Composition for enhancing immune responses by utilizing the activation function of dendritic cells of stromal vascular components isolated from adipose tissue

Abstract

This invention relates to an immune response enhancement composition utilizing the activation function of dendritic cells of the stromal vascular component isolated from adipose tissue, and particularly discloses an immune response enhancement composition for antitumor use.

Description

Technical Field

[0001] This invention relates to an immune response enhancement composition utilizing the activation function of dendritic cells of the stromal vascular component isolated from adipose tissue, and more particularly to an immune response enhancement composition for antitumor purposes. Background Technology

[0002] Dendritic cells (DCCs) can strongly induce antigen-specific T-cell responses in adaptive immunity. The promotion of DCCs in T-cell responses is particularly important for anti-tumor immune responses. Dendritic cells can capture tumor antigens specific to tumor cells and deliver them to the nearest draining lymph node. In the lymph node, DCCs can cross-present the captured tumor antigens to cytotoxic T lymphocytes. Based on their important role in anti-tumor efficacy and the safety profile of clinical applications, DCCs are suitable for cancer immunotherapy. Although the first clinical trials were conducted as early as the 1990s, the response rate has remained below 15% for over 20 years. To improve the low response rate, dendritic cell therapy needs to overcome several obstacles. First, creating opportunities for DCCs to encounter T cells is crucial for them to migrate to lymph nodes and initiate an adaptive immune response. Several papers have indicated that less than 15% of DCCs migrate to lymph nodes, with the majority remaining at the injection site. In particular, the tumor microenvironment, in addition to suppressing the tumor site, also inhibits the anti-tumor immune response in nearby lymph nodes, thereby suppressing the therapeutic efficacy of dendritic cell-based immunotherapy. Therefore, in order to enhance antigen-specific immune responses and fully realize the potential of dendritic cell-based therapies, it is essential to utilize residual dendritic cells at the injection site and mitigate the effects of the tumor microenvironment.

[0003] Dendritic cells encounter T cells in specialized compartments known as "lymphoid organs," which are used to activate appropriate T cells. These organs are composed of various CD45-dependent non-hematopoietic stromal cells, including fibroblastic reticular cells (FRCs) and ednothelial cells. Stromal cells can generally be distinguished by the expression of the fibroblast marker platelet protein (PDPN, gp38) and the endothelial marker platelet endothelial cell adhesion molecule-1 (PECAM-1). PDPN+CD31-FRCs can form a conduit system for delivering soluble antigens and create a three-dimensional cellular network that allows immune cells to move, thus providing structural support. Chemokines CCL19 and CCL21, generated by FRCs, can chemotactically attract CCR7+ T cells and dendritic cells. As a result, the interaction between T cells and dendritic cells increases and promotes dendritic cell maturation, thereby enabling antigen presentation using dendritic cells. Furthermore, FRCs can regulate the homeostasis of T cells and dendritic cells by secreting interleukin (IL)-7 and IL-15. In addition, PDPN expressed by FRCs can promote dendritic cell motility. Recently, IL-6 secreted from FRCs has been shown to induce enhanced survival and metabolism of CD8+ T cells and generate resident memory T cells through chromatin remodeling. PDPN+CD31+ lymphatic endothelial cells (LECs) can constitute afferent and efferent lymphatic vessels that regulate the movement of leukocytes and lymph, and regulate T cell differentiation. PDPN-CD31+ blood endothelial cells (BECs) can constitute high endothelial venules (HEVs) and blood vessels. BECs intervene in the extravasation of lymphocytes in the bloodstream. That is, because stromal cells can provide structural and functional assistance, dendritic cell-based immunotherapy requires the assistance of stromal cells that can induce a complete anti-tumor T-cell response. However, since the content of stromal cells within lymph nodes is less than 5%, it is practically impossible to isolate stromal cells as described above and apply them directly to therapeutic agents.

[0004] This invention discloses that stromal vascular fraction (SVF), isolated from adipose tissue abundant in the body, can enhance anti-tumor T-cell responses induced by dendritic cells, similar to those stromal cells of lymph nodes. Summary of the Invention

[0005] The purpose of this invention is to provide an immune-enhancing composition that utilizes the activating function of dendritic cells of the stromal vascular component isolated from adipose tissue.

[0006] Other or specific objects of the present invention will be set forth below.

[0007] As described in the following embodiments, the inventors confirmed that the stromal vascular fraction (SVF) isolated from visceral adipose tissue and subcutaneous adipose tissue and the mixture is composed of cells that are phenotypically or functionally similar to lymph node stromal cells. They also confirmed that SVF spheroids obtained by three-dimensional culture of the SVF can form blood vessels serving as channels for the movement of immune cells. Furthermore, they confirmed that SVF spheroids can highly express various factors (such as Spp1, Ccl8, Cxcl5, Postn, Cxcl16, and Ccl17) and osteopontin (Spp1), a known factor regulating the maturation and survival of dendritic cells. Additionally, they confirmed that SVF spheroids can attract immune cells, including dendritic cells, in vitro, and enhance the interaction between dendritic cells and T cells when transplanted into the body together with dendritic cells in vivo. Furthermore, the inventors confirmed that when two-dimensional cultures of SVF are co-cultured with dendritic cells in vitro or transplanted into vivo with dendritic cells loaded with a specific antigen (ovalbumin, OVA), the two-dimensional cultures of SVF can promote the activation of dendritic cells and improve their survival rate, thereby effectively activating T cells and enhancing the response of antigen-specific T cells. They also confirmed that injecting SVF spheroids and dendritic cells loaded with the aforementioned antigen (OVA) together into mice that have been transplanted with B16 melanoma cells expressing the specific antigen (OVA) (B16-OVA) can inhibit tumor growth and improve the survival rate of the experimental animals.

[0008] This invention is provided based on the experimental results described above. One aspect of this invention is an immune response enhancement composition comprising, as an active ingredient, stromal vascular components isolated from adipose tissue, particularly spheroids of said stromal vascular components, and antigen-loaded dendritic cells. Herein, the immune response refers to an adaptive immune response, preferably a cell-mediated immune response, and more preferably a T-cell-based immune response.

[0009] In this invention, the stromal vascular component isolated from adipose tissue can be used in the form of a two-dimensional culture or a spherical form of a three-dimensional culture, preferably in the form of a spherical form.

[0010] The immune-enhancing composition of the present invention can be an antiviral immune-enhancing composition for treating or preventing symptoms of viral infection when the antigen loaded on the dendritic cells is a virus-derived antigen; when the antigen loaded on the dendritic cells is a pathogenic microorganism-derived antigen such as bacteria; when the antigen loaded on the dendritic cells is a pathogenic microorganism-derived antigen such as bacteria; when the antigen loaded on the dendritic cells is a tumor cell-derived antigen; and when the antigen loaded on the dendritic cells is a tumor cell-derived antigen; when the antigen loaded on the dendritic cells is a tumor-derived immune-enhancing composition for treating or preventing tumors.

[0011] The immune-enhancing composition of the present invention can be used for the treatment or prevention of symptoms of any viral infection and symptoms of infection of any bacteria or other microorganisms, and preferably for the treatment or prevention of any tumor.

[0012] In this invention, dendritic cells can be autologous dendritic cells obtained from the recipient of the immune-enhancing composition of this invention, or dendritic cells of the same type obtained from other individuals, or dendritic cells of different types obtained from different types of subjects. When the recipient of the immune-enhancing composition of this invention is a human, the dendritic cells obtained from different types of subjects can be obtained from mammals such as mice, rats, rabbits, monkeys, pigs, horses, cattle, sheep, antelopes, dogs, and cats. The dendritic cells described above can be derived from sources such as bone marrow, peripheral blood, and umbilical cord blood.

