Novel method

Abstract

To provide an ex vivo method for obtaining tolerogenic antigen-presenting cells (APCs) having the ability to induce tolerance to an antigen in the immune system.SOLUTION: The method comprises (a) isolating monocytes from a sample obtained from the mammal; and (b) culturing the isolated monocytes in a cell culture to induce differentiation of the monocytes into antigen-presenting cells having a tolerogenic phenotype, wherein the cell culture comprises (I) retinoic acid and TGFbeta, (ii) retinoic acid, TGFbeta and an AhR agonist, or (iii) retinoic acid and an AhR agonist.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to, inter alia, tolerogenic antigen-presenting cells capable of tolerizing to an antigen. an ex vivo method for obtaining anti-inflammatory cells (APCs); and tolerogenic APCs obtained by the method of the present invention. Tolerogenic APCs themselves, their uses, and unwanted immune reactions to antigens The present invention relates to methods for treating immune rejection of allogeneic (allo)grafts and methods for preventing immune rejection of allogeneic (allo)grafts. [Background technology]

[0002] BACKGROUND OF THE INVENTION Tolerogenic antigen-presenting cells (APCs) exploit the mechanisms of antigen presentation in a tolerogenic manner Based immunotherapy is used to treat immune disorders or to prevent rejection of transplants, e.g., allografts. They represent a promising non-toxic method for preventing HIV infection. They can be used as monotherapy or in combination with immunosuppression. Can be used as an add-on to other types of therapy, such as in combination with drugs or other immunomodulatory therapies This plan aims to generate cell therapy to circumvent immune disorders with the goal of inducing antigen-specific tolerance. Therefore, such APC-based immunotherapy is based on the ex vivo manipulation and transfer of APCs. The ultimate goal of the method is to deliver inhibitory signals to effector cells in vivo and induce regulatory T cells ( The goal is to induce tolerance by inducing and expanding Tregs. For example, type 1 diabetes, arthritis, Patients with autoimmune diseases such as rheumatoid arthritis and multiple sclerosis require such tolerogenic may benefit from treatment based on APC-based therapy.

[0003] Induction of antigen-specific immune responses requires the use of major histocompatibility complex (MHC) molecules and membrane-bound co-interacting antigens. It requires the involvement of professional APCs that express stimulatory and secreted inflammatory molecules. Furthermore, such APCs take up antigens, process them, and present them in association with MHC molecules. It must be possible to do so.

[0004] Similarly, the induction of antigen-specific immune tolerance also requires the presentation of the antigen in the context of the MHC. However, unlike initiating an immune response, tolerance induction involves the production of tolerogenic molecules and secretions. High cell surface expression of anti-inflammatory mediators, along with membrane-bound costimulatory molecules and secretory inflammatory mediators Requires low expression of the sex molecule.

[0005] The main types of professional APCs in the immune system are dendritic cells (DCs), macrophages, and specific At the immature stage, APCs are derived from the endothelial cells by phagocytosis or pinocytosis. They take up extracellular antigens and transport them to endocytic sites such as endosomes and phagosomes. In the cis compartment, the peptides are processed into peptides, which are then bound to the MHC class. They are also bound to exogenous IL-1 molecules, a process called "cross-presentation." They have the unique ability to load peptides from proteins into the MHC class I presentation pathway. With appropriate differentiation signals, APCs can develop into tolerogenic or non-tolerogenic APCs. Tolerogenic APCs can mediate downregulation or prevention of immune responses, maintaining peripheral tolerance. is thought to play an important role in

[0006] Hemophilia A (HA) is a condition characterized by a factor V gene that interferes with the expression of translated proteins or procoagulant function. X staining caused by various mutations in the F8 gene encoding factor III (FVIII) FVIII is primarily expressed in the liver and endothelial vascular beds. Patients lack sufficient procoagulant activity and are therefore at increased risk of complications, including increased morbidity and mortality. The FVIII database currently lists 5,4 2,015 unique FVIII variants have been identified based on case reports from 72 individuals. The proportion of point mutations (66.5%), deletions (23.2%) and others (duplications, polymorphisms, insertions, indels and complexes) was high. A large array of FVIII treatments results in a variety of clinical outcomes. They can be treated acutely (on demand) or prophylactically.

[0007] The immune system is not fully tolerant to the specific sequence of normal FVIII, A significant number of patients develop so-called "inhibitors" or anti-drug antibodies (ADAs) that block the activity of administered FVIII. The production of inhibitors is currently seen in HA patients. It is the most serious and significant complication of treatment.

[0008] Currently, once inhibitors have formed, the only proven method of eradication is frequent high-dose FVIII Infusion-mediated immune tolerance induction (ITI), however, this treatment regimen fails in 20–40% of patients.

[0009] The problem of ADA is not limited to hemophiliacs who develop defective FVIII. ADA can affect other living organisms. ADA production can occur in subjects treated with anti-cancer drugs. ADA production results from a lack of central T cell tolerance. Tolerance to self-proteins is an important part of the development of the immune system. proteins encountered later in life are usually expressed differently depending on the context in which they are presented. are recognized as foreign and lead to the development of an antibody response.

[0010] The first step in the immune response to FVIII is thought to be the uptake of FVIII by APCs. Following endocytosis of FVIII by APCs, FVIII is processed into small peptides. The peptide is then loaded onto an MHC class II molecule and the resulting MHC class II-peptide complex is then formed. However, FVIII-specific CD4 + These T cells are transported to the cell surface for presentation to T cells. Activation requires additional activation signals provided by APCs. The activating signals are mediated by costimulatory molecules such as CD40, CD80, and CD86 on the plasma membrane of APCs and by T cells, for example. The membrane-associated interactions between CD28, CD154, and CTLA-4 on these receptor / ligand complexes are In addition to interactions, APCs also mediate T cell responses via secreted cytokines such as IL-12 or IL-10. The combination of signals determines the direction of differentiation of activated T cells. T helper 1 (Th1) cells generally induce cytotoxic immune responses, while Th2 cells induce B cell responses. Tregs induce cell-mediated antibody responses, while Tregs suppress activated B and T cells, acting in tandem. It can induce immune suppression / tolerance through activation and other mechanisms. The activated FVIII-specific T cells activate FVIII-specific B cells, leading to the release of immunoglobulins in the B cells. It can induce affinity maturation and class switching of the antibody gene, resulting in the development of anti-FV antibodies. III antibody-secreting plasma cells and circulating FVIII-specific memory B cells are generated, which reactivate FVIII When exposed, they produce antibodies.

