Method for culturing dendritic cells

Abstract

The present invention relates to a composition for inhibiting the self-activation of primary dendritic cells derived from lymphoid tissues isolated from the spleen, which contains as active ingredients PHSRN, RGD and LDV peptides derived from the integrin-binding domain of fibronectin, and peptides containing these amino acids. The present invention relates to the effect of a combination of peptides for inhibiting self-activation, which provides a culture method in which the self-activation of primary dendritic cells isolated from lymphoid tissues is inhibited by the addition of fibronectin protein during culture, but activation is not induced by immune activating substances. However, the primary dendritic cells cultured with the addition of peptides can be normally activated by immune stimulants. Primary lymphoid tissue dendritic cells activated while being cultured with the combination of peptides are characterized by enhancing the proliferation of T cells and inducing the production of cytokines such as interferon when co-cultured with T cells. The compound can be usefully used as a composition for preventing or treating cancer, autoimmune diseases and inflammatory diseases by culturing dendritic cells, inducing the activation of dendritic cells, and proliferating T cells by co-culture with dendritic cells.

Description

[Technical field]

[0001] The present invention relates to a method for culturing immature primary dendritic cells isolated from lymphoid tissues, and more specifically, to a culture method that enables culturing while maintaining a state in which activation does not occur by using a combination of peptides derived from fibronectin and a linker peptide composed of these peptides. [Background technology]

[0002] Dendritic cells (DCs) are antigen-presenting cells that provide a link between innate and adaptive immune responses. Dendritic cells are widely distributed throughout the body and are divided into several subtypes in the spleen, lymph nodes, mucosa, intestine, and epidermis. In the spleen, DC subtypes are well characterized and distinct from macrophage and monocyte types.

[0003] Non-activated dendritic cells express low levels of major histocompatibility class II (MHC-II) surface molecules and costimulatory molecules, and produce few proinflammatory cytokines. Dendritic cells can directly detect pathogens by pattern recognition receptors such as scavenger receptors, Toll-like receptors, and mannose receptors, and phagocytose harmful signals derived from pathogens and damaged cells and antigens in their vicinity by phagocytosis. Dendritic cells activated by these receptors are transformed into a cell type with reduced antigen uptake capacity, increased expression of MHC-II, increased expression of costimulatory molecules such as CD80, CD86, and CD40, and the chemoreceptor CCR7, secrete various types of cytokines, and increase migration to lymph nodes. These activated dendritic cells strongly induce T cell responses against specific antigens.

[0004] The process of phagocytosis and processing of antigens is carried out by inactivated dendritic cells. The biggest problem in measuring the immunological function of these stable dendritic cells and using them as cell therapy is the isolation and culture of tissue-derived primary dendritic cells (primary DCs). Primary dendritic cells isolated for cell culture undergo spontaneous activation from inactivated to activated within 12 hours in culture. Spontaneously activated dendritic cells cannot be cultured for long periods because their survival period is very short. It has been observed that spontaneous activation is somewhat reduced when the number of dendritic cells is reduced under culture conditions or when they are cultured in semi-solid gels, etc., and it has been reported that spontaneous activation occurs due to binding or contact between dendritic cells, but the specific factors involved in dendritic cell activation and the technology to maintain and culture the inactivated form have not yet been reported.

[0005] The three-dimensional structure and extracellular matrix of the spleen and lymph nodes regulate cell interactions and play an important role in cell-cell cohesion within the tissues. Only a small amount of collagen is present in the paracortical region of lymph nodes, and fibronectin is expressed as the main extracellular matrix in lymphoid tissues such as the spleen, with very little laminin expression. It has been reported that dendritic cells differentiated from monocytes using cytokines change morphology and remain in an inactive form when cultured on plates coated with fibronectin or laminin.

[0006] Due to the various technical limitations mentioned above, techniques using dendritic cell lines transformed by gene conversion or dendritic cells differentiated from precursor cells are applied rather than culturing primary dendritic cells isolated from lymphoid tissues. Currently, the dendritic cells most commonly used for culture are dendritic cells differentiated from bone marrow cells or monocytes, which are cells differentiated from these precursor cells in the presence of inflammatory factors such as granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor (TNF-α). Therefore, dendritic cells differentiated from these precursor cells can be induced by cell culture of bone marrow cells or monocytes, but belong to the inflammatory dendritic cells that emerge in vivo from single cells in the blood to tissues under inflammatory conditions. However, in this case, dendritic cells activated in the process of differentiation from monocytes, rather than primary dendritic cells present in lymph nodes, can maintain relatively stable functions, unlike dendritic cells that are not stimulated in culture. In addition, a culture method has been reported that uses a cell culture structure based on nano-micro hybrid fibers to stably culture dendritic cells isolated from the spleen while maintaining them in an inactive state, as well as a method for analyzing inactive dendritic cells.

[0007] The present invention provides a technique for maintaining the survival of primary dendritic cells isolated from immune tissues, allowing them to exist in a functionally inactive state, phagocytose antigens, and become active when stimulated by immune or inflammatory stimuli, thereby enabling the immunological responses of dendritic cells that may occur in vivo to be realized in a cultured state. The primary dendritic cells isolated in this way are highly likely to be developed as immune cell therapeutic agents for diseases such as cancer, viral infection, autoimmune diseases, organ transplants, allergies, and rheumatoid arthritis by inducing the proliferation of T cells or by co-culturing them with T cells to induce cytokine secretion. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a composition for suppressing the autoactivation of dendritic cells, which comprises a specific peptide capable of maintaining primary dendritic cells isolated from lymphoid tissues in an inactive form.

[0009] Another object of the present invention is to provide a method for activating immune cells, comprising the step of co-culturing the dendritic cells cultured by the above-mentioned culture method with immune cells.

[0010] Another object of the present invention is to provide a dendritic cell composition comprising isolated dendritic cells maintained in a non-activated state for 24 hours or more, and a pharmaceutical composition for treating or preventing cancer comprising the same as an active ingredient. [Means for solving the problem]

[0011] 1. A composition for inhibiting the autoactivation of dendritic cells, comprising a peptide derived from fibronectin that binds to an integrin binding site between dendritic cells.

[0012] 2. A composition for suppressing the autoactivation of dendritic cells according to item 1 above, wherein the peptide is a peptide consisting of an amino acid sequence comprising any one of SEQ ID NOs: 1 to 3.

[0013] 3. A composition for inhibiting dendritic cell autoactivation according to item 1 above, wherein the peptide comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 3 and has a length of 3 to 11 aa.

[0014] 4. A composition for suppressing the autoactivation of dendritic cells according to item 1 above, wherein the peptide is at least one selected from the group consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 7.

[0015] 5. A composition for suppressing dendritic cell autoactivation according to item 1 above, wherein the peptide comprises two or more amino acid sequences each including any one of SEQ ID NOs: 1 to 3 linked together via a linker.

[0016] 6. A composition for inhibiting dendritic cell autoactivation according to item 5 above, wherein the linker consists of amino acids G and S and has a length of 2 to 8 aa.

[0017] 7. A composition for inhibiting the autoactivation of dendritic cells according to item 5 above, wherein the linker comprises an amino acid sequence including any one of SEQ ID NOs: 8 to 11.

