Novel regulatory macrophages and their use

Abstract

This invention provides novel immunomodulatory macrophage cells (Mregs) useful for treating a variety of immunological and non-immunological diseases and conditions. [Solution] Novel immunomodulatory macrophage cells are characterized by specific markers and activity patterns that distinguish them from other cells. Novel immunomodulatory macrophage cells have high phagocytic capacity and can suppress T cell proliferation. The present invention also provides a novel method for preparing immunomodulatory macrophage cells from blood monocytes in suspension culture. This method is suitable for high automation. In a further aspect, the present invention relates to a pharmaceutical composition comprising the immunomodulatory macrophage cells of the present invention.

Description

[Technical Field]

[0001] This invention relates to novel immunomodulatory macrophage cells useful for the treatment of various immunological and non-immunological diseases and conditions. These cells are characterized by specific markers and activity patterns that distinguish them from other cells. The novel immunomodulatory macrophage cells have high phagocytic capacity and can suppress T cell proliferation. This invention also provides a novel method for preparing immunomodulatory macrophage cells from blood monocytes in suspension culture. This method is suitable for high automation. In a further aspect, this invention relates to pharmaceutical compositions comprising the immunomodulatory macrophage cells of the present invention. [Background technology]

[0002] Regulatory macrophages (referred to as "Mreg" in this specification and literature) represent an intrinsic state of macrophage differentiation, distinguished from other activated macrophages by their robust phenotype and their immunosuppressive, anti-inflammatory, and angiogenic properties.

[0003] Human Mreg cells have been found to be particularly effective as immunomodulatory therapy in solid organ transplantation. Most notably, these cells suppress mitogen-stimulated T cell proliferation in vitro, which may be due to interferon (IFN)γ-induced indoleamine 2,3-dioxygenase activity and contact-dependent deletion of activated T cells. Furthermore, Mreg cells promote the development of activation-induced regulatory T cells, suppress effector T cell proliferation, and inhibit dendritic cell maturation. Therefore, when Mreg cells are administered to the recipient, a feedforward loop of immunomodulatory regulation is initiated, leading to long-term immunological changes in expat transplantation. It is hypothesized that this will lead to tolerance or prevention of immunopathology. Mreg-containing cell preparations have been administered to kidney transplant recipients as a form of adjunctive immunosuppressive treatment in a series of case studies and two early clinical trials [1]-[5]. These studies demonstrated the feasibility of Mreg in suppressing immunological responses that lead to organ rejection. Transplant studies using ex vivo-produced Mreg have shown that these cells are safe and well-tolerated [6]. Mreg has also been used to treat other clinical conditions such as non-healing diabetic foot ulcers associated with peripheral arterial dysfunction. In this regard, International Publication 2019 / 053091 describes the use of Mreg for the treatment of macrovascular or microvascular complications of the lower extremities. Furthermore, Mreg has been recommended for ischemia or reperfusion-related diseases [7].

[0004] Several protocols using blood monocytes as a starting material for preparing Mreg cells are known in the art. Mononuclear leukocytes are isolated from peripheral blood samples by commonly known methods such as leukocyte apheresis. After isolation of monocytes, these cells are incubated in a medium containing macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) and human serum, such as human AB serum (HABS). After 3–6 days, these cells are further stimulated with interferon-gamma (IFN-γ) for 18–24 hours, after which Mreg cells are harvested.

[0005] Mregs were typically prepared in tissue culture flasks.[2][4] However, monocytes and their derivatives adhere firmly to hard plastic or glass surfaces, requiring the Mregs to be scraped off the surface for recovery, which results in a significant loss of viable cells that can be used for therapeutic purposes. Thus, this approach is not feasible for preparing large quantities of Mregs for clinical use. International Publication 2017 / 153607 describes an improved method for producing Mregs using gas-permeable differentiation bags. Differentiating monocytes in these bags yields a homogeneous population of Mregs that differ in phenotypic and functional characteristics from their flask-cultured counterparts. Nevertheless, these cells proliferate semi-adherently within the bags, and as a result, recovering the cells from the bags without an undesirable decrease in viability remains somewhat cumbersome.

[0006] As a result, there is a need in this field for an improved method that can be used to prepare Mreg cells from blood monocytes. This method must be GMP compliant and should involve minimal interventions that could affect the sterility of the resulting therapeutic cell product. Furthermore, in order to enable the economical production of large quantities of therapeutic cell product, the method must be suitable for upscaling and automation. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2019 / 053091 [Patent Document 2] International Publication No. 2017 / 153607 [Overview of the project]

[0008] This invention provides a novel method for producing Mregs that is highly effective in the production of Mregs and minimizes sterility and recovery issues. The method allows an entire batch of cells obtained from monocyte enrichment to be subjected to differentiation in a suspension within a single bioreactor bag. Therefore, the method can be easily upscaled to industrial scale, avoiding additional enzymes or harsh recovery methods, thereby ensuring high product quality. Furthermore, the method can also be applied to all types of regulatory macrophages in the art, including those described in, for example, International Publication No. 2017 / 153607. As had been thought, regulatory macrophages did not differentiate early in suspension, requiring at least temporary adhesion to the surface of the culture system in order to obtain the phenotype.[6]

[0009] Surprisingly, as described below, this method generates a novel type of Mreg cell. Specifically, the inventors found that when monocytes used to prepare Mreg cells are cultured in suspension, i.e., without significant adhesion to the surface of the cell vessel, Mreg cells with a unique phenotype exhibiting immunomodulatory properties that make them highly suitable for cell-based therapeutic approaches are obtained. These cells are referred to herein as "Mreg-sc" to distinguish them from known Mreg cells. In particular, the cells obtained by the method of the present invention do not exist naturally in the human body. Rather, they This is a unique cell type that develops in response to the culture of monocytes in the presence of growth factors and cytokines as described below in this specification.

[0010] Therefore, in a first aspect, the present invention relates to a method for producing Mreg cells, comprising culturing monocytes from a target blood sample in suspension culture in the presence of M-CSF / GM-CSF, CD16 ligand (such as immunoglobulin), and IFN-γ, without a significant portion of the cells adhering to the inner surface of the culture vessel.

[0011] In a second aspect, the present invention relates to a novel type of Mreg cell, namely Mreg-sc cells, which can be obtained by the method described in the first aspect of the present invention. Mreg-sc cells have a unique phenotype not found in the prior art. Mreg-sc cells mediate biological activities that are unique to this cell type and give useful therapeutic properties not described in the prior art. Specifically, Mreg-sc cells exhibit immunosuppressive, anti-inflammatory, and tissue repair properties that distinguish them from naturally occurring macrophages and make them highly attractive for therapeutic purposes.

[0012] In a third aspect, the present invention relates to a pharmaceutical composition comprising Mreg-sc cells or an intracellular fraction of said cells according to a second aspect of the present invention. The pharmaceutical composition containing the novel cell type of the present invention may also contain further active ingredients or excipients as needed.

[0013] In a fourth aspect, the present invention relates to the use of Mreg-sc cells or their intracellular fraction according to a second aspect of the present invention, or a pharmaceutical composition according to a third aspect of the present invention, for therapeutic purposes, particularly for the suppression of immunological adverse reactions.

[0014] In a fifth aspect, the present invention relates to a method for preparing an intracellular fraction of Mreg-sc cells according to a second aspect of the present invention by degrading Mreg-sc cells under appropriate conditions.

[0015] Finally, in a sixth aspect, the present invention relates to a method for preparing immunomodulatory T cells by co-culturing T cells from a target blood sample with Mreg-sc cells or their intracellular fraction according to a second aspect of the present invention. [Modes for carrying out the invention]

[0016] According to the present invention, Mreg cells are derived from human CD14+ circulating monocytes. To induce the characteristic biological properties of Mreg-sc cells, monocytes are grown in suspension culture and treated with specific combinations of growth factors, cytokines, and receptor ligands. Cells obtained by the method of the present invention are characterized by a unique phenotype that distinguishes them from circulating monocytes, other types of monocyte-derived macrophages, monocyte-derived dendritic cells, and other suppressor myelomonocytic cell products described in the prior art.

[0017] Therefore, in the first embodiment, the present invention relates to a method for preparing immunomodulatory macrophage cells, (a) A step of isolating CD14-positive monocytes from the target blood sample, (b) A step of culturing monocytes in a medium containing (i) M-CSF and / or GM-CSF, and (ii) CD16 ligand, (c) A step of bringing monocytes or monocyte-derived cells into contact with IFN-γ, (d) a step of obtaining immunomodulatory macrophage cells from a culture medium, A method wherein steps (b) and (c) are performed in a vessel that is agitated to minimize or effectively avoid adhesion of cells to the surface of the vessel. The agitation of the vessel to minimize or effectively avoid adhesion of cells to the surface of the vessel is continued for at least 4 days, at least 5 days, at least 6 days, or at least 7 days of cell culture. Preferably, the agitation of the vessel is continued for the entire culture period, i.e., from the introduction of monocytes into the culture vessel until Mreg-sc is harvested.

[0018] The method of the present invention uses blood monocytes as a starting material. While the method of the present invention is preferably used to generate Mreg cells from human blood monocytes, the present invention is not limited to the differentiation of human-derived cells. In fact, the present invention is also applicable to other types of non-human cells, specifically vertebrate cells, such as non-human primate or porcine cells. Thus, the present invention makes an important contribution to the field of xenotransplant medicine.

[0019] In a preferred embodiment, the method of the present invention is used to differentiate CD14-positive monocytes from a human donor into Mreg cells. The monocytes serving as the starting material for the method of the present invention are obtained from the peripheral blood of a human donor. The donor may be a healthy subject or a patient suffering from one or more diseases. In a preferred embodiment, the monocyte donor is also the recipient of Mreg cells (autologous approach). In another preferred embodiment, the monocyte donor is a different person from the recipient of Mreg cells (allogeneic approach). In the latter case, the donor and recipient may or may not be genetically related. In another embodiment, the monocyte donor is Simultaneously, organs are provided to the recipient of Mreg cells. The preferred relationship between the donor and recipient depends on the clinical application. In some cases, the use of autologous Mreg cells is preferable to avoid certain adverse reactions. Therefore, the use of autologous Mreg cells is preferable in regenerative therapy or anti-inflammatory therapy. In transplantation settings, donor antigen-expressing cells are more effective than recipient-derived cells, so it is preferable to use donor-derived Mreg cells as immunosuppressive therapy.

[0020] Various methods for concentrating mononuclear cells from peripheral blood are known in the art, and each of these methods can be used in connection with the present invention. For example, blood obtained by venous puncture can be treated with an anticoagulant and then separated using a separation medium such as Ficoll-Paque Plus. For this purpose, the anticoagulant-treated blood sample is layered in a Ficoll-Paque Plus solution and centrifuged to form layers containing different cell types. The bottom layer contains red blood cells that have agglutinated and settled with the Ficoll-Paque Plus reagent. The layer immediately above the red blood cell layer mainly contains Ficoll-Paque Plus The sample contains granulocytes that have migrated through the layers. Due to lower densities, monocytes and lymphocytes are found at the interface between the plasma and Ficoll-Paque Plus. Concentration of the mononuclear cell fraction can be achieved by isolating the layers and subsequently washing and centrifugating them.

[0021] Another method routinely used to separate mononuclear leukocytes from blood samples involves leukocyte apheresis. Leukocyte apheresis is a specific type of apheresis in which leukocytes are obtained from peripheral blood in a continuous process according to their relative density. In this procedure, the blood in question is passed through a special centrifuge that collects a predetermined fraction of leukocytes and returns the remaining blood cells and plasma to the donor. Currently, leukocyte apheresis is a routine clinical method for obtaining leukocytes or stem cells from peripheral blood. Various apparatuses that can be used to perform leukocyte apheresis in connection with the present invention are available from several manufacturers. For example, Terumo BCT's COBE® Spectra Apheresis System is one such example. When leukocyte apheresis is performed using the COBE® Spectra Apheresis System, it is preferable to use the manual protocol provided by the manufacturer, as this protocol has been found to yield better quality monocytes compared to the AutoPBSC protocol.

[0022] The above-described method and apparatus for leukocyte concentration provide a cell fraction that includes lymphocytes in addition to monocytes. According to the present invention, monocytes can be further concentrated before the cells are introduced into the preparation method of the present invention and separated from lymphocytes by known methods, for example, by magnetic bead separation, sorting by flow cytometry, elution, filtration, or plastic adhesion. However, it is not essential to use a homogeneous monocyte fraction in the method of the present invention. In fact, the presence of lymphocytes in an amount of 0.1 to 20%, more preferably 0.1 to 1%, in the monocyte fraction can have a positive effect on the differentiation of monocytes into regulatory macrophages.

[0023] In a preferred embodiment of the present invention, the monocyte fraction used in the method of the present invention is essentially pure and contains less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of non-monocyte nucleated blood cells, such as lymphocytes or granulocytes. To obtain a mononuclear cell preparation enriched with monocytes, peripheral blood mononuclear cells can be brought into contact with CD14 microbeads to which, for example, CD14-positive monocytes bind. In one embodiment, the monocytes from step (a) are isolated by leukocyte apheresis and subsequently subjected to a separation step using a CD14 affinity molecule, preferably a CD14 antibody. Such a purification step greatly reduces contamination of the starting material containing non-monocytes. The risk of T-cell contamination is significantly reduced. Reducing T-cell contamination is highly beneficial from a patient safety perspective because it minimizes the potential risk of donor-recipient reactions.

[0024] In preferred embodiments of the present invention, CD14-positive monocytes used in the method of the present invention are isolated using cell manufacturing equipment such as that of CliniMACS® Technology (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). For example, a GMP-compliant fully closed LP14 process can be used in accordance with the manufacturer's instructions for CD14 monocyte enrichment (LP-14 System User Manual). This method is based on labeling of monocytes with iron-dextran particles conjugated with an anti-CD14 antibody, and further sorting by a magnetic separation column. Particularly preferred embodiments of the present invention In one embodiment, CD14 monocytes from a peripheral blood sample are separated or concentrated using a CliniMACS Prodigy® device.