[0013] In this invention, dendritic cells can be immature or mature, preferably mature. Compared to immature dendritic cells, mature dendritic cells can express MHC type II molecules, T cell co-stimulatory factors such as CD80, CD86, and CD83, immunostimulatory factors such as cytokines IL-12 and IL-10, and TNF at high concentrations on their surface. Morphologically, immature dendritic cells have a smooth surface, while mature dendritic cells have a rough surface and possess multiple pseudopodia. Techniques for maturing immature dendritic cells are well-known in the industry. Examples of such methods include culturing undifferentiated precursor cells, such as CD34+ hematopoietic cells (which can be isolated from bone marrow, peripheral blood, and umbilical cord blood and differentiated into macrophages and dendritic cells), together with dendritic cell maturation factors such as INF-α (European Patent No. 922,758); culturing with dendritic cell maturation factors such as GM-CSF, TNF-α, and IL-3 (European Patent No. 663,930); and culturing with a mixture of hematopoietic growth factors and cytokines (WO). 95 / 28479) and methods for culturing with LPS (lipopolysaccharide) (Am. J. Physiol. Cell Physiol., 2011, 300, C1205-C1214; Annu. Rev. Immunol., 2003, 21, 335-376), etc. All references cited in this specification, including those mentioned above, are considered part of this specification.

[0014] In this invention, the stromal vascular component (SVF) isolated from adipose tissue can be obtained by enzymatic isolation methods and mechanical isolation methods.

[0015] In the enzymatic separation method, adipose tissue or lipospirate is first washed 2-3 times with an aqueous salt solution such as PBS, LRS (Lactated Ringer's solution), or HBSS (Hank's Balanced Salt Solution). Then, the washed adipose tissue or lipospirate is treated with enzymes that degrade the extracellular matrix (ECM). This enzymatic treatment, as described above, is typically performed at 37°C with stirring for 30 minutes to 2 hours. Following enzymatic treatment, centrifugation is performed. Centrifugation separates the enzymatically treated adipose tissue or lipospirate into (i) an adipose tissue or oil layer, (ii) an aqueous solution layer, and (iii) a pellet layer. Only the pellet layer is collected, and the other layers are discarded. These pellets contain stromal vascular components.

[0016] In the enzyme separation method described above, examples of enzymes that can be used for the separation of extracellular matrix, etc., include type I or type II collagenase isolated from *Clostridium histolyticum*, and neutral protease (dispase) isolated from *P. polymyxa*, thermolysin isolated from *G. stearothermophilus* or *B. thermoproteolyticus*. Furthermore, commercially available mixtures for the purposes described above, such as a mixture of type I or type II collagenase and neutral protease (i.e., CIzyme), can also be used. TM Liberase is a mixture of type I or II collagenase (Vitacyte LLC, Indianapolis, Indiana) and thermophilic bacterial protease. TM Research Grade (Roche Diagnostic, Based, Switzerland), etc.

[0017] In addition to using collagenases, fusion enzymes such as DNA degraders can also be used to improve the efficiency of cell separation by degrading chromosomes that flow out of dead cells during cell separation.

[0018] The mechanical separation method is the same as the enzymatic separation method, which is also carried out by recovering the particles after washing and centrifugation. Before centrifugation, the washings can be shaken / vibrated to facilitate easy recovery of the particles.

[0019] For more specific details regarding methods for separating stromal vascular components from adipose tissue, see references [Aronowitz et al. SpringerPlus (2015) 4:713], etc.

[0020] As described above, the stromal vascular component is composed of various cell types, including lymphocytes, adipocyte precursors, adipocyte-derived stromal cells, fibroblasts, endothelial cells, and pericytes (Stem Cell Res Ther 2017 Jun 15; 8(1):145). By culturing SVF, lymphocytes will die and be removed, thereby obtaining various types of stromal cells with adhesion (Cytotherapy 15, 641-648, 2013).

[0021] In this invention, the stromal vascular component can be obtained from the adipose tissue of any mammal, preferably from the adipose tissue of a human, mouse, rat, rabbit, monkey, pig, horse, cow, sheep, antelope, dog or cat, more preferably from a human, mouse, rat or monkey, and most preferably from a human or mouse.

[0022] Furthermore, in this invention, the stromal vascular component can be obtained from any adipose tissue. It can be obtained from subcutaneous fat tissue or visceral fat tissue surrounding organs, or from brown or white adipose tissue.

[0023] Furthermore, in this invention, the two-dimensional culture of the stromal vascular component isolated from adipose tissue refers to a product in which one side of the cell culture is attached to the culture surface of a culture dish or the like by culturing the stromal vascular component in a two-dimensional monolayer.

[0024] Furthermore, in this invention, the spherical body of the matrix vascular component isolated from adipose tissue refers to the spherical product obtained by three-dimensional culture of the matrix vascular component isolated from adipose tissue. Various three-dimensional culture methods for obtaining the spherical body as described above are well known in the art. Examples of the methods described above include hanging-drop cultures (Biotechnol Bioeng 2003, 83(2):173-80; Trends Biotechnol 2004, 22(4):195-202; Crit Rev Oncol Hematol 2000, 36(2-3):107-22), culture using microwell plates with non-adhesive surfaces (Cancer Res 1977, 37(10):3639-43; In Vitro Cell Dev Biol Anim 2001, 37(10):656-67), and culture using microfabricated microstructures (Biomed Microdevices 2008, 10(2):197-202; Tissue Eng 2007,13(8):2087-94), culture method using rotating bioreactor (In Vitro Cell Dev Biol Anim 1997,33(6):459-66; NatMed 1998,4(8):901-7), culture method using surface modified substrates or scaffolds (Tissue Eng 2007,13(7):1455-68; Tissue Eng 2006,12(5):1357-68), culture method using external forces (Cell Transplant 2006,15(6):521-32; Biotechnol Bioeng 2007,98(3):694-700), and culture method using spheroid on a chip (Biomaterials 2007,28(3):559-66; Biomaterials 2006, 27(36):6032-42, etc.For more specific details regarding the three-dimensional culture method for obtaining spheroids by culturing stromal vascular components isolated from adipose tissue, please refer to the literature [Korean JOtorhinolaryngol-Head Neck Surg 2020,63(6):245-51].

[0025] Furthermore, in this invention, antigen loading on dendritic cells aims to induce the activity of T cells and the like, which selectively recognize T cell receptors, by presenting antigen peptides on the surface of dendritic cells in the form of a complex with MHC through antigen processing, thereby enhancing immune responses such as antiviral, antimicrobial, and antitumor reactions. As described above, methods for antigen loading on dendritic cells, such as introducing and using the peptide antigen itself or by processing it with cell-penetrating peptides such as antigen expression genes (e.g., RNA), antigen expression viral vectors, and HIV (human immunodeficiency virus) Tat peptides bound to the antigen, to express antigen proteins within dendritic cells, are well-known in the art. As in the embodiments of this invention, Fe3O4-ZnO core-shell nanoparticles (FZ-NPs) can also be used. For more specific details regarding antigen loading methods on dendritic cells, please refer to the literature [Immunol Res. 2017, 65: 798-810] etc.

[0026] Furthermore, in this invention, the antigen can be a virus-derived antigen, a pathogenic microorganism-derived antigen such as bacteria, or a tumor-derived antigen. The tumor antigen can be any antigen derived from cancer cells, such as surface-expressed proteins, surface-expressed receptors, peptides or proteins inside tumor cells, or lysates of tumor cells.