[0018] 8. A composition for suppressing the autoactivation of dendritic cells according to item 5 above, wherein the peptide comprises any one of the amino acid sequences of SEQ ID NOs: 12 to 14.

[0019] 9. In the above-mentioned item 1, the peptide is administered to 2×10 dendritic cells to be treated. 5 A composition for inhibiting the self-activation of dendritic cells, comprising a concentration of 200 μg / mL or more per mL of dendritic cell.

[0020] 10. A composition for inhibiting the activation of dendritic cells according to item 1 above, wherein the dendritic cells are derived from mononuclear cells, the spleen or lymph nodes.

[0021] 11. A composition for inhibiting dendritic cell activation according to item 1 above, which is intended for in vitro use.

[0022] 12. A method for culturing dendritic cells, comprising the step of treating isolated dendritic cells with a composition according to any one of items 1 to 11 above.

[0023] 13. The method for culturing dendritic cells according to item 12, further comprising the step of treating the dendritic cells treated with the composition with an immunostimulant to induce activation of the dendritic cells.

[0024] 14. The method for culturing dendritic cells according to item 13, wherein the immunostimulant is at least one selected from the group consisting of a bacterial preparation, CD40 ligand, TNF-α, IL-1β, LPS and poly(I:C).

[0025] 15. A step of treating isolated dendritic cells with a composition according to any one of items 1 to 11 and culturing the dendritic cells in an inactive state; activating the dendritic cells; and co-culturing the activated dendritic cells with immune cells.

[0026] 16. A dendritic cell composition comprising a peptide derived from fibronectin that binds to an integrin-binding site between dendritic cells according to any one of items 1 to 11 above, and dendritic cells mixed therewith.

[0027] 17. A pharmaceutical composition for treating or preventing cancer, an infectious disease, a chronic inflammatory disease, or an autoimmune disease, comprising the cell composition according to item 16 as an active ingredient. Effect of the Invention

[0028] The present invention relates to a cell culture composition for maintaining the inactivity of dendritic cells, which comprises as an active ingredient a combination of peptides including a sequence of a cell adhesion domain of the fibronectin protein sequence, and relates to an effect as a culture medium for dendritic cells derived from lymphatic tissues such as the spleen and lymph nodes. The combination of peptides maintains the inactivity of dendritic cells or activates the cells with an immunostimulant, thereby inducing the proliferation and activation of T cells, and can be usefully used as a composition for immunological diagnosis or immune cell therapy of cancer, autoimmune diseases, and inflammatory diseases. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 shows the distribution of spleen dendritic cells (sDCs) cultured without the addition of the peptide mixture of the present invention, as observed by confocal microscopy, and the degree of cell clustering. [Diagram 2]FIG. 2 shows the results of flow cytometry of splenic dendritic cells cultured without the addition of the peptide mixture of the present invention or a stimulant, confirming that expression of MHC-II, CD86, CD80 and CD40 increased on the surface of dendritic cells in a time-dependent manner. [Diagram 3] FIG. 3 shows the distribution of splenic dendritic cells cultured with the addition of fibronectin or laminin in the absence of the peptide mixture of the present invention, as observed by confocal microscopy. [Figure 4] FIG. 4 shows the results of flow cytometry measuring the expression levels of CD86 and MHC-II on the surface of splenic dendritic cells after culturing for 6 hours with various concentrations of fibronectin or laminin without the addition of the peptide mixture of the present invention. [Diagram 5] FIG. 5 shows the results of flow cytometry to measure the expression levels of CD86, MHC-II, CD80, and CD40 on the surface of splenic dendritic cells after culturing for 6 hours with fibronectin or laminin at a concentration of 200 μg / mL without the addition of the peptide mixture of the present invention. [Figure 6] FIG. 6 shows the amino acid sequences of the peptides and the types of peptides used in the present invention. [Figure 7] FIG. 7 shows the results of flow cytometry in which the expression level of CD86 was measured after splenic dendritic cells were cultured for 6 hours in the presence of the peptide mixture of the present invention. [Figure 8] FIG. 8 shows the distribution of splenic dendritic cells observed by confocal microscopy after 6 or 24 hours of culture with the addition of the peptide mixture of the present invention. [Figure 9] FIG. 9 shows the results of flow cytometry in which the expression level of CD86 was measured after splenic dendritic cells were cultured for 6 hours in the presence of a peptide mixture containing P3. [Figure 10]FIG. 10 shows the results of flow cytometry that measured the expression levels of MHC-II, CD86, CD80, and CD40 in splenic dendritic cells after culturing for 6 or 24 hours with various combinations of three peptide mixtures, compared with culture conditions with the addition of fibronectin. [Figure 11] FIG. 11 shows the results of culturing dendritic cells in the presence of the peptide mixture of the present invention, fluorescently staining the expression of CD86 with an antibody, and then observing the cells with a confocal microscope. [Figure 12] FIG. 12 shows the results of an enzyme-linked immunosorbent assay (ELISA) measuring the amount of TNF-α secreted into the culture medium of dendritic cells cultured without the addition of the peptide mixture of the present invention over time. [Figure 13] FIG. 13 shows the results of ELISA in which the amount of secreted TNF-α was measured after 24 hours of culture in the presence of a mixture of the three peptides. [Figure 14] Figure 14 shows the results of flow cytometry to measure the level of CD86 expression in splenic dendritic cells after 24 hours of culture on a culture plate containing a mixture of three peptides or fibronectin, with or without the addition of the immunostimulant LPS (lipopolysaccharide). [Figure 15] Figure 15 shows the results of flow cytometry to measure the level of CD86 expression in splenic dendritic cells after 24 hours of culture on a PCL (polycaprolactone) nanofiber membrane loaded with a peptide mixture or fibronectin, with or without the addition of the immunostimulant LPS. [Figure 16] Figure 16 shows the results of ELISA that measured the amounts of TNF-α and IL-12p40 secreted by splenic dendritic cells after 24 hours of culture on a culture plate containing a mixture of three peptides or fibronectin, with or without the addition of the immunostimulant LPS. [Figure 17]Figure 17 shows the results of flow cytometry to measure the level of CD86 expression in bone marrow-derived dendritic cells (BMDCs) after 24 hours of culture on a culture plate containing a mixture of three peptides or fibronectin, with or without the addition of the immunostimulant LPS. [Figure 18] FIG. 18 shows the results of flow cytometry to measure the level of CD86 expression in bone marrow-derived dendritic cells after 24 hours of culture in a culture plate containing a mixture of three peptides or fibronectin at various concentrations, in the presence of LPS. [Figure 19] Figure 19 shows the results of ELISA to measure the amounts of TNF-α and IL-12p40 secreted by bone marrow-derived dendritic cells after 24 hours of culture in a culture plate containing various concentrations of a mixture of three peptides or fibronectin, in the presence of LPS. [Figure 20] Figure 20 shows the results of flow cytometry in which the level of CD86 expression in splenic dendritic cells was measured in comparison with that in LPS after 24 hours of culture in a culture plate containing a mixture of three peptides or fibronectin and then the immunostimulant poly(I:C). [Figure 21] Figure 21 shows the results of flow cytometry of T cell proliferation in a mixed lymphocyte reaction between splenic dendritic cells cultured for 24 hours in a culture plate containing a mixture of three peptides or fibronectin, and the immunostimulants LPS or poly(I:C), and T cells isolated from xenogeneic mice and fluorescently stained with CFSE (carboxyfluorescein succinimidyl ester). [Figure 22] Figure 22 shows the results of measuring the amount of cytokines secreted by ELISA after co-culturing splenic dendritic cells for 4 days with T cells isolated from xenogeneic mice, which were cultured for 24 hours in a culture plate containing a mixture of three peptides or fibronectin and the immunostimulants LPS or poly(I:C). [Figure 23]FIG. 23 shows the results of measuring the viability of primary splenic dendritic cells cultured for 6 hours in a culture plate containing a mixture of three types of peptides or fibronectin, by flow cytometry using 7-AAD (7-aminoactinomycin D) staining. [Figure 24] FIG. 24 shows the results of flow cytometry measurement of the level of CD86 expression in primary splenic dendritic cells cultured with the addition of the linker peptide of the present invention. [Diagram 25] FIG. 25 shows the results of flow cytometry measuring the expression level of CD86 in primary splenic dendritic cells cultured with the addition of the scrambled linker peptide of the present invention. [Figure 26] FIG. 26 shows the results of confirming the expression of CD86 by confocal microscopy after adding linker peptide to splenic dendritic cells and culturing them for 24 hours. [Figure 27] FIG. 27 shows the results of ELISA in which the concentration of secreted tumor necrosis factor (TNF-α) was measured after adding linker peptide or LPS and culturing primary splenic dendritic cells for 24 hours. [Figure 28] Figure 28 shows the results of flow cytometry in which splenic dendritic cells that do not self-activate after incubation with the linker peptide or dendritic cells obtained by activating these cells with LPS were co-cultured with OT-1 T cells fluorescently stained with CFSE for 4 days, after which T cell proliferation was measured. [Figure 29] Figure 29 shows the results of ELISA that measured the concentrations of interferon-gamma (IFN-γ) and TNF-α secreted into the culture medium after co-culture of splenic dendritic cells cultured with the linker peptide with OT-1 T cells for 4 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The present invention provides a composition for suppressing the autoactivation of dendritic cells, which comprises a peptide derived from fibronectin that binds to an integrin binding site between dendritic cells.