[0011] Autoimmune diseases occur when a specific adaptive immune response is mistakenly initiated against self-antigens. As a result, immune effector pathways induce chronic inflammatory injury in tissues, which Autoimmunity is driven by the activation of autoantigen-specific T cells and the production of autoantibodies. Specific genes found within the MHC and other immune regulatory loci may Individuals who develop autoimmune diseases due to their ability to regulate adaptive T cell and B cell immune responses T cell responses to self-antigens play an important role in determining the susceptibility of cells to autoantigens. Cytotoxic T cell responses, inappropriate activation of other effector cells, and inappropriate activation of B cells They can cause tissue damage with the help of T cells. An immune system that ceases to recognize “self” can lead to the production of pathological autoantibodies. Autoantibodies that recognize the antigen as a foreign substance initiate further immune responses, leading to increased T cell infiltration, inflammation, and These actions of T and B cells result in the production of proinflammatory cytokines and widespread tissue damage. Diseases in which this may be important include rheumatoid arthritis, type 1 diabetes mellitus, and multiple sclerosis. Included.

[0012] A graft from a genetically unrelated donor of the same species is called an allograft. It is considered the last resort for the treatment of chronic organ failure. Even with the help of a transplant, the main complication after transplantation is rejection. Despite this analysis, rejection occurs to varying degrees in almost all transplant recipients. Presence of pre-existing antibodies (due to pregnancy, blood transfusion and / or previous transplant) Apart from hyperacute rejection caused by graft rejection, transplant rejection can be broadly divided into two types: acute and chronic. Acute rejection is considered to be solely an immunological reaction, while chronic rejection is Sexual rejection involves both immunological and non-immunological mechanisms.

[0013] Allo-recognition is the presentation of graft antigens (alloantigens) and has two main modes: direct and indirect. DCs and other professional APCs that migrate from the graft directly initiates non-self recognition, where recipient T cells are directly stimulated by allogeneic MHC-bound peptides The recipient APCs then transfer the donor-derived graft or dying cells to the recipient. They pick up alloantigens shed from the cells and convert the processed alloantigens into self-MHC. They bind to and present to recipient T cells (indirect allo-recognition). Through a process called gocytosis, recipient APCs deliver ATP to their cell surface. A third subtype, semi-direct non-self recognition, involving passive acquisition of the inner MHC has also been proposed. Naive CD4 + T helper cells are among the first immune cells to be activated after transplantation. Activated naive CD4 + T Hell Patient cells develop into either Th1 (pro-inflammatory) or Th2 (anti-inflammatory) subtypes. The types modulate characteristic immune response profiles (each is mutually inhibitory). In the presence of transforming growth factor beta (TGF beta) and IL-6, naive CD4 + T Helper cells can differentiate into Th17 cells, a subset of Th cells that secrete IL-17. is further stimulated by IL-23.

[0014] Ex vivo generation of APCs with appropriate immune tolerogenic functions is essential for the prevention of anti-drug responses, autoimmune diseases, and other conditions. It can be performed as a therapeutic treatment for the prevention of adverse immune responses and for the induction of transplant tolerance. The suppression of CD4 + and CD8 + This involves the induction of tolerance in both ex vivo and T cells. Tolerogenic APCs generated in vivo enhance anti-drug responses and are useful for the treatment of autoimmune diseases and should have the same properties for the prevention of transplant rejection.

[0015] Retinoic acid, a vitamin A metabolite, plays a key role in cell proliferation, differentiation, organogenesis, and regeneration. Retinoic acid plays an important role in the mucosal immune response. 3 + Enhances differentiation of inducible and IL-10-producing Treg cells and promotes gut-homing specificity in T cells It has been reported that it induces oxidative stress (Bakdash et al., 2015). In addition, its regulatory activity Retinoic acid plays an important role in regulating inflammatory diseases not only in the intestine but also in other tissues. It has been reported that this is a significant benefit (Oliveira et al., 2018).

[0016] Transforming growth factor beta (TGF beta) is a growth factor found in vertebrate and invertebrate organisms. It is a pleiotropic cytokine present in the brain and plays a role in many physiological and pathological processes. TGF-beta affects all cells of the immune system and is one of three known TGF-beta isoforms. Among the forms, TGF-beta 1 is the predominant isoform expressed in immune cells. Data 1 regulates all immune cell functions, especially T cell development and induction of immune tolerance in DCs. DCs secrete TGF-beta, which plays a crucial role in the It regulates immune function, including immunosuppression, by promoting immune function through the activation of ATP (Esebanmen et al., 2017).

[0017] The aryl hydrocarbon receptor (AhR) is activated by several exogenous and endogenous ligands. Among its several physiological effects, AhR promotes immunomodulatory functions. It contributes to immune homeostasis by promoting the 6-formylindrolase activity during DC differentiation and maturation. (3,2b) Activation of the AhR via agonist ligands such as carbazole (FICZ) induces the activation of the enzyme IDO. leading to increased expression and decreased production of inflammatory cytokines such as IL-6 and TNF-alpha One study showed that FICZ-treated DCs stimulated the CD4 + CD25 高 Foxp3 + This indicates that the differentiation of human DCs into Treg cells was induced, and the activation of AhR in human DCs was It has been demonstrated that inactivation promotes a tolerogenic phenotype ( Jurado-Manzano et al., 2017 ). .

[0018] VIII against autoantigens in autoimmune diseases and alloantigens in transplanted grafts -tolerogenic expression useful for treating undesired immune responses to biological agents such as steroids - Patents.com There is a need to provide alternative methods for the generation of tolerogenic APCs with the required type. Summary of the Invention [Problem to be solved by the invention]

[0019] (Summary of the Invention) The inventors of the present application have surprisingly found that the combination of retinoic acid, TGF-beta and AhR agonists A specific cell culture method involving the use of a specific combination has been identified, which advantageously involves the use of monocytic starting cells. This allows for the generation of tolerogenic APCs with unique profiles derived from cell populations. [Means for solving the problem]

[0020] The present inventors have demonstrated that monocytes can be cultured in cell cultures containing specific combinations of components. We have discovered that tolerogenic APCs can be generated by a method comprising:

[0021] Advantageously, the tolerogenic APCs obtained by the method of the present invention are capable of inhibiting the response of a mammalian subject to an antigen. Targeted immunotherapy that has or is at risk of having an unwanted immune response to The antigen may be, for example, a biologic or an autoantigen. Furthermore, the tolerogenic APCs obtained by the method of the present invention are capable of inhibiting immune responses in recipient subjects. It is expected that this technology can be used to prevent immune rejection of transplanted allografts.

[0022] Therefore, in a first aspect of the present invention, an immunotolerant antibody capable of inducing tolerance to an antigen is provided. 1. An ex vivo method for obtaining enterogenic APCs, comprising: (a) isolating monocytes from a sample obtained from a mammal; and (b) Cultivating the isolated monocytes in cell culture to differentiate them into APCs with a tolerogenic phenotype. Inducing differentiation, the cell culture comprising: (i) retinoic acid and TGF-beta; (ii) retinoic acid, TGF-beta; and an AhR agonist, or (iii) retinoic acid and an AhR agonist. It is served.