[0027] As mentioned above, it is preferable that the tumor antigens are expressed only in cancer cells or overexpressed in cancer cells compared to normal cells. Examples include EGFRvⅢ (epidermal growth factor receptor variant Ⅲ) expressed in glioblastoma; EGFR (epidermal growth factor receptor) overexpressed in undifferentiated thyroid cancer, breast cancer, lung cancer, and glioma; metastin receptor overexpressed in papillary thyroid cancer; ErbB family receptor tyrosine kinases overexpressed in breast cancer; and HER2 (human epidermal growth factor receptor) overexpressed in breast cancer, bladder cancer, gallbladder cancer, cholangiocarcinomas, and esophagogastric junction cancers. 2) Tyrosine kinase-18 receptor (c-Kit) overexpressed in papillary renal cell carcinoma, etc.; HGF receptor c-Met overexpressed in esophageal adenocarcinoma, etc.; CXCR4 or CCR7 overexpressed in breast cancer, etc.; endothelin-A receptor overexpressed in prostate cancer; PPAR-δ (peroxisome proliferator activated receptor δ) overexpressed in rectal cancer, etc.; PDGFR-α (Platelet-derived growth factor receptor α) overexpressed in ovarian cancer, etc.; CD133 overexpressed in liver cancer and multiple myeloma, etc.; CEA (carcinoembryonic antigen) overexpressed in lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, breast cancer, rectal cancer, colon cancer, and medullary thyroid carcinoma, etc.; EpCAM (epithelial cell adhesion retardation) overexpressed in liver cancer, gastric cancer, colorectal cancer, pancreatic cancer, and breast cancer, etc. molecule); GD2 (disialoganglioside) overexpressed in neuroblastoma, etc.; GPC3 (Glypican 3) overexpressed in hepatocellular carcinoma, etc.; PSMA (Prostate Specific Membrane Antigen) overexpressed in prostate cancer, etc.TAG-72 (tumor-associated glycoprotein 72) is overexpressed in ovarian cancer, breast cancer, colon cancer, lung cancer, and pancreatic cancer; GD3 (disialoganglioside) is overexpressed in melanoma; HLA-DR (human leukocyte antigen-DR) is overexpressed in hematologic malignancies and solid tumors; MUC1 (Mucin 1) is overexpressed in advanced solid tumors; NY-ESO-1 (New York esophagealsquamous cell carcinoma 1) is overexpressed in advanced non-small-cell cancer; LMP1 (Latent membrane protein 1) is overexpressed in nasopharyngeal neoplasms; and TRAILR2 (tumor-necrosis factor-related apoptosis-inducing ligand) is overexpressed in lung cancer, non-Hodgkin's lymphoma, ovarian cancer, colon cancer, colorectal cancer, and pancreatic cancer. The receptors include: vascular endothelial growth factor receptor (VEGFR2); hepatocyte growth factor receptor (HGFR), which is overexpressed in hepatocellular carcinoma, etc.; and surface antigens of cancer stem cells, such as CD44 and CD166. There are numerous known antigens in this field that are overexpressed in cancer cells compared to normal cells. For more specific information on other tumor antigens besides the examples mentioned above, please refer to the following references: [Anne T Collins et al. Prospective Identification of Tumorigenic Prostate Cancer Stem Cells. Cancer Res. 2005 Dec 1; 65(23):10946-51], [Chenwei Li et al. Identification of Pancreatic Cancer Stem Cells. Cancer Res. 2007 Feb 1; 67(3):1030-7], and [Shuo Ma et al. Current Progress in CAR-T Cell Therapy for Solid Tumors. Int J Biol Sci. 2019 Sep 7;][15(12):2548-2560] and references [Dhaval S Sanchala et al. Oncolytic Herpes Simplex Viral Therapy: A Stride Toward Selective Targeting of Cancer Cells. Front Pharmacol. 2017 May 16; 8:270] etc.

[0028] When the immune-enhancing composition of the present invention is used for antitumor purposes, it can be used against any cancer cells expressing antigens loaded on dendritic cells. Cancer cells can be any type of cancer, such as esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, melanoma, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, multiple myeloma, and blood cancer.

[0029] In this invention, anti-tumor means the suppression or delay of pathological symptoms of cancer, the inhibition of cancer cell metastasis, and the inhibition of cancer cell recurrence, achieved by killing cancer cells, reducing the survival ability of cancer cells, and inhibiting the proliferation of cancer cells.

[0030] The immune-enhancing composition of the present invention can be used in combination with or mixed with any approved antiviral agent, any antimicrobial agent (i.e., antibiotic), and any anticancer agent. When used in combination with or mixed with an anticancer agent, the anticancer agent includes any anticancer agent that exhibits cytotoxicity to cancer cells, such as metabolic antagonists, alkylating agents, topoisomerase antagonists, microtubule antagonists, and plant-derived alkaloids; any cytokine drug; any antibody drug; any immune checkpoint inhibitor; and any cell therapy agent (car-T cell therapy and car-NK cell therapy). Specific examples may include paclitaxel, nitrogen mustard, imatinib, oxaliplatin, gefitinib, bortezomib, sunitinib, carboplatin, cisplatin, rituximab, erlotinib, sorafenib, IL-2 drugs, INF-α drugs, INF-γ drugs, trastuzumab, bonatetumab, ipilimumab, pembrolizumab, nivolumab, atezolizumab, durvalumab, bevacizumab, cetuximab, tisagenlecleucel (Kymriah), and tisagenlecleucel (Axicabtagene Ciloleucel (Yescarta), etc., and other anticancer agents known in the art, in addition to the examples described, may be used in combination or mixed with the compositions of the present invention without limitation.

[0031] The compositions of the present invention may contain pharmaceutically permissible carriers or excipients and are formulated into pharmaceutical compositions by means of general methods known in the art according to the route of administration.

[0032] As described above, pharmaceutically permissible carriers or excipients must not be particularly toxic to humans while not hindering the activity or properties of the drug. They may be, for example, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water (e.g., saline and sterile water), syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, Ringer's solution, buffer solution, maltodextrin solution, glycerol, ethanol, dextran, albumin, or any combination thereof. In particular, when the pharmaceutical composition of the present invention is formulated into a liquid solution, one or more of the following components, such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, glucose solution, maltodextrin solution, glycerol and ethanol, can be used alone or in combination as a suitable carrier or excipient. Antioxidants, buffer solutions and antibacterial agents and other general pharmaceutical additives can also be added as needed.

[0033] The pharmaceutical compositions of the present invention can be formulated into non-oral administration agents, especially injections, and, as described above, can be manufactured into single-dose ampoules or multiple-dose formulations. The pharmaceutical compositions of the present invention can particularly be formulated into solutions and suspensions.

[0034] The pharmaceutical composition of the present invention can be formulated into a unit-dose formulation suitable for intra-patient administration using general methods in the pharmaceutical field, and can be administered via non-oral routes such as skin, intralesional, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intracardiac, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intradigestive tract, local, sublingual, intravaginal, rectal, and intrarenal capsule.

[0035] The dosage (effective amount) of the pharmaceutical composition of the present invention can be selected from various formulations based on factors such as formulation method, route of administration, patient's age, weight, sex, disease state, diet, administration time, route of administration, excretion rate, and reaction sensitivity. Furthermore, those skilled in the art can appropriately determine the dosage taking into account the factors described above. In a preferred embodiment, the pharmaceutical composition of the present invention can be manufactured as an injection in unit volume form. When manufactured as an injection in unit volume form, the content of the stromal vascular component globular or dendritic cells contained in the unit volume of the pharmaceutical composition of the present invention can be 10... 2 ~10 8 The range of cells.