[0031] Dendritic cells act as antigen-transmitting cells by processing pathogenic material with immune cells and displaying antigens on their surface for other cells of the immune system, and thus act as intermediaries between the innate and adaptive immune responses. Types of dendritic cells include, but are not limited to, monocyte-derived dendritic cells, spleen-derived dendritic cells, and lymphoid tissue-derived dendritic cells.

[0032] The integrin binding site between dendritic cells may be a site that plays an important role in cell-substrate and cell-cell binding and interaction. When the peptide binds to such an integrin binding site, it can inhibit cell-cell interaction, and inhibit intercellular signal transduction, autoactivation, etc.

[0033] The peptide derived from fibronectin consists of a part of the sequence of fibronectin, which is one of the extracellular matrix proteins, and can further contain other amino acid sequences other than the part of the fibronectin sequence.

[0034] That is, the present invention includes a composition for inhibiting the autoactivation of dendritic cells, which comprises a peptide comprising the amino acid sequence of the site of fibronectin that binds to the integrin-binding site between dendritic cells.

[0035] Specifically, the sequence of fibronectin can include the sequence of a cell adhesion domain among the domains constituting fibronectin.

[0036] The peptide may consist of a sequence including RGD (SEQ ID NO: 1), LDV (SEQ ID NO: 2) or PHSRN (SEQ ID NO: 3), for example, RGD (SEQ ID NO: 1), LDV (SEQ ID NO: 2), PHSRN (SEQ ID NO: 3), KGRGDS (SEQ ID NO: 4), GRGDS (SEQ ID NO: 5), GGPEILDVPST (SEQ ID NO: 6) or EILDVPST (SEQ ID NO: 7).

[0037] The length of the peptide may be, but is not limited to, 3 to 11 aa.

[0038] The peptide may have two or more amino acid sequences containing RGD, LDV or PHSRN linked together via a linker.

[0039] The linker may be 2 to 8 aa (amino acid) in length and composed of the amino acids G and S. For example, the linker may be composed of an amino acid sequence containing GS (SEQ ID NO: 8), GGGS (SEQ ID NO: 9), GGGGS (SEQ ID NO: 10), or GGGSGGGS (SEQ ID NO: 11).

[0040] For example, the peptide may comprise the amino acid sequence of PHSRNGSRGDGSLDV (SEQ ID NO: 12), PHSRNGGGSRGDGGGSLDV (SEQ ID NO: 13), or PHSRNGGSGGGSRGDGGGSLDV (SEQ ID NO: 14).

[0041] In the embodiments of the present specification, the term "linker peptide" refers to a peptide in which multiple peptides are linked by linkers as described above.

[0042] The composition may contain two or more of the peptides.

[0043] The composition of the present invention is administered in a concentration of 2×10 5 The peptide can be contained at a specific concentration or more relative to dendritic cells per mL of the antibody, and the concentration is, for example, 50 μg / mL or more, 100 μg / mL or more, 200 μg / mL or more, 300 μg / mL or more, 400 μg / mL or more, or 500 μg / mL or more, specifically, 50 to 800 μg / mL, 50 to 500 μg / mL, 100 to 800 μg / mL, 100 to 600 μg / mL, 100 to 500 μg / mL, 100 to 300 μg / mL, 100 to 200 μg / mL, 150 to 250 μg / mL, 200 to 800 μg / mL, 200 to 500 μg / mL, or 300 to 500 μg / mL, but is not limited thereto.

[0044] The compositions of the present invention can modulate dendritic cell-mediated immune responses, specifically, but not limited to, suppressing the autoactivation of dendritic cells or modulating the ability of dendritic cells to present autoantigens.

[0045] "Autoactivation of dendritic cells" refers to the spontaneous activation phenomenon in which isolated dendritic cells are converted from inactive to active form within 12 hours in culture. This phenomenon has been reported to occur due to binding or contact between dendritic cells. The activated dendritic cells have a drawback in that they have a very short life span and cannot be cultured for a long period of time. Therefore, inhibition of activation allows isolated dendritic cells to be cultured in an inactive form that can phagocytose and process antigens.

[0046] The composition for inhibiting activation of the present invention can be used in vivo or in vitro, but is preferably for use in vitro.

[0047] The present invention also provides a method for culturing dendritic cells, which comprises the step of treating the cells with a composition for inhibiting the autoactivation of dendritic cells.

[0048] The composition for inhibiting autoactivation is as described above, and the treatment concentration and treatment time of the composition are not limited to a specific concentration and time as long as the composition can inhibit the autoactivation of dendritic cells. 5 The concentration is 50 to 500 μg / mL, 100 to 400 μg / mL, or 0 to 300 μg / mL per mL, and the treatment time may be, for example, 2 to 48 hours, 6 to 48 hours, 4 to 24 hours, 6 to 18 hours, 4 to 12 hours, 6 to 8 hours, or 6 to 24 hours, but is not limited to these.

