatga acaacgtgac cgtaacgctc catgatgcca ccatccaggc gtacctttcc 540 aacagcagct tcagccgggg agagacacgc tgtgaacaag acaggccttc cccaaccaca 600 gcgccccctg cgccacccag cccctcgccc tcacccgtgc ccaagagccc ctctgtggac 660 aagtacaacg tgagcggcac caacgggacc tgcctgctgg ccagcatggg gctgcagctg 720 aacctcacct atgagaggaa ggacaacacg acggtgacaa ggcttctcaa catcaacccc 780 aacaagacct cggccagcgg gagctgcggc gcccacctgg tgactctgga gctgcacagc 840 gagggcacca ccgtcctgct cttccagttc gggatgaatg caagttctag ccggtttttc 900 ctacaaggaa tccagttgaa tacaattctt cctgacgcca gagaccctgc ctttaaagct 960 gccaacggct ccctgcgagc gctgcaggcc acagtcggca attcctacaa gtgcaacgcg 1020 gaggagcacg tccgtgtcac gaaggcgttt tcagtcaata tattcaaagt gtgggtccag 1080 gctttcaagg tggaaggtgg ccagtttggc tctgtggagg agtgtctgct ggacgagaac 1140 agcatgctga tccccatcgc tgtgggtggt gccctggcgg ggctggtcct catcgtcctc 1200 atcgcctacc tcgtcggcag gaagaggagt cacgcaggct accagactat ctag 1254 <210> 8 <211> 1278 <212> DNA <213> Artificial Sequence <220> <223> Serpin B12 <400> 8 atggactctc ttgttacagc aaacaccaaa ttttgctttg atctttttca agagataggc 60 aaagatgatc gtcataaaaa catatttttc tctcccctga gcctctcagc tgcccttggt 120 atggtacgct tgggtgctag aagtgacagt gcacatcaga ttgatgaggt actacacttc 180 aacgaatttt cccagaatga aagcaaagaa cctgaccctt gtctgaaaag caacaaacaa 240 aaagtgctgg ctgacagctc tctggagggg cagaaaaaaa cgacagagcc tctggatcag 300 caggctgggt ccttaaacaa tgagagcgga ctggtcagct gctactttgg gcagcttctc 360 tccaaattag acaggatcaa gactgattac acactgagta ttgccaacag gctttatgga 420 gagcaggaat tcccaatctg tcaggaatac tagatggtg tgattcaatt ttaccacacg 480 acgattgaaa gtgttgattt ccaaaaaaac cctgaaaaat ccagacaaga gattaacttc 540 tgggttgaat gtcaatccca aggtaaaatc aaggaactct tcagcaagga cgctattaat 600 gctgagactg tgctggtact ggtgaatgct gtttacttca aggccaaatg ggaaacatac 660 tttgaccatg aaaacacggt ggatgcacct ttctgtctaa atgcgaatga aaacaagagt 720 gtgaagatga tgacgcaaaa aggcctctac agaattggct tcatagagga ggtgaaggca 780 cagatcctgg aaatgaggta caccaagggg aagctcagca tgttcgtgct gctgccatct 840 cactctaaag ataacctgaa gggtctggaa gagcttgaaa ggaaaatcac ctatgaaaaa 900 atggtggcct ggagcagctc agaaaacatg tcagaagaat cggtggtcct gtccttcccc 960 cggttcaccc tggaagacag ctatgatctc aattccattt tacaagacat gggcattacg 1020 gatatctttg atgaaacgag ggctgatctt actggaatct ctccaagtcc caatttgtac 1080 ttgtcaaaaa ttatccacaa aacctttgtg gaggtggatg aaaacggtac ccaggcagct 1140 gcagccactg gggctgttgt ctcggaaagg tcactacgat cttgggtgga gtttaatgcc 1200 aaccaccctt ttctcttttt cattagacac aacaaaaccc aaaccattct cttttatggc 1260 agggtctgct ctccttaa 1278 <210> 9 <211> 2132 <212> DNA <213> Artificial Sequence <220> <223> Lactotransferrin <400> 9 tgaaacttgt cttcctcgtc ctgctgttcc tcggggccct cggactgtgt ctggctggcc 60 gtaggaggag tgttcagtgg tgcgccgtat cccaacccga ggccacaaaa tgcttccaat 120 ggcaaaggaa tatgagaaaa gtgcgtggcc ctcctgtcag ctgcataaag agagactccc 180 ccatccagtg tatccaggcc attgcggaaa acagggccga tgctgtgacc cttgatggtg 240 gtttcatata cgaggcaggc ctggccccct acaaactgcg acctgtagcg gcggaagtct 300 acgggaccga aagacagcca cgaactcact attatgccgt ggctgtggtg aagaagggcg 360 gcagctttca gctgaacgaa ctgcaaggtc tgaagtcctg ccacacaggc cttcgcagga 420 ccgctggatg gaatgtccct atagggacac ttcgtccatt cttgaattgg acgggtccac 480 ctgagcccat tgaggcagct gtggccaggt tcttctcagc cagctgtgtt