[0025] The CD14-positive monocyte fraction isolated as described above can be used directly for differentiation by incubation with M-CSF and / or GM-CSF and CD16 ligand, or it can be stored in autologous plasma supplemented with the anticoagulant dextrose citrate solution (ACD-A) or any other suitable buffer until use. If the monocyte fraction must be transported to different sites where the differentiation process takes place, the differentiation of cells by incubation with M-CSF / GM-CSF should occur within 24 hours after cell isolation, preferably within 18 hours, 12 hours, 6 hours, 4 hours, or 2 hours after monocyte isolation. Care must be taken to ensure that the process is started within the allotted time. For long-term storage, the monocyte fraction can be resuspended in a suitable cryopreservation solution and stored for an extended period at a temperature below 20°C, preferably below 80°C.

[0026] To initiate the conversion of concentrated monocytes to Mreg cells, the cells are incubated in the presence of M-CSF / GM-CSF and CD16 ligand. Preferably, the cells can be introduced into a medium containing M-CSF and / or GM-CSF and CD16 ligand. Alternatively, M-CSF / GM-CSF and CD16 ligand can be added some time after the start of cell culture. The medium used in step (b) of the above method may be any medium described in the literature as suitable for use in the culture of monocytes and / or macrophages. A suitable culture medium is, for example, PromoCell Macrophage Examples of media include Di-Cell medium (PromoCell GmbH, Heidelberg, Germany), Dulbecco's Modified Eagle Medium (DMEM), DMEM:F12 blend, Medium 199, or RPMI-1640 medium. The medium is preferably a chemically defined medium. Apart from M-CSF / GM-CSF, the medium may contain other factors to promote Mreg survival and differentiation, growth factors and cytokines such as epidermal growth factor (EGF) or IL-4, fatty acids, cholesterol and other lipids, vitamins, transferrin and trace elements, insulin, glucocorticoids, cholecalciferol or ergocalciferol and other hormones. It may contain mon, nonspecific immunoglobulins, and other plasma proteins. In preferred embodiments of the present invention, the culture medium is RPMI-1640 or a culture medium derived therefrom.

[0027] The culture medium used to incubate isolated CD14-positive monocytes contains M-CSF (also known as CSF1), GM-CSF (also known as CSF2), or both. M-CSF is known in the art as a hematopoietic growth factor that influences the proliferation, differentiation, and survival of monocytes, macrophages, and myeloid progenitor cells. GM-CSF is a monomeric glycoprotein that functions as a cytokine and is secreted by macrophages, T cells, mast cells, NK cells, endothelial cells, and fibroblasts. M-CSF and GM-CSF proteins from various species have been described and can be purchased from various manufacturers. This is possible. The selection of M-CSF and / or GM-CSF used in the method of the present invention depends on the origin of the monocytes to differentiate into Mreg cells. For example, when differentiating human monocytes into Mreg using the process described herein, the culture medium used contains human M-CSF and / or human GM-CSF, preferably recombinant human M-CSF and / or recombinant human GM-CSF. Similarly, when porcine monocytes are used in the differentiation method, the M-CSF and / or GM-CSF added to the culture medium will be of porcine origin. In a particularly preferred embodiment of the present invention, the M-CSF and / or GM-CSF are recombinant human M-CSF and / or GM-CSF. These are of human origin, and the monocytes are human monocytes.

[0028] Those skilled in the art will be able to determine, using routine methods, the amount of M-CSF and / or GM-CSF suitable for differentiating monocytes into Mreg-sc at a high rate. Typically, the concentration of M-CSF in the culture medium in step (b) of the above method is in the range of 1 to 100 ng of protein per ml of medium. For example, as taught in International Publication No. 2017 / 153607, a time-course experiment to measure the amount of M-CSF in the culture medium shows that a culture with an initial dose of 5 ng / ml of M-CSF contains below physiological concentrations by day 2 of culture, while a culture with an initial dose of 25 ng / ml of M-CSF contains below physiological concentrations throughout a 7-day culture period. The study revealed that M-CSF is consumed or degraded over time, such as by maintaining a concentration of more than 10 ng / ml. Therefore, in preferred embodiments of the present invention, the concentration of M-CSF in the culture medium is in the range of 20-75 ng / ml, 20-50 ng / ml, or 20-25 ng / ml. A concentration of at least 25 ng of M-CSF per 1 ml of culture medium is particularly preferred. Preferably, the above concentrations refer to recombinant human M-CSF.

[0029] When using GM-CSF instead of M-CSF, the same concentrations outlined above in relation to M-CSF can be used in the culture medium. Since GM-CSF appears to be more potent than M-CSF, a GM-CSF concentration of 0.1–100 ng of protein per ml of medium is recommended herein. When using both M-CSF and GM-CSF in the culture medium, the overall concentration including these two growth factors is within the ranges mentioned above, namely 20–75 ng / ml, 20–50 ng / ml, or 20–25 ng / ml. An overall concentration of 25 ng of M-CSF and GM-CSF per ml of medium is particularly preferred.

[0030] In addition to M-CSF and / or GM-CSF, the culture medium used in step (b) of the above method also contains a CD16 ligand. It is known that stimulation of the CD16 cell surface receptor on monocytes is necessary to induce their differentiation into Mreg-sc cells. More specifically, experiments have shown that monocytes grown in a medium supplemented with human AB serum (HABS) develop into Mreg cells, while monocytes grown in a medium supplemented with fetal bovine serum (FCS) do not develop into Mreg cells. Monocytes grown in an equal mixture of both serums express the Mreg phenotype. Therefore, HABS has positive Mreg-inducing activity. By removing the chloroform-extractable fraction of S, it was revealed that the Mreg-inducing activity of HABS is mainly located in the chloroform-resistant fraction, and therefore is likely to be a protein. Size fractionation revealed that the major protein component of HABS responsible for Mreg development is over 100 kDa, leading to the hypothesis that the unknown factor is immunoglobulin (Ig). HABS depleted of Ig using protein A / G Sepharose could not support the development of Mreg morphology and DHRS9 mRNA expression. Re-addition of eluted Ig (or IVIg) to Ig-deficient serum induced DHRS9 expression. The ability to do so was restored. Similarly, when monocytes were cultured in FCS supplemented with human Ig, increased DHRS9 mRNA expression was observed compared to FCS alone as a control, and normal Mreg morphology was obtained. Monocytes treated with anti-FcγRIII antibody expressed significantly lower levels of DHRS9 mRNA and did not express Mreg morphology compared to monocytes treated with anti-FcγRI(CD64), anti-FcγRIIa / b(CD32a / b), or control antibodies. Blocking either FcγRIIb or DC-SIGN alone, or both receptors together, did not affect DHRS9 expression. +Mreg generation was not affected. To support the view that FcγRIII is required for Mreg generation, FcγRIII expression was silenced using siRNA. Transient suppression of FCGR3A and FCGR3B transcript expression was achieved in newly isolated monocytes cultured in 10% HABS, and importantly, FCGR2B expression was not reduced by this procedure. Protein-level knockdown of FcγRIII was demonstrated by flow cytometry (35.2% ± 4.4 CD16 using negative control siRNA). + Cell-to-FCGR3 siRNA was used (15.3% ± 3.7 cells; n=4, p=0.002). Silencing of FcγRIII expression (not suppression of MAPK1 expression or treatment with negative control siRNA) resulted in significant downregulation of DHRS9 mRNA expression.

[0031] It was concluded that serum Ig acts via FcγRIII(CD16) to induce the Mreg phenotype. Due to the dependence of Mreg differentiation on FcγRIII, Mreg differs significantly from other Ig complex-induced macrophage types described in the prior art. Specifically, due to its derivation mechanism, FcγRIII-induced Mreg differs significantly from FcγRIIb-induced macrophages, FcγRI-induced macrophages, and macrophages generated in the absence of immunoglobulins, as described in the prior art.

[0032] Since stimulation of the CD16 cell surface receptor is important for differentiation into the desired Mreg phenotype, the method of the present invention includes incubation of monocytes with a CD16 ligand in step (b). The ligand that binds to the receptor is preferably human or non-human immunoglobulin, more preferably human immunoglobulin or a fragment thereof. The immunoglobulin fragment may be, for example, an Fc fragment of immunoglobulin. The immunoglobulin or immunoglobulin fragment is preferably added to serum-free medium. Alternatively, a recombinant protein containing the sequence of immunoglobulin or immunoglobulin fragment, for example, the sequence of human immunoglobulin, may be used. In one embodiment, a non-human or human antibody or fragment thereof that specifically binds to CD16 via its antigen recognition domain is used to promote Mreg-sc differentiation. In yet another embodiment, a small molecule is used to stimulate the CD16 signaling pathway to promote Mreg-sc differentiation.

[0033] In preferred embodiments, the culture medium used to generate Mreg cells contains 1-20% human serum or an equivalent amount of a specific serum component, such as immunoglobulin. More preferably, the medium is supplemented with 10% serum. When using a serum-containing medium to carry out the method of the present invention, the medium contains 5-15%, preferably 10%, human serum. A medium containing 10% human AB serum is particularly preferred. In other words, the serum is preferably added at a concentration of about 0.01 to 10%, preferably about 0.1 to 1%, more preferably about 1%. When using immunoglobulin or immunoglobulin fragments as CD16 ligands, slightly lower Any concentration can be used. If immunoglobulin or other CD16 ligands are immobilized on the tissue culture surface, beads, or other physical matrix, substantially lower concentrations may be used. Human serum, such as AB serum, is preferably derived from a male donor. If serum from a female donor is used, it is preferable that the donor does not use progesterone or progesterone-estrogen contraceptives. The above medium is even more preferably free from any intermediate forms, including antibodies against monocytes or Mreg cells, or antibodies against major histocompatibility molecules.

[0034] It has been found that antibiotics in the culture medium do not have a measurable effect on the viability, yield, phenotype, or inhibitory function of Mreg-sc produced by the method of the present invention. Nevertheless, it is preferable that the culture medium used in step (b) of the method of the present invention does not contain antibiotics.

[0035] If Mreg-sc cells are intended for use in therapeutic applications where induction of angiogenesis is desired (see below), the culture medium used to culture monocytes in step (b) of the method of the present invention may contain, in addition to M-CSF / GM-CSF and CD16 ligands, Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS), monophosphoryl lipid A (MPLA), or high-mobility group box protein 1 (HMGB1) to enhance the production of angiogenic factors such as VEGF-A. The TLR ligand may be present in a concentration range of 10 ng / ml to 1 μg / ml, preferably 50 to 500 ng / ml, e.g., 100 ng / ml, 200 ng / ml. It can be added to the culture medium at g / ml, 300 ng / ml, or 400 ng / ml. If two or more TLR ligands are added, the total concentration including all of these ligands must be within the range listed above. The TLR ligand can be added at any stage of the manufacturing process. It may be present in the initial medium used to culture monocytes, i.e., on day 0 of culture, or it may be added at a later stage, for example, on day 5, 6, or 7 of culture. Preferably, the TLR ligand is added at the same time as the addition of IFN-γ.

[0036] According to the present invention, the steps of culturing cells in the presence of M-CSF / GM-CSF and CD16 ligand, and culturing cells in the presence of IFN-γ, are performed in a container that is agitated to avoid cell adhesion to the surface of the container. Therefore, the cells are cultured non-adherently in suspension culture. This is achieved by performing steps (b) and (c) of the method of the present invention in a bioreactor in which the container (e.g., a bag) containing the cells is slowly and continuously agitated so that the cells do not adhere to the inner surface of the container. A wave-type bioreactor has been found to be particularly useful for this purpose. Here, a wave-type bioreactor is used for suspension culture. It is preferable to do so. A wave bioreactor mixes cells and culture medium by the wave-like motion of the culture medium. An example of a wave bioreactor for use in the method of the present invention is the Xuri® cell proliferation system (GE Healthcare). This wave bioreactor was first developed and validated for T cell proliferation, and its mechanism is based on the principle that the cell culture is constantly moving.

[0037] Once the monocytes are suspended in a suitable medium, the suspension can be transferred to a suitable container adapted to accept the cell culture and used to culture the cells under specified conditions. The container may be made of a material suitable for cell culture, such as glass or plastic. In a preferred embodiment, the container is made of plastic, such as ethylene vinyl alcohol (EVOH), ethylene vinyl acetate copolymer (EVA), polyolefin, etc. In another preferred embodiment, the container is made of polyethylene, such as low-density polyethylene (LDPE) or linear low-density polyethylene (LLDPE). Yet another In this embodiment, the container is a plastic bag made from one of the above materials.

[0038] The volume of the container (e.g., bag) used to differentiate monocytes into Mreg-sc cells is at least 0.5 L, preferably at least 1.0 L, at least 1.5 L, at least 2.0 L, at least 2.5 L, at least 3.0 L, at least 3.5 L, at least 4.0 L, at least 4.5 L, or at least 5.0 L or more. The ratio of the volume of the cell suspension to the volume of the container (e.g., bag) is at least about 1:6, at least 1:5, at least 1:4, at least 1:3, or at least 1:2. A ratio of 1:2 means that 1 L of cell suspension is used in a 2 L container. For example, when using a 2 L bag, the volume of the cell suspension is 0.3 L to 1 L, with 0.5 to 0.6 L being particularly preferred. When using a 10 L bag, the volume of the cell suspension is 1.5 L to 5 L, with 2.5 to 3.0 L being particularly preferred. When using a 20L bag, the volume of the cell suspension is 3.0L to 10L, with 5.0 to 6.0L being particularly preferred. In a particularly preferred embodiment, the Xuri® cell proliferation system is used with bags of 2L, 10L, or 20L volume.

[0039] After transferring the monocytes to a container (e.g., a culture bag), the cells are incubated in the container for at least 3 days prior to IFN-γ stimulation in the presence of M-CSF / GM-CSF and CD16 ligand (e.g., human serum or human immunoglobulin). As used herein, a "1-day" culture period refers to a 24-hour culture. Therefore, a "3-day" culture period refers to a culture of 72 hours or more. The optimal duration of IFN-γ stimulation is at least 12 hours, preferably 18 hours, and more preferably 24 hours. According to the present invention, the total culture period, i.e., the period from the introduction of monocytes into the culture vessel to the collection of Mreg-sc, is at least 4 days, preferably at least 5 days, at least 6 days, at least 7 days, or at least 8 days. Preferably, the period from the introduction of monocytes into the culture vessel to the collection of Mreg-sc is 9 days or less. In other words, the total culture period is 4 to 8 days, preferably 6 to 8 days, and more preferably 7 days. Monocytes in a container are incubated under conditions that allow for their proliferation and differentiation into Mreg-sc cells. General conditions for culturing monocytes or macrophages are known to those working in the field of cell culture.