[0049] The method for culturing dendritic cells of the present invention may further include a step of inducing activation of dendritic cells by treating with an immunostimulant. The immunostimulant refers to a substance that exposes antigens to dendritic cells and induces their conversion into cells that display antigens on their surface. Examples of the immunostimulant include, but are not limited to, bacterial preparations, CD40 agonists TNF-α, IL-1β, LPS, and poly(I:C).

[0050] The dendritic cells can be cultured three-dimensionally on a structure made of polycaprolactone (PCL) nanofibers, but is not limited thereto.

[0051] The present invention also provides a method for activating immune cells.

[0052] The method for activating immune cells includes the steps of treating isolated dendritic cells with the composition and culturing the dendritic cells in a non-activated state, activating the dendritic cells, and co-culturing the activated dendritic cells with immune cells.

[0053] The immune cells are cells involved in immune responses in the immune system and may refer to cells used as cell therapeutic agents, including, but not limited to, T cells, B cells, natural killer cells (NK cells), natural killer T cells (NKT cells), mast cells, or bone marrow-derived phagocytes.

[0054] The step of treating the composition and culturing dendritic cells in a non-activated state is as described in the method for culturing dendritic cells.

[0055] The step of activating dendritic cells can be carried out by treating non-activated dendritic cells with a maturation factor or an immunostimulant. The maturation factor or immunostimulant is not limited to any particular type, so long as it is a substance that induces increased expression of MHC-II, CD86, CD80 or CD40 on the surface of dendritic cells, and may be, for example, a bacterial preparation, CD40 agonist TNF-α, IL-1β, LPS or poly(I:C).

[0056] The step of co-culturing activated dendritic cells and immune cells is intended to induce proliferation of immune cells and secretion of cytokines. The ratio of the number of immune cells co-cultured based on the number of activated dendritic cells is, for example, 1 to 50, 4 to 30, 5 to 25, 10 to 30, or 15 to 25, and the co-culture time may be, but is not limited to, 2 to 144 hours, 4 to 120 hours, 12 to 96 hours, 24 to 96 hours, 48 ​​to 96 hours, or 48 to 72 hours.

[0057] The present invention also provides a dendritic cell composition comprising the peptide and dendritic cells mixed therewith.

[0058] The dendritic cells of the composition maintain a non-activated state for, for example, 6 hours or more, 12 hours or more, 24 hours or more, or 48 hours or more, and preferably, for 6 to 24 hours, 6 to 48 hours, 12 to 48 hours, or 12 to 24 hours, but are not limited to these.

[0059] The cell composition contains dendritic cells maintained in a non-activated state by the above-mentioned composition for inhibiting dendritic cell activation, and the dendritic cells may specifically be dendritic cells in which increased expression of MHC-II, CD86, CD80 or CD40 factors has been inhibited.

[0060] The dendritic cells remain viable until they are stimulated and activated, and can functionally phagocytose antigens. Unlike activated dendritic cells, they can be cultured for a long time. Therefore, by inducing the proliferation of T cells in vitro or by co-culturing them with T cells to promote cytokine secretion, it is possible to develop immune cell therapeutic agents for various diseases.

[0061] The present invention also provides a pharmaceutical composition for treating or preventing cancer, bacterial and viral infectious diseases, chronic inflammatory diseases, or autoimmune diseases, which comprises the dendritic cell composition as an active ingredient.

[0062] Examples of the cancer include, but are not limited to, lung cancer, laryngeal cancer, gastric cancer, colon / rectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, prostate cancer, kidney cancer, carcinoma derived from epithelial cells, bone cancer, muscle cancer, fat cancer, sarcoma derived from connective tissue cells, leukemia, lymphoma, blood cancer derived from hematopoietic cells such as multiple myeloma, tumors occurring in nervous tissue, thyroid cancer, colon cancer, testicular cancer, brain tumor, head and neck cancer, bladder cancer, epithelial cancer, adenocarcinoma, esophageal cancer, mesothelioma, skin cancer, and melanoma.

[0063] The infectious disease is a disease caused by infection with a pathogen such as bacteria, spirochetes, rickettsia, virus, fungus, or parasite, but is not limited to these.

[0064] The chronic inflammatory disease is a disease that occurs due to the long-term accumulation of inflammation, and is known to cause cellular aging and transformation, and to disrupt the immune system by excessively activating the immune response. It has been reported that this can lead to metabolic diseases such as obesity and diabetes, or even autoimmune diseases.

[0065] The autoimmune disease is a disease that occurs when the immune system inside the human body attacks normal cells inside the body instead of external antigens, and examples of the disease include, but are not limited to, rheumatoid arthritis, atopy, lupus, Crohn's disease, type 1 diabetes, ulcerative colitis, and multiple sclerosis.

[0066] In addition to the above-mentioned cancers, infectious diseases, or immune diseases, as long as immune cells can be used as a therapeutic agent for a disease, regardless of the type of disease, the cell composition of the present invention can be used as an active ingredient and as a pharmaceutical composition for treating or preventing the disease.

[0067] The present invention will be described in detail below with reference to examples. The following examples are for illustrating the present invention, but are not intended to limit the scope of the present invention.

[0068] Example 1. Isolation and culture of splenic dendritic cells Mouse splenic dendritic cells were prepared by adding EDTA-BSS (Balanced Salt Solution) buffer and collagenase solution (DMEM containing 100 units of collagenase type IV, a medium containing low glucose) to the spleen of a female C57BL / 6 mouse, cutting the spleen into small pieces with surgical scissors, mixing with 6 ml of collagenase treatment solution, and incubating for 30 minutes. The isolated whole spleen tissue was filtered using a 70 μM cell strainer (Falcon) and centrifuged at 1800 rpm for 5 minutes to separate the cells. Red blood cells were removed by adding red blood cell lysis buffer (Sigma Aldrich), and the dendritic cells were washed twice with RPMI-1640 medium containing 10% fetal bovine serum (FBS). Dendritic cells were identified as CD11c + The cells were reacted with antibody-conjugated microbeads and separated using CD11c Microbeads UltraPure (MACS, Milteni Biotec, USA).

[0069] In Figure 1, primary dendritic cells isolated from the spleen were dispensed into a 48-well plate and cultured for 24 hours in a cell chamber maintained at 37°C and 5% CO2, and the degree of clustering, in which the cells were distributed as single cells or gathered together, was observed using differential interference contrast (DIC) images taken with a confocal microscope (Olympus confocal microscope FV1000, Olympus Corporation; n = 5). In this case, it was shown that cell aggregation further increased with the passage of culture time. This phenomenon occurred when the number of cells reached 2 × 10 5 / mL than 1 × 10 6 The number of single cells and clusters of cells was measured using ImageJ software (NIH, 6 Bethesda). * indicates statistical significance, P < 0.05 compared to 0h.

[0070] Example 2. Measurement of dendritic cell activation using flow cytometry Unstimulated, steady-state dendritic cells have a non-activated phenotype and express very low levels of phenotypic factors such as MHC-II and costimulatory molecules such as CD80, CD86, and CD40, whereas activated dendritic cells show a marked increase in the expression of these markers.