[0036] In another embodiment, the present invention relates to an immune enhancement method comprising the step of administering the immune-enhancing pharmaceutical composition of the present invention, as described above, to a patient or other subject at an effective dose. When the immune-enhancing composition of the present invention comprises dendritic cells loaded with viral antigens and is used for antiviral purposes, the immune enhancement method may be used for the treatment or prevention of viral infection symptoms. When the immune-enhancing composition of the present invention comprises a dendritic composition loaded with pathogenic microorganism antigens such as viruses and is used for antimicrobial purposes, the immune enhancement method may be used for the treatment or prevention of pathogenic microorganism infection symptoms. And when the immune-enhancing composition of the present invention comprises dendritic cells loaded with tumor antigens and is used for antitumor purposes, the immune enhancement method may be used for antitumor (cancer treatment or prevention) purposes.

[0037] The immune enhancement method of the present invention can exhibit antiviral, antimicrobial, and antitumor activities by activating antigen-specific T cells through the spheroids of the stromal vascular component and the antigen-loaded dendritic cells contained in the immune enhancement composition of the present invention. Therefore, the immune enhancement method of the present invention can be applied to any viral infection symptoms containing the antigen, microbial infection symptoms containing the antigen, and any cancer containing the antigen.

[0038] When the immune-enhancing method of the present invention is used for anti-tumor purposes to treat or prevent cancer, it can be used in combination with other cancer treatment methods without limitation. For example, it can be used in combination with the cytotoxic anticancer agents, cytokine drugs, antibody drugs, immune checkpoint inhibitors and cell therapy agents (car-T celltherarpy and car-NK celltherapy), radiotherapy and surgical therapy, etc., before, after or simultaneously with the composition of the present invention.

[0039] The effective dosage in the treatment method of the present invention refers to the amount of the composition of the present invention that, when administered to its intended recipients, i.e., patients, during the period of administration recommended by medical experts or other relevant practitioners, can produce the expected medical effects such as cancer treatment or prevention. The dosage can be appropriately determined by medical experts or other relevant practitioners based on factors such as the patient's age, weight, gender, and disease status, as described above.

[0040] In another aspect, the present invention may be an immune-enhancing composition comprising, as an active ingredient, stromal vascular components isolated from adipose tissue, particularly spheroids and dendritic cells of said stromal vascular components, or an immune-enhancing method comprising the step of administering the composition as described above to a patient or other subject at an effective dose.

[0041] As described above, the inventors have confirmed through the following embodiments that when spheroids of stromal vascular components isolated from adipose tissue are transplanted together with dendritic cells into an organism, the interaction between dendritic cells and T cells can be enhanced. Furthermore, it has been confirmed that when two-dimensional cultures of stromal vascular components isolated from adipose tissue are co-cultured with dendritic cells in vitro, T cells can be effectively activated by promoting the activation of dendritic cells and improving their survival rate.

[0042] The results described above indicate that administering the globular sclerotia and dendritic cells of the stromal vascular component isolated from adipose tissue together can enhance the immune response, specifically the cell-mediated immune response, and more specifically, the T-cell-based immune response.

[0043] The immune response enhancement composition of the present invention and the immune enhancement method of administering the composition thereon are described above in the same manner as the immune response enhancement composition of the present invention comprising vascular matrix components and antigen-loaded dendritic cells, and can be used for antiviral, antimicrobial, or antitumor purposes. Moreover, the composition and method can also be implemented and used in the manner described above.

[0044] In another aspect, the present invention relates to a dendritic cell maturation composition comprising, as an active ingredient, a spheroidal body containing a matrix vascular component isolated from adipose tissue, particularly said matrix vascular component.

[0045] As described above, the inventors have confirmed through the following examples that the spheroids of the stromal vascular component isolated from adipose tissue can highly express various factors (such as Spp1, Ccl8, Cxcl5, Postn, Cxcl16, and Ccl17) that are used for chemotaxis and activation of immune cells, and can also highly express osteopontin, a factor known to regulate the maturation and survival of dendritic cells. Furthermore, it has been confirmed that when co-cultured with dendritic cells in vitro, the two-dimensional culture of the stromal vascular component can promote the activation of dendritic cells and improve their survival rate.

[0046] The results described above indicate that globular stromal vascular components isolated from adipose tissue can induce dendritic cell maturation, thereby inducing activation and improving survival.

[0047] To utilize dendritic cells for anti-tumor and other applications in immunotherapy, in addition to techniques for differentiating and manufacturing dendritic cells in vitro (ex vivo), maturation techniques for effectively inducing T-cell immune responses are also essential. As mentioned above, mature dendritic cells can express large amounts of cytokines such as IL-12, IL-10, and TNF, which directly interfere with the induction of T-cell immune responses. By employing the aforementioned changes during maturation, the T-cell immune response induction capacity of dendritic cells can be significantly enhanced.

[0048] The dendritic cell maturation composition of the present invention, as a maturation factor for dendritic cells, may additionally contain IFN-α, IL-1β, IL-6, IL-3, TNF-α, IFN-γ, PGE2, and OK432 (Picibanil), which are known in the art, and can be added to media such as RPMI (Roswell Park Memorial Institute) 1640 and serum-free media X-VIVO 15.

[0049] The dendritic cell maturation composition of the present invention can be manufactured into a pharmaceutical composition for administration to patients or other subjects, or it can be used for the purpose of maturation and proliferation of dendritic cells in vitro (ex vivo).

[0050] As described above, the present invention can provide an immune response enhancement composition utilizing the activation function of dendritic cells of a matrix vascular component isolated from adipose tissue. Specifically, it can provide an immune response enhancement composition comprising, as an active ingredient, a matrix vascular component isolated from adipose tissue, particularly spheroids of the matrix vascular component, and dendritic cells loaded with antigens.

[0051] The compositions of the present invention can be used for purposes such as antiviral, antimicrobial, and antitumor applications. Attached Figure Description

[0052] Figures 1 to 5 This indicates that the stromal vascular components isolated from adipose tissue exhibit similar properties and functions to lymph node stromal cells when cultured.

[0053] Figure 6 This results in spherical structures, which are stromal vascular components separated from adipose tissue, forming blood vessels that serve as channels for the movement of immune cells.

[0054] Figures 7 to 11 This is because the spheroids, which show the stromal vascular components isolated from adipose tissue, can chemotact dendritic cells and enhance the interaction between dendritic cells and T cells, and most of the T cells can differentiate into CD4+ effector T cells and carry out immune responses.

[0055] Figure 12 as well as Figure 13This demonstrates that two-dimensional cultures or spheroids of stromal vascular components isolated from adipose tissue can enhance the survival rate of dendritic cells and, together with antigen-loaded dendritic cells, enhance the immune response of antigen-specific T cells.

[0056] Figure 14 The results showed that administering globular stromal vascular components isolated from adipose tissue, along with dendritic cells loaded with tumor antigen (OVA), to mice that had been transplanted with B16 melanoma cells (B16-OVA) expressing tumor antigen (OVA) could reduce tumor size and improve mouse survival.

[0057] Figure 15 This is a conceptual diagram of the present invention. Detailed Implementation

[0058] The present invention will now be described in detail with reference to the embodiments. However, the scope of the present invention is not limited to the embodiments described.

[0059] Example

[0060] 1. Samples and test methods

[0061] 1.1 Mice

[0062] C57BL / 6 wild-type mice (Koatech, Seoul, Republic of Korea) and C57BL / 10NAGCSAni-(KO)Rag2(H-2b) mice (Taconic Biosciences, NY, US) were used in the specific pathogen-free animal facility located at Seoul National University College of Medicine. All mice were male and aged 6–12 weeks. All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (IACUC Number: SNU-150115-2-7, SNU-160212-2-7, SNU-171123-4-7).