[0023] In another aspect of the present invention, there is provided a method for producing an immunotolerance antigen obtainable or obtained by the method of the present invention. A sexual APC or population thereof is provided.

[0024] In yet another aspect of the invention, the cells or population are resistant to stimulation, either unstimulated or stimulated with an immunizing agent such as LPS. Upon stimulation with an immunogenic stimulus, they express CD103, with high expression of CD141, GARP, and ILT3, and low expression of CD Tolerogenic APCs or populations thereof having CD83 and CD86 are provided.

[0025] In a further aspect of the invention, a mammal having or at risk of having an immune response to an antigen is Tolerogenic APCs or populations thereof according to the present invention for use in methods of treating mammalian subjects administering to a mammalian subject tolerogenic APCs, thereby increasing immune tolerance to the antigen. The method further comprises establishing a volume.

[0026] In yet a further aspect of the invention, a subject having or at risk of having an immune response to an antigen is 1. A method of treating a mammalian subject comprising: (i) an isolated antibody derived from a mammalian subject, which has the ability to induce tolerance to an antigen according to the present invention; obtaining a tolerogenic APC or a population thereof obtained from the sample of monocytes; (ii) administering the tolerogenic APCs or population thereof back to the mammalian subject to detect the antigen Establishing immune tolerance to The present invention provides a tolerogenic APC or population thereof for use in a method comprising:

[0027] In another aspect of the present invention, immune rejection of a donor-derived allograft is inhibited by a method for treating a recipient's immune system. A method for preventing leukemia in an animal, comprising: The resulting tolerogenic APCs are administered to the recipient subject, thereby infecting the allograft. - Tolerogenicity according to the invention for use in a method comprising establishing tolerance to a APCs or populations thereof are provided.

[0028] In yet another aspect of the present invention, immune rejection of a donor-derived allograft is induced by a recipient A method for preventing a disease in a recipient subject, comprising: and administering to the recipient subject tolerogenic APCs obtained from the monocytes, thereby and (c) establishing tolerance to said allograft. Tolerogenic APCs or populations thereof are provided. [Brief explanation of the drawings]

[0029] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows the expression of the maturation marker CD83 on DCs cultured under various conditions (Figures (A) and (B)). DCs were either unstimulated (gray bars) or stimulated with lipopolysaccharide (LPS) (black bars) (see Example 1). [Figure 2] Figure 2 shows the expression of the activation marker CD86 on DCs cultured under various conditions (Fig. (A) and (B)). DCs were unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 2). [Figure 3] Figure 3 shows the expression of the tolerogenic marker ILT3 on DCs cultured under various conditions (Figures (A) and (B)). DCs were unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 3). [Figure 4] Figure 4 shows the tolerogenic index, defined as the ratio of ILT3 / CD86 expression (MFI) on DCs cultured under various conditions: DCs were unstimulated (Figure (A)) or stimulated with LPS (Figure (B)) (see Example 4). [Figure 5]Figure 5 shows the ability of DCs to stimulate CD4+ T cell proliferation in a mixed lymphocyte reaction (MLR). DCs were either unstimulated (gray bars) or stimulated with either LPS (Figure (A)) or a proinflammatory cytokine cocktail (Figure (B)) before co-culture with T cells (black bars) (see Example 5). [Figure 6] Figure 6 shows the frequency of CD141 and GARP co-expressing cells among DCs cultured under various conditions (Figures (A) and (B)). DCs were unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 6). [Figure 7] Figure 7 shows the production of IL-23 by DCs cultured under various conditions (Figures (A) and (B)). DCs were unstimulated (gray bars) or stimulated (black bars) with either LPS (Figure (A)) or a proinflammatory cytokine cocktail (Figure (B)) (see Example 7). [Figure 8] Figure 8 shows the expression of the marker CD103 on DCs cultured under various conditions (Figures (A) and (B)). DCs were either unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 8). [Figure 9] Figure 9 shows the expression levels of the tolerogenic markers MERTK (Panel A), BTLA (Panel B), LAP (Panel C), HLA-G (Panel D), and CD49b (Panel E) on DCs cultured under various conditions (see Example 9). [Figure 10] Figure 10 shows the ability of DCs to induce CD4+CD25highFoxp3+ Tregs in an extended MLR (Figures (A) and (B)). DCs were unstimulated (gray bars) or stimulated with LPS (black bars) before co-culture with T cells (see Example 10). [Figure 11]Figure 11 shows the expression of maturation marker CD83 (Figure A), activation marker CD86 (Figure B), tolerogenic marker ILT3 (Figure C), tolerogenic index defined as the ILT3 / CD86 expression (MFI) ratio (Figure D), frequency of CD141 and GARP co-expressing cells (Figure E), expression of the marker CD103 (Figure F), expression level of the tolerogenic marker LAP (Figure G), IL-23 production (Figure H), T cell proliferation (Figure I), and induction of CD4+CD49b+LAG3+Tr1 cells (Figure J) when DCs were generated from CD14+ monocytes isolated from the blood of healthy donors (black bars) or hemophilia patients (striped bars) and cultured in the presence of RA, TGFb, and AhR agonist (see Example 11). [Figure 12] 12 shows the viability (Fig. A), expression of the maturation marker CD83 (Fig. B), expression of the tolerogenic marker ILT3 (Fig. C), expression of the activation marker CD86 (Fig. D), the tolerogenic index defined as the ratio of ILT3 / CD86 expression (MFI) (Fig. E), expression level of the tolerogenic marker LAP (Fig. F), and expression of the marker CD103 (Fig. G) when DCs were cultured in the presence of RA, TGFb, and an AhR agonist and then frozen / thawed (dark gray bars). The phenotype of the frozen / thawed cells was compared to that of fresh cells (light gray bars) (see Example 12). [Figure 13] Figure 13 shows the expression of maturation marker CD83 (Fig. A), activation marker CD86 (Fig. B), tolerogenic marker ILT3 (Fig. C), tolerogenic index defined as the ILT3 / CD86 expression (MFI) ratio (Fig. D), expression of marker CD103 (Fig. E), frequency of CD141 and GARP co-expressing cells (Fig. F), T cell proliferation (Fig. G), and generation of CD4+CD25highFoxp3+ Treg cells (Fig. H) when DCs were cultured under various conditions. DCs were unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 13). [Figure 14] Figure 14 shows T cell proliferation when DCs cultured in the presence of RA + TGFb + AhR agonist were loaded with tetanus toxoid and subsequently cultured in the presence of T cells. DCs were either unloaded (black bars) or loaded with TT (gray bars) (see Example 14). [Figure 15] Figure 15 shows the expression of maturation marker CD83 (Figure A), the expression level of the tolerogenic marker LAP (Figure B), the expression of the marker CD103 (Figure C), the expression of the tolerogenic marker ILT3 (Figure D), the expression of the activation marker CD86 (Figure E), and the tolerogenic index (defined as the ILT3 / CD86 expression (MFI) ratio) of DCs cultured in the presence of RA, TGFb, and AhR agonist and subsequently stimulated with CD40L (black bars) or not (gray bars). Figure G shows the induction of T cell proliferation by unstimulated and CD40L-stimulated RA, TGFb, and AhR agonist-treated DCs (see Example 15). [Figure 16] Figure 16 shows antigen loading of DCs cultured in the presence of RA + TGFb + AhR agonist and antigen (Figures A-B), expression of CD11c after antigen loading (Figure C), expression of maturation marker CD83 after antigen loading (Figure D), expression of activation marker CD86 after antigen loading (Figure E), tolerogenic marker ILT3 after antigen loading (Figure F), expression of marker CD103 after antigen loading (Figure G), expression of tolerogenic marker GARP after antigen loading (Figure H), and expression level of tolerogenic marker LAP after antigen loading (Figure I) (see Example 16). [Figure 17] Figure 17 shows the induction of B regulatory cells in cultures containing RA+TGF beta+AhR agonist-treated DCs, B cells, and T cells (Figure A), the ability of RA+TGF beta+AhR agonist-treated DCs, B cells, and T cells to stimulate T cell proliferation (Figure B), and the ability of RA+TGF beta+AhR agonist-treated DCs, B cells, and T cells to induce T cell activation (Figure C). [Figure 18] Figure 18 shows the ability of RA+TGFbeta+AhR agonist-treated DCs to induce T cell proliferation when co-cultured with allogeneic PBMCs (Figure A) and the ability of RA+TGFbeta+AhR agonist-treated DCs to induce Tregs in a mixed lymphocyte reaction (MLR) with allogeneic PBMCs (Figure B). DETAILED DESCRIPTION OF THE INVENTION