cccggtgcag 540 ataaaggaca gttccccaac ctgtgtcgcc tgtgtgcggg gacaggggaa aacaaatgtg 600 ccttctcctc ccaggaaccg tacttcagct actctggtgc cttcaagtgt ctgagagacg 660 gggctggaga cgtggctttt atcagagaga gcacagtgtt tgaggacctg tcagacgagg 720 ctgaaaggga cgagtatgag ttactctgcc cagacaacac tcggaagcca gtggacaagt 780 tcaaagactg ccatctggcc cgggtccctt ctcatgccgt tgtggcacga agtgtgaatg 840 gcaaggagga tgccatctgg aatcttctcc gccaggcaca ggaaaagttt ggaaaggaca 900 agtcaccgaa attccagctc tttggctccc ctagtgggca gaaagatctg ctgttcaagg 960 actctgccat tgggttttcg agggtgcccc cgaggataga ttctgggctg taccttggct 1020 ccggctactt cactgccatc cagaacttga ggaaaagtga ggaggaagtg gctgcccggc 1080 gtgcgcgggt cgtgtggtgt gcggtgggcg agcaggagct gcgcaagtgt aaccagtgga 1140 gtggcttgag cgaaggcagc gtgacctgct cctcggcctc caccacagag gactgcatcg 1200 ccctggtgct gaaaggagaa gctgatgcca tgagtttgga tggaggatat gtgtacactg 1260 caggcaaatg tggtttggtg cctgtcctgg cagagaacta caaatcccaa caaagcagtg 1320 accctgatcc taactgtgtg gatagacctg tggaaggata tcttgctgtg gcggtggtta 1380 ggagatcaga cactagcctt acctggaact ctgtgaaagg caagaagtcc tgccacaccg 1440 ccgtggacag gactgcaggc tggaatatcc ccatgggcct gctcttcaac cagacgggct 1500 cctgcaaatt tgatgaatat ttcagtcaaa gctgtgcccc tgggtctgac ccgagatcta 1560 atctctgtgc tctgtgtatt ggcgacgagc agggtgagaa taagtgcgtg cccaacagca 1620 acgagagata ctacggctac actggggctt tccggtgcct ggctgagaat gctggagacg 1680 ttgcatttgt gaaagatgtc actgtcttgc agaacactga tggaaataac aatgaggcat 1740 gggctaagga tttgaagctg gcagactttg cgctgctgtg cctcgatggc aaacggaagc 1800 ctgtgactga ggctagaagc tgccatcttg ccatggcccc gaatcatgcc gtggtgtctc 1860 ggatggataa ggtggaacgc ctgaaacagg tgttgctcca ccaacaggct aaatttggga 1920 gaaatggatc tgactgcccg gacaagtttt gcttattcca gtctgaaacc aaaaaccttc 1980 tgttcaatga caacactgag tgtctggcca gactccatgg caaaacaaca tatgaaaaat 2040 atttgggacc acagtatgtc gcaggcatta ctaatctgaa aaagtgctca acctcccccc 2100 tcctggaagc ctgtgaattc ctcaggaagt aa 2132 <210> 10 <211> 606 <212> DNA <213> Artificial Sequence <220> <223> Alpha-1-acid glycoprotein 1 <400> 10 atggcgctgt cctgggttct tacagtcctg agcctcctac ctctgctgga agcccagatc 60 ccattgtgtg ccaacctagt accggtgccc atcaccaacg ccaccctgga ccggatcact 120 ggcaagtggt tttatatcgc atcggccttt cgaaacgagg agtacaataa gtcggttcag 180 gagatccaag caaccttctt ttacttcacc cccaacaaga cagaggacac gatctttctc 240 agagagtacc agacccgaca ggaccagtgc atctataaca ccacctacct gaatgtccag 300 cgggaaaatg ggaccatctc cagatacgtg ggaggccaag agcatttcgc tcacttgctg 360 atcctcaggg acaccaagac ctacatgctt gcttttgacg tgaacgatga gaagaactgg 420 gggctgtctg tctatgctga caagccagag acgaccaagg agcaactggg agagttctac 480 gaagctctcg actgcttgcg cattcccaag tcagatgtcg tgtacaccga ttggaaaaag 540 gataagtgtg agccactgga gaagcagcac gagaaggaga ggaaacagga ggagggggaa 600 tcctag 606 <210> 11 <211> 1044 <212> DNA <213> Artificial Sequence <220> <223> CD5 antigen-like <400> 11 atggctctgc tattctcctt gatccttgcc atttgcacca gacctggatt cctagcgtct 60 ccatctggag tgcggctggt ggggggcctc caccgctgtg aagggcgggt ggaggtggaa 120 cagaaaggcc agtggggcac cgtgtgtgat gacggctggg acattaagga cgtggctgtg 180 ttgtgccggg agctgggctg tggagctgcc agcggaaccc ctagtggtat tttgtatgag 240 ccaccagcag aaaaagagca aaaggtcctc atccaatcag tcagttgcac aggaacagaa 300 gatacattgg ctcagtgtga gcaagaagaa gtttatgatt gttcacatga