[0063] 1.2 Isolation of stromal vascular components (SVF) and culture of spheroids

[0064] After harvesting adipose tissue from the viscera and skin, the tissue was minced and then cultured for 1 hour at 37°C in a sterile Hank's balanced salt solution (Intron, Seoul, Republic of Korea) containing 0.8 mg / ml collagenase, 100 μg / ml DNA degrading enzyme I (DNase I), 3% bovine serum albumin (BSA) (MP biomedicals, CA, US), 1.0 mM CaCl2 (Sigma-Aldrich, MO, US), and 0.8 mM MgCl2 (Sigma-Aldrich, MO, US) at 170 rpm. Following centrifugation at 500 g for 3 minutes, the supernatant was removed, and the stromal vascular fraction (SVF) particles were separated. Figure 1 a). Next, the cells were cultured in Dubco's Modified Eagle Medium (Welgene, Daegu, Republic of Korea) containing 10% fetal bovine serum (Gibco, MA, US) and 1% penicillin / streptomycin (Gibco, MA, US). SVFs cultured for 6–10 days were treated with trypsin and counted to generate spheroids. The cultured SVFs were then transferred to a spheroid membrane (Incyto, Cheonan, Republic of Korea). After confirming the formation of 361 (19 × 19) SVF-spheroids on the spheroid membrane, the cells were cultured for at least two more days before being used in experiments.

[0065] 1.3 Production of recombinant ZnO-binding peptide (ZBP)-ovalbumin (OVA)

[0066] To produce recombinant ZnO-binding peptide (ZBP)-ovalbumin (OVA) (ZBPOVA), genomic DNA was first extracted from the B16MO5 cell line using DNeasyBlood & Tissue Kits (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was then amplified by PCR (forward primer: 5'-TTT). GAA TTC ATG GGC TCC ATC GGT GCA-3', sequence number 1, reverse primer: 5'-AAA CTC GAG The gene was AGG GGA AAC ACA TCT GCC-3', EcoRI, XhoI restriction sites underlined, and cloned into the pET23d-ZBP vector encoded by 3xZBP (BamHI-RPHRKGGDARPHRKGGDARPHRKGGDA-EcoRI, sequence number 3). The cloned vector pET23d-ZBPOVA was transformed into *E. coliHIT Competent* BL21 strain according to the manufacturer's instructions. ZBPOVA was purified using a modified protocol from the previously described method (Nature methods 3, 55-64, 2006; Biochemistry and Molecular Biology Education 39, 28-37, 2011). In short, E. coli culture induced with protein expression by isopropylthiogalactoside (IPTG; 0.1 mM) was dissolved in ice-cold buffer (300 mM NaCl, 50 mM phosphate, pH = 8.0) containing a mixture of lysozyme (1 mg / ml) and a protease inhibitor for 30 minutes, followed by sonication. The dissolved material was centrifuged, the supernatant was collected and filtered, and then processed using AKTAstart (GE Healthcare, IL, US) and HisTrap. TM Column purification was performed using FF (GE Healthcare, IL, US). The protein was dialyzed overnight with PBS at 4°C. Endotoxins were removed from the purified protein using Triton X-114, following previous instructions (Microbial cellfactories 10, 57, 2011). Endotoxin contamination of the purified protein was confirmed using the Pierce LAL Chromogenic Endotoxin Quantification Kit (Thermo Fisher Scientific, MA, US) according to the manufacturer's guidelines. After quantification of the purified ZBPOVA protein using SDS-PAGE, it was stored at -80°C before use.

[0067] 1.4 Isolation, antigen delivery, and maturation of bone marrow-derived dendritic cells

[0068] Following the previous instructions (Nat Nanotechnol 6,675-682, 2011), dendritic cells were differentiated from bone marrow through isolation. In simple terms, bone marrow from knock-out Rag2 mice was isolated and cultured in Iscove's modified Eagle medium (IMDM) (Gibco, MA, US) supplemented with 10% FBS, 1.5 ng / ml recombinant mouse GM-CSF (PeproTech, NJ, Us), 1.5 ng / ml mouse IL-4 (PeproTech, NJ, Us), 1% penicillin / streptomycin, 50 μg / ml gentamicin (Gibco, MA, US), 2 mM L-glutamine (Gibco, MA, US), and 50 nM β-mercaptoethanol (Gibco, MA, US). Immature dendritic cells were cultured for 6–8 days, with the culture medium changed every other day. Fe3O4-ZnO core-shell nanoparticles (FZ-NPs) were used to deliver internal antigens to immature bone marrow-derived dendritic cells (BMDCs). 100 μg of FZ-NPs was washed three times with PBS, and then cultured at room temperature for 1 hour to allow the FZ-NPs to bind with 20 μg of ZBP-OVA. Next, the ZBP-OVA and FZ-NPs (NP-OVA) were washed three times with IMDM. Finally, 1 × 10⁻⁶ cells were cultured... 6 Dendritic cells were cultured at 37°C with NP-OVA for 1 hour. For maturation, the dendritic cells were treated with 1 μg / ml lipopolysaccharide (LPS) for 16–18 hours. The LPS-matured dendritic cells were then washed three times with IMDM prior to the experiment.

[0069] 1.5 Flow cytometry and antibody analysis

[0070] Cells were blocked using a super-block solution consisting of 10% goat serum (Thermo Fisher Scientific, MA, US), 10% rat serum (Thermo Fisher Scientific, MA, US), 10% mouse serum (Sigma-Aldrich, MO, US), and 10 μg / ml of anti-CD16 / CD32 (2.4G2) antibody (BD Pharmingen, NJ, US). Next, in ice, the following anti-CD45 conjugated to APC / Cy7, anti-CD11c conjugated to BV405, anti-CD11c conjugated to Alexa488, anti-B220 conjugated to APC, anti-CD11b conjugated to PE, anti-PDPN conjugated to APC, anti-ICAM-1 conjugated to FITC, anti-CD31 conjugated to PE / Cy7, anti-LTβR conjugated to PE, anti-VCAM-1 conjugated to PE, anti-CD140α conjugated to PerCP / Cy5.5, and anti-CD8 conjugated to PE / Cy7 were used. 6. Surface marker staining was performed for 30 minutes on APC-conjugated anti-I-Ab, BV605-conjugated anti-Ki67, PE-conjugated anti-DC-SIGN (Biolegend, CA, US), PE-conjugated anti-CD3, BV421-conjugated anti-CD3, PerCP-conjugated anti-CD4, FITC-conjugated anti-CD8, APC-conjugated anti-Gr-1, BV605-conjugated anti-CD62L, FITC-conjugated anti-I-Ab, PE-conjugated anti-CD40 (BDPharmingen, NJ, US), PE / Cy7-conjugated anti-CD4, eFlour 450-conjugated anti-CD3, PE / Cy7-conjugated anti-F4 / 80, and APC-conjugated anti-CD80 (eBioscience, CA, US). Surviving cells were stained individually using Zombi Aqua (Biolegend, CA, US) or 7-aminoactinomycin D (7-AAD) (BD Pharmingen, NJ, US).Cellular measurements were performed using an LSR Fortessa X-20 flow cytometer, a BD LSR II flow cytometer (BD Pharmingen, NJ, US), and a CytoFLEX S (Beckman Coulter, CA, US). Data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

[0071] 1.6 Transplantation to the renal capsule

[0072] Following the previously described method, SVF-globules or dendritic cells were transplanted into the renal capsule (BiochemBiophys Res Commun 514, 1081-1086, 2019). In short, after anesthetizing the mice, the hair on the side to be operated on was removed. Next, after disinfecting the skin, an incision was made to expose the kidney. The renal capsule was torn open using a 30-gauge needle (BD Pharmingen, NJ, US), and the transplant was slowly injected using a threaded plunger (Hamilton, NV, US). The incision was cauterized using a high-temperature cauterizer (Bovie Medical Corporation, NY, US) and then sutured. The kidney was reinserted and the skin was sutured.