[0040] For example, the bag containing the suspension can be transferred to an incubation chamber that allows for the selection of specified conditions for temperature, humidity, and CO2. Suitable conditions include a temperature in the range of 30-40°C, preferably 32-38°C, more preferably 37-38°C, for example 37°C. The humidity used for cultivation is typically in the range of 30-70%, preferably 40-60%, more preferably 50-60%, for example 60% humidity. The incubation chamber may contain up to 10% CO2. Particularly preferred CO2 content is up to 5%, up to 4%, up to 3%, up to 2%, or up to 1%.

[0041] All culture steps in the method of the present invention are carried out so that the majority of cells in the container remain suspended and do not adhere to the surface of the container, for example, the bottom of the container. Preferably, the culture is carried out so that 10% or less of the cells in the container, more preferably 5% or less or 1% or less of the cells in the container adhere to the inner surface of the container, for example, the bottom of the container. This is achieved by constantly stirring the container to the extent that cell adhesion is minimized. The required stirring speed depends on the size of the cell container and the bioreactor device used for culture and differentiation. Those skilled in the art will be able to easily determine the minimum stirring speed that prevents adhesion and keeps the cells in suspension based on routine experiments. For example, when using the Xuri® cell proliferation system for culture and differentiation, stirring speeds of 2 to 10 rpm, more preferably 2 to 6 rpm, and even more preferably 2 to 4 rpm can be used. The tray angle is preferably adjusted to 2 to 12°, more preferably 2 to 8°, and even more preferably 2 to 4°.

[0042] In step (c) of the method of the present invention, cells are brought into contact with the cytokine interferon-gamma (IFN-γ). It is known in the art that cytokines alter the transcription of more than 30 genes, thereby producing a variety of physiological and cellular responses. IFN-γ proteins have been isolated from various species and can be purchased from various manufacturers. The selection of IFN-γ used in the method of the present invention depends on the origin of the monocytes subjected to the method of the present invention. For example, when differentiating human monocytes into Mreg-sc using the process described herein, the IFN-γ added is human IFN-γ, preferably compound IFN-γ. This is recombinant human IFN-γ. Similarly, when porcine monocytes are used in the differentiation method, the IFN-γ added to the culture medium will be of porcine origin. In a particularly preferred embodiment of the present invention, the IFN-γ is human IFN-γ, more preferably recombinant human IFN-γ.

[0043] Any amount of IFN-γ effective in inducing the expression of indoleamine 2,3-dioxygenase (IDO1) by monocytes in the culture can be added. Preferably, the amount of IFN-γ added to the monocyte culture is in the range of 5 to 100 ng / ml, more preferably 10 to 80 ng / ml, and even more preferably 20 to 50 ng / ml. In this specification, an amount of 25 ng of IFN-γ per 1 ml of medium is particularly preferred.

[0044] IFN-γ can be added to the culture medium simultaneously with M-CSF / GM-CSF and CD16 ligand, meaning that cytokines can be added, for example, when monocytes are introduced into the vessel where differentiation takes place. In such embodiments, monocytes differentiated by the method of the present invention are cultured in the presence of M-CSF / GM-CSF, CD16 ligand, and IFN-γ for the entire culture period. However, the culture period in the presence of IFN-γ is preferably significantly shorter than the culture period in the presence of M-CSF / GM-CSF, meaning that the cells are cultured only after being cultured in the presence of M-CSF / GM-CSF for at least 3 days. This means that IFN-γ is added. In a preferred embodiment, IFN-γ is added after culturing cells in the presence of M-CSF / GM-CSF for 3 to 6 days. Preferably, cells are cultured in the presence of M-CSF / GM-CSF for at least 3, at least 4, at least 5, or at least 6 days before the addition of IFN-γ. In a particularly preferred embodiment, IFN-γ is added after culturing cells in the presence of M-CSF / GM-CSF for 3 to 6 days, and then the culture is continued for a further 18 to 72 hours.

[0045] In a particularly preferred embodiment of the present invention, monocytes are cultured on day 7 in a medium containing, for example, M-CSF / GM-CSF and CD16 ligand, followed by 18-24 hours of IFN-γ stimulation, and differentiated cells are harvested 6 days later. If several containers are cultured in parallel, the contents of the containers may be pooled at the end of the culture process. Differentiated macrophages may be washed with a buffer suitable for use with macrophages. For example, preferably Ringer's solution supplemented with 5% human serum albumin or phosphate-buffered saline (PBS) is used to wash the cells by continuous exchange of buffer by centrifugation and decantation of the supernatant. This is possible. Since the cells do not adhere to the surface of the container, the recovery step of the method of the present invention does not involve the addition of trypsin.

[0046] Preferably, the recovery step (d) of the method of the present invention does not involve mechanical detachment of cells from the surface of the container, e.g., scraping of cells from the inner surface of the container. In preferred embodiments, cell recovery is carried out by the use of a countercurrent centrifuge, such as a Gibco CTS Rotea countercurrent centrifuge system. Such an apparatus uses countercurrent techniques to separate cells from the culture medium and form a compact pellet (see, for example, U.S. Patent No. 1,0099,228). The cells are suspended within the liquid bed and are therefore protected from shear stress more than during conventional centrifugation. By adjusting both the centrifugal force and the countercurrent velocity... It is possible to separate debris and dead cells from live Mreg-sc cells, thereby purifying the final product.

[0047] In certain preferred embodiments, the countercurrent centrifuge may also be used to transfer monocytes from the concentrator to the bioreactor. For example, if a CliniMACS Prodigy® apparatus is used for monocyte concentration, the monocytes can be transferred to a wave reactor, such as a Xuri® cell proliferation system. For this purpose, the bags for culturing monocytes in the Xuri® cell proliferation system are sterile-connected to the tubing system of the countercurrent centrifuge. In this way, Mreg differentiation can be carried out as a fully closed process.

[0048] After cell harvesting, the quality of the differentiation process can be determined by measuring IP-10 (interferon-gamma-inducible protein 10). IP-10 protein is produced and secreted in the surrounding culture medium by Mreg cells in response to IFN-γ stimulation. Therefore, the concentration of IP-10 in the culture medium is related to the number of differentiated Mreg-sc cells. Thus, the concentration of IP-10 in the culture medium can be used as a quality control marker for the preparation process. In this way, it is not necessary to use cells resulting from the process for quality control purposes. In a preferred embodiment, IP-10 is 5 per 1 ml of culture medium. Concentrations exceeding 000 pg indicate that monocytes have sufficiently differentiated into Mreg-sc cells. Preferably, concentrations exceeding 10,000 pg, 15,000 pg, 20,000 pg, 25,000 pg, or 30,000 pg per ml of culture medium indicate sufficient differentiation.

[0049] These Mreg-sc cells can be transferred and stored in blood bags, glass infusion devices, or other closed-system containers that allow the cells to be transported to a treatment center or the patient's bedside. For this purpose, the differentiated cells are suspended in a suitable storage medium. The storage medium may be, for example, Ringer's solution supplemented with 5% human serum albumin. In a particularly preferred embodiment, the storage medium is a serum-free and / or protein-free sterile-prepared medium. Suitable commercially available sterile-prepared media include HypoThermosol® FRS (Stemcell Technologies SARL, Cologne, D). The medium preferably has a pH of 6.5 to 8.0, more preferably 7.0 to 7.5, for example, 7.4. The cell solution should be stored at 4°C to minimize energy consumption and cell adhesion. Alternatively, Mreg-sc cells may be resuspended in a cryopreservation solution such as 10% dimethyl sulfoxide + human AB serum (DMSO-HABS) and stored in a frozen state until final use.

[0050] The phenotypic and functional stability of the differentiated macrophage cells of the present invention depend on the selection of excipients and storage temperature. When resuspended in Ringer's solution supplemented with human serum albumin, the macrophages of the present invention are stable at 20°C to 25°C for up to 24 hours after cell harvesting. When resuspended in Plasmalyte / PBS supplemented with 5% human serum albumin, the macrophages can be stored at 2–8°C, preferably 4°C, for at least 48 hours after cell harvesting. If a longer storage period is required, the cells can be frozen or cryopreserved. In general, the cells of the present invention have been found to be stable in their immunosuppressive phenotype.

[0051] The method for preparing Mreg-sc cells according to the present invention can be automated according to conventional methods, for example, by using a GMP-compliant platform that provides an integrated solution to streamline the cell processing workflow. The method is preferably carried out in a “closed system” that takes advantage of sealed, disposable products, optional customization of tube sets, buffers and reagents, multiple input lines with sterile filters, optional output lines for in-process control, and significantly reduced cleanroom requirements. For example, the platform may include a cell separation system that enables the separation of monocytes from the leukocyte fraction. The isolation system should be able to isolate monocytes from apheresate or whole peripheral blood in a starting volume of 100-1000 ml. Monocytes in the isolated mononuclear leukocyte fraction can then be isolated, for example, using magnetic beads bound to CD14+ cells. These cells are then cultured in appropriate culture medium. This platform allows for the supply of culture medium, growth factors, and / or cytokines to the cell culture via multiple input ports. At the end of the culture process, the cells are automatically washed, harvested, and transferred to appropriate sterile delivery bags. Aseptic sealing of PVC and EVA tubing is also possible. Customized tube sealers can be used. Cell products may be barcoded, and the entire manufacturing process may be monitored online for quality control purposes.

[0052] In a second aspect, the present invention relates to a novel type of Mreg cell called Mreg-sc cells (abbreviated as “Mreg derived from suspension culture”), which can be obtained by the method of the first aspect of the present invention. As used herein, regulatory macrophages or “Mreg” are macrophage cells that can prevent the proliferation of mitogen-stimulated allogeneic T cells or autologous T cells. The cells provided by the present invention are monocyte-derived human macrophages and therefore express common leukocyte markers and macrophage lineage markers, specifically CD45 and CD33. Mreg-sc cells also express the marker CD85 h expresses CD258 and CD206.

[0053] Mreg-sc cells were found to consistently express the characteristic markers CD16, CD163, and syndecan-3. This is surprising, as all other Mregs have been reported to lack CD16 and CD163 expression. Therefore, these markers are very useful for distinguishing Mreg-sc cells from previously known Mregs. Furthermore, Mreg-sc cells also express the markers CD51, CD72, and CD11c. All human Mregs cultured in bags, flasks, or suspensions showed relatively high levels of DHRS9, i.e., retinol dehydrogenase S. It expresses retinol dehydrogenases of the DR family.

[0054] Similarly, all human Mreg cells express indoleamine 2,3-dioxygenase (IDO1), which is not typically observed in other monocyte-derived macrophage types described in the conventional art. Previous studies have shown that IDO1 is involved in immunomodulation, particularly effector T cell-mediated immunomodulation. It has been identified as one of the key molecules in suppressing harmful inflammatory responses [8]-

[10] . Proposed mechanisms include direct elimination of effector T cells by tryptophan deficiency

[11] -

[12] and indirect immunomodulation by Stat3-mediated induction of regulatory T cells. Thus, it has been identified as one of the primary targets for measuring the potency and therapeutic potential of monocyte-derived cell therapy products. IFN-γ is a potent inducer of IDO1 gene transcription. IFN-γ is added while both Mreg-bc and Mreg-sc are produced, as well as while pro-inflammatory M1 macrophages are generated, but anti-inflammatory It is not added to M2a cells. As a result, IDO1 expression is not detected at the protein level in M2a cells and appears to be downregulated at the level of transcribed mRNA compared to CD14+ monocytes.

[0055] Accordingly, in one embodiment, the present invention provides a novel type of immunomodulatory macrophage characterized by the expression of markers CD16, CD163 and syndecan-3. In one embodiment, a macrophage expressing markers CD16, CD51, CD163 and syndecan-3 is provided. In another embodiment, a macrophage expressing markers CD16, CD11c, CD163 and syndecan-3 is provided. In yet another embodiment, a macrophage expressing markers CD16, CD11c, CD51, CD163 and syndecan-3 is provided. In yet another embodiment, markers CD16, CD1 Macrophages expressing 1c, CD51, CD72, CD163, and syndecan-3 are provided. Preferably, the macrophages also express the marker IDO1.

[0056] Here, cells are negative for a particular surface marker if their fluorescence intensity, as measured by flow cytometry, is below the 99th percentile fluorescence intensity of the corresponding isotype control stained sample.

[0057] Mreg-sc cells are further negative for CD38 or express low levels of CD38. During Mreg-sc cell development, the initial population of monocytes downregulates CD38 expression on the cell surface. Downregulation of CD38 during Mreg-bc development can be expressed as the percentage of CD38 expression in Mreg-sc cells at day 7 compared to monocytes at day 0 (d0) of culture. CD38 expression is proportional to the difference in mean fluorescence intensity between isotype-controlled stained cells and specific CD38 signals. Therefore, downregulation % = 100 - 100 × (CD38d7 - Isood7) / (CD38d0 - Isood0) (where CD38d7 is day 7) is a specific signal, Isod7 is the isotype control signal on day 7, CD38d0 is the specific signal on day 0, and Isod0 is the isotype control signal on day 0). The downregulation of CD38 by Mreg-sc cells can be easily determined by using a standard flow cytometry method. In a preferred embodiment, the expression of CD38 by Mreg-sc cells is downregulated by more than 50%, more preferably more than 60%, more than 70%, more than 80%, more than 90%, more than 95% or more than 99% compared to the initial expression of CD38 by monocytes on day 0 of culture.