tgaagatgct 360 ggggcatcgt gtgagaaccc agagagctct ttctccccag tcccagaggg tgtcaggctg 420 gctgacggcc ctgggcattg caagggacgc gtggaagtga agcaccagaa ccagtggtat 480 accgtgtgcc agacaggctg gagcctccgg gccgcaaagg tggtgtgccg gcagctggga 540 tgtgggaggg ctgtactgac tcaaaaacgc tgcaacaagc atgcctatgg ccgaaaaccc 600 atctggctga gccagatgtc atgctcagga cgagaagcaa cccttcagga ttgcccttct 660 gggccttggg ggaagaacac ctgcaaccat gatgaagaca cgtgggtcga atgtgaagat 720 ccctttgact tgagactagt aggaggagac aacctctgct ctgggcgact ggaggtgctg 780 cacaagggcg tatggggctc tgtctgtgat gacaactggg gagaaaagga ggaccaggtg 840 gtatgcaagc aactgggctg tgggaagtcc ctctctccct ccttcagaga ccggaaatgc 900 tatggccctg gggttggccg catctggctg gataatgttc gttgctcagg ggaggagcag 960 tccctggagc agtgccagca cagattttgg gggtttcacg actgcaccca ccaggaagat 1020 gtggctgtca tctgctcagg atag 1044 <210> 12 <211> 5235 <212> DNA <213> Artificial Sequence <220> <223> Complement C4-B <400> 12 atgaggctgc tctgggggct gatctgggca tccagcttct tcaccttatc tctgcagaag 60 cccaggttgc tcttgttctc tccttctgtg gttcatctgg gggtccccct atcggtgggg 120 gtgcagctcc aggatgtgcc ccgaggacag gtagtgaaag gatcagtgtt cctgagaaac 180 ccatctcgta ataatgtccc ctgctcccca aaggtggact tcacccttag ctcagaaaga 240 gacttcgcac tcctcagtct ccaggtgccc ttgaaagatg cgaagagctg tggcctccat 300 caactcctca gaggccctga ggtccagctg gtggcccatt cgccatggct aaaggactct 360 ctgtccagaa cgacaaacat ccagggtatc aacctgctct tctcctctcg ccgggggcac 420 ctctttttgc agacggacca gcccatttac aaccctggcc agcgggttcg gtaccgggtc 480 tttgctctgg atcagaagat gcgcccgagc actgacacca tcacagtcat ggtggagaac 540 tctcacggcc tccgcgtgcg gaagaaggag gtgtacatgc cctcgtccat cttccaggat 600 gactttgtga tcccagacat ctcagagcca gggacctgga agatctcagc ccgattctca 660 gatggcctgg aatccaacag cagcacccag tttgaggtga agaaatatgt ccttcccaac 720 tttgaggtga agatcacccc tggaaagccc tacatcctga cggtgccagg ccatcttgat 780 gaaatgcagt tagacatcca ggccaggtac atctatggga agccagtgca gggggtggca 840 tatgtgcgct ttgggctcct agatgaggat ggtaagaaga ctttctttcg ggggctggag 900 agtcagacca agctggtgaa tggacagagc cacatttccc tctcaaaggc agagttccag 960 gacgccctgg agaagctgaa tatgggcatt actgacctcc aggggctgcg cctctacgtt 1020 gctgcagcca tcattgagtc tccaggtggg gagatggagg aggcagagct cacatcctgg 1080 tattttgtgt catctccctt ctccttggat cttagcaaga ccaagcgaca ccttgtgcct 1140 ggggccccct tcctgctgca ggccttggtc cgtgagatgt caggctcccc agcttctggc 1200 attcctgtca aagtttctgc cacggtgtct tctcctgggt ctgttcctga agtccaggac 1260 attcagcaaa acacagacgg gagcggccaa gtcagcattc caataattat ccctcagacc 1320 atctcagagc tgcagctctc agtatctgca ggctccccac atccagcgat agccaggctc 1380 actgtggcag ccccaccttc aggaggcccc gggtttctgt ctattgagcg gccggattct 1440 cgacctcctc gtgttgggga cactctgaac ctgaacttgc gagccgtggg cagtggggcc 1500 accttttctc attactacta catgatccta tcccgagggc agatcgtgtt catgaatcga 1560 gagcccaaga ggaccctgac ctcggtctcg gtgtttgtgg accatcacct ggcaccctcc 1620 ttctactttg tggccttcta ctaccatgga gaccacccag tggccaactc cctgcgagtg 1680 gatgtccagg ctggggcctg cgagggcaag ctggagctca gcgtggacgg tgccaagcag 1740 taccggaacg gggagtccgt gaagctccac ttagaaaccg actccctagc cctggtggcg 1800 ctgggagcct tggacacagc tctgtatgct gcaggcagca agtcccacaa gcccctcaac 1860 atgggcaagg tctttgaagc tatgaacagc tatgacctcg gctgtggtcc tgggggtggg 1920 gacagtgccc ttcaggtgtt ccaggcagcg