[0071] In Figure 2, to determine whether autoactivation occurs when primary dendritic cells isolated from spleens are cultured, dendritic cells were plated at 2 × 10 5The cells were cultured in RPMI-1640 medium at 37°C and 5% CO2 for 6 hours, and the expression levels of MHC-II, CD86, CD80, and CD40 on the surface of dendritic cells were analyzed using flow cytometry (MACSQuant VYB flow cytometer, Miltenyi Biotec). The results of the flow cytometry were analyzed using FlowJo v10 (Tree Star, Ashland). The expression levels of CD86, CD80, and CD40 were expressed as the percentage of cells stained with CD11c, and the expression level of MHC-II was expressed as the mean fluorescence intensity (MFI). The results showed that the expression levels of MHC-II, CD86, CD80, and CD40 on the surface of dendritic cells increased in a time-dependent manner.

[0072] Example 3. Cultivation of splenic dendritic cells in medium supplemented with fibronectin In Figure 3, fibronectin or laminin was added to RPMI-1640 medium at a concentration of 200 μg / mL, and 2 × 10 primary CD11c+ dendritic cells isolated from the spleen were cultured. 5 After 6 hours of incubation, the distribution of cells was observed by confocal microscopy. When fibronectin was added, cell aggregation was significantly reduced. This suggests that fibronectin may be an extracellular matrix factor that inhibits aggregation between dendritic cells. * indicates statistical significance at P<0.05 compared to None.

[0073] In Figure 4, splenic dendritic cells were cultured for 6 hours with various concentrations of fibronectin or laminin, and the expression levels of CD86 and MHC-II were measured by flow cytometry. As a result, fibronectin at a concentration of 200 μg / mL did not induce autoactivation, and laminin at a concentration of 200 μg / mL or higher did not suppress autoactivation. From this, it was confirmed that in the present invention, fibronectin at a concentration of 200 μg / mL is the most effective for the autoactivation of splenic dendritic cells. * indicates statistical significance, P<0.05 or less compared to 0 μg / mL concentration.

[0074] In Figure 5, fibronectin at a concentration of 200 μg / mL inhibited CD11c + It was shown that when splenic dendritic cells were cultured for 6 hours, the expression of not only CD86 and MHC-II but also CD80 and CD40 did not increase.

[0075] Example 4. Cultivation of splenic dendritic cells in a medium supplemented with a peptide prepared from the amino acid sequence of fibronectin protein In FIG. 6, in the amino acid base sequence of fibronectin, one of the extracellular matrix proteins that mediate cell adhesion, arginine-glycine-aspartate (Arg-Gly-Asp, RGD) (hereinafter referred to as "P1", sequence number 1) and the like correspond to the site that binds to the integrin protein of the cell membrane, and other base sequences Leu-Asp-Val (LDV) (hereinafter referred to as "P2", sequence number 2) and Pro-His-Ser-Arg-Asn (PHSRN) (hereinafter referred to as "P3", sequence number 3) are also known to play a similar role, so peptides consisting of these base sequences were manufactured by requesting PEPTRON, INC. Motifs such as RGD in the fibronectin protein bind to integrins and act on cell-substrate and cell-cell binding. Similar peptides other than RGD peptide were produced and used, including Lys-Gly-Arg-Gly-Asp-Ser (KGRGDS) (hereinafter referred to as "P1-1"; sequence number 4), GRGDS (hereinafter referred to as "P1-2"; sequence number 5), Gly-Gly-Pro-Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr (GGPEILDVPST) (hereinafter referred to as "P2-1"; sequence number 6), and Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr (EILDVPST) (hereinafter referred to as "P2-2"; sequence number 7). As controls, Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER) (hereinafter referred to as "P4"; sequence number 18) was prepared as an amino acid sequence corresponding to the integrin binding motif in collagen, and Tyr-Ile-Gly-Ser-Arg (YIGSR) (hereinafter referred to as "P5"; sequence number 19) and Ile-Lys-Val-Ala-Val (hereinafter referred to as "P6"; sequence number 20) were prepared as amino acid sequences which are one of the integrin binding motifs in laminin and used.

[0076] In Figure 7, when splenic dendritic cells were cultured, the prepared peptides were added at a concentration of 200 μg / mL, and 6 hours later, the expression level of CD86 was measured. When each peptide was treated alone, the expression of CD86 in splenic dendritic cells was not suppressed, but when the P1, P1-2, and P2-1 peptides were treated together, the expression of CD86 was significantly reduced. Compared to when the cells were treated with these three peptides, the expression was not suppressed when the two peptides were treated together. * indicates statistical significance, P<0.05 or less compared to the P1, P1-2, and P2-1 conditions.

[0077] In FIG. 8, the produced P3 peptide was added to the splenic dendritic cell culture medium at a concentration of 200 μg / mL in combination with P1 (200 μg / mL) and P2 (200 μg / mL), and the cells were cultured for 6 or 24 hours. In this case, cell cluster formation was significantly suppressed. In comparison, it was shown that the simultaneous addition of three peptides, P4, P5, and P6, which are peptides derived from collagen and laminin proteins, did not suppress cluster formation, and no suppression phenomenon was observed when P5 and P6, which are peptides derived from laminin, were added. * indicates statistical significance, P<0.05 or less compared to None.

[0078] In FIG. 9, the expression level of CD86 was measured by flow cytometry after adding three combinations of P1, P2, and peptides containing amino acids of these peptides together with P3 peptide to the splenic dendritic cell culture medium and culturing for 6 hours. When the other two peptides were added at a concentration of 200 μg / mL each while containing P3 (200 μg / mL), the expression of CD86 was significantly reduced in all measurements. In particular, the most significant inhibitory effect was obtained when P1, P2, and P3 peptides were added, or when P1-2, P2-1, and P3 peptides were added. In comparison, no inhibitory effect was observed when P3 was used alone at a concentration of 600 μg / mL. * indicates P<0.05 or less compared to None, indicating statistical significance.

[0079] In Figure 10, three types of peptides were added to splenic dendritic cell cultures, and the expression of MHC-II, CD80, and CD40 other than CD86 was suppressed by flow cytometry after culturing for 6 or 24 hours. As a result, it was confirmed that the expression of these peptides was significantly suppressed when P1, P2, and P3 peptides were added together with fibronectin, or when P1-2, P2-1, and P3 peptides were added. In contrast, when P4, P5, and P6 peptides were added at a concentration of 200 μg / mL, no suppressive effect was observed, and treatment with laminin alone had no effect. * indicates P<0.05 or less compared to None, indicating statistical significance.

[0080] Example 5. Measurement of Fluorescent Staining for CD86 Expression in Splenic Dendritic Cells Cultured with Peptides In Figure 11, splenic dendritic cells (2 × 10 5 The peptides were added at a concentration of 200 μg / mL when the cells were cultured at 100 μg / mL. After 6 hours, the expression level of CD86 was measured by fluorescent staining using PE-conjugated anti-CD86 antibody (eBioscience) and confocal microscopy. After culturing primary splenic dendritic cells for a certain period of time, intracellular CD86, actin (F-actin), and nucleus staining were performed. Actin expression was stained with FITC-conjugated phalloidin (eBioscience), and nucleus staining was performed with Hoescht 33342 (Sigma-Aldrich). For this purpose, the cells cultured in a 48-well plate were placed in 4% paraformaldehyde fixative and fixed at room temperature for 20 minutes.