[0030] (Detailed Description of the Invention) The different applications of the disclosed products and methods are tailored to meet specific needs in the art. It is understood that the terms used herein may be adjusted to suit the particular implementation of the present invention. It is for illustrative purposes only and is not intended to be limiting. Additionally, as used in this specification and the appended claims, the singular "a," "an," "the ... The terms "a," "an," and "the" refer to plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen" includes two or more such antigens. .

[0031] definition "Monocytes" are large mononuclear phagocytes of peripheral blood. Monocytes are typically 10-30 μm in diameter. The nucleus-to-cytoplasm ratio is typically in the range of 2:1 to 1:1. It is often band-shaped (horseshoe-shaped), or kidney-shaped (kidney-shaped). The nucleoli are not visible, and the chromatin pattern is finely The cytoplasm is abundant and contains many fine azurophilic chromosomes. It appears blue-gray with soluble granules and gives a ground-glass appearance when stained with Giemsa. Expression of specific surface markers is often used to determine whether the cells are monocytes or not. For example, monocytes express CD14 (a monocyte marker), CD1c (a DC marker), CD56 (a N K cell marker), CD19 (B cell marker), CD3 (T cell marker), CD16b (neutrophil marker) or do not express CD66b (a neutrophil marker).

[0032] "Antigen-presenting cells" (APCs) process antigens for recognition by specific lymphocytes such as T cells. Heterogeneous groups of immune cells mediate cellular immune responses by sequestering and presenting APCs present antigens complexed with MHC on their surface. T cells then bind to their T These complexes can be recognized using a TCR. Classical APCs include DCs, macrophages, and These include phage, Langerhans cells and B cells.

[0033] "Dendritic cells" (DCs) are APCs that exist in vivo, in vitro, ex vivo, or DCs are APCs that can be derived from hematopoietic stem cells, hematopoietic progenitor cells, or monocytes. DCs and their precursors are Isolation from various lymphoid organs, such as the spleen, lymph nodes, as well as from bone marrow and peripheral blood DCs have thin sheets (lamellipodia) that extend in multiple directions away from the DC body. DCs have a characteristic morphology that constitutively express both MHC class I and class II molecules. CD8 respectively + and CD4 + It presents peptide antigens to T cells. In addition, it is also found in human skin and mucous membranes. DCs also express the CD1 gene family, an MHC class I-related molecule that presents microbial lipid or glycolipid antigens. DC membranes also express molecules that allow T cell adhesion (e.g., intercellular adhesion molecule 1). or CD54) or B7-1 and B7-2 (also known as CD80 and CD86, respectively). It is rich in molecules that costimulate sexualization.

[0034] "Tolerance" is the result of a tolerogenic mechanism of the immune response to a specific antigen, directly or indirectly preventing, silencing, or down-regulating the adaptive immune response to one or more antigens or downregulation. Substances capable of inducing tolerance, i.e., tolerogenic substances The IL-1 receptor modulates APCs, e.g., DCs, to become tolerogenic, resulting in immune responses. Tolerogenic DCs can mediate tolerance induction.

[0035] "Tolerogenicity" refers to the lack of antigen-specific T cell responses, nonresponsiveness, or Induction of anergy and tolerance mediated through mechanisms such as active suppression "Tolerization" refers to the ability of a cell or substance to tolerate an antigen. This refers to the tolerance that has been achieved, and tolerogenic mechanisms are mediated to induce immunosuppressive effects. This means that there is

[0036] "Immunogenicity" refers to the ability to directly or indirectly activate the adaptive immune response to an antigen. means a cell or substance that can

[0037] "Tolerogenic APCs" are those of microbial origin, components of mammalian cells, and cytokines. immunotolerance, which may be a combination of hormones, vitamins and other biological or pharmaceutical agents; It refers to APCs that have acquired the ability to induce tolerance through exposure to tolerogenic stimuli. Although resistant APCs have a low capacity to induce immunogenic responses, they induce activated Tregs and other tolerogenic responses. They have a high ability to do so.

[0038] "Tolerogenic DCs" are composed of microbial origin, mammalian cell components, cytokines, Immune tolerance, which can be a combination of hormones, vitamins, and other biological or pharmaceutical agents DCs that have acquired the ability to induce tolerance upon exposure to tolerogenic stimuli. Although it has a low ability to induce immunogenic responses, it has the ability to induce activated Treg and other tolerogenic responses. It has great power.

[0039] An "autoimmune disease" is a pathological condition in which the adaptive immune system is directed against self-antigens in a destructive manner. It means attitude.

[0040] A "self-antigen" (or "autoantigen") is a protein that is present in a disease state. Interacts with antigens in the induction of unwanted immune responses (e.g., antibodies or effector T cell responses) However, any portion of a mammal (e.g., a human) to which the mammal's healthy immune system is tolerant. The terms "self-antigen" and "autoantigen" refer to a molecule or chemical group. )" should be considered synonymous and are used interchangeably herein.