[0073] 1.7 Quantitative PCR

[0074] RNA was extracted using Trizol reagent according to standard specifications. Complementary DNA (cDNA) was synthesized sequentially using a reverse transcript premix kit (Intron, Seoul, Republic of Korea). To confirm whether SVFs could secrete the major chemokines of lymph node stromal cells, mRNA expression was measured by quantitative PCR (qPCR) using the SYBR Green master mix (Life Technologies, CA, US) and the CFX Real-Time PCR Detection System (Bio-Rad Laboratories, CA, US). Each sample was tested twice. Relative mRNA expression was calculated based on β-actin (Actb) levels. Primers are listed in Table 1.

[0075] Table 1

[0076]

[0077] 1.8 mRNA base sequence analysis

[0078] To confirm the gene expression characteristics of SVF and SVF-globules, anti-CD45 microbeads and magnetic cell separation (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) were used to classify CD45 from SVF. - Cells were used to isolate total RNA. RNA quality was determined using an RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, Netherlands) and an Agilent 2100 bioanalyzer, while RNA quantity was quantified using an ND-2000 Spectrophotometer (Thermo Fisher Scientific, MA, US). Libraries were generated from total RNA using the SMARTer Stranded RNA-Seq Kit (Clontech Laboratories, CA, US). Polyadenylated mRNA was specifically isolated using the Poly(A)RNA Selection Kit (LEXOGEN, Vienna, Austria), followed by cDNA synthesis, fragmentation, and shearing for PCR amplification. Average fragment size was determined using an Agilent 2100 bioanalyzer and quantified using the StepOne Real-Time PCR System (Life Technologies, CA, US). The library's base sequence was analyzed using HiSeq 2500 (Illumina, CA, US) with paired-end 100-bp decoding. TopHat was used to visualize the sequence data. ExDEGA (E-biogen, Seoul, Republic of Korea) was used for data analysis, and Prism 8 (GraphPad, CA, US) was used for visualization.

[0079] 1.9 Enzyme-linked immunosorbent assay (ELISA)

[0080] Following the manufacturer's guidelines, the expression levels of CCL21 and osteopontin in cell culture supernatants were determined using ELISAM kits purchased from Research and Diagnostic Systems (MN, US) and Lifespan Biosciences (WA, US), respectively.

[0081] 1.10 Chemotaxis Analysis

[0082] To confirm the chemical deactivation capability of SVF-globules, a chemotaxis assay was performed using the μ-Slide Chemotaxis kit (Cat#80326; Ibidi, Germany) according to the manufacturer's instructions. 1.5 mg / ml of Type I Collagen solution (Corning, NY, US) was injected into the observation area. SVF-globules or culture vessels in the culture medium were injected into the left space, while spleen cells were inserted into the right space. During the 24-hour culture period, images of surviving cells were captured using an FV1000 (Olympus Corporation, Tokyo, Japan).

[0083] 1.11 Co-culture of stromal vascular components (SVF) with dendritic cells (DCs) or spleen cells

[0084] Prepare 1×10⁻⁶ wells in a 24-well plate. 6 / Dendrite cells of pore, 1×10 6 / pore of spleen cells, 1×10 6 / Mature dendritic cells (mature Dc) per pore or 1×10 6 / Spleen cells from the pore and cultured 2×10 4 Cells were co-cultured with SVF in / wells. After culture, the cells were analyzed using flow cytometry.

[0085] 1.12 In vitro and in vivo imaging

[0086] In vitro cultured SVFs were observed using a microscope (Olympus Corporation, Tokyo, Japan) and VisiView software (Visitron Systems, Puchheim, Germany). To confirm the structure of in vivo renal capsule transplantation, graft sections were collected two weeks after surgery and rapidly cooled in liquid nitrogen in frozen section media (Leica Biosystems, IL, US). After preparing frozen sections of 5–6 μm thickness, the samples were stored at -80°C before the experiment. Hematoxylin-eosin (H&E) staining was performed for histological analysis. To perform confocal imaging, slide samples were fixed in 4% paraformaldehyde and stained with FITC-labeled anti-CD3, FITC-labeled anti-CD8 (BD Pharmingen, NJ, US), Alexa 647-labeled anti-CD31, Alexa 594-labeled anti-PDPN, Alexa 647-labeled anti-CD11c, Alexa 647-labeled anti-CD4 (Biolegend, CA, US), Alexa 594-labeled anti-VEGFR3 (Bioss Antibodies, MA, US), and 4',6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, MA, US). Confocal images were acquired using an FV3000 (Olympus corporation, Tokyo, Japan) and analyzed using IMARIS version 9.3 (Bitplane, Zurich, Switzerland).

[0087] 1.13 Assessment of antigen-specific immune response

[0088] To determine whether injecting SVF-spheroids, dendritic cells, and the aforementioned mixture could effectively induce an antigen-specific immune response, each combination (1×10⁻⁶) was tested. 6 Dendritic cells / mouse or 361 SVF-globules / mouse or both were transplanted twice, once at different weeks, into the renal capsule of each mouse. In each group, the injected dendritic cells were inhibited after NP-OVA uptake into the cells, while the SVF-globule transplantation group received an equal amount of NP-OVA simultaneously. Five weeks after the first immunization, spleen cells isolated from each mouse were separately immunized via H-2K to facilitate the spread of OVA-specific T cells.b Major Histocompatibility Complex (MHC) Type I and IA b Cells were cultured in MHCII-presented OVA peptides 257-264 and 339-323 (InvivoGen, CA, US). After 24 hours of culture, cells were stained at 4°C for 30 minutes with anti-MHC tetramer (tetramer), followed by a 30-minute staining of the cell surface, and then flow cytometry analysis was performed. The APC-labeled tetramer SIINFEKL-H-2K was also used. b And the PE-labeled tetramer AHAEINEA-IA b Obtained from the National Institutes of Health Tetramer Core Facility in the United States.

[0089] 1.14 Tumor inoculation and examination of anti-tumor efficacy

[0090] To inoculate the tumor, OVA-expressing malignant melanoma B16MO5 (5×10⁻⁶) was used. 4 Cells / mouse (mice) were injected into the left lateral side of mice, respectively. Seven days later, each combination was administered 1×10⁻⁶ cells / mice. 6 Dendritic cells / mouse and 361 SVF spheroids / mouse were inserted into the subcapsular region of the kidney three times a week. Each group consisted of 5 mice. The anti-tumor effect was investigated, and tumor size and survival were assessed. Tumor size was measured every 2–3 days. Tumor size (mm) 3 The value is calculated as 1 / 2 × [(minor axis) × (major axis)]. 2 The calculation was performed using the method described above. When the tumor size grows to 2000 mm... 3 When the above conditions are met, the mouse is considered dead.

[0091] 1.15 Statistical Analysis

[0092] For statistical analysis, the data were analyzed using GraphPad Prism software. RNA base sequence analysis was performed using ExDEGAversion 2.5.

[0093] 2. Test Results

[0094] 2.1 Characteristics of SVF isolated from adipose tissue

[0095] SVF extracted from adipose tissue initially consisted mostly of small (FSC < 250K, FSC: forward scatter, SCC: side scatter) round cells on day 0 (D0). However, during culture (D3: day 3 of culture, D6: day 6 of culture), they transformed into larger (FSC > 250K) and longer cells. Figure 1 (b~1d). Flow cytometry analysis was performed to confirm the cellular changes described above in detail. The results showed that the CD45+ hematopoietic cell population decreased during culture (23.4% → 5.94%), while the CD45- non-hematopoietic cell population increased (76.0% → 93.7%). Figure 1 e, 1f).