[0081] Next, the cells were washed twice with phosphate buffer solution, infiltrated with 0.1% Triton X-100 (Sigma-Aldrich) solution at room temperature for 5 minutes, and washed twice with phosphate buffer solution. To remove non-specific staining, the cells were reacted with 0.2% bovine serum albumin (Sigma-Aldrich) solution at room temperature for 20 minutes, and then washed twice with phosphate buffer solution. Then, CD86 antibody and phalloidin were diluted with 0.2% bovine serum albumin solution at a ratio of 1:40 and reacted for 40 minutes, washed three times with Tris buffer solution, and placed on a slide glass. For cell nuclei staining, the cells were counter-stained with Hoechst 33342 solution, and then covered with a cover glass to mount the cells. The cells were then observed using a confocal laser microscope (K1 Nanoscope).

[0082] Example 6. Secretion of TNF-α from cultured splenic dendritic cells and effects of peptides In Figure 12, 1 × 10 primary splenic dendritic cells were 6 After 24 h of incubation, the concentration of TNF-α secreted by these cells in the culture medium was measured, and it was shown that the secretion increased in a time-dependent manner. The concentration of TNF-α was measured at 450 nm using an ELISA kit (R&D systems) and a microplate reader (Synergy H1, Biotek). However, when dendritic cells were cultured at 2 × 10 5 / mL increased CD86 expression but not TNF-α secretion (results not presented). * indicates statistical significance, P < 0.05 compared to 3h.

[0083] In Figure 13, 1 × 10 primary splenic dendritic cells were 6The secretion of TNF-α was significantly decreased when the P1, P2 and P3 peptides or the P1-2, P2-1 and P3 peptides were added to the culture medium at a concentration of 100 μg / mL and cultured for 24 hours. In addition, when fibronectin was added and cultured, almost no TNF-α was measured in the culture medium, and when the P4, P5 and P6 peptides were added at a concentration of 200 μg / mL, respectively, the secretion of TNF-α was not suppressed. From this, it is seen that the activation of primary dendritic cells is not directly related to the secretion of TNF-α, but the amount of TNF-α secreted from activated cells is effectively suppressed by the combination of fibronectin and peptides. * indicates P<0.05 or less compared to 3h, indicating statistical significance.

[0084] Example 7. Induction of activation of splenic dendritic cells cultured with peptides and fibronectin In FIG. 14, the CD86 expression level of splenic primary dendritic cells cultured for 24 hours was measured similarly to the CD86 expression level increased by treatment with E. coli LPS, an activation inducer, at a concentration of 1 μg / mL, and it was shown that the CD86 expression did not increase further even when LPS was added to the culture conditions where self-activation occurs. The CD86 expression of splenic dendritic cells cultured with fibronectin did not increase even when treated with 1 μg / mL LPS, but the CD86 expression of dendritic cells with suppressed self-activation cultured with peptides P1, P2, and P3 or peptides P1-2, P2-1, and P3 was significantly increased by LPS stimulation. * indicates statistical significance at P<0.05 or less compared to None. ** indicates statistical significance at P<0.05 or less compared to -LPS.

[0085] Example 8. Degree of activation of splenic dendritic cells cultured on nanofiber membranes and effects of peptides and LPS FIG. 15 shows that primary splenic dendritic cells cultured for 24 hours on a nanofiber membrane made of polycaprolactone (PCL) also experienced spontaneous activation, as in the case of culture plates, and that CD86 expression increased significantly. Nanofiber structures were produced by adding polycaprolactone (Sigma, MW 80,000) to a chloroform solvent at a ratio of 15% (w / v), and electrospinning this solution (5 ml) at a spinning speed of 8 μl / min and a spinning distance of 20 cm using a 25G metal syringe at 10 KV using an electrospinning machine (Nano-NC). The effects of peptide combinations and LPS on CD86 expression in splenic dendritic cells cultured on polycaprolactone nanofibers showed results similar to those obtained on culture plates shown in FIG. 14. Thus, the degree of spontaneous activation of splenic dendritic cells under three-dimensional culture conditions such as nanofiber membranes showed a similar aspect to that of two-dimensional culture. * indicates statistical significance at P<0.05 or less compared to None, and ** indicates statistical significance at P<0.05 or less compared to -LPS.

[0086] Example 9. Cytokine secretion ability of splenic dendritic cells cultured with peptide and fibronectin Figure 16 shows the results of measuring whether inflammatory cytokines and Th1-inducing cytokines were normally secreted when splenic primary dendritic cells cultured for 24 hours with the addition of a combination of peptides were stimulated with LPS. Cells cultured with the addition of peptides maintained inactive forms and secretion of TNF-α and IL-12p40 was barely measured, but after treatment with 1 μg / mL LPS, high concentrations were measured in the culture medium. In contrast, splenic dendritic cells cultured with the addition of fibronectin were shown to be unable to produce cytokines when stimulated with LPS. This shows that fibronectin can maintain the inactivation of splenic dendritic cells, but has the disadvantage of suppressing the activation effect of stimulants. * indicates P<0.05 or less compared to -LPS, indicating statistical significance.

[0087] Example 10. Investigation of the effects of peptides and fibronectin on stimulatory activation of bone marrow-derived dendritic cells We measured how peptide combinations and the addition of fibronectin affected LPS stimulation of bone marrow derived dendritic cells (BMDCs) compared to splenic dendritic cells.

[0088] Murine bone marrow-derived dendritic cells were obtained by differentiation from mouse bone marrow progenitor cells. The femurs and tibias of female C57BL / 6 mice were removed, and bone marrow was isolated using 10 mL of EDTA:BSS (balanced salt solution) buffer containing 2% FBS. Red blood cells were removed using red blood cell lysis buffer (Sigma Aldrich), and after washing with EDTA:BSS buffer, the cells were filtered using a 70 μm cell strainer (Falcon). 3 × 10 6 The cells were dispensed into a 6-well plate at 1000μg / mL, and cultured for 7 days with interleukin-4 (20ng / mL) (JW Creagene) and GM-CSF (20ng / mL) (JW Creagene) to induce cell differentiation. The differentiated dendritic cells were reacted with microbeads bound to CD11c antibody (eBioscience), isolated using CD11c Microbeads Ultrapure (MACS, Milteni Biotec), and then cultured.

[0089] In Figure 17, 2 x 10 5 100μg / mL bone marrow-derived dendritic cells were cultured for 24 hours, and the cells were stimulated with LPS (1μg / mL) in the presence of either peptides P1, P2, or P3 at 200μg / mL or fibronectin at 200μg / mL. When peptides were included, LPS significantly increased CD86 expression, but fibronectin did not.

[0090] In Figure 18, when bone marrow-derived dendritic cells were cultured for 24 hours and fibronectin was administered at various concentrations, it was shown that the increase in CD86 expression caused by LPS (1 μg / mL) was suppressed from a concentration of 50 μg / mL. * indicates statistical significance, P<0.05 or less compared to a concentration of 0 μg / mL.