[0058] Therefore, the Mreg-sc cells provided by the method of the present invention are macrophages that can be described by one of the following marker patterns. (1). CD16 + , CD163 + , syndecan-3 + (2). CD16 + , CD51 + , CD163 + , syndecan-3 + (3). CD16 + , CD11c + , CD163 + , syndecan-3 + (4). CD16 + , CD51 + , CD11c + , CD163 + , syndecan-3 + (5). CD16 + , CD163 + , syndecan-3 + , CD72 + (6). CD16 + , CD51 + , CD163 + , syndecan-3 + , CD72 + (7). CD16 + , CD11c+ CD163 + , Cindecan-3 + CD72 + (8). CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + CD72 + (9). CD16 + CD163 + , Cindecan-3 + IDO1 + (10). CD16 + CD51 + CD163 + , Cindecan-3 + IDO1 + (11). CD16 + CD11c + CD163 + , Cindecan-3 + IDO1 + (12). CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + IDO1 + (13). CD16 + CD163 + , Cindecan-3 + CD72 + IDO1 + (14). CD16 + CD51 + CD163 + , Cindecan-3 + CD72, IDO1 + (15). CD16 + CD11c + CD163 + , Cindecan-3 + CD72 + IDO1 + (16). CD16 + 、CD51 + 、CD11c + 、CD163 + 、 Syndecan-3 + 、CD72 + 、IDO1 + (17). CD16 + 、CD163 + 、 Syndecan-3 + 、CD86 low (18). CD16 + 、CD51 + 、CD163 + 、 Syndecan-3 + 、CD86 low (19). CD16 + 、CD11c + 、CD163 + 、 Syndecan-3 + 、CD86 low (20). CD16 + 、CD51 + 、CD11c + 、CD163 + 、 Syndecan-3 + 、CD86 low (21). CD16 + 、CD163 + 、 Syndecan-3 + 、CD72 + 、CD86 low (22). CD16 + 、CD51 + 、CD163 + 、 Syndecan-3 + 、CD72 + 、CD86 low (23). CD16 + 、CD11c + 、CD163 + 、 Syndecan-3 + 、CD72 + 、CD86 low (24). CD16 +CD51 + CD11c + CD163 + , Cindecan-3 + CD72 + CD86 low (25).CD16 + CD163 + , Cindecan-3 + IDO1 + CD86 low (26).CD16 + CD51 + CD163 + , Cindecan-3 + IDO1 + CD86 low (27).CD16 + CD11c + CD163 + , Cindecan-3 + IDO1 + CD86 low (28).CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + IDO1 + CD86 low (29).CD16 + CD163 + , Cindecan-3 + CD72 + IDO1 + CD86 low (30).CD16 + CD51 + CD163 + , Cindecan-3 + CD72, IDO1 + CD86 low (31). CD16 + CD11c + CD163 + , Cindecan-3 + CD72+ IDO1 + CD86 low (32).CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + CD72 + IDO1 + CD86 low (33).CD16 + CD163 + , Cindecan-3 + , CD83 low (34).CD16 + CD51 + CD163 + , Cindecan-3 + , CD83 low (35).CD16 + CD11c + CD163 + , Cindecan-3 + , CD83 low (36).CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + , CD83 low (37).CD16 + CD163 + , Cindecan-3 + CD72 + , CD83 low (38).CD16 + CD51 + CD163 + , Cindecan-3 + CD72 + , CD83 low (39).CD16 + CD11c + CD163 + , Cindecan-3 + CD72+ , CD83 low (40).CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + CD72 + , CD83 low (41). CD16 + CD163 + , Cindecan-3 + IDO1 + , CD83 low (42).CD16 + CD51 + CD163 + , Cindecan-3 + IDO1 + , CD83 low (43).CD16 + CD11c + CD163 + , Cindecan-3 + IDO1 + , CD83 low (44).CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + IDO1 + , CD83 low (45).CD16 + CD163 + , Cindecan-3 + CD72 + IDO1 + , CD83 low (46).CD16 + CD51 + CD163 + , Cindecan-3 + CD72, IDO1 + , CD83 low (47).CD16 + CD11c+ CD163 + , Cindecan-3 + CD72 + IDO1 + , CD83 low (48).CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + CD72 + IDO1 + , CD83 low (49).CD16 + CD163 + , Cindecan-3 + CD370 low (50).CD16 + CD51 + CD163 + , Cindecan-3 + CD370 low (51).CD16 + CD11c + CD163 + , Cindecan-3 + CD370 low (52).CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + CD370 low (53).CD16 + CD163 + , Cindecan-3 + CD72 + CD370 low (54).CD16 + CD51 + CD163 + , Cindecan-3 + CD72 + CD370 low (55).CD16 +CD11c + CD163 + , Cindecan-3 + CD72 + CD370 low (56).CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + CD72 + CD370 low (57).CD16 + CD163 + , Cindecan-3 + IDO1 + CD370 low (58).CD16 + CD51 + CD163 + , Cindecan-3 + IDO1 + CD370 low (59). CD16+, CD11c+, CD163+, Syndecan-3+, IDO1+, CD370low, (60).CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + IDO1 + CD370 low (61).CD16 + CD163 + , Cindecan-3 + CD72 + IDO1 + CD370 low (62).CD16 + CD51 + CD163 + , Cindecan-3 + CD72, IDO1 + CD370 low (63).CD16 +CD11c + CD163 + , Cindecan-3 + CD72 + IDO1 + CD370 low (64).CD16 + CD51 + CD11c + CD163 + , Cindecan-3 + CD72 + IDO1 + CD370 low

[0059] Marker expression can be determined at the mRNA or protein level. For marker determination at the protein level, the marker profile of Mreg-sc cells can be easily determined using standard flow cytometry. Flow cytometry is a widely used method for analyzing the expression of cell surface markers and intracellular molecules. It is routinely used for applications such as cell counting, cell sorting, and biomarker profiling. In particular, it can be used to define different cell types in heterogeneous cell populations. Flow cytometry is typically used to analyze markers on the cell surface. This involves measuring the fluorescence intensity produced by a fluorescently labeled antibody that detects the substance. While it can also be used for detecting intracellular markers, such detection is generally undesirable because the antibody, which usually kills cells, penetrates the cells. This hinders the detection of intracellular markers in cell sorting applications aimed at providing a homogeneous population of viable cells. Therefore, intracellular markers such as IDO1 are not suitable for detection at the mRNA level, for example, by commonly known methods that allow for quantitative or semi-quantitative detection at the mRNA level, such as quantitative RT-PCR (e.g., TaqMan® RT-PCR), real-time RT-PCR, or Northern Blocking. It is determined by methods such as t-analysis.

[0060] According to the present invention, several genes have been identified in transcriptome analysis that can be used to distinguish Mreg from other macrophages, such as M0, M1, or M2a macrophages. These genes include the ARMH1 gene (armadillo-like helical domain containing 1-NCBI reference sequence: NM_001145636.2-SEQ ID NO: 1), the CA11 gene (carbonic anhydrase 11-NCBI reference sequence: NM_001217.5-SEQ ID NO: 2), and the SMARCD3 gene (SWI / SNF-related, matrix-related, actin-dependent regulator of chromatin, subfamily d, member 3-NCBI). This includes the reference sequence: NM_001003801.2 (SEQ ID NO: 3), and the HLA-DOA gene (major histocompatibility complex, class II, DO alpha - NCBI reference sequence: NM_002119.4 (SEQ ID NO: 4)). The genes ARMH1, CA11, and SMARCD3 are expressed at the same level in M0, M1, and M2a cells, but they are expressed more than 4 times more strongly in Mregs. HLA-DOA has more than 8 times higher expression in Mregs compared to other macrophages. Therefore, in another preferred embodiment, any of the surface marker patterns (1) to (64), more preferably CD16 + CD163 + , Cindecan-3 + The following are provided: Mreg cells that express and, furthermore, significantly strongly express at least one of the genes shown in SEQ ID NOs: 1-4, preferably expressing 2- or 3-fold more strongly than quiescent macrophages (M0) as measured by quantitative RT-PCR. In other words, any of the surface marker patterns (1)-(64), more preferably CD16 + CD163 + , Cindecan-3 + Furthermore, when expressing and quantitatively measuring at least one of the genes shown in Sequence IDs 1-4 by RT-PCR, compared to quiescent macrophages (M0), the expression was at least 50%, at least 100%, at least 200%, and at least Mreg cells are provided that express the gene strongly by 300%, at least 400%, at least 500%, at least 600%, at least 700%, or at least 800%.

[0061] Transcriptome analysis further revealed several genes that could be used to distinguish Mreg-sc cells from other Mreg cells, such as Mreg-bc cells. These genes include the SELENOP gene (selenoprotein P - NCBI reference sequence: NM_005410.4 - SEQ ID NO: 5), RBASE1 (ribonuclease A family member 1 - NCBI reference sequence: NM_002933.5 - SEQ ID NO: 6), the C1QC gene (complement C1qC chain - NCBI reference sequence: NM_172369.5 - SEQ ID NO: 7), and NRA4A3 (nuclear receptor subfamily group 4A member 3 - NCBI reference sequence: NM This includes (_006981.4 - SEQ ID NO: 8). Compared to Mreg-bc cells, Mreg-sc cells express the SELENOP, RBASE1, and C1QC genes more strongly, while the NRA4A3 gene is downregulated in Mreg-sc cells. Therefore, in another preferred embodiment, Mreg cells are provided that express at least one of the genes shown in SEQ ID NOs: 5-7 significantly more strongly, preferably two or three times more strongly than other types of Mreg cells such as Mreg-bc cells, as measured by quantitative RT-PCR. In other words, at least one of the genes shown in SEQ ID NOs: 5-7 is expressed more strongly, Mreg cells are provided that, when measured by quantitative RT-PCR, express at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, or at least 800% more strongly than other types of Mreg cells, such as Mreg-bc cells.

[0062] In yet another preferred embodiment, Mreg cells are provided that express the gene shown in SEQ ID NO: 8 at a significantly lower level, preferably at a level 1 / 2 or 1 / 3 lower than other types of Mreg cells, such as Mreg-bc cells, as measured by quantitative RT-PCR. In other words, when the gene shown in SEQ ID NO: 8 is measured by quantitative RT-PCR, it is expressed at a level at least 1 / 50%, at least 1 / 100%, at least 1 / 200%, or at least 1 / 300%, at least 1 / 400%, at least 1 / 500%, or less than other types of Mreg cells, such as Mreg-bc cells. Mreg cells are provided that express the gene at least 1 / 600%, at least 1 / 700%, or at least 1 / 800% less.

[0063] Mreg-sc cells are particularly well-suited for therapeutic use, as will be described in more detail below. Conceptually, Mreg-sc therapy is a gain-of-function therapy in which the administration of Mreg-sc cells, which possess immunosuppressive, anti-inflammatory, or tissue repair functions, compensates for deficiencies in those cellular functions in the recipient. By administering appropriately large doses, it may be possible to restore or even enhance the above activities in the recipient. In transplantation and autoimmune models, Mreg-sc treatment has therapeutic effects that persist beyond the recipient's lifespan. This persistence is due to... This can be explained by the effect of Mreg-sc treatment on the recipient's T cells. Administration of Mreg-sc cells can affect the recipient's T cell response in three complementary ways.

[0064] (a) Mreg-sc cells directly interact with recipient T cells, resulting in the deletion of specific T cells or their conversion to activation-induced regulatory T cells (iTreg).

[0065] (b) Mreg-sc cells alter the behavior of recipient dendritic cells through direct interaction with or release of anti-inflammatory mediators. One important function of Mreg-sc cells may be to die in a well-regulated environment and deliver antigens to recipient dendritic cells, thereby specifically suppressing recipient T cells.

[0066] (c) Mreg-sc cells or their intracellular fraction exert active or passive nonspecific suppression through the release of soluble mediators that can act or exert effects directly via recipient myeloid monocytic cells.

[0067] In a third aspect, the present invention relates to a pharmaceutical composition comprising Mreg-sc cells or their intracellular fraction according to a second aspect of the present invention. The pharmaceutical composition comprises, as a first component, an effective amount of the Mreg-sc cells or their intracellular fraction according to the present invention. When used herein, the effective amount of Mreg-sc cells administered intravenously to a patient is about 1 × 10¹⁶ per kg of body weight. 4 From approximately 1 x 10 8 / kg, preferably about 1 × 10 5 ~Approx. 1×10 7 / kg, more preferably about 1 × 10 6 ~Approx. 9×10 6 The range is / kg, for example, approximately 1 × 10 per kg of the patient's body weight being treated. 6 / kg, approximately 2 x 10 6 / kg, approximately 3 x 10 6 / kg, approximately 4 x 10 6 / kg, approximately 5 x 10 6 / kg, approximately 6 x 10 6 / kg, approximately 7 x 10 6 / kg, or approximately 8 x 10 6 / kg Similarly, if the present invention involves the administration of intracellular fractions of Mreg-sc cells, these fractions are prepared based on the amount of cells corresponding to one of the ranges described above in relation to the administration of cells. As used herein, intracellular fractions of Mreg-sc cells may include necrotic cell particles, apoptotic cell particles, or exosomes containing major histocompatibility (MHC) molecules of the cells. Cell lysates prepared by treatment of cells with hypotonic solutions, dissolution using surfactants or acids, freeze-thaw or heating, sonication, irradiation, mechanical destruction or long-term storage can also be used. Intracellular fractions also include whole cell proteins, membrane proteins, and cells. It may contain cell extracts containing high-quality proteins or purified MHC molecules. The amount of cells will differ when the pharmaceutical composition is formulated for topical administration, such as intradermal administration.

[0068] Apart from the cells or intracellular fractions of the cells, the pharmaceutical composition may contain further excipients such as buffers, pH adjusters, and preservatives. The properties and amounts of excipients contained in the pharmaceutical composition of the present invention depend on the intended route of administration. Generally, various routes of administration are feasible for administering the Mreg-sc cells or their intracellular fractions of the present invention to patients in need of treatment. Preferably, the pharmaceutical composition of the present invention is formulated for parenteral administration, such as subcutaneous, intramuscular, intravenous, or intradermal administration. Mreg-sc cells or their intracellular fractions, or compositions containing such cells or fractions, are particularly preferably administered to patients by intravenous administration.

[0069] The formulation of Mreg-sc cells or their intracellular fractions into pharmaceutical compositions according to the present invention can be achieved by applying routine methods known in the field of pharmaceutical formulation. Suitable methods are described, for example, in standard textbooks. Pharmaceutical compositions suitable for intravenous administration by injection or infusion typically comprise a sterile aqueous solution or suspension, and a sterile powder for immediate preparation of the sterile solution or suspension. Compositions for injection must be sterile and fluid to facilitate handling in a syringe or infusion bag.