ggcctggcct tttctgatgg agaccagtgg 1980 accttatcca gaaagagact aagctgtccc aaggagaaga caacccggaa aaagagaaac 2040 gtgaacttcc aaaaggcgat taatgagaaa ttgggtcagt atgcttcccc gacagccaag 2100 cgctgctgcc aggatggggt gacacgtctg cccatgatgc gttcctgcga gcagcgggca 2160 gcccgcgtgc agcagccgga ctgccgggag cccttcctgt cctgctgcca atttgctgag 2220 agtctgcgca agaagagcag ggacaagggc caggcgggcc tccaacgagc cctggagatc 2280 ctgcaggagg aggacctgat tgatgaggat gacattcccg tgcgcagctt cttcccagag 2340 aactggctct ggagagtgga aacagtggac cgctttcaaa tattgacact gtggctcccc 2400 gactctctga ccacgtggga gatccatggc ctgagcctgt ccaaaaccaa aggcctatgt 2460 gtggccaccc cagtccagct ccgggtgttc cgcgagttcc acctgcacct ccgcctgccc 2520 atgtctgtcc gccgctttga gcagctggag ctgcggcctg tcctctataa ctacctggat 2580 aaaaacctga ctgtgagcgt ccacgtgtcc ccagtggagg ggctgtgcct ggctgggggc 2640 ggagggctgg cccagcaggt gctggtgcct gcgggctctg cccggcctgt tgccttctct 2700 gtggtgccca cggcagccac cgctgtgtct ctgaaggtgg tggctcgagg gtccttcgaa 2760 ttccctgtgg gagatgcggt gtccaaggtt ctgcagattg agaaggaagg ggccatccat 2820 agagaggagc tggtctatga actcaacccc ttggaccacc gaggccggac cttggaaata 2880 cctggcaact ctgatcccaa tatgatccct gatggggact ttaacagcta cgtcagggtt 2940 acagcctcag atccattgga cactttaggc tctgaggggg ccttgtcacc aggaggcgtg 3000 gcctccctct tgaggcttcc tcgaggctgt ggggagcaaa ccatgatcta cttggctccg 3060 acactggctg cttcccgcta cctggacaag acagagcagt ggagcacact gcctcccgag 3120 accaaggacc acgccgtgga tctgatccag aaaggctaca tgcggatcca gcagtttcgg 3180 aaggcggatg gttcctatgc ggcttggttg tcacggggca gcagcacctg gctcacagcc 3240 tttgtgttga aggtcctgag tttggcccag gagcaggtag gaggctcgcc tgagaaactg 3300 caggagacat ctaactggct tctgtcccag cagcaggctg acggctcgtt ccaggacctc 3360 tctccagtga tacataggag catgcagggg ggtttggtgg gcaatgatga gactgtggca 3420 ctcacagcct ttgtgaccat cgcccttcat catgggctgg ccgtcttcca ggatgagggt 3480 gcagagccat tgaagcagag agtggaagcc tccatctcaa aggcaagctc atttttgggg 3540 gagaaagcaa gtgctgggct cctgggtgcc cacgcagctg ccatcacggc ctatgccctg 3600 acactgacca aggcccctgc ggacctgcgg ggtgttgccc acaacaacct catggcaatg 3660 gcccaggaga ctggagataa cctgtactgg ggctcagtca ctggttctca gagcaatgcc 3720 gtgtcgccca ccccggctcc tcgcaaccca tccgacccca tgccccaggc cccagccctg 3780 tggattgaaa ccacagccta cgccctgctg cacctcctgc ttcacgaggg caaagcagag 3840 atggcagacc aggctgcggc ctggctcacc cgtcagggca gcttccaagg gggattccgc 3900 agtacccaag acacggtgat tgccctggat gccctgtctg cctactggat tgcctcccac 3960 accactgagg agaggggtct caatgtgact ctcagctcca caggccggaa tgggttcaag 4020 tcccacgcgc tgcagctgaa caaccgccag attcgcggcc tggaggagga gctgcagttt 4080 tccttgggca gcaagatcaa tgtgaaggtg ggaggaaaca gcaaaggaac cctgaaggtc 4140 cttcgtacct acaatgtcct ggacatgaag aacacgacct gccaggacct acagatagaa 4200 gtgacagtca aaggccacgt cgagtacacg atggaagcaa acgaggacta tgaggactat 4260 gagtacgatg agcttccagc caaggatgac ccagatgccc ctctgcagcc cgtgacaccc 4320 ctgcagctgt ttgagggtcg gaggaaccgc cgcaggaggg aggcgcccaa ggtggtggag 4380 gagcaggagt ccagggtgca ctacaccgtg tgcatctggc ggaacggcaa ggtggggctg 4440 tctggcatgg ccatcgcgga cgtcaccctc ctgagtggat tccacgccct gcgtgctgac 4500 ctggagaagc tgacctccct ctctgaccgt tacgtgagtc actttgagac cgaggggccc 4560 cacgtcctgc tgtattttga ctcggtcccc acctcccggg agtgcgtggg ctttgaggct 4620 gtgcaggaag tgccggtggg gctggtgcag