[0091] In Figure 19, when bone marrow-derived dendritic cells were cultured for 24 hours and fibronectin was administered at various concentrations, it was shown that the secretion of TNF-α and IL-12p40 was suppressed by LPS (1 μg / mL) from a concentration of 100 μg / mL. * indicates P<0.05 or less compared to the 0 μg / mL concentration, indicating statistical significance.

[0092] In Figure 20, poly(I:C) (polyinosinic:polycytidylic acid), which is known as an immunostimulant that resembles structural double-stranded RNA and binds to Toll-like receptor-3, was administered at a concentration of 50 μg / mL, in comparison with LPS, which binds to Toll-like receptor-4 on immune cells and inflammatory cells, including dendritic cells. When fibronectin was added and cultured, the increase in CD86 expression in splenic dendritic cells was suppressed, but when peptides P1, P2, and P3 were added at a concentration of 200 μg / mL, there was no effect. * indicates P<0.05 or less compared to None, indicating statistical significance.

[0093] Example 11. Increase in T cell proliferation and cytokine secretion by co-culture with splenic dendritic cells cultured with peptides Since the main role of activated dendritic cells is to induce antigen-specific T cell activation and proliferation through their stimulatory properties, we investigated whether cultured autoactivated splenic dendritic cells and dendritic cells cultured with peptides and activated with LPS could induce T cell proliferation in a mixed lymphocyte reaction assay. Splenic dendritic cells isolated from C57B / L6 mice were either autoactivated for 24 hours or activated by LPS when cultured with peptides or fibronectin. Splenic T cells from Balb / c mice were CD4 + / CD8 +After isolation using a T cell isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany) and a MACS column (Miltenyi Biotec), the cells were stained with 10 μM CFSE (Molecular Probes; Thermo Fisher Scientific, USA). Activated dendritic cells and CFSE-stained T cells were cocultured at a ratio of 1:20 for 4 days, and the degree of T cell proliferation was measured as the percentage of cells with reduced CFSE.

[0094] In Figure 21, compared with the control splenic dendritic cells not treated with peptide or fibronectin, dendritic cells activated with LPS or poly(I:C) slightly increased the proliferation of co-cultured splenic T cells, and splenic dendritic cells activated after peptide treatment also effectively increased T cell proliferation. However, splenic dendritic cells cultured with the addition of fibronectin were not activated by LPS and could not induce T cell proliferation. * indicates statistical significance, P<0.05 or less compared with untreated sDC. In Figure 22, the amount of secreted cytokines was measured by ELISA after co-culture of splenic dendritic cells and T cells for 4 days. When dendritic cells activated with LPS and poly(I:C) were co-cultured with T cells in the control group or with the addition of peptides, high concentrations of interferon-gamma (IFN-γ), TNF-α, IL-6, and IL-5 were secreted, whereas when splenic dendritic cells activated with fibronectin were co-cultured with T cells, the secretion of cytokines was extremely low. * indicates statistical significance, P<0.05 or less, compared to untreated sDCs.

[0095] Example 12. Increased viability of splenic dendritic cells cultured with peptides Dendritic cells are characterized by the fact that they undergo autoactivation and die rapidly during culture. We measured whether the survival rate of dendritic cells changes with the inhibition of autoactivation. Cell survival was measured by measuring the number of cells that were not stained with fluorescence using flow cytometry while culturing cells stained with 7-AAD.

[0096] In Figure 23, the cell viability of splenic dendritic cells decreased significantly after 6 hours of culture, but the number of 7-AAD-negative viable cells increased significantly when the cells were cultured with the addition of peptides P1, P2, and P3 or fibronectin. * indicates statistical significance at P<0.05 or less compared to untreated sDC.

[0097] Example 13: Determination of the effect of a linker peptide composed of three peptides on the expression of CD86 in cultured primary dendritic cells As linkers, the amino acid linker GS and the flexible linker GGGSGGGS were used to link three peptides, RGD (P1), LDV (P2), and PHSRN (P3). GS RGD GS LDV (referred to as "LP1"; SEQ ID NO: 12), PHSRN GGGS RGD GGGS LDV (referred to as "LP2"; SEQ ID NO: 13), PHSRN GGGSGGGS RGDGGGSLDV (referred to as "LP3"; SEQ ID NO: 14) was produced. The linker peptide was produced by Peptron, Inc. (Daejeon, Korea) on a SHIMADZU LCMS-2020. As controls, scrambled peptides S-P1 (SEQ ID NO: 15), S-P2 (SEQ ID NO: 16) and S-P3 (SEQ ID NO: 17) were produced by mixing the amino acid sequences constituting the P1, P2 and P3 peptides, and linker peptides S-LP1 (SEQ ID NO: 21), S-LP2 (SEQ ID NO: 22) and S-LP3 (SEQ ID NO: 23) composed of these scrambled peptides were produced and used. The scrambled peptides and scrambled linker peptides have the same number of amino acids, molecular weight, isoelectric point and hydrophilicity as the original peptides. The sequences of these peptides are shown in Table 1.

[0098] [Table 1]

[0099] In FIG. 24, various concentrations of LP1, LP2, and LP3 were added up to a concentration of 800 μg / mL, and splenic dendritic cells (2×10 5 After culturing 100 μg / mL of CD86 for 6 or 24 hours, the expression of CD86 was measured by flow cytometry (MACSQuant VYB flow cytometer, Miltenyi Biotec). The results were analyzed using FlowJo v10 (Tree Star, Ashland). The increase in CD86 expression due to autoactivation after culture was inhibited by LP1, LP2, and LP3 in a concentration-dependent manner, with LP3 showing the greatest inhibitory effect on autoactivation. * indicates P<0.05 or less compared to none, indicating statistical significance.

[0100] In Figure 25, 2 × 10 primary splenic dendritic cells 5 It was shown that the increase in CD86 expression was not suppressed when scrambled peptides S-LP1, S-LP2 and S-LP3 were added at a concentration of 800 μg / mL and cultured for 24 hours. Furthermore, the expression of CD86 did not decrease even when scrambled peptides S-P1, S-P2 and S-P3 were simultaneously added at a concentration of 600 μg / mL.

[0101] Example 14. Measurement of fluorescent staining for CD86 expression in splenic dendritic cells cultured with the addition of linker peptide In FIG. 26, splenic dendritic cells (2×10 51 μg / mL) was dispensed into a 48-well plate, and the linker peptide was added at a concentration of 400 μg / mL and cultured for 24 hours. LPS was added to the culture medium at 1 μg / mL to induce activation of splenic dendritic cells. The cultured cells were placed in 4% paraformaldehyde fixative and fixed at room temperature for 20 minutes. Fluorescent staining was performed using PE-conjugated anti-CD86 antibody (eBioscience) and then measured under a confocal microscope (K1 NanoScope, Seoul). Expression of intracellular actin was stained with FITC-conjugated phalloidin (eBioscience), and nuclear staining was performed with Hoescht 33342 (Sigma-Aldrich). CD86 antibody and phalloidin were diluted 1:40 in 0.2% bovine serum albumin solution. When splenic dendritic cells were cultured alone in the culture medium, the expression of CD86 was significantly increased, but when fibronectin or the three peptides were added, as well as the linker peptides LP1, LP2, and LP3, almost no cells expressing CD86 were observed. When LPS was added or fibronectin was included, almost no cells expressing CD86 were measured, but when the combination of the three peptides (P1+P2+P3) or the linker peptides were added, the number of cells expressing CD86 was significantly increased by LPS treatment.