[0070] The composition must be stable under administration conditions and preferably protected against microbial contamination such as bacteria and fungi by including, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. For intravenous administration, suitable carriers may include physiological saline, bacteriostatic water, Cremophor EL® (BASF), or phosphate-buffered saline (PBS). The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. Motility can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and by the use of surfactants. Sterile injection solutions can be prepared by incorporating cells or intracellular fractions in the required amount with one or more of the above components into a suitable solvent, followed by sterile filtration. Generally, suspensions are prepared by incorporating the active compound, i.e., cells or their intracellular fractions, into a sterile vehicle containing the underlying dispersion medium and other necessary components from the above. In the case of sterile powders for the preparation of sterile injection solutions, the preparation method involves pre-sterilizing the powder of cells or their intracellular fractions and any additional desired components by sterile filtration. This involves vacuum drying and freeze-drying of the resulting solution.

[0071] The composition for injection or injection has a volume of 50 to 500 ml, with a volume of 90 ml to 250 ml being particularly preferred, and a volume of 90 ml to 150 ml being even more preferred. In the case of local administration, such as intradermal administration, the volume per injection is in the range of 0.1 to 1.0 ml.

[0072] Mreg-sc cells can be administered to patients requiring treatment with various dosing regimens. For example, when cells or cell fractions are administered to a patient by intravenous infusion, the total amount of Mreg-sc cells or Mreg-sc cell fractions administered may be delivered in one or more infusions. In a preferred embodiment, Mreg-sc cells or cell fractions are delivered to the patient via an infusion set equipped with a 200 μm filter. The suspension containing Mreg-sc cells or Mreg-sc cell fractions may be primed with 0.9% NaCl. The suspension is administered in a single infusion, more preferably within less than 60 minutes, e.g., 60 minutes, 30 minutes, 20 minutes. It can be administered by short-term infusion within 15 minutes or less. Preferably, a central venous catheter is used to administer the Mreg-sc suspension.

[0073] Administration of Mreg-sc cells or cell fractions may be preceded, simultaneously with, or subsequently accompanied by the administration of other active agents. For example, if the Mreg-sc cells or cell fractions of the present invention are administered to prevent an immune response in a patient undergoing organ transplantation, immunosuppressants may be administered together with the cells or cell fractions of the present invention. Immunosuppressants routinely used in the field of transplant medicine include, but are not limited to, cyclosporine A (CSA), tacrolimus, azathioprine (AZA), mycophenolate mofetil, rapamycin, and steroids (STE). Generally, immunosuppression in the recipient's blood The presence of the drug does not affect the efficacy of the cells or cell fractions of the present invention.

[0074] Mreg-sc cells obtained by the method described in the first aspect of the present invention exhibit a stable phenotype; however, for safety reasons, it is recommended that Mreg-sc cells or intracellular fractions obtained therefrom be administered within 24 hours of being harvested from the cell culture. Preferably, the cells are administered within 20 hours, 16 hours, 12 hours, 8 hours, or 4 hours of being harvested from the culture.

[0075] In a fourth aspect, the present invention relates to the therapeutic administration of Mreg-sc cells or their intracellular fraction according to a second aspect of the present invention, or a composition according to a third aspect of the present invention. As shown elsewhere in this specification, the Mreg-sc cells provided by the present invention exhibit many pharmacological properties, such as immunosuppressive, immunomodulatory, angiogenic, and anti-inflammatory properties, making them highly suitable for use in immunosuppressive therapy, anti-inflammatory therapy, or tissue repair therapy. For example, the artificially induced Mreg-sc cells of the present invention are T cell suppressive and mediate active deletion of activated T cells. Therefore, these cells are suitable for various immunotherapy applications, such as organ transplantation. It is highly suitable for use as adjuvant immunosuppressive therapy in disease-mediated illnesses.

[0076] Accordingly, in one embodiment of the present invention, Mreg-sc cells or their intracellular fraction according to the second aspect of the present invention, or a pharmaceutical composition according to the third aspect of the present invention, are used in a method for suppressing transplant rejection and / or extending transplant survival in a recipient. Thus, the present invention relates to a method for suppressing transplant rejection and / or extending transplant survival in a recipient, and comprises (i) administration of an effective amount of Mreg-sc cells or their intracellular fraction according to the second aspect of the present invention, or (ii) administration of a pharmaceutical composition according to the third aspect of the present invention. Preferably, the transplant is the transplantation of organs, tissues, or cells. The type of organ is not limited by the present invention, but is preferably the kidney, liver, heart, lung, or pancreas. It is particularly preferable that the organ transplanted to the recipient is a human organ.

[0077] The Mreg-sc of the present invention can also be used to suppress transplant rejection and / or extend the survival time of the graft even when the transplant is a tissue transplant rather than an organ transplant. In this case as well, the tissue transplanted to the recipient is not particularly limited. Rejection of any tissue derived from an allogeneic donor in the recipient can be prevented or mitigated by the Mreg-sc of the present invention. The transplanted tissue is preferably human tissue, such as intestinal tissue, corneal tissue, skin tissue, composite tissue, bone marrow tissue, or pancreatic islet tissue.

[0078] Mreg-sc cells prepared according to the method of the present invention can also support cell transplantation to the recipient by suppressing the immune response in the recipient. When transplantation is cell transplantation, the properties of the transplanted cells are not generally limited, but it is preferable that the transplanted cells be selected from the group consisting of adult stem cell transplantation, isolated hepatocyte transplantation, or leukocyte transplantation. In a preferred embodiment of the present invention, Mreg-sc cells are used to promote engraftment of hematopoietic stem cells (HSCs) after bone marrow transplantation or HSC transplantation.

[0079] To suppress transplant rejection in recipients and induce acceptance of allogeneic organ, tissue, or cell transplants, the Mreg-sc cells of the present invention or a pharmaceutical composition containing Mreg-sc cells or their intracellular fraction can be administered intravenously by injection or infusion as described above. The injection or infusion can be performed either preoperatively or postoperatively. When Mreg-sc cells are administered preoperatively, they are administered to the recipient at least once, preferably twice, more preferably three times, before surgery. Mreg-sc cells are administered up to one week before surgery, for example, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day before surgery. It is preferable to administer it to the recipient. When Mreg-sc cells are administered postoperatively, the first dose is preferably given within 24 hours postoperatively, more preferably within 36, 48, 60, or 72 hours postoperatively. Alternatively, in stably immunosuppressed transplant recipients, Mreg-sc therapy can be administered at any time post-transplant. Alternatively, Mreg-sc may be administered to transplant recipients who experience acute or chronic transplant rejection. Mreg-sc subsequently resists the recipient's immune system's T-cell response to the transplant, and the recipient It can persist in the recipient's body (especially the spleen, liver, lungs, and bone marrow) for a sufficiently long period to provide long-term transplant acceptance to the ent.

[0080] When using Mreg-sc of the present invention to suppress transplant rejection or extend transplant survival in a recipient, the transplant is usually an allogeneic transplant, i.e., a transplant from a donor that is genetically different but belongs to the same species as the recipient. In this case, Mreg-sc cells are generated from blood monocytes obtained from the donor. Monocytes can be obtained from living or deceased donors. In the case of a deceased donor, i.e., a donor's body, the donor's body is usually washed with perfusion medium by opening the main artery for the purpose of organ preservation. Venous blood is taken from the body and Mreg-sc cells are prepared according to the method described herein. g-sc can be collected for preparation. Alternatively, Mreg-sc can be prepared from bone marrow mononuclear cells isolated from the donor's spleen. In the case of postoperative administration of Mreg-sc cells prepared from a deceased donor, organ rejection can be prevented by administering immunosuppressants routinely used for this purpose during organ transplantation.

[0081] In another embodiment, Mreg-sc cells or their intracellular fraction according to a second aspect of the present invention, or a pharmaceutical composition according to a third aspect of the present invention, are used in a method to promote or maintain the engraftment or effect of a regulatory T cell-based drug. Thus, the present invention also relates to a method for promoting or maintaining the engraftment or effect of a regulatory T cell-based drug in a subject, comprising (i) administration of an effective amount of Mreg-sc cells or their intracellular fraction according to a second aspect of the present invention, or (ii) administration of a pharmaceutical composition according to a third aspect of the present invention.

[0082] Apart from its immunomodulatory and immunosuppressive properties, the Mreg-sc cells of the present invention possess anti-inflammatory properties that enable the suppression of chronic inflammatory immune processes. Therefore, the Mreg-sc cells provided herein are also useful in treating diseases or disorders characterized by disordered immune states or excessive inflammatory responses, particularly chronic inflammatory diseases. Such diseases or disorders include, for example, autoimmune diseases, inflammatory diseases, and hypersensitivity reactions.

[0083] In another embodiment, Mreg-sc cells or their intracellular fraction according to a second aspect of the present invention, or a pharmaceutical composition according to a third aspect of the present invention, are used in methods for treating or preventing autoimmune diseases, inflammatory diseases, or hypersensitivity reactions.

[0084] When Mreg-sc cells are used to treat autoimmune diseases, the disease may be (a) primarily T cell-mediated, (b) primarily antibody-mediated, or (c) primarily mediated by other cellular components of the immune system. The disease may be a local or systemic autoimmune condition. Types of autoimmune conditions treated with Mreg-sc therapy include, but are not limited to, systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, polymyositis, dermatomyositis, and other systemic autoimmune conditions; rheumatoid arthritis (RA), juvenile rheumatoid arthritis, and other inflammatory arthritis; ulcerative colitis, Crohn's disease, and other inflammatory conditions Intestinal diseases; autoimmune hepatitis, primary biliary cirrhosis, and other autoimmune liver diseases; cutaneous vasculitis of small vessels, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, Behçet's disease, thromboangiitis obliterans, Kawasaki disease, and other large, medium, or small vessel vasculitis of autoimmune etiologies; multiple sclerosis (MS) and neuroimmunological disorders; type 1 diabetes mellitus, autoimmune thyroid dysfunction, autoimmune pituitary dysfunction, and other autoimmune endocrine disorders; hemolytic anemia, thrombocytopenic purpura, and other autoimmune disorders of the blood and bone marrow; psoriasis, pemphigus vulgaris, bullous pemphigoid, and other autoimmune skin diseases.

[0085] Mreg-sc cells are also effective in treating acute or chronic inflammatory diseases, and diseases with pathophysiologically significant inflammatory components. The inflammatory diseases treated may be local or systemic. The types of inflammatory diseases or conditions that may benefit from treatment with Mreg-sc are not limited to, but include: arterial occlusive diseases, such as peripheral artery occlusive disease (pAOD), severe limb ischemia, arteriosclerosis, cerebral infarction, myocardial infarction, renal infarction, intestinal infarction, angina pectoris, and other conditions caused by arterial occlusion or stenosis; microvascular angina, also known as cardiac syndrome X; systemic inflammation associated with metabolic disorders, including type II diabetes and obesity-related metabolic syndromes; and skin diseases, including eczema. Examples of diseases include, but are not limited to, those listed below. Preferably, the inflammatory diseases to be treated are characterized by chronic inflammation of the intima of the arterial wall, such as myocardial infarction, stroke, severe ischemic limb vasculitis, and pAOD.

[0086] If treatment of a hypersensitivity reaction is desired, the hypersensitivity reaction is preferably selected from the group of asthma, eczema, allergic rhinitis, angioedema, drug hypersensitivity, and mastocytosis.

[0087] These cells may also be useful for treating pAOD. pAOD in patients unsuitable for revascularization due to the degree or location of arterial occlusion or significant comorbidities is a common, severely debilitating condition where amputation is known to be the only treatment option. Amputation remains a last resort, associated with a relatively high mortality rate, and only a small number of patients subsequently regain full mobility. Mreg-sc cells obtained by the methods described herein, as their counterparts described in the prior art, are basal to pro-angiogenic growth factors, such as VEGF, FGF (VEGF-D), PDGFB, and MDK. Through expression and stimulated expression, neovascularization, i.e., the formation of new blood vessels, can be actively promoted.

[0088] In one embodiment, Mreg-sc cells are injected intramuscularly or subcutaneously into an ischemic limb. In ischemic tissue, Mreg-sc cells are inevitably exposed to microbial and necrotic tissue components (e.g., HMGB1) that act as TLR4 agonists. Therefore, Mreg-sc can be used to promote tissue regeneration through local secretion of pro-angiogenic growth factor. In another embodiment, Mreg-sc cells may be ex vivo stimulated with a TLR ligand during the manufacturing process to ensure their high levels of pro-angiogenic growth factor production. Examples of TLR ligands include, but are not limited to, lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA). The pAOD treated with Mreg-sc of the present invention may be of any grade or category. For example, the pAOD may be grade I, category 1-4, or grade II-IV.

[0089] Mreg-sc cells prepared according to the method of the present invention can also be used to treat foot ulcers, specifically chronic foot ulcers. More specifically, the cells can be used to treat microvascular and macrovascular disorders of the lower extremities of a subject. As used herein, microvascular disorders are vascular diseases that affect small blood vessels and capillaries in the body, such as small blood vessels in the brain, coronary arteries, or legs. During microvascular disorders, the capillary basement membrane thickens and hardens, causing occlusion or rupture of capillaries or arteries, resulting in tissue necrosis and loss of function. In contrast, macrovascular disorders are vascular diseases that affect larger blood vessels, such as arteries. Occlusion of larger arteries results in heart attacks, strokes, and a high incidence of peripheral vascular disease in diabetic patients. Occlusion of leg arteries often results in ulcers in the feet and legs that do not heal easily. Peripheral vascular disease also causes intermittent claudication, i.e., pain during walking, and significantly impairs mobility. In many cases, macrovascular complications, particularly in diabetic patients, necessitated the amputation of one or both legs.

[0090] One cause of microvascular and macrovascular complications is long-term diabetes mellitus. In diabetic patients, high blood glucose levels cause vascular endothelial cells to absorb more glucose than normal. These cells then form more glycoproteins on their surface than normal, and further cause the basement membrane of the blood vessel wall to grow abnormally thick and weak. As a result, the blood vessel walls become more prone to leakage, blood flow through the body slows down, and some tissues are damaged because they do not receive enough oxygen.