ccggccagcg caaccctgta cgactactac 4680 aaccccgagc gcagatgttc tgtgttttac ggggcaccaa gtaagagcag actcttggcc 4740 accttgtgtt ctgctgaagt ctgccagtgt gctgagggga agtgccctcg ccagcgtcgc 4800 gccctggagc ggggtctgca ggacgaggat ggctacagga tgaagtttgc ctgctactac 4860 ccccgtgtgg agtacggctt ccaggttaag gttctccgag aagacagcag agctgctttc 4920 cgcctctttg agaccaagat cacccaagtc ctgcacttca ccaaggatgt caaggccgct 4980 gctaatcaga tgcgcaactt cctggttcga gcctcctgcc gccttcgctt ggaacctggg 5040 aaagaatatt tgatcatggg tctggatggg gccacctatg acctcgaggg acacccccag 5100 tacctgctgg actcgaatag ctggatcgag gagatgccct ctgaacgcct gtgccggagc 5160 acccgccagc gggcagcctg tgcccagctc aacgacttcc tccaggagta tggcactcag 5220 gggtgccagg tgtga 5235 <210> 13 <211> 2208 <212> DNA <213> Artificial Sequence <220> <223> Mannan-binding lectin serine protease 1 <400> 13 atgagaccta cattcatgtc tttcaggtgg ctgcttctct attatgctct gtgcttctcc 60 ctgtcaaagg cttcagccca caccgtggag ctaaacaata tgtttggcca gatccagtcg 120 cctggttatc cagactccta tcccagtgat tcagaggtga cttggaatat cactgtccca 180 gatgggtttc ggatcaagct ttacttcatg cacttcaact tggaatcctc ctacctttgt 240 gaatatgact atgtgaaggt agaaactgag gaccaggtgc tggcaacctt ctgtggcagg 300 gagaccacag acacagagca gactcccggc caggaggtgg tcctctcccc tggctccttc 360 atgtccatca ctttccggtc agatttctcc aatgaggagc gtttcacagg ctttgatgcc 420 cactacatgg ctgtggatgt ggacgagtgc aaggagaggg aggacgagga gctgtcctgt 480 gaccactact gccacaacta cattggcggc tactactgct cctgccgctt cggctacatc 540 ctccacacag acaacaggac ctgccgagtg gagtgcagtg acaacctctt cactcaaagg 600 actggggtga tcaccagccc tgacttccca aacccttacc ccaagagctc tgaatgcctg 660 tataccatcg agctggagga gggtttcatg gtcaacctgc agtttgagga catatttgac 720 attgaggacc atcctgaggt gccctgcccc tatgactaca tcaagatcaa agttggtcca 780 aaagttttgg ggcctttctg tggagagaaa gccccagaac ccatcagcac ccagagccac 840 agtgtcctga tcctgttcca tagtgacaac tcgggagaga accggggctg gaggctctca 900 tacagggctg caggaaatga gtgcccagag ctacagcctc ctgtccatgg gaaaatcgag 960 ccctcccaag ccaagtattt cttcaaagac caagtgctcg tcagctgtga cacaggctac 1020 aaagtgctga aggataatgt ggagatggac acattccaga ttgagtgtct gaaggatggg 1080 acgtggagta acaagattcc cacctgtaaa attgtagact gtagagcccc aggagagctg 1140 gaacacgggc tgatcacctt ctctacaagg aacaacctca ccacatacaa gtctgagatc 1200 aaatactcct gtcaggagcc ctattacaag atgctcaaca ataacacagg tatatatacc 1260 tgttctgccc aaggagtctg gatgaataaa gtattgggga gaagcctacc cacctgcctt 1320 ccagagtgtg gtcagccctc ccgctccctg ccaagcctgg tcaagaggat cattgggggc 1380 cgaaatgctg agcctggcct cttcccgtgg caggccctga tagtggtgga ggacacttcg 1440 agagtgccaa atgacaagtg gtttgggagt ggggccctgc tctctgcgtc ctggatcctc 1500 acagcagctc atgtgctgcg ctcccagcgt agagacacca cggtgatacc agtctccaag 1560 gagcatgtca ccgtctacct gggcttgcat gatgtgcgag acaaatcggg ggcagtcaac 1620 agctcagctg cccgagtggt gctccaccca gacttcaaca tccaaaacta caaccacgat 1680 atagctctgg tgcagctgca ggagcctgtg cccctgggac cccacgttat gcctgtctgc 1740 ctgccaaggc ttgagcctga aggcccggcc ccccacatgc tgggcctggt ggccggctgg 1800 ggcatctcca atcccaatgt gacagtggat gagatcatca gcagtggcac acggaccttg 1860 tcagatgtcc tgcagtatgt caagttaccc gtggtgcctc acgctgagtg caaaactagc 1920 tatgagtccc gctcgggcaa ttacagcgtc acggagaaca tgttctgtgc tggctactac 1980 gagggcggca aagacacgtg ccttggagat agcggtgggg cctttgtcat