[0041] "Stimulus" refers to exposure to any artificial or natural compound that results in cell signaling. means.

[0042] method The present invention provides a method for obtaining tolerogenic APCs that have the ability to make the immune system tolerant to an antigen. The present invention provides a method for the treatment of monocytes comprising culturing isolated monocytes under specific culture conditions. Monocytes can be isolated from a number of sources. APCs are obtained according to the present invention. The APCs obtained according to the present invention include DCs and macrophages, preferably DCs. The DCs obtained by the method of the present invention may be myeloid DCs. This method typically involves: It is carried out under ex vivo or in vitro conditions, preferably under ex vivo conditions. The isolated monocytes are typically present in a sample taken from a mammal. The mammal is typically a human (Homo sapiens). The sample is typically a blood sample. More preferably, the sample is a peripheral blood mononuclear cell (PBMC) sample prepared from blood. Typically, the cells comprise mononuclear cells, preferably monocytic cells, and in particular monocytes. , comprising isolating a population of monocytes from a sample, e.g., a PBMC sample. Preferably, the isolated The cells are CD14 + The method further comprises culturing the isolated monocytic cells in cell culture. This includes:

[0043] In one embodiment, the cell culture of the methods of the invention comprises retinoic acid and TGF-beta. In another embodiment of the invention, the cell culture of the method of the invention is and an AhR agonist. In a further embodiment of the invention, the cell culture of the method of the invention comprises The cell culture preferably comprises retinoic acid and an AhR agonist. and even more preferably, retinoic acid, TGF beta, and an AhR agonist. nothing.

[0044] Retinoic acid is a metabolic product of vitamin A1. The cell culture contains retinoic acid. Retinoic acid can be administered in cell culture in any form, including all-trans-retinoic acid, 13-cis- -retinoic acid and / or 9-cis-retinoic acid, in particular all-trans-retinoic acid Retinoic acid can be prepared from retinoic acid precursors, such as retinol and / or in situ retinoic acid. Vitamin A may be provided as a variant form of vitamin A that produces acetylcholine, such as retinal.

[0045] The AhR agonist used in the cell culture of the present invention may be any AhR agonist, e.g., AhR agonists disclosed in WO2012 / 050500, the contents of which are incorporated by reference in their entirety. The AhR agonist may be any AhR agonist, either a full agonist or a The AhR agonist used in the methods of the present invention is preferably or N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxo quinoline-3-carboxamide (IMA-06201) (Mahiout et al., 2017), herein The AhR agonist is alternatively 6-formylindolo[3,2-b]carbazole (F ICZ), 2-(1H-indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE) , N-acetyl-N-phenyl-4-acetoxy-5-chloro-1,2-dihydro-1-methyl-2-oxoquino Phosphorus-3-carboxamide ("C2"), N-(4-trifluoromethylphenyl)-1,2-dihydro-4- Hydroxy-5-methoxy-1-methyl-2-oxoquinoline-3-carboxamide ("C3") or N-amino Cetyl-N-(4-trifluoromethylphenyl)-4-acetoxy-1,2-dihydro-5-methoxy-1- Other potentially useful AHs include methyl-2-oxoquinoline-3-carboxamide ("C4"). R agonists are described in Denison and Nagy, all of which are incorporated by reference in their entirety. Ann. Rev. Pharmacol. Toxicol., 43:309-34, 2003 and the references cited therein. This is described in the literature.

[0046] The TGF beta used in the cell culture of the present invention may be any TGF beta isoform, e.g. It can be TGF beta 1, TGF beta 2 or TGF beta 3, preferably TGF beta 1. The beta isoform may be used alone or in combination with any other TGF beta isoform. It can be used in combination.

[0047] The cell culture typically contains GM-CSF and IL-4. Alternatively, the cell culture may contain GM-CSF The cell culture may contain GM-CSF alone, for example without IL-4, or M-CSF. GM-CSF and IL-4 are preferably administered to isolated monocytes, APCs, preferably DCs. M-CSF is added to cell cultures to induce differentiation of isolated monocyte APCs, preferred Alternatively, it may be added to cell cultures to induce differentiation into macrophages.

[0048] Typically, GM-CSF and IL-4 or GM-CSF or M-CSF are used in combination with TGF-beta, AhR agonists and receptors. The cell culture was added either before or at the same time as the addition of any one of the thiamin and thiamin mononitrates. Preferably, GM-CSF and IL-4 are administered in combination with any of TGF-beta, AhR agonist, and retinoic acid. Typically, the AhR is added to the cell culture before or at the same time as the AhR is added to the cell culture. The agonist is added to the cell culture at the same time that GM-CSF or M-CSF and IL-4 are first added. A first dose of TGF-beta and a second dose of an AhR agonist are added to the cell culture. The dose is added to the cell culture after the first dose of AhR agonist is added to the cell culture. Furthermore, typically, a second dose of TGF-beta and an AhR agonist is added to the cell culture. After the addition of GM-CSF or M-CSF and IL-1, retinoic acid is added to the cell culture. 4 is added to the cell culture in a second dose. Preferably, the second dose of GM-CSF or M-CSF and IL-4 A dose of TGF-beta and a second dose of AhR agonist are added to the cell culture at the same time. is added to the cell culture.

[0049] GM-CSF or M-CSF is a compound that inhibits the growth of cells by either TGF-beta, AhR agonist, or retinoic acid. IL-4 may be added to the cell culture before or at the same time as the addition of IL-4 to the culture medium. Typically, a first dose of TGF-β is added to the cell culture after the first dose of TGF-β is added to the cell culture. The second dose of AhR agonist was administered at 100 μg / ml after the first dose of AhR agonist was added to the cell culture. Typically, retinoic acid is added to the cell culture after being added to the cell culture medium. A second dose of the hR agonist is added to the cell culture after which GM-CSF is added to the cell culture. Alternatively, M-CSF may be added to the cell culture in a second dose. Preferably, GM-CSF or M-CSF A second dose of TGF-beta AhR agonist is added to the cell culture at the same time that a second dose of TGF-beta AhR agonist is added to the cell culture. is added to the cell culture.

[0050] The cell culture may further comprise an antigen or an epitope-containing fragment of the antigen. The epitope-containing fragment can bind to or be expressed on isolated monocytes in culture. The antigen or its epitope-containing fragment may be expressed on the cell surface of a monocyte or APC. or an epitope-containing fragment thereof may be intracellular. may be an antigen or epitope-containing fragment that is not native to monocytes, e.g., The cells may take up the antigen or epitope-containing fragments thereof from other cells before being isolated. The spheres or APCs are transfected with antigen-encoding mRNA to express the antigen or its epitope-containing fragments. Methods for transfecting cells with antigen-encoding mRNA are known in the art. It is well known in the art.