[0096] The results of biomarker analysis on cell types during the aforementioned cell changes revealed a rapid decrease in CD+ T cells, CD8+ T cells, and B220+ B cells, while CD11c+ dendritic cells remained unchanged until day 10 of culture. Figure 2 (a~2d), while CD11b+F4 / 80+ macrophages and GR-1+ neutrophils decreased ( Figure 2 e, 2f). In Figure 2 In the diagram, P#0, P#1, and P#2 represent the first, second, and third generation successions, respectively.

[0097] Lymph node stromal cell subtypes can be classified using PDPN and CD31 markers: PDPN+CD31- are classified as fibroblastic reticular cells (FRCs), PDPN+CD31+ as lymphatic endothelial cells (LECs), and PDPN-CD31+ as blood endothelial cells (BECs). Flow cytometry analysis of SVF isolated from adipose tissue confirmed that the CD45- cell population exhibits phenotypes similar to those of lymph node stromal cells, classifying them as PDPN+CD31-, PDPN+CD31+, and PDPN-CD31+. Figure 3a~3e). In particular, it can be confirmed that more than 60% of SVFs exhibit the FRC phenotype when cultured for more than 5 days, and more than 80% when cultured for more than 10 days (PDPN+CD31-). Figure 3 b).

[0098] Further confirmation of SVF exhibiting a PDPN+CD31-FRC-like phenotype using FRC-specific markers confirmed that these cells express FRC-specific markers such as LTβR (Lymphotoxinβ receptor), CD140α (Platelet-derived growth factor α), VCAM-1 (vascular cell adhesion molecule-1), and ICAM-1 (Intracellular adhesion molecule-1). Figure 4 ).exist Figure 4 In the diagram, P#0, P#1, and P#2 represent the first, second, and third generation successions, respectively.

[0099] In addition to the phenotypes described above, to determine whether SVFs function similarly to lymph node stromal cells, they were treated with multiple stimuli—TLR3-agonist (PolyI:C, plc), TLR7-agonist (Imiquimod), αLTβR (αLT), and LTβR ligand (anti-LTβR, LIGHT, L)—for 24 hours. Quantitative PCR was then used to quantify the mRNA levels. This confirmed the expression of representative chemokines secreted by lymph node stromal cells, namely Ccl19, Ccl21, Cxcl13, and Cxcl12 (…). Figure 5 a). Furthermore, it was confirmed that when cultured using a three-dimensional cell culture method, i.e., spheroidal cell culture (SVF spheroids, SPH), significantly more chemokines, namely Ccl19, Ccl21, and Cxcl13, were expressed compared to SVF cultured in two dimensions. Figure 5 b).

[0100] The results described above indicate that SVF isolated from adipose tissue is composed of cells similar to lymph node stromal cells in both phenotype and function.

[0101] 2.2 Angiogenesis capacity of SVF spheroids

[0102] Comparison of gene expression in SVF spheroids (SPH) cultured for 8 days (D8) using mRNA analysis with those cultured in a two-dimensional (monolayer) manner confirmed the expression of 11 genes related to angiogenesis during SVF spheroidization. Figure 6 (a) and (b) showed an increase, and flow cytometry analysis confirmed an increase in CD31+ cells (PDPN-CD31+BEC, PDPN+CD31+LEC). Figure 6 c). In Figure 6 In this context, >10 represents a fold change in gene expression that is more than 10 times greater than the average value of the control group (SVF(D8)), <0.1 represents a fold change of less than 0.1 times, and P#0 and P#1 represent the 0th and 1st generation, respectively.

[0103] To confirm whether angiogenesis is actually possible in vivo, when SVF spheroids were transplanted into the renal capsule of mice, it was confirmed that blood vessels formed on the transplant surface two weeks after transplantation. Figure 6 d), and confirmed that the generated blood vessels were positive for lymphocytes and endothelial cell markers VEGFR3 and CD31, respectively. Figure 6 e). Furthermore, frozen section images confirm the interaction and migration of CD3+ T cells with CD31+ cells. Figure 6 f).

[0104] The results described above indicate that SVF spheroids can form blood vessels that serve as channels for the movement of immune cells.

[0105] 2.3 The enhanced chemotactic capacity of dendritic cells in SVF spheroids and the enhanced interaction between T cells and dendritic cells.

[0106] The results of mRNA analysis confirming changes in gene expression of chemokines, cytokines, and growth factors in three-dimensional cultured spheroidal fungi (SPH) showed that, compared with two-dimensional cultured SVF, the expression of factors used to chemotactically attract or activate immune cells was statistically significantly increased on day 0 (D0) (p < 0.05). Figure 7 a). In Figure 7In a, >10 represents a fold change value exceeding 10 times the average of the control group (DVF(D0)), and <0.1 represents less than 0.1 times. Specifically, the expression of genes inducing angiogenesis, namely Vegfa (vascular endothelialgrowth factor α) and Pdgfc (platelet-derived growth factor C), was increased. Furthermore, the expression of Mmp 8, 10, and 13 (Matrix metalloproteinase 8, 10, 13) genes, which open cell migration channels by breaking down the extracellular matrix, was also increased. In addition, the expression of genes involved in chemotaxis and activation of immune cells, namely Spp1, Ccl8, Cxcl5, Postn, Cxcl16, and Ccl17, was also increased. In particular, the expression of the osteopontin gene Spp1, a known regulator of dendritic cell maturation and survival, was increased, and quantification of osteopontin in the culture supernatant confirmed a significant increase in its concentration in SVF spheroids. Figure 7 b).

[0107] The results described above indicate that SVF spheroids can express various factors that facilitate chemotaxis and activate immune cells.

[0108] To confirm whether chemokines secreted from the SVF can actually attract immune cells, a 24-hour migration assay was performed using the collagen layer. The results confirmed that right-side splenocytes migrate over time towards the left-side SVF spheroids (APHs). Figure 8 a). Regarding the chemotactic ability of SVF spheroids, migration assays performed on CD3+ T cells and dendritic cells (BMDCs) confirmed that while CD3+ T cells could not migrate, BMDCs could migrate into SVF spheroids. Figure 8 b, 8c, and 8d).

[0109] To confirm whether SVF spheroids possess chemotactic ability against immune cells in vivo, the results of SVF spheroids (SPH) showed that although they could not chemotize CD3+ T cells, a large number of CD3+ T cells could be chemotized when mature dendritic cells (mDCs), i.e., BMDCs, were simultaneously transplanted. This also confirmed the coexistence of CD3+ T cells with PDPN+SVF cells. Figure 9 a: SVF spheroid transplantation group alone; 9b: Mature dendritic cell transplantation group alone; 9c: Transplantation group simultaneously injected with SVF spheroids and mature dendritic cells. It was also confirmed that while CD3+ T cells could be chemotactically attracted even with only dendritic cells (BMDCs) transplanted, more CD3+ T cells could be chemotactically attracted when dendritic cells were transplanted together with SVF spheroids. Figure 9 d).

[0110] The T cells chemotactic by the SVF globules include CD4+, CD8+ T cells, and CD11c+ dendritic cells. Figure 10 (a) and (10b) Quantitative results by flow cytometry confirmed that the groups with transplanted mature dendritic cells (mDC) and SVF spheroids (SPH+mDC) had the highest number of CD3+ T cells and CD4+ T cells, and the highest number of CD8+ T cells and CD11c+ dendritic cells. Figure 10 c~10j). The results described above indicate that the coexistence of SVF globules and dendritic cells can enhance the infiltration of CD3+ T cells.