ctttgatgac 2040 ttgagccagc gctgggtggt gcaaggcctg gtgtcctggg ggggacctga agaatgcggc 2100 agcaagcagg tctatggagt ctacacaaag gtctccaatt acgtggactg ggtgtgggag 2160 cagatgggct taccacaaag tgttgtggag ccccaggtgg aacggtga 2208 <210> 14 <211> 741 <212> DNA <213> Artificial Sequence <220> <223> Proteasome subunit alpha type-6 <400> 14 atgtcccgtg gttccagcgc cggttttgac cgccacatta ccattttttc acccgagggt 60 cggctctacc aagtagaata tgcttttaag gctattaacc agggtggcct tacatcagta 120 gctgtcagag ggaaagactg tgcagtaatt gtcacacaga agaaagtacc tgacaaatta 180 ttggattcca gcacagtgac tcacttattc aagataactg aaaacattgg ttgtgtgatg 240 accggaatga cagctgacag cagatcccag gtacagaggg cacgctatga ggcagctaac 300 tggaaataca agtatggcta tgagattcct gtggacatgc tgtgtaaaag aattgccgat 360 atttctcagg tctacacaca gaatgctgaa atgaggcctc ttggttgttg tatgatttta 420 attggtatag atgaagagca aggccctcag gtatataagt gtgatcctgc aggttactac 480 tgtgggttta aagccactgc agcgggagtt aaacaaactg agtcaaccag cttccttgaa 540 aaaaaagtga agaagaaatt tgattggaca tttgaacaga cagtggaaac tgcaattaca 600 tgcctgtcta ctgttctatc aattgatttc aaaccttcag aaatagaagt tggagtagtg 660 acagttgaaa atcctaaatt caggattctt acagaagcag agattgatgc tcaccttgtt 720 gctctagcag agagagacta a 741 <210> 15 <211> 600 <212> DNA <213> Artificial Sequence <220> <223> Peroxiredoxin-1 <400> 15 atgtcttcag gaaatgctaa aattgggcac cctgccccca acttcaaagc cacagctgtt 60 atgccagatg gtcagtttaa agatatcagc ctgtctgact acaaaggaaa atatgttgtg 120 ttcttctttt accctcttga cttcaccttt gtgtgcccca cggagatcat tgctttcagt 180 gatagggcag aagaatttaa gaaactcaac tgccaagtga ttggtgcttc tgtggattct 240 cacttctgtc atctagcatg ggtcaataca cctaagaaac aaggaggact gggacccatg 300 aacattcctt tggtatcaga cccgaagcgc accattgctc aggattatgg ggtcttaaag 360 gctgatgaag gcatctcgtt caggggcctt tttatcattg atgataaggg tattcttcgg 420 cagatcactg taaatgacct ccctgttggc cgctctgtgg atgagacttt gagactagtt 480 caggccttcc agttcactga caaacatggg gaagtgtgcc cagctggctg gaaacctggc 540 agtgatacca tcaagcctga tgtccaaaag agcaaagaat atttctccaa gcagaagtga 600 600 <210> 16 <211> 285 <212> DNA <213> Artificial Sequence <220> <223> Neutrophil defensin 3 <400> 16 atgaggaccc tcgccatcct tgctgccatt ctcctggtgg ccctgcaggc ccaggctgag 60 ccactccagg caagagctga tgaggttgct gcagccccgg agcagattgc agcggacatc 120 ccagaagtgg ttgtttccct tgcatgggac gaaagcttgg ctccaaagca tccaggctca 180 aggaaaaaca tggactgcta ttgcagaata ccagcgtgca ttgcaggaga acgtcgctat 240 ggaacctgca tctaccaggg aagactctgg gcattctgct gctga 285 <210> 17 <211> 2232 <212> DNA <213> Artificial Sequence <220> <223> CD44 antigen <400> 17 atggacaagt tttggtggca cgcagcctgg ggactctgcc tcgtgccgct gagcctggcg 60 cagatcgatt tgaatataac ctgccgcttt gcaggtgtat tccacgtgga gaaaaatggt 120 cgctacagca tctctcggac ggaggccgct gacctctgca aggctttcaa tagcaccttg 180 cccacaatgg cccagatgga gaaagctctg agcatcggat ttgagacctg caggtatggg 240 ttcatagaag ggcacgtggt gattccccgg atccacccca actccatctg tgcagcaaac 300 aacacagggg tgtacatcct cacatccaac acctcccagt atgacacata ttgcttcaat 360 gcttcagctc cacctgaaga agattgtaca tcagtcacag acctgcccaa tgcctttgat 420 ggaccaatta ccataactat tgttaaccgt gatggcaccc gctatgtcca gaaaggagaa 480 tacagaacga atcctgaaga catctacccc agcaacccta ctgatgatga cgtgagcagc 540 ggctcctcca gtgaaaggag cagcacttca ggaggttaca tcttttacac