[0051] In the method for inducing tolerance to an allograft, the antigen or epitope-containing fragment thereof is Binding to or expressed on monocytes isolated from samples taken from transplant donors. or may be expressed by APCs during or after culture using the methods of the present invention. Using the method of the invention, monocytes isolated from a sample taken from a cultured donor are immunotolerant. Such tolerogenic APCs can be generated by the donor. It may be administered to the recipient to prevent immune rejection of the seed transplant.

[0052] In the case of monocytes isolated from a sample taken from a recipient, the monocytes are immunotolerogenic. The cells may be cultured using the methods of the present invention to allow tolerogenic APCs to be produced. PCs may be loaded with donor antigens so as to present donor-derived antigens. Tolerogenic APCs do not have to be loaded with donor antigens ex vivo. Such tolerogenic APCs, which may or may not express immune responses, may prevent immune rejection of the allograft from the donor. The recipient may be given another dose to prevent an adverse reaction.

[0053] Alternatively, the antigen or epitope-containing fragment thereof is a component that is added to the cell culture. Therefore, the cell culture further comprises an antigen or an epitope-containing fragment thereof. The antigen or epitope-containing fragment may be added before, at the same time as, or after retinoic acid is added to the cell culture. The antigen or epitope-containing fragment thereof is added to the cell culture as part of a mixture of antigens. For example, cells, tissues or samples containing one or more antigens may be added to the culture. You may.

[0054] The antigen or epitope-containing fragment may be a single antigen or an epitope-containing fragment thereof (e.g. , purified form), or a pool or multiple antigens and / or multiple epitopes thereof The antigen or epitope-containing fragment may be derived from a cell, blood, tissue sample, or any of these. In the case of a method for inducing tolerance to an allograft, the antigen or its extract may be used. The epitope-containing fragment may be a sample of the graft or a sample of the graft or other drug. The antigen or its epitope-containing fragment may be derived from a tissue, cell, or blood sample from a donor. The pieces can be added directly to the cell culture.

[0055] The antigen or epitope-containing fragment thereof may optionally not be added directly to the cell culture. Alternatively, the antigen or epitope-containing fragment thereof may be present in vivo. The tolerogenic APCs are optionally loaded ex vivo with the antigen or an epitope-containing fragment thereof. Instead, the antigen or its antigens may be expressed in vivo when tolerogenic APCs are administered. Thus, tolerogenic APCs have the ability to be loaded with a mitochondrial fragment containing a mitogen-activated antigen receptor (APC). The antigen or its epitope-containing fragment is recognized and processed by the antibody.

[0056] The antigen or epitope-containing fragment thereof may be or be derived from a biopharmaceutical. Therefore, biological agents can be used as all or part of an antigen (i.e., the part containing the epitope). It can be used.

[0057] The antigen or epitope-containing fragment thereof may be or be derived from an autoantigen.

[0058] Generation of regulatory T cells The method of the present invention involves ex vivo culturing the obtained tolerogenic APCs with T cells, Therefore, the method may further comprise inducing the generation of Tregs. C induces Tregs when cultured ex vivo with T cells. The induced Tregs can be expressed by any Treg. , e.g., CD4 + CD25 + Foxp3 + Treg and / or type 1 regulatory T (Tr1) CD4 + CD49b + LAG3 + Cells, preferred Or CD4 + CD25 + Foxp3 + Tregs or a population thereof can be obtained by the method of the present invention. It may be possible or obtainable.

[0059] Tregs or populations thereof may be used in methods of treating a mammalian subject or in methods of generating an immune response to an antigen. The compounds may be used in methods of treating mammalian subjects suffering from or at risk of suffering from a pulmonary embolism.

[0060] As shown in Figures 5(A) and 5(B), the tolerogenic DCs of the present invention are capable of activating immune cells when unstimulated or after e.g. Upon stimulation with immunogenic stimuli such as LPS or inflammatory cytokine cocktails, low T cell The combination of retinoic acid, TGF-beta and AhR agonist showed the ability to induce proliferation. The combination of retinoic acid and TGF-beta and the combination of retinoic acid and AhR agonist stimulated T cells more effectively. It was also superior in reducing the ability to induce cell proliferation.

[0061] Therefore, suitable tolerogenic APCs of the present invention (for example, DCs of the present invention) when unstimulated are 100% or less, e.g., 90% or less, e.g., 80% or less, e.g., 70% or less, of that of control cells at stimulation Furthermore, the T cell proliferation-inducing ability is, for example, LPS or an inflammatory cytokine cocktail. Suitable tolerogenic APCs of the invention (e.g., DCs of the invention) upon stimulation with an immunogenic stimulus such as , e.g., 70% or less, e.g., 60%, of that of control cells upon stimulation with an immunogenic stimulus such as LPS. The control cells have a T cell proliferation induction ability of, for example, 50% or less. The same type of APCs from the same subjects not exposed to the substance.

[0062] As shown in Figures 10(A) and 10(B), the tolerogenic DCs of the present invention have a high Treg induction ability. The combination of retinoic acid, TGF-beta and AhR agonists is induces Tregs better than the combination of retinoic acid and an AhR agonist or the combination of retinoic acid and an AhR agonist. It was excellent on the

[0063] Therefore, suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated: At least 130%, for example at least 150%, for example at least 150% of that of unstimulated control cells Furthermore, the Treg induction ability is 175%, for example, at least 200%. Suitable tolerogenic APCs of the invention (e.g., DCs of the invention) upon stimulation with an immunogenic stimulus of: Suitably, at least 130% of that of control cells upon stimulation with an immunogenic stimulus, such as LPS. , for example, at least 150%, for example, at least 200% Treg inducibility. are the same type of APCs from the same subject who have not been exposed to the tolerogenic compound.

[0064] As shown in Figure 11(J), the tolerogenic DCs of the present invention were able to attract CD14+ monocytes from the blood of hemophilia patients. When isolated, it has high Treg inducibility.

[0065] Therefore, suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated: At least 130%, e.g., at least 150%, e.g., at least 150% of that of unstimulated control cells Furthermore, the Treg inducibility of the LP is at least 175%, for example, at least 200%. Suitable tolerogenic APCs of the invention (e.g., DCs of the invention) upon stimulation with an immunogenic stimulus such as S. is suitably at least as high as that of control cells upon stimulation with an immunogenic stimulus such as, for example, LPS. and the Treg inducibility is at least 130%, e.g., at least 150%, e.g., at least 200%. Control cells were APCs of the same type from the same subject that had not been exposed to the tolerogenic compound. is.

[0066] Tolerogenic antigen-presenting cells Monocytes differentiate into DCs. During differentiation, a proportion of DCs become polarized toward a tolerogenic phenotype. The methods of the present invention typically involve the use of immunoglobulins having tolerogenic cell surface marker expression. It allows the generation of tolerogenic APCs, for example tolerogenic DCs.