[0091] According to the present invention, the microvascular disorders to be treated are preferably selected from the group of diseases consisting of vasculitis, arteritis, angioplasia, tinea atrophy, sclerosis cutaneously, Disterman syndrome, diabetic vascular disorders, endovascular occlusion, erythromelitis, fibromuscular dysplasia, perforating malm, Mönkenberg sclerosis, Osler disease, compartment syndrome, Paget-von Schleier syndrome, Raynaud's syndrome, and foot ulcers. In a particularly preferred embodiment, the microvascular or macrovascular disorder to be treated according to the present invention is a foot ulcer. As used herein, foot ulcers include diabetic foot ulcers and venous foot ulcers.

[0092] According to the present invention, the macrovascular disorder to be treated is preferably selected from the group consisting of aneurysms, dissections, atherosclerosis, atherothrombosis, peripheral artery occlusive disease (PAD), intermittent claudication, necrosis and gangrene, vascular malformations, Lulish syndrome, or compression syndrome.

[0093] The administration of Mreg cells or cell fractions may be preceded, simultaneously with, or subsequently accompanied by the administration of other active agents. For example, when Mreg cells or cell fractions are administered to patients with microvascular or macrovascular complications, compounds that reduce blood hyperviscosity, such as calcium dobesylate, or compounds that exert capillary stabilizing effects, such as naphthazone, may be administered before, simultaneously with, or after the administration of Mreg cells.

[0094] In another embodiment, the Mreg-sc cells of the present invention can be used to promote the healing of surgical wounds, traumatic wounds, or other wounds in a subject. Mreg therapy may be used in conjunction with conventional treatments (i.e., washing, suturing, and dressing) as needed to promote the healing of acute or chronic wounds. Wounds may be open or closed. Open wounds may include incisions, lacerations, abrasions, delaminations, penetrating injuries, or punctures. Closed wounds may include crush injuries or hematomas. Incisions may be traumatic or iatrogenic (i.e., surgical incisions). Mreg may be used to promote the engraftment of autologous or allogeneic skin grafts. Mreg can be used in combination with conventional treatments to promote the healing of burns that may be caused by exposure to heat, extreme cold, chemicals, friction, radiation, or electric currents. The descriptions made in relation to the treatment of ulcers also apply to the treatment of wounds or burns.

[0095] As described above, since the Mreg-sc cells of the present invention may possess angiogenic properties, this specification intends to use them to treat diseases or conditions requiring angiogenesis. Accordingly, the present invention also relates to Mreg-sc cells or their intracellular fractions according to a second aspect of the present invention, or to pharmaceutical compositions according to a third aspect of the present invention, used in methods for inducing angiogenesis or vascularization in hypoxic tissue, methods for promoting tissue repair processes by participating in tissue remodeling and tissue regeneration, methods for preventing or alleviating fibrosis, methods for reducing ischemic pain, or methods for avoiding limb amputation. Accordingly, The present invention relates to a method for inducing angiogenesis or angiogenesis in hypoxic tissue, promoting tissue repair processes by participating in tissue remodeling and tissue regeneration, preventing or alleviating fibrosis, reducing ischemic pain, or avoiding limb amputation, comprising (i) administering an effective amount of Mreg-sc cells or their intracellular fraction according to a second aspect of the present invention, or (ii) administering a pharmaceutical composition according to a third aspect of the present invention.

[0096] Treatment of autoimmune diseases, inflammatory diseases, or hypersensitivity reactions can be achieved using either Mreg-sc derived from allogeneic monocytes in the patient, or autologous monocytes in patients requiring treatment, as described above in relation to transplant applications. Where possible, treatment of autoimmune diseases, inflammatory diseases, or hypersensitivity reactions is performed using autologous monocytes. For this purpose, Mreg-sc can be administered intravenously, either concurrently with or without local intramuscular injection.

[0097] In yet another embodiment, Mreg-sc cells or their intracellular fraction according to a second aspect of the present invention, or a pharmaceutical composition according to a third aspect of the present invention, are used as a vehicle for delivering gene therapy. Thus, the present invention relates to a method for delivering gene therapy, comprising (i) administration of an effective amount of Mreg-sc cells according to a second aspect of the present invention containing a transgene, or (ii) administration of a pharmaceutical composition containing Mreg-sc cells according to a second aspect of the present invention containing a transgene.

[0098] According to a fifth aspect, the present invention relates to a method for preparing an intracellular fraction of immunomodulatory macrophage cells, (a) A step of providing an immunomodulatory macrophage as described in relation to a first aspect of the present invention, (b) A step of degrading immunomodulatory macrophage cells in order to provide an intracellular fraction, (c) The present invention relates to a method comprising the step of obtaining an intracellular fraction.

[0099] The Mreg-sc cells of the present invention can be degraded according to conventional methods. For example, Mreg-sc cells can be lysed by treating them with a hypotonic solution, a surfactant, or an acid. Alternatively, the cells can be degraded by freeze-thaw or heating, sonication, irradiation, mechanical destruction, or long-term storage. In the final step of this method, intracellular fractions of the cells, such as the total protein fraction, membrane protein fraction, and cytoplasmic protein fraction, are obtained. These fractions can be used for the therapeutic purposes described above in place of live Mreg-sc cells. Alternatively, these fractions can be further purified to obtain specific proteins such as MHC proteins. The quality can be isolated.

[0100] According to a sixth aspect, the present invention relates to a method for preparing immunomodulatory T cells, (a) A step to obtain the target T cells, (b) T cells are Mreg-sc cells as defined above, i.e., Mre cells derived from suspension culture. A step of co-culturing with G cells or their intracellular fraction, (c) The present invention relates to a method comprising the step of obtaining immunomodulatory T cells from a culture medium.

[0101] As described elsewhere in this specification, T cells co-cultured with Mreg-sc inhibit T cell proliferation. Therefore, immunomodulatory T cells obtained by the above method can be used alone or in combination with the Mreg-sc cells of the present invention to treat any of the diseases or disorders discussed elsewhere in this specification. In the first step, T cells are obtained from a blood sample of interest. These cells can be obtained, for example, from a blood sample or apheresate, or from a tissue of interest, such as bone marrow or spleen. These cells can be obtained by conventional methods, for example, from blood cells. These can be obtained by venipuncture. The T cells used in the above method are, for example, CD3+ T cells or a subset thereof. Before co-culturing with Mreg-sc cells, CD3+ T cells can be purified or concentrated by conventional methods, such as magnetic microbead separation or flow cytometry sorting.

[0102] Next, T cells are brought into contact with the Mreg-sc of the present invention. These cells can be brought into contact at different Mreg:Treg ratios. For example, cell fractions can be brought into contact at an Mreg:Treg ratio of 1:5 to 5:1, preferably 1:2 to 2:1. More preferably, the Mreg:Treg ratio is about 1:1. Various culture media can be used in the co-culture method. The medium may be the media described above in relation to the method for preparing Mreg-sc cells. In a preferred embodiment, the medium is Lonza's X-vivo10. The medium contains M-CSF and / or GM-CSF, preferably human recombinant M-CSF and / or The medium may contain further additives such as GM-CSF. The amount of M-CSF and / or GM-CSF is within the range mentioned elsewhere in this specification, for example, 5 to 100 ng / ml, preferably 20 to 25 ng / ml. The medium may also contain other additives such as Glutamax in an amount of 1 to 5 mM, preferably 2 mM.

[0103] The cells are co-cultured for 1 to 8 days, preferably at least 3 days, at least 4 days, or at least 5 days. After the predetermined culture period, the T cells are converted by conventional methods, for example, CD4 + CD25 + TIGIT + FoxP3 + Treg cells can be re-isolated by enriching them. If necessary, the cells can be further formulated into pharmaceuticals. [Brief explanation of the drawing]

[0104] [Figure 1] Figure 1 shows a comparison of the expression of 23 extracellular markers in Mreg-sc and Mreg-bc cells. (A) The background normalized median fluorescence intensity of each marker is shown in a dot blot, with each dot representing one batch, i.e., a donor. (B) In the waterfall plot, the bars represent the logarithmic rate of change of channel normalized median fluorescence intensity (nMFI). nMFI was calculated at Rv3.5 by grouping assays and channels and then subtracting the FMO MFI measurements obtained within those groups. (C) Ten EC markers were expressed to a statistically significant degree in two different Mreg products. (D) A representative diagram of the gating strategy for macrophage phenotyping. For intracellular analysis, CD45 was replaced with CD33, and a fixable viability-determining dye was used for APC channels. The gating logic was the same when using 7-AAD. To assess the degree of non-target cell contamination, the gate was extended to cover small cells such as lymphocytes. [Figure 2]Figure 2 shows the results of gene expression analysis in various macrophages. (A) IDO1 mRNA expression by RT-qPCR, (B) IDO1 protein expression by flow cytometry, (C) correlation between IDO1 protein expression and detected mRNA levels, (D) DHRS9 mRNA expression by RT-qPCR. Statistical significance of the difference in expression between two products with p<0.0001 is indicated by ***, p<0.001 by **, and p<0.01 by *. In panels (A) and (D), the dashed line at Log2[Rq]=0 indicates no change in mRNA expression, values ​​above the line indicate mRNA upregulation, and values ​​below the line indicate mRNA downregulation. In panels (B) and (C), the dashed line at MFI=2 indicates the preliminary detection threshold at which IDO1 protein is expressed by macrophages at least twice as high as background fluorescence. [Figure 3] Figure 3 shows the isolation of regulatory macrophages from M1 and M2a macrophages based on DHRS9 and IDO1 mRNA expression. [Figure 4] Figure 4 depicts preliminary stability data (n=2) showing that Rotea-formulated Mreg cells maintain high viability for at least 48 hours when stored at +4°C. (A) Mreg-sc, (B) Mreg-bc, and (C) control cells heat-treated in 5% HSA plasma light for 0, 24, and 48 hours. Cell viability and quality were determined using the Annexin V Apoptosis Kit and analyzed by the MUSE® Cell Analyser. [Figure 5] Figure 5 shows that the basal secretome profiles of Mreg-bc and Mreg-sc are very similar. Of the 27 factors measured, only IP-10 was differentially secreted into the culture medium. The estimated concentration pg / mL is shown on the y-axis. [Figure 6]Figure 6 shows the results of a phagocytic assay using pHrodo Green E. coli. Mreg cells were incubated with E. coli particles for 1 hour and analyzed by flow cytometry. (A) Representative histogram of phagocytosed particles, measured as pHrodo Green fluorescence intensity in Mreg-bc. The dashed light gray plot represents a control without particles incubated at +37°C, the light gray plot represents a control with particles incubated on ice, and the dark gray plot represents a sample with particles incubated at +37°C. (B) Dot plot of the percentage of cells with incorporated labeled E. coli particles. Results are shown as mean ± SD percentage of pHrodo Green-positive cells within the live CD45-positive population (live CD45+CD3+ for T cells). [Figure 7] Figure 7 clearly demonstrates that Mreg-sc cells can inhibit T cell (CD3+) proliferation in co-culture (72 hours). Results are shown as mean ± SD from 3–5 Mreg batches (i.e., 3–5 donors). Statistical significance is indicated as *p<0.05, ***p<0.001. Addition of the IDO1 inhibitor (1mM1-methylDL-tryptophan) to the co-culture restored T cell proliferation to levels observed in unsuppressed monocytes. Legend: 1st bar = monocyte, 2nd bar = Mreg-bc, 3rd bar = Mreg-sc. [Figure 8] Figure 8 shows the results of CD72 expression in Mreg-bc and Mreg-sc macrophages, as well as M0, M1, and M2a macrophages. [Figure 9] Figure 9 shows the results of transcriptome analysis. Mreg-sc, Mreg-bc, M1, and M2a were compared with M0 macrophages. The log2 change rates of gene transcripts are shown for the genes ARMH1, CA11, SMARCD3, and HLA-DOA. ARMH1, CA11, and SMARCD3 are expressed at the same levels in M0, M1, and M2a cells. These genes are expressed more than four times higher in Mreg cells compared to M0, M1, and M2a cells (a change of more than approximately log2). HLA-DOA has three times higher expression in Mreg cells compared to other macrophages. [Figure 10] Figure 10 shows that the gene transcripts of SELENOP, RBASE1, C1QC, and NRA4A3 can be used as specific markers for Mreg-sc. In Mreg-sc, NRA4A3 is downregulated, while SELENOP, RASE1, and C1QC are upregulated.

Example

[0105] Mreg was manufactured in accordance with current GMP principles for the production of sterile pharmaceuticals. Attention was paid to protecting products, materials, and equipment from contamination and impurities in all processing steps.

[0106] Peripheral blood monocytes were isolated from leukapheresis products of healthy volunteers collected by approved blood sampling. Informed consent for the provision of apheresis was obtained in accordance with the Helsinki Declaration, which complies with the legal regulations replacing Directive 2002 / 98 / EC and 2004 / 23 / EC. Example 1: Monocyte Concentration

[0107] To concentrate monocytes, CD14+ monocytes were isolated using a GMP-compliant fully closed LP14 process of CliniMACS Prodigy (registered trademark) (Miltenyi Biotec GmbH) in accordance with the manufacturer's instructions (LP-14 System User Manual issued in March 2015). Prior to the process, a very small part of the leukapheresis product was sampled, and the total cell count and viability were determined using a NucleoCounter (registered trademark) NC-200TM automatic cell counter (ChemoMetec), and the percentages of CD14+ cells and CD3+ lymphocytes from all CD45+ white blood cells were determined by flow cytometry. Similarly, after the separation process, the purity, cell recovery rate, and viability of the target cell population were confirmed. Example 2: Mreg Differentiation

[0108] To differentiate the monocytes obtained in Example 1 into Mregs, the monocytes were divided into two different fractions. The first fraction was differentiated into Mregs by suspension culture in the Xuri (trademark) cell growth system according to the method of the present invention, thereby obtaining Mreg-sc cells. The second fraction was differentiated into Mregs in a gas-permeable bag to obtain Mreg-bc cells. The important parameters of the two processes are shown in Table 1 below. [Table 1] Table 1: Important parameters of the manufacturing process of Mreg cells. Differentiation into Mreg-bc

[0109] The monocytes were differentiated in gas-permeable MACS GMP differentiation bags (Miltenyi Biotec) as described in [6]. When the monocytes were differentiated in these bags, the Mregs became a semi-adherent and more uniform cell population than their flask-cultured counterparts. Briefly, 1.8×10 8 monocytes were seeded into a 3L differentiation bag with 180 ml (i.e., 1×10 6 cells / ml) of medium. The same medium was used for both processes (Table 1): RPMI1640 supplemented with 10% human AB serum (HABS), 2 mM GlutaMAX (Invitrogen, Germany), 100 U / ml penicillin, 100 μg / ml streptomycin (Invitrogen) and 25 ng / ml recombinant human M-CSF (R&D Systems, Germany). The bags were placed in an incubator at a humidified atmosphere of 37 °C containing 5% CO2. The next day, the bags were turned over to provide more surface for the monocytes to attach. The cell density on the bag surface was 0.27×10 6 cells / cm 2 (when counting only one side (672 cm 2 ) or 0.13×10 6 cells / cm 2 (both sides (1344 cm 2(This was the result when counting ). On day 6 (18-24 hours before collection), human recombinant interferon-gamma (IFN-γ, Merck, Germany) was added and the bag was turned inside out again. No culture medium was changed during the 7-day incubation period.