cttttctact 600 gtacacccca tcccagacga agacagtccc tggatcaccg acagcacaga cagaatccct 660 gctaccactt tgatgagcac tagtgctaca gcaactgaga cagcaaccaa gaggcaagaa 720 acctgggatt ggttttcatg gttgtttcta ccatcagagt caaagaatca tcttcacaca 780 acaacacaaa tggctggtac gtcttcaaat accatctcag caggctggga gccaaatgaa 840 gaaaatgaag atgaaagaga cagacacctc agtttttctg gatcaggcat tgatgatgat 900 gaagatttta tctccagcac catttcaacc acaccacggg cttttgacca cacaaaacag 960 aaccaggact ggacccagtg gaacccaagc cattcaaatc cggaagtgct acttcagaca 1020 accacaagga tgactgcaga tgtagacaga aatggcacca ctgcttatga aggaaactgg 1080 aacccagaag cacaccctcc cctcattcac catgagcatc atgaggaaga agagacccca 1140 cattctacaa gcacaatcca ggcaactcct agtagtacaa cggaagaaac agctacccag 1200 aaggaacagt ggtttggcaa cagatggcat gagggatatc gccaaacacc caaagaagac 1260 tcccattcga caacagggac agctgcagcc tcagctcata ccagccatcc aatgcaagga 1320 aggacaacac caagcccaga ggacagttcc tggactgatt tcttcaaccc aatctcacac 1380 cccatgggac gaggtcatca agcaggaaga aggatggata tggactccag tcatagtata 1440 acgcttcagc ctactgcaaa tccaaacaca ggtttggtgg aagatttgga caggacagga 1500 cctctttcaa tgacaacgca gcagagtaat tctcagagct tctctacatc acatgaaggc 1560 ttggaagaag ataaagacca tccaacaact tctactctga catcaagcaa taggaatgat 1620 gtcacaggtg gaagaagaga cccaaatcat tctgaaggct caactacttt actggaaggt 1680 tatacctctc attacccaca cacgaaggaa agcaggacct tcatcccagt gacctcagct 1740 aagactgggt cctttggagt tactgcagtt actgttggag attccaactc taatgtcaat 1800 cgttccttat caggagacca agacacattc caccccagtg gggggtccca taccactcat 1860 ggatctgaat cagatggaca ctcacatggg agtcaagaag gtggagcaaa cacaacctct 1920 ggtcctataa ggacacccca aattccagaa tggctgatca tcttggcatc cctcttggcc 1980 ttggctttga ttcttgcagt ttgcattgca gtcaacagtc gaagaaggtg tgggcagaag 2040 aaaaagctag tgatcaacag tggcaatgga gctgtggagg acagaaagcc aagtggactc 2100 aacggagagg ccagcaagtc tcaggaaatg gtgcatttgg tgaacaagga gtcgtcagaa 2160 actccagacc agtttatgac agctgatgag acaaggaacc tgcagaatgt ggacatgaag 2220 attggggtgt aa 2232 <210> 18 <211> 993 <212> DNA <213> Artificial Sequence <220> <223> Arginase-1 <400> 18 atgagcgcca agtccagaac catagggatt attggagctc ctttctcaaa gggacagcca 60 cgaggagggg tggaagaagg ccctacagta ttgagaaagg ctggtctgct tgagaaactt 120 aaagaacaag taactcaaaa ctttttaatt ttagagtgtg atgtgaagga ttatggggac 180 ctgccctttg ctgacatccc taatgacagt ccctttcaaa ttgtgaagaa tccaaggtct 240 gtgggaaaag caagcgagca gctggctggc aaggtggcag aagtcaagaa gaacggaaga 300 atcagcctgg tgctgggcgg agaccacagt ttggcaattg gaagcatctc tggccatgcc 360 agggtccacc ctgatcttgg agtcatctgg gtggatgctc acactgatat caacactcca 420 ctgacaacca caagtggaaa cttgcatgga caacctgtat ctttcctcct gaaggaacta 480 aaaggaaaga ttcccgatgt gccaggattc tcctgggtga ctccctgtat atctgccaag 540 gatattgtgt atattggctt gagagacgtg gaccctgggg aacactacat tttgaaaact 600 ctaggcatta aatacttttc aatgactgaa gtggacagac taggaattgg caaggtgatg 660 gaagaaacac tcagctatct actaggaaga aagaaaaggc caattcatct aagttttgat 720 gttgacggac tggacccatc tttcacacca gctactggca caccagtcgt gggaggtctg 780 acatacagag aaggtctcta catcacagaa gaaatctaca aaacagggct actctcagga 840 ttagatataa tggaagtgaa cccatccctg gggaagacac cagaagaagt aactcgaaca 900 gtgaacacag cagttgcaat aaccttggct tgtttcggac ttgctcggga gggtaatcac 960 aagcctattg actaccttaa cccacctaag taa 993

Claims (8)