[0067] CD83 is an integral membrane protein and an activation / maturation marker. The expression of stimulated APCs, especially DCs, does not differ significantly between tolerogenic and non-tolerogenic cells. However, upon stimulation with an immunogenic stimulus such as LPS, APCs, particularly DCs may exhibit elevated levels of CD83 expression. Therefore, CD83 expression levels are typically , higher in stimulated non-tolerogenic cells than in stimulated tolerogenic cells Desirable tolerogenic APCs, especially DCs, show a low CD83 expression level even after stimulation. Desirable tolerogenic APCs, especially DCs, have low immunogenicity with respect to upregulation of CD83 expression. It is as resistant as possible to stimulation, for example, by LPS.

[0068] The tolerogenic APCs of the present invention (e.g., DCs of the present invention) are, in at least some embodiments, is the percentage of cells stimulated with an immunogenic stimulus, such as LPS, compared with correspondingly stimulated control cells. The control cells have the advantage of lower CD83 expression compared to the control cells. The APCs are the same type from the same subject who has not been exposed to the antibody.

[0069] Suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated are 100% or less, e.g., 90% or less, e.g., 80% or less, e.g., 70% or less, of that of control cells , for example, have an expression level of CD83 that is 60% or less, for example, 50% or less.

[0070] Suitably, the tolerogenic APCs of the invention (e.g., upon stimulation with an immunogenic stimulus such as LPS) For example, DCs of the present invention have a cell proliferation rate 70% or more than that of control cells upon stimulation with an immunogenic stimulus such as LPS. % or less, e.g., 60% or less, e.g., 50% or less. As described in more detail in the previous section and shown in FIG. 1, the immune tolerogens used When the active compound is retinoic acid, TGF beta, and an AhR agonist, the APC of the present invention When produced by the methods of the invention, stimulated APCs exhibit a higher activity than corresponding control cells. The greatest reduction in CD83 expression levels was observed in retinoic acid, TGF-beta, and AhR agonists. The use of a combination of retinoic acid and TGF-beta is superior to the use of retinoic acid and TGF-beta. The use of retinoic acid and TGF-beta was superior to the use of retinoic acid and an AhR agonist.

[0071] The tolerogenic APCs of the present invention (e.g., DCs of the present invention) are, in at least some embodiments, have the added benefit of having CD83 expression levels that are resistant to further upregulation upon stimulation. Has.

[0072] Suitably, the tolerogenic APCs of the invention (e.g., upon stimulation with an immunogenic stimulus such as LPS) For example, the DCs of the present invention have a cell density of 200% or less, for example, 175% or less, of that of the corresponding cells in the absence of stimulation. , e.g., have an expression level of CD83 that is 150% or less, e.g., 125% or less. The tolerogenic agent used is described in more detail in and shown in FIG. When the compound is retinoic acid, TGF beta, and an AhR agonist, the APC of the present invention can be used in combination with the in stimulated APCs compared to corresponding unstimulated cells when produced by the method The greatest reduction in CD83 expression levels was observed in retinoic acid, TGF-beta, and AhR agonists. The use of the combination of retinoic acid and TGF-beta is superior to the use of retinoic acid and TGF-beta. The use of retinoic acid and TGF-beta was superior to the use of retinoic acid and AhR agonists.

[0073] CD86 is an integral membrane protein, an activation / maturation marker and a costimulatory molecule. In general, unstimulated APCs, especially DCs, show a significant difference between tolerogenic and non-tolerogenic cells. However, stimulation with an immunogenic stimulus such as LPS did not significantly affect the expression of CD86. Occasionally, APCs, particularly DCs, may exhibit elevated levels of CD86 expression. is increased to a greater extent in non-tolerogenic cells than in tolerogenic cells. Desirable tolerogenic APCs, especially DCs, express low levels of CD86 upon stimulation. Tolerogenic APCs, especially DCs, upregulate CD86 expression in response to immunogenic stimuli, e.g., LPS. It is as tolerant as possible to stimuli.

[0074] The tolerogenic APCs of the present invention (e.g., DCs of the present invention) are, in at least some embodiments, indicates an increase in CD86 expression upon stimulation with an immunogenic stimulus, such as LPS, compared to control cells upon stimulation. Control cells have the advantage of being low in expression. It is the same type of APC from the same target.

[0075] Suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated are 100% or less, e.g., 90% or less, e.g., 80% or less, e.g., 70% or less, of that of control cells , for example, has an expression level of CD86 of 60% or less, for example, 50% or less.

[0076] Suitably, the tolerogenic APCs of the invention (e.g., upon stimulation with an immunogenic stimulus such as LPS) For example, DCs of the present invention have a cell proliferation rate 80% or more than that of control cells upon stimulation with an immunogenic stimulus such as LPS. % or less, for example, 70% or less, for example, 60% or less, for example, 50% or less, As described in more detail in the Examples section and shown in FIG. When the tolerogenic compounds used are retinoic acid, TGF-beta, and AhR agonists, When the APCs of the present invention are produced by the method of the present invention, the APCs are compared with corresponding control cells upon stimulation. In comparison, retinoin resulted in the greatest reduction in CD83 expression levels in stimulated APCs. The use of a combination of retinoic acid, TGF-beta and AhR agonists is The use of retinoic acid and TGF-beta is superior to the use of retinoic acid and AhR agonists. was better than using

[0077] The tolerogenic state of DCs is due to low costimulatory potential and high expression of inhibitory molecules. ILT3 is an inhibitor that can be expressed by tolerogenic APCs, preferably DCs. DCs overexpressing ILT3 have lower phosphorylated levels of NF-kappa B. Instead of Treg differentiation, they stimulate the complete program that triggers Th proliferation and maturation. ILT3 expression and upregulation are typically associated with LPS-induced inflammatory responses (Vlad et al., 2009). Typically, non-tolerogenic and tolerogenic APCs, In particular, DCs do not differ significantly between unstimulated and stimulated conditions (e.g., with an immunogenic stimulus such as LPS). The level of ILT3 expression is typically higher than in non-tolerogenic cells. The desired tolerogenic APCs, especially DCs, are immune-competent cells. have high levels of ILT3 expression that is not downregulated upon stimulation with an immunogenic stimulus, e.g., LPS .

[0078] The tolerogenic APCs of the present invention (e.g., DCs of the present invention) are, in at least some embodiments, are measured unstimulated or stimulated with an immunogenic stimulus such as LPS, respectively. The advantage of these cells is that they have high expression of ILT3 compared to the control cells. The same type of antigen-presenting cells from the same subject who has not been exposed to the tolerogenic compound.