[0110] At the end of the 7-day culture period, the cells in the bag were vigorously shaken to remove them from the inside of the bag. The cells were then transferred to a centrifuge tube using a syringe (recovery 1), and the bag was then rinsed with DPBS to collect the remaining cells (recovery 2). The two recovered fractions were kept separate, and the tubes were centrifuged at 300 × g for 10 minutes at room temperature. Next, the supernatant of the first recovery was collected and stored at -80°C until Multiplex ELISA analysis. The cell pellets were resuspended in DPBS / culture medium / final formulation buffer (as required for subsequent analysis). The total cell count and viability of both fractions were determined by NC-200. Subsequently, these cells were pooled, or only one fraction of the recovered material was used for further testing. These "conventionally formulated" Mreg-bc cells were assigned to flow cytometry, phenotypic characterization by qPCR, and further functional assays (see below). The recovered "conventionally formulated" Mreg-bc cells were over 85% viable, with a maximum recovery rate of 70%. Differentiation into Mreg-sc

[0111] Monocytes were differentiated using the Xuri® cell proliferation system (GE Healthcare). This is a wave-type bioreactor, the first to be developed and validated for the proliferation of T cell-based cellular immunotherapy, and its mechanism is based on the principle that the cell culture is constantly moving. Mreg-sc cells were differentiated in a 2L Xuri Cellbag (catalog no. 29-1054-92, GE Healthcare). For bags of this size, the final usable volume range of the culture medium is 300-1000mL. The seeding density was 1.0 × 10⁶ cells in the medium shown in Table 1 above. 6 The cell count was calculated as cells / ml (the cell volume was doubled and the test was run once). The final process parameters that were ultimately established are shown in Table 2 below. More than 10 batches of Mreg-sc cells were prepared according to these parameters. [Table 2] Table 2: Parameters of the Xuri (trademark) differentiation process

[0112] During the process development phase and after the establishment of final parameters, recovered samples 1 and 2 were stored separately to determine cell viability and recovery rates in order to better understand the process and ensure that low-quality cells were not used for further analysis. Furthermore, since it was unclear whether Mreg (recovered sample 1), which quickly becomes suspended, would exhibit a different extracellular marker expression pattern than that which adheres to the bag surface and requires incubation with cold buffer with increased agitation parameters (recovered sample 2), immunophenotyping was initially performed separately on these two samples. For secretome analysis, culture medium samples were taken from recovered sample 1. Final analysis (Mreg-sc and below) was performed on the recovered material after centrifugation, resuspension, and pooling.

[0113] From a manufacturing perspective, the Xuri bioreactor solved many of the obstacles related to the production of several separate cell differentiation bags. Table 3 shows the results from bag-based and Xuri-based differentiation processes. This calculation theoretically yields 0.541–3.55 × 10¹⁶ cells from a single leukocyte apheresis. 9 This is based on the assumption that the product of each monocyte is obtained for further Mreg differentiation (data from more than 30 LPs processed by Prodigy in the inventors' laboratory), and the average monocyte yield is 0.850 × 10⁶. 9 The process recovery rate and survival rate were determined in 2019 after the final process parameters for Xuri were established. The data is calculated from batches (n=6, with over 10 batches as of today, 2020). It is noteworthy that in this comparison, the Mreg-bc figures were obtained from only one 3L differentiation bag collected by an experienced operator. Therefore, the data obtained from these “small sub-batch” Mregs do not fully represent a real “full-size clinical Mreg-bc batch.” For both Mreg types, recovery and viability were calculated from collected, centrifuged, and buffered samples. [Table 3] Table 3: Comparison of Mreg-sc process and Mreg-bc process

[0114] The Xuri® bioreactor-based method appears to have a lower Mreg cell recovery rate compared to the bag-based approach. The main advantage of the bag-based method is the amount of Mreg cells recovered. On the other hand, the Xuri® bioreactor-based method yields relatively high-quality Mreg cells. Furthermore, this process has a higher degree of automation and is therefore not dependent on the specific skills of individual operators. Being suspension-based rather than adhesion-based allows for the production of an entire batch within a single compartment, i.e., a single bag. Example 3: Preparation of comparative macrophages

[0115] In the same gas-permeable MACS GMP differentiation bag described above in relation to Mreg-bc, M1 and M2a macrophages were produced with slight modifications to the protocol described in

[11] . The same medium as for Mreg-bc was used, but instead of HABS, 5 ng / ml M-CSF and 20% fetal bovine serum (FBS, Gibco) were used to differentiate the cells for 6 days. On day 6, a complete medium change was performed, and the serum concentration was reduced to 5% FBS. Simultaneously, M1 cells were polarized with 100 ng / ml lipopolysaccharide (LPS) and 25 ng / ml IFN-γ derived from Escherichia coli (Sigma-Aldrich), while M2a cells were polarized. The cells were polarized with 20 ng / ml recombinant human IL-4 (R&D Systems). Similar to Mreg-bc, the cells were harvested on day 7. Example 4: Analysis of extracellular and intracellular markers

[0116] Intracellular staining for indoleamine 2,3-dioxygenase (IDO1) was performed. Specifically, cells were stained with a fixable live-dead dye and blocked with an FcR blocking reagent. Next, the cells were stained with CD33-PE. After incubation, the cells were fixed, permeabilized, and then blocked again with a 10% FcR blocking reagent. Finally, the cells were divided into three reaction mixtures, and intracellular antibodies were added as follows: Tube 1: IDO1-PerCP / eFluor710, Tube 2: IgG1-PerCP / eFluor710 isotype control, or Tube 3: no antibody. Analysis was performed after incubation and washing.

[0117] For extracellular markers, a minimum of 2 × 10⁶ 4 Individual viable cells (defined as CD45-positive, SYTOX Green-negative events) were analyzed using CytoFLEX S. Initial SSC / FSC gates were set to exclude the majority of debris and dead cells. Viable cells were then plotted on a histogram, each showing one phenotypic marker. Background levels were set based on CD45+ / SYTOX Green+ double-stained cells to account for background autofluorescence. Initially, the level of nonspecific mAb binding was estimated using isotype and fluorescent dye-matched nonspecific antibodies (isotype controls). For each analysis, Prior to the test, quality control (QC) was performed and passed. Furthermore, the target MFI value was set using the same assay parameters and Beckman-Coulter Daily QC Beads in an external analysis layout to verify the instrument's performance.

[0118] Next, after removing the debris, the same gating strategy as shown in Figure 1D is used to reduce the volume to a minimum of 1 × 10⁻⁶. 4IDO1 signals were analyzed by flow cytometry from individual CD33-positive, Fixable Live-Dead staining-negative events. CD33 and isotype controls for live / dead staining were used to determine background and non-specific fluorescence levels. Prior to each analysis, QC was performed on the CytoFLEX S flow cytometer and passed.

[0119] Flow cytometry data were analyzed using FCS Express6 Flow Research Edition (DeNovo Software, Glendale, CA, USA). The median fluorescence intensity (MFI) was used as the main parameter to describe the intensity of phenotypic marker expression. For extracellular markers, the MFI of the control sample was subtracted from the stained sample to obtain the background-adjusted MFI value for each sample. For IDO1, the index of specific MFI / isotype MFI was used.

[0120] The percentages of CD14+ monocytes and CD3+ lymphocytes from all CD45+ white blood cells were determined by flow cytometry from the first leukapheresis sample and then again after monocytes were enriched with Prodigy for purity checking. For phenotypic analysis, cells were stained with SYTOX Green dead cell dye according to the manufacturer's instructions. The cells were then divided into seven reaction mixtures and stained with the markers described in Table 4 below. According to the kit's instructions, cells were blocked with FcR blocking reagent (Miltenyi Biotec) in all flow cytometry experiments.

Table 4

[0121] The antibodies used for flow cytometry and intracellular staining are shown in Table 5 below. All antibodies listed in the table above, with the exception of the last three antibodies used for intracellular staining, were used for extracellular staining of CD14+ monocyte purity or for characterizing the phenotype of regulatory macrophages. [Table 5] Table 5: Antibodies used for flow cytometry and intracellular staining

[0122] Results: The results obtained from marker analysis are shown in Figure 1. Both Mreg processes consistently yielded relatively homogeneous macrophage populations, and certain clear differences can be observed between the two processes. Mreg-bc cells showed marked downregulation of several activation-related markers, including CD38, CD40, and CD80, as well as elevated levels of markers CD86 and CD71, as determined by flow cytometry. Notable factors in the Mreg-sc process were high levels of CD11c, CD14, CD16, CD51, CD163, and syndecan-3. Thus, multiple markers were observed in the two macrophages. This enables reliable differentiation between process products. Furthermore, the two processes were distinguished by differential expression of IDO1, which was significantly higher in Mreg-sc (Figure 2B). A direct correlation was observed between the degree of upregulation of IDO1 mRNA and IDO1 protein expression (Figure 2C), but a certain level of mRNA accumulation is required before IDO1 can be detected at the protein level. As a result, not all batches of Mreg-bc showed IDO1 expression at the protein level, even though mRNA was upregulated at least 50 times compared to CD14+. Therefore, the IDO1 expression level This method may allow us to distinguish between different types of macrophages, and even between Mreg-sc and Mreg-bc. Example 5: Characterization of gene expression by RT-qPCR

[0123] For RT-qPCR, 5 × 10 6The cells were resuspended in 500 μl of RNAprotect Cell reagent (Qiagen) and frozen at -20°C. Total RNA was extracted from the cells using the RNeasy Protect Cell Mini Kit (74624, Qiagen) together with a QIAshredder disposable cell lysate homogenizer (79654, Qiagen). RNA quantity was quantified by NanoDrop OD260 measurement. 4 μg of RNA from each sample was collected from the DNA according to the manufacturer's instructions. The samples were treated with se I (DNASE-50 PrimerDesign or 18068015 Invitrogen). After adjusting the sample concentration to 50 ng / μl, the results were verified by Qubit® RNA HS Assay Kit (Q32855, Invitrogen) and Qubit® Fluorometer (Invitrogen). The relative expression of DHRS9 and IDO1 mRNA was measured using TaqPath1-Step Multiplex Master Mix Kit (A28526). The expression was measured by RT-qPCR using the Applied Biosystems QuantStudio5 Real-time PCR system (Applied Biosystems) according to the manufacturer's instructions. Taqman assays, namely human IDO1 assay number Hs00984148_m1 (FAM-MGB), human DHRS9 Hs00608375_m1 (FAM-MGB), and human GAPDH Hs03929097_g1 (VIC-MGB), were purchased from Applied Biosystems. GAPDH mRNA expression was used as an endogenous control for data normalization. The RNA was used as the starting gene. Each amplification reaction contained 50 ng of RNA and was performed in triplicate. The obtained amplification data was analyzed using QuantStudio Design and Analysis desktop Software (Applied Biosystems). Changes in expression were calculated by comparing them with the expression of the corresponding mRNA in the starting material, i.e., CD14+ monocytes.

[0124] Results: DHRS9 expression was significantly increased in Mreg-bc, Mreg-sc, and M2a cells (Figure 2D). In M1 cells, DHRS9 expression was significantly lower, and some M1 batches showed downregulation of DHRS9 mRNA compared to the starting CD14+ monocyte population. Thus, the set of two genes, IDO1 and DHRS9, can help identify regulatory macrophages from M1 and M2a phenotypes. Regulatory macrophages show elevated expression of both IDO1 and DHRS9, while M1 pro-inflammatory macrophages have elevated expression of IDO1 only, and M2a anti-inflammatory macrophages express only DHRS9. (Figure 3) Example 6: Cytokine and Growth Factor Secretion Profiles

[0125] Cell culture samples were collected during recovery for secretome analysis. All samples were stored at -80°C until analysis with the Bio-Plex Pro® Human Cytokine 27plex Assay kit (Bio-Rad Laboratories, Hercules, California, USA, kit number M500KCAF0Y). This kit analyzes anti-inflammatory factors IL-1ra, IL-4, IL-10, IL-13, and pro-inflammatory factors TNF-α, IL-1β, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12, IL-15, IL-17, RANTES, eotaxin, M This allows for the detection of IP-1α, MIP-1β, MCP-1[MCAF], INF-γ, IP-10, and growth factors such as PDGFbb, VEGF, G-CSF, GM-CSF, and bFGF. Although classified previously, some of these factors are multifaceted, and therefore their function depends on the affected cell type and the microenvironment in which they are expressed.

[0126] The experimental procedure was carried out according to the manufacturer's instructions. Briefly, magnetic beads coated with capture antibody were incubated with pre-mixed standard material or sample supernatant on a shaker for 30 minutes. Then, detection antibody was added and incubated as described above. After washing, streptavidin-PE was added and incubated for 10 minutes. After washing, the beads were resuspended in assay buffer and the results were read using the Bio-Plex® 200 system. The data was analyzed using Bio-Plex Manager® software version 4.1.1. Non-irritating (basic) samples were collected from the culture medium sample at the time of recovery. We determined the secretory profiles, i.e., secretomes, of Mreg-sc and Mreg-bc cells at the basal level.