[0102] Example 15: Effect of linker peptide on secretion of TNF-α from cultured splenic dendritic cells In Figure 27, 2 × 10 primary splenic dendritic cells 5When dendritic cells were cultured for 24 hours, the concentration of TNF-α secreted into the culture medium was measured using an ELISA kit (R&D systems). The concentration was measured at 450 nm using a microplate reader (Synergy H1, Biotek). When dendritic cells were cultured alone, the TNF-α concentration was measured to be 75 ± 10 pg / mL, and was significantly increased by LPS stimulation. When fibronectin, the combination of three peptides (P1 + P2 + P3), or the linker peptides LP1, LP2, and LP3 were added, there was almost no secretion of TNF-α. When treated with LPS, the secretion of TNF-α was significantly increased when the linker peptide was added as well as the combination of three peptides (P1 + P2 + P3) compared to fibronectin. * is P < 0.05 or less compared to none, and has statistical significance.

[0103] Example 16. Measurement of T cell activation by splenic dendritic cells cultured with linker peptide In Figure 28, we investigated whether splenic dendritic cells that do not undergo self-activation by the linker peptide during culture and dendritic cells activated by LPS can induce T cell proliferation. Splenic dendritic cells (2 × 10 6 ) were allowed to autoactivate for 24 h in 96-well plates or their autoactivation was suppressed by the addition of a linker peptide, and then either not treated with LPS or stimulated with LPS for 24 h. Dendritic cells were cultured with 10 μM of the ovalbumin (OVA) peptide SIINFEKL (InvivoGen, San Diego, California, USA) for 1 h. CD8 + T cells were isolated from OT-I transgenic mice using a CD8 T cell isolation kit (Miltenyi Biotec) and stained with 10 μM carboxyfluorescein succinimidyl ester (CFSE) for 10 min. + T cells (1×10 5 ) and OVA peptide-treated DCs (5 × 10 3) were co-cultured at a ratio of 1:20 for 4 days, and the degree of T cell proliferation was measured as the percentage of cells negative for CFSE fluorescent staining.

[0104] Autoactivated dendritic cells did not induce T cell proliferation in the absence of LPS treatment, and when stimulated with LPS, autoactivated cells and dendritic cells cultured with fibronectin were only weakly able to induce T cell proliferation, whereas dendritic cells cultured with the triple peptide combination or the linker peptide significantly increased T cell proliferation.

[0105] In Figure 29, splenic dendritic cells and T cells were co-cultured for 4 days under the same conditions as those for measuring T cell proliferation, and the concentrations of interferon-gamma (IFN-γ) and TNF-α secreted into the culture medium were measured by ELISA. Autologous activated dendritic cells secreted about 2.5 times more interferon-gamma and TNF-α when co-cultured with T cells after stimulation with LPS, whereas dendritic cells cultured with the combination of three peptides or with the LP1, LP2, and LP3 linker peptides secreted more than 4.5 times more cytokines when co-cultured with T cells after stimulation with LPS. * indicates P<0.05 or less compared to none, indicating statistical significance.

Claims

1. A composition for inhibiting the autoactivation of dendritic cells, comprising a peptide derived from fibronectin that binds to an integrin binding site between dendritic cells, The composition for suppressing dendritic cell self-activation comprises a peptide having an amino acid sequence including SEQ ID NO: 1, a peptide having an amino acid sequence including SEQ ID NO: 2, and a peptide having an amino acid sequence including SEQ ID NO:

3.

2. A composition for inhibiting the self-activation of dendritic cells as described in claim 1, wherein the peptide consisting of an amino acid sequence including sequence number 1, the peptide consisting of an amino acid sequence including sequence number 2, and the peptide consisting of an amino acid sequence including sequence number 3 each have a length of 3 to 11 aa.

3. A composition for inhibiting the self-activation of dendritic cells as described in claim 1, wherein the peptide consisting of an amino acid sequence including SEQ ID NO: 1 is a peptide consisting of the amino acid sequence of SEQ ID NO: 1, 4 or 5, the peptide consisting of an amino acid sequence including SEQ ID NO: 2 is a peptide consisting of the amino acid sequence of SEQ ID NO: 2, 6 or 7, and the peptide consisting of an amino acid sequence including SEQ ID NO: 3 is a peptide consisting of the amino acid sequence of SEQ ID NO:

3.

4. 2. The composition for suppressing dendritic cell self-activation according to claim 1, wherein the peptide comprises a peptide having an amino acid sequence including SEQ ID NO:1, a peptide having an amino acid sequence including SEQ ID NO:2, and a peptide having an amino acid sequence including SEQ ID NO:3, linked by a linker.

5. The composition for inhibiting dendritic cell autoactivation according to claim 4, wherein the linker consists of the amino acids G and S and is 2 to 8 aa in length.

6. The composition for suppressing dendritic cell self-activation according to claim 4, wherein the linker comprises an amino acid sequence comprising any one of SEQ ID NOs: 8 to 11.

7. The composition for suppressing dendritic cell autoactivation according to claim 4, wherein the peptide comprises any one of the amino acid sequences of SEQ ID NOs: 12 to 14.

8. The peptide was administered to dendritic cells to be treated at a concentration of 2×10 ５ 2. The composition for inhibiting the self-activation of dendritic cells according to claim 1, wherein the composition is contained at a concentration of 200 μg / mL or more per mL of the antibody.

9. The composition for suppressing the autoactivation of dendritic cells according to claim 1 , wherein the dendritic cells are derived from mononuclear cells, the spleen or lymph nodes.

10. The composition for inhibiting the autoactivation of dendritic cells according to claim 1, which is for in vitro use.

11. A method for culturing dendritic cells, comprising the step of treating isolated dendritic cells with a composition according to any one of claims 1 to 10.

12. The method for culturing dendritic cells according to claim 11, further comprising the step of treating the dendritic cells treated with the composition with an immunostimulant to induce activation of the dendritic cells.

13. The method for culturing dendritic cells according to claim 12, wherein the immunostimulant is at least one selected from the group consisting of a bacterial preparation, CD40 ligand, TNF-α, IL-1β, LPS and poly(I:C).

14. A step of treating isolated dendritic cells with a composition according to any one of claims 1 to 10 and culturing the dendritic cells in a non-activated state; activating the dendritic cells; and co-culturing the activated dendritic cells with immune cells.

15. A dendritic cell composition comprising a peptide derived from fibronectin that binds to an integrin binding site between dendritic cells according to any one of claims 1 to 10, and dendritic cells mixed therewith.

16. A pharmaceutical composition for treating or preventing cancer, an infectious disease, a chronic inflammatory disease, or an autoimmune disease, comprising the cell composition according to claim 15 as an active ingredient.

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