Galectin-3, Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7), Galectin-7 (GAL-7) , Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B), Mannan- The expression of the gene family consisting of the binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 (ARG1) in adults A composition for immunosuppression comprising, as an active ingredient, an exosome derived from cord blood plasma, which is higher than that of plasma-derived exosome.
According to claim 1,
Immunosuppression is associated with graft-versus-host disease, autoimmune disease, hyperproliferative skin disease, chronic obstructive pulmonary disease (COPD), allergic asthma, bronchitis, allergic rhinitis, and autoimmune hepatitis. ) for the improvement, prevention or treatment of an immune disease selected from, the composition for immunosuppression.
Galectin-3, Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7), Galectin-7 (GAL-7) , Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B), Mannan- The expression of the gene family consisting of the binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 (ARG1) in adults A medium composition for inhibiting differentiation of Th1 and Th17 cells comprising exosomes derived from cord blood plasma as an active ingredient, which is higher than that of plasma-derived exosomes.
Galectin-3, Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7), Galectin-7 (GAL-7) , Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B), Mannan- The expression of the gene family consisting of the binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 (ARG1) in adults A method for inhibiting differentiation of Th1 and Th17 cells in vitro comprising culturing in vitro exosomes derived from cord blood plasma and naive CD4 + T cells, which are higher than those derived from plasma.
Derived from HLA and MIC gene deficient cell lines, galectin-3, Matrix metalloproteinase (MMP-9), Heat shock protein 72 (HSP72), Prolactin-inducible protein (PIP), Protein S100-A7 (S100A7) ), Galectin-7 (GAL-7), Lysosome-associated membrane glycoprotein 1 (LAMP1), Serpin B12 (SERPINB12), Lactotransferrin (LTF), Alpha-1-acid glycoprotein (ORM1), CD5 antigen-like (CD5L), Complement C4-B (C4B), Mannan-binding lectin serine protease 1 (MASP1), Proteasome subunit alpha type-6 (PSMA6), Peroxiredoxin-1 (PRDX1), Neutrophil defensin 3 (DEFA3), CD44 antigen and Arginase-1 ( A umbilical cord blood plasma exosome mimic expressing at least one selected from the group consisting of ARG1).
6. The method of claim 5,
The cell line deficient in HLA and MIC genes is H1ME-5 (Accession No.: KCTC 13602BP), an umbilical cord blood plasma exosome mimetic.
A composition for immunosuppression comprising the umbilical cord blood plasma exosome mimetic of claim 5.
8. The method of claim 7,
Immunosuppression includes graft-versus-host disease, autoimmune disease, hyperproliferative skin disease, chronic obstructive pulmonary disease (COPD), allergic asthma asthma, bronchitis, allergic rhinitis and autoimmune hepatitis. hepatitis) for the improvement, prevention or treatment of an immune disease selected from, an immunosuppressive composition.

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