[0127] Results: Secretome profiles of Mreg-sc (n=3, generated in 2019 using final Xuri process parameters) and Mreg-bc (n=11, samples collected from batches 2018-2019) were analyzed by BioRad multiplex enzyme-coupled immunoassay. Of the 27 factors analyzed, eight distinct releases were found from Mreg-bc and Mreg-sc when Welch's two-sample method was used in R (data not shown). However, further examination and simultaneous comparison of secretions from M1 and M2a macrophages revealed that IFN-γ-inducible protein 10, IP- Only secretion of 10 (Figure 5) was found to be significantly secreted from Mreg-sc rather than from Mreg-bc

[13] . In conclusion, the basal secretion profiles of these two Mreg products appear to be very similar to each other. Example 7: Phagocytic Assay

[0128] The functional capacity of macrophages was evaluated by performing flow cytometry-based phagocytosis assays using Mreg-sc (cultured in suspension) and Mreg-bc (cultured in bags). This assay was performed using harvested macrophages and cryopreserved and thawed CD14+ monocytes and CD3+ T cells, with slight modifications to the manufacturer's protocol using the pHrodo Green Escherichiacoli (Escherichia coli) BioParticles Phagocytosis Kit for Flow Cytometry (Invitrogen, P35381 by ThermoFisher Scientific). The procedure was carried out using the added components. Briefly, 1 million macrophages / monocytes and 500,000 T cells, attached for at least 1 hour after harvesting, were incubated with pHrodo Green E. coli BioParticles Conjugate (1:20 dilution in medium) at +37°C for 1 hour, and then on ice to inhibit particle uptake. After washing, the cells were harvested and suspended in 100 μl of FACS buffer. The cells were stained with 5 μl of 7-AAD viability dye (BD Biosciences, no. 559925) and 1 μl of CD45-PE / Cy7 (Biolegend, no. 304015), and T cell samples were further stained with 1 μl of CD3-BV421 (Biolegend, no. 317344). After incubation at room temperature in the dark for 15 minutes, unbound antibody was removed by washing with 2.5 ml of FACS buffer before acquisition using a CytoFLEX S flow cytometer (Beckman Coulter, USA). Minimum 4 × 10⁶ 4 Individual viable cells (doublet-recognized CD45 positive, 7-AAD negative) were obtained. Flow cytometry data were analyzed using FlowJo software (TreeStar, USA). Phagocytosis was compared with that observed in the control sample. This was determined by an increase in (trademark) Green fluorescence, incubated on ice, and calculated as the frequency (positive percentage) of pHrodo(trademark) Green-positive cells that phagocytosed particles.

[0129] Results: The results are shown in Figure 6. Flow cytometry analysis showed that both Mreg types (and monocytes) actively phagocytosed E. coli (Figure 6A). Detection of phagocytic events was confirmed by a negative control containing target particles and incubated on ice, which inhibited phagocytosis (Figure 6A). After 1 hour, 85±4% of Mreg-sc macrophages and 75±7% of Mreg-bc macrophages (83±4% monocytes) had ingested particles (Figure 6B). Example 8: T cell suppression assay

[0130] IDO1 expression was identified as the primary cause of the Mreg-mediated inhibitory effect on T cell proliferation, likely due to induced tryptophan depletion.[6]

[11] As described above, Mreg-sc expresses higher levels of indoleamine IDO1 than Mreg-bc, both at the mRNA and protein levels. Therefore, we investigated whether Mreg-sc exerts stronger Mreg-mediated inhibition of T cell proliferation. The inhibition test was performed according to a protocol with minor modifications to the one described in

[11] . One day prior to Mreg harvesting, CD3+ T cells from allogeneic donors were converted to C14 cells according to the manufacturer's protocol. CytoTell®-labeled T cells were fluorescently labeled with CytoTell Green (22253, AAT Bioquest). CytoTell-labeled T cells and unlabeled control T cells were activated using MACSbeads (1 bead per 2 cells, 130-091-441, Miltenyi Biotec). The cells were cultured overnight (+37°C, 5% CO2) in RPMI growth medium supplemented with 5% HABS, 1% Glutamax, 100 IU / ml penicillin, and 100 μg / ml streptomycin, together with unactivated control CytoTell®-labeled T cells. The following day, freshly harvested macrophages and thawed cryopreserved monocytes were plated into the wells of a 48-well plate in the above medium. After allowing the cells to adhere for 2 hours (+37°C, 5% CO2), T cells were added. The total amount of seeded monocytes / macrophages was 6.25 × 10⁶ per well. 4 ~3.75×10 5 Since it varied in cells, 1.25 × 10 5When individual T cells were added to the wells, the resulting co-cultures had effector cell ratios of 3:1, 2:1, 1:1, and 1:2. To investigate the role of IDO1 in Mreg-mediated suppression of T cell proliferation, an IDO1 inhibitor (1-methyl DL-tryptophan, 860646, Sigma Aldrich) was added to individually specified 3:1 co-cultures. The following procedure was performed: After 72 hours in co-culture (+37°C, 5% CO2), non-adherent cells (mainly T cells) were harvested and identified by labeling with anti-CD3-APC (300312, BioLegend). 7-AAD (559925, BD Biosciences) exclusion was used to distinguish between viable and dead cells. The degree of T cell proliferation was evaluated by flow cytometry using CytoFLEX S. Flow cytometry data was exported as an FCS3.0 file, and proliferation analysis was performed using FCS Express6 Flow Research Edition. The process was run on n to calculate the growth index (PI) of the live T cell population (CD3-positive cells, 7-AAD-negative). The growth index in FCS expression represents the growth rate during culture (ratio of final cell number to starting cell number) and is calculated according to the following formula.

number

[0131] Results: No significant difference in T cell proliferation was observed in 1:2 and 1:1 co-cultures with Mreg-sc, Mreg-bc, or monocytes. However, at higher cell ratios, 2:1 and 3:1, T cells co-cultured with both Mreg-sc and Mreg-bc proliferated to a lower degree than T cells cultured alone (data not shown) or T cells co-cultured with monocytes (Figure 7). The observed suppression of T cell proliferation was exacerbated with Mreg-sc. Addition of an IDO1 inhibitor (1 mM 1-methylDL-tryptophan) to the 3:1 co-culture reduced the inhibitory effect, and T cell proliferation recovered to the level observed in unsuppressed monocytes. This confirmed that the inhibitory effect on T cell proliferation is related to the IDO1 expression level by regulatory macrophages, rather than to nonspecific nutrient restriction in co-culture. literature [1]Hutchinson JA, Riquelme P, Sawitzki B, et al.Cutting edge:immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients.J Immunol 2011;187:2072-8. [2]Hutchinson JA, Riquelme P, Brem-Exner BG, et al. Tranplant acceptance-inducing cells as an immune-conditioning therapy in renal transplantation. Transpl Int 2008;21:728-41. [3]Hutchinson JA, Brem-Exner BG, Riquelme P, et al.A cell-based approach to the minimization of immunosuppression in renal transplantation.Transpl Int 2008;21:742 -54. [4]Hutchinson JA, Roelen D, Riquelme P, et al.Preoperative treatment of a pre-sensitized kidney transplant recipient with donor-derived transplant acceptance-inducing cells.Transpl Int 2008;21:808-13. [5]Hutchinson JA,Govert F,Riquelme P,et al.Administration of donor-derived transplant acceptance-inducing cells to the recipients of renal transplants from deceased donors is technically feasible.Clin Transplant 2009;23:140-5. [6]Hutchinson JA,Ahrens N,Geissler EK.MITAP-compliant characterization of human regulatory macrophages.Transplant International 2017;30(8),765-775. [7]Hummitzsch L,Zitta K,Rusch R,et al.Characterization of the Angiogenic Potential of Human Regulatory Macrophages(Mreg)after Ischemia / Reperfusion Injury In Vitro.Stem Cells International 2019;Jun 25. [8]de Araujo EF,Medeiros DH,Galdino NA et al.Tolerogenic Plasmacytoid Dendritic Cells Control Paracoccidioides brasiliensis Infection by Inducting Regulatory T Cells in an IDO-Dependent Manner.PLoS Pathogens 2016;12(12),1-29. [9]Yun TJ,Lee,JS,Machmach K,et al.Indoleamine 2,3-Dioxygenase-Expressing Aortic Plasmacytoid Dendritic Cells Protect against Atherosclerosis by Induction of Regulatory T Cells.Cell Metabolism 2016;23(5),852-866.

[10] Zhao Y,Wu T,Shao S,et al.Phenotype,development,and biological function of myeloid-derived suppressor cells.Oncoimmunology 2016;5(2),e1004983

[11] Hutchinson JA,Riquelme P,Geissler,EK,Fuandrich,F.Human regulatory macrophages.Methods Mol Biol 2011;677,181-192.

[12] Munn DH,Shafizadeh E,Attwood JT,et al.Inhibition of T Cell Proliferation by Macrophage Tryptophan Catabolism.The Journal of Experimental Medicine 1999;189(9),1363-1372.

[13] Shanmugam N,Reddy MA,Guha M,et al.High glucose-induced expression of proinflammatory cytokine and chemokine genes in monocytic cells.Diabetes 2003;52(5),1256-1264

Claims

1. A method for preparing immunomodulatory macrophage cells, (a) A step of isolating CD14-positive monocytes from the target blood sample, (b) A step of culturing the monocytes in a medium containing (i) M-CSF and / or GM-CSF, and (ii) CD16 ligand, (c) A step of bringing the monocyte or monocyte-derived cells into contact with IFN-γ, (d) a step of obtaining the immunomodulatory macrophage cells from the culture medium, A method wherein steps (b) and (c) are performed in the container, which is stirred in such a way that the cells do not adhere to the surface of the container.

2. The method according to claim 1, wherein step (d) does not involve the mechanical removal of cells from the surface of the container.

3. The method according to claim 1 or 2, wherein steps (b) and (c) are performed such that 10% or less, preferably 5% or less of the cells adhere to the surface of the container.

4. The method according to any one of claims 1 to 3, wherein the culture medium in step (b) comprises human serum, for example, human AB serum.

5. The method according to any one of claims 1 to 4, wherein the concentration of M-CSF and / or GM-CSF in step (b) is in the range of 5 to 100 ng / ml, preferably in the range of 20 to 25 ng / ml.

6. The method according to any one of claims 1 to 5, wherein the monocytes of step (b) are cultured for at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days before being brought into contact with IFN-γ.

7. The method according to any one of claims 1 to 6, wherein the container is made of plastic, preferably ethylene vinyl alcohol (EVOH), ethylene vinyl acetate copolymer (EVA), or polyolefin.

8. The method according to any one of claims 1 to 7, wherein the concentration of IFN-γ in step (c) is in the range of 5 to 100 ng / ml, preferably in the range of 20 to 25 ng / ml.

9. Immunomodulatory macrophage cells that can be obtained by the methods described in claims 1 to 8.

10. Immunomodulatory macrophage cells expressing the following markers: CD16, CD163, and syndecan-3.

11. The immunomodulatory macrophage cells according to claim 10, wherein the cells further express at least one of the following markers: CD51, CD11c, CD72, and IDO1.

12. A pharmaceutical composition comprising immunomodulatory macrophage cells or intracellular fraction thereof according to any one of claims 9 to 11.

13. The pharmaceutical composition according to claim 12, wherein at least 70%, preferably at least 80%, and more preferably at least 90% of the cells in the composition are immunomodulatory macrophage cells according to any one of claims 9 to 11.

14. An immunomodulatory macrophage cell or intracellular fraction thereof according to any one of claims 9 to 11, or a pharmaceutical composition according to any one of claims 12 to 13, for use in a method for suppressing transplant rejection and / or extending transplant survival in a subject undergoing transplantation.

15. Immunomodulatory macrophage cells or their intracellular fractions, or pharmaceutical compositions, for use in the method according to claim 14, wherein the transplantation is an allogeneic transplant.

16. An immunomodulatory macrophage cell or intracellular fraction thereof according to any one of claims 9 to 11, or a pharmaceutical composition according to any one of claims 12 to 13, for use in a method for promoting or maintaining the engraftment or effect of a regulatory T cell-based pharmaceutical.

17. An immunomodulatory macrophage cell or intracellular fraction thereof according to any one of claims 9 to 11, or a pharmaceutical composition according to any one of claims 12 to 13, for use in a method of treating or preventing an autoimmune disease, an inflammatory disease, or a hypersensitivity reaction.

18. The aforementioned autoimmune diseases include systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, polymyositis, dermatomyositis, and other systemic autoimmune conditions; rheumatoid arthritis (RA), juvenile rheumatoid arthritis, and other inflammatory arthritis; ulcerative colitis, Crohn's disease, and other inflammatory bowel diseases; autoimmune hepatitis, primary biliary cirrhosis, and other autoimmune liver diseases; cutaneous vasculitis of small vessels, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, Behçet's disease, thromboangiitis obliterans, Kawasaki disease, and other large, medium, or small vessel vasculitis of autoimmune etiologies; multiple sclerosis (MS) and neuroimmune disorders; type 1 diabetes mellitus, autoimmune thyroid dysfunction, autoimmune Immunomodulatory macrophage cells or intracellular fractions thereof, or pharmaceutical compositions for use in the method of claim 17, selected from the group consisting of pituitary dysfunction and other autoimmune endocrine disorders; hemolytic anemia, thrombocytopenic purpura, and other autoimmune disorders of the blood and bone marrow; psoriasis, pemphigus vulgaris, bullous pemphigoid, and other autoimmune skin diseases.

19. Immunomodulatory macrophage cells or intracellular fractions thereof, or pharmaceutical compositions for use in the method of claim 17, selected from the group consisting of arterial occlusive diseases, such as peripheral arterial occlusive disease (pAOD), severe limb ischemia, arteriosclerosis, cerebral infarction, myocardial infarction, renal infarction, intestinal infarction, angina pectoris, and other conditions caused by arterial occlusion or stenosis; microvascular angina, also known as cardiac syndrome X; inflammation systemically associated with metabolic disorders, including type II diabetes mellitus and obesity-associated metabolic syndromes; and skin diseases, including eczema.

20. An immunomodulatory macrophage cell or intracellular fraction thereof, or a pharmaceutical composition, for use in the method of claim 17, wherein the hypersensitivity reaction is selected from the group consisting of asthma, eczema, allergic rhinitis, angioedema, drug hypersensitivity, and mastocytosis.

21. An immunomodulatory macrophage cell or intracellular fraction thereof according to any one of claims 9 to 11, or a pharmaceutical composition according to any one of claims 12 to 13, for use in a method of promoting a tissue repair process by being involved in tissue remodeling, tissue regeneration, angiogenesis, vascularization, or prevention / limitation of fibrosis.

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