[0111] To determine whether the chemotactically attracted T cells, when both SVF spheroids and dendritic cells were present, belonged to the CD62L-CD44+ effector T cell, CD62L+CD44+ memory T cell, or CD62L+CD44- naive T cell category, further detailed analysis was performed using flow cytometry. The results confirmed that CD4+ effector T cells constituted the highest proportion of the T cells aggregated on the graft slice. Figure 11 a, 11b).

[0112] The results described above indicate that SVF spheroids can chemotact with dendritic cells and enhance the interaction between dendritic cells and T cells, and that most of the T cells can differentiate into CD4+ effector T cells and carry out immune responses.

[0113] 2.4 The complex of SVF spheroids and dendritic cells can enhance antigen-specific T cell responses.

[0114] To confirm the mechanism by which SVF (a two-dimensional culture of SVF) regulates the interaction between dendritic cells and T cells, co-culturing SVF with mature dendritic cells (mDCs) revealed that the expression of dendritic cell activation markers, namely CD40, CD80, MHCII, and DC-SIGN, was actually increased compared to culturing immature dendritic cells (iDCs) alone or mature dendritic cells alone. Figure 12 a). Furthermore, the results of co-culturing two-dimensional culture (SVF) and mature dendritic cells for one day and observing the degree of cell apoptosis using 7-AAD staining confirmed that the survival rate of mature dendritic cells (SVF+mDC) co-cultured with SVF was improved compared with the cases of culturing immature dendritic cells (iDC) and mature dendritic cells (mDC) alone. Figure 12 b). Furthermore, the results of co-culturing two-dimensional culture (SVF) with spleen cells for 7 days and staining with 7-AAD confirmed that spleen cells co-cultured with SVF for 7 days (SVF+Spl) exhibited approximately 8-fold higher survival rates compared to spleen cells cultured alone (Spl). Figure 12 c).

[0115] To confirm whether the SVF spheroids and mDC complex could enhance antigen-specific T cell responses, mice were divided into four groups: (i) untreated (UT), (ii) SVF spheroids and OVA (ovabulbumin) transplantation group (SPH), (iii) mature dendritic cells (mDC) loaded with OVA transplantation group (mDC), and (iv) SVF spheroids and mature dendritic cells (mDC) loaded with OVA transplantation group (SPH+mDC) (n=5). Two transplantation procedures were performed in mice at the beginning of the renal capsule assay and at week two. Figure 13 a) Subsequently, after four weeks, staining of the OVA peptide presented in the MHC tetramer and observation of the proportion of OVA antigen-specific CD4+ and CD8+ T cells confirmed that the proportion of CD4+ and CD8+ T cells was most significantly increased in the mDC transplantation group using SVF spheroids and OVA-activated cells. Figure 13 (b) In this case, the OVA, acting as an antigen, was loaded into dendritic cells after being bound to Fe3O4-ZnO nanoparticles.

[0116] The results described above indicate that co-culturing or co-transplanting mature dendritic cells with SVF two-dimensional culture or their spheroids can promote dendritic cell activation and improve survival rate, which can more effectively activate T cells and enhance antigen-specific T cell responses.

[0117] Antitumor activity of the 2.5SVF spheroid-dendritic cell complex

[0118] As described above, to confirm whether the increased antigen-specific T-cell immune response actually has an anticancer effect by inhibiting the growth of cancer cells, after injecting B16 melanoma cells expressing OVA (B16-OVA) into the rib area of ​​mice, they were divided into (i) untreated group (UT), (ii) SVF spheroids and OVA (ovabulbumin) transplantation group (SPH), (iii) OVA-loaded mDC transplantation group (mDC), and (iv) SVF spheroids and OVA-loaded mDC transplantation group (SPH+mDC) (n=5) as described above, and transplanted into the renal capsule of mice three times at one-week intervals. Figure 14 a). In this study, OVA, acting as an antigen, was also loaded onto dendritic cells after being bound to Fe3O4-ZnO nanoparticles. After 32 days of testing, the mDC transplantation group utilizing SVF spheroids and OVA-activated cells showed the best inhibitory effect on tumor growth, and the survival rate remained at 100% during the trial. Figure 14 (b, 14c, and 14d). This demonstrates that SVF spheroids and dendritic cells have increased efficacy compared to injecting dendritic cells alone.

[0119] As described above, it can be confirmed that when stromal vascular component (SVF) isolated from adipose tissue is transplanted together with dendritic cells to the dendritic cell transplantation site, the dendritic cells can be activated at the transplantation site and ultimately enhance the T cell response.

Claims

1. An immune response enhancement composition comprising, as an active ingredient, a stromal vascular fraction (SVF) isolated from adipose tissue and dendritic cells loaded with antigen; wherein the stromal vascular fraction is a two-dimensional culture or a spheroid obtained from a three-dimensional culture; and wherein the dendritic cells are mature dendritic cells.

2. The composition according to claim 1, characterized in that: The immune response is a T-cell-based immune response.

3. The composition according to claim 1, characterized in that: The antigen is a pathogenic microorganism-derived antigen or a tumor-derived antigen.

4. The composition according to claim 3, characterized in that: The antigen is a virus-derived antigen.

5. The composition according to claim 1, characterized in that: The composition is an antimicrobial immune-enhancing composition for treating or preventing symptoms of infection caused by pathogenic microorganisms, or an antitumor immune-enhancing composition for treating or preventing tumors.

6. The composition according to claim 5, characterized in that: The composition is an antiviral immune-enhancing composition used to treat or prevent symptoms of viral infection.

7. The composition according to claim 1, characterized in that: The dendritic cells are autologous dendritic cells obtained from the individual receiving the composition.

8. The composition according to claim 1, characterized in that: The adipose tissue is either subcutaneous fat tissue or visceral fat tissue.

9. The composition according to claim 1, characterized in that: The antigen is a tumor-derived antigen. The tumor-derived antigen is a protein expressed on the surface of tumor cells, a peptide inside tumor cells, or a protein inside tumor cells.

10. The composition according to claim 9, characterized in that: The tumor-derived antigen is a receptor expressed on the surface of tumor cells.

11. The composition according to claim 9, characterized in that: The tumor-derived antigen is a lysate of tumor cells.

12. The composition according to claim 1, characterized in that: The antigen is a tumor-derived antigen. The tumor-derived antigens mentioned are EGFRvIII, EGFR, metastin receptor, receptor tyrosine kinases, HER2 (Human epidermal growth factor receptor 2), CXCR4, CCR7, endothelin-A receptor, PPAR-δ (peroxisome proliferator activated receptor δ), PDGFR-α (Platelet-derived growth factor receptor α), CD133, CEA (carcinoembryonic antigen), EpCAM (epithelial cell adhesion molecule), GD2 (disialoganglioside), GPC3 (Glypican 3), PSMA (Prostate Specific Membrane Antigen), TAG-72 (tumor-associated glycoprotein 72), GD3 (disialoganglioside), HLA-DR (human leukocyte antigen-DR), MUC1 (Mucin 1), and NY-ESO-1 (New York esophagealsquamous cell carcinoma). 1), LMP1 (Latent membrane protein 1), TRAILR2 (tumor-necrosis factor-related apoptosis-inducing ligand receptor), VEGFR2 (vascularendothelial growth factor receptor 2), HGFR (hepatocyte growth factor receptor), CD44 or CD166.

13. The composition according to claim 1, characterized in that: The composition is a pharmaceutical composition.

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