[0079] Suitably, the immune tolerance of the present invention upon stimulation with an immunogenic stimulus, such as LPS, and without stimulation. The immunogenic APCs (e.g., DCs of the present invention) are expressed in the presence or absence of an immunogenic stimulus such as LPS. at least 150%, e.g., at least 175%, e.g., at least 150% of that of control cells upon stimulation with an expression level of ILT3 that is at least 200%, for example, at least 250%, for example, at least 300%; As described in more detail in the Examples section and shown in FIG. The tolerogenic compounds used are retinoic acid, TGF-beta, and AhR agonists. In this case, when the APCs of the present invention are produced by the method of the present invention, compared to control cells, The greatest increase in ILT3 expression levels was observed in unstimulated and stimulated APCs. The use of a combination of retinoic acid, TGF-beta and AhR agonists has been shown to be effective in reducing the use of retinoic acid and TGF-beta. The use of retinoic acid and TGF-beta is superior to the use of retinoic acid and AhR agonists. This was superior to using

[0080] The tolerogenic potential of APCs, especially DCs, may be determined by the ratio of ILT3 and CD86 expression levels The ILT3 / CD86 ratio is typically higher in tolerogenic cells than in non-tolerogenic cells. Desirable tolerogenic APCs, especially DCs, are either unstimulated or immunogenic. They have a high ILT3 / CD86 ratio regardless of whether they are stimulated by a stimulus, such as LPS.

[0081] Tolerogenic APCs of the present invention (e.g., upon stimulation with an immunogenic stimulus such as LPS) can be used to For example, DCs of the present invention may, at least in some embodiments, be either unstimulated or, e.g., , and have a higher ILT3 / CD86 ratio compared to control cells upon stimulation with immunogenic stimuli such as LPS. Control cells are obtained from the same subject who has not been exposed to the tolerogenic compound. It is the same type of APC as these.

[0082] Suitably, the APCs of the present invention (eg, DCs of the present invention) when unstimulated are those of control cells when unstimulated. At least 150%, for example, at least 200%, for example, at least 250%, for example, At least 300%, for example at least 350%, for example at least 400%, for example at least As described in more detail in the Examples section, As shown in FIG. 4, the tolerogenic compounds used were retinoic acid, TGF-beta, and When the APC of the present invention is produced by the method of the present invention, The greatest increase in the ILT3 / CD86 ratio was observed in unstimulated APCs compared to control cells. The use of a combination of retinoic acid, TGF-beta and AhR agonists is The use of retinoic acid and TGF-beta is superior to the use of retinoic acid and AhR agonists. was better than using

[0083] Suitably, the APCs of the invention (e.g., the APCs of the invention) upon stimulation with an immunogenic stimulus such as LPS. DCs) exhibit a cell density at least 150% higher than that of control cells upon stimulation with an immunogenic stimulus such as LPS. %, for example at least 200%, for example at least 250%, for example at least 300%, For example, at least 350%, for example, at least 400%, for example, at least 500% LT3 / CD86 ratio. As described in more detail in the Examples section and shown in FIG. Thus, the tolerogenic compounds used are retinoic acid, TGF-beta, and AhR agonists. When the APCs of the present invention are produced by the method of the present invention, stimulated control cells The greatest increase in ILT3 / CD86 ratio in stimulated APCs was obtained compared to retinoic acid. The use of a combination of TGF-beta and an AhR agonist is more effective than the use of retinoic acid and TGF-beta. The use of retinoic acid and TGF-beta is also superior to the use of retinoic acid and AhR agonists. It was better than I expected.

[0084] CD141 and GARP, when co-expressed, are tolerogenic APC markers. Tolerogenic APCs co-expressing P have been shown to be associated with an enhanced ability to induce Tregs. CD141 and GARP are expressed on both unstimulated and stimulated APCs, especially DCs (Agrawal et al., 2016). Both can be co-expressed on the cell surface. Co-expression and up-regulation of CD141 and GARP on tolerogenic APCs The regulation may not be dependent on immunogenic stimuli, such as LPS. The degree of immunosuppression is typically much higher between tolerogenic cells than between non-tolerogenic cells. Preferred tolerogenic APCs, particularly DCs, are those that are unstimulated or stimulated with an immunogenic stimulus, such as LPS. When treated with HCl, they have a high frequency of CD141 and GARP co-expressing cells.

[0085] Tolerogenic APCs of the present invention (e.g., upon stimulation with an immunogenic stimulus such as LPS) can be used to For example, DCs of the present invention, at least in some embodiments, can be expressed either unstimulated or stimulated, respectively. Compared with control cells, these cells have the advantage of having high levels of CD141 and GARP co-expression. Control cells were APCs of the same type from the same subject that had not been exposed to the tolerogenic compound. do.

[0086] Suitably, the APCs of the present invention (eg, DCs of the present invention) when unstimulated are those of control cells when unstimulated. At least 400%, for example, at least 450%, for example, at least 500%, for example, At least 600%, for example at least 700%, for example at least 800%, for example at least a high frequency of cells co-expressing CD141 and GARP, e.g., at least 900%, e.g., at least 1000%. As described in more detail in the Examples section and shown in Figure 6, the immunogens used When the immunotolerogenic compounds are retinoic acid, TGF beta and AhR agonists, When APCs are produced by the methods of the present invention, the unstimulated APCs are increased in number compared to unstimulated control cells. The greatest increase in the frequency of CD141 and GARP co-expressing cells was observed among PCs. The combined use of retinoic acid and an AhR agonist was superior to the use of retinoic acid and an AhR agonist. The use of retinoic acid and AhR agonists is more effective than the use of retinoic acid and TGF-beta. The combined use of retinoic acid, TGF-beta and AhR agonists was also superior. was superior to the use of acetylcholinesterase inhibitors and TGF-beta.

[0087] Suitably, the APCs of the invention (e.g., the APCs of the invention) upon stimulation with an immunogenic stimulus such as LPS. DCs) are at least 200%, e.g., at least 250%, e.g., at least 200% of that of control cells upon stimulation. For example, at least 300%, at least 400%, for example, at least 450%, for example, at least The frequency of CD141 and GARP co-expressing cells is 500%. As shown in FIG. 6, the tolerogenic compound used is retinoic acid. , TGF beta and AhR agonist, the APC of the present invention is produced by the method of the present invention Co-expression of CD141 and GARP among stimulated APCs compared to stimulated control cells when The greatest increase in cell frequency was obtained with the combination of retinoic acid, TGF-beta and AhR agonists. The use of retinoic acid and TGF-beta was superior to the use of retinoic acid and TGF-beta.

[0088] IL-23 is a pro-inflammatory cytokine released by APCs, especially DCs, during inflammation. This cytokine, produced by phage, inhibits the production of IL-17 and related cytokines by lymphocytes. IL-23 promotes host protection against mucosal pathogens through the induction of cytokines. It is only released from APCs, especially DCs, upon cell activation. Typically, it is released from unstimulated APCs, In particular, DCs produce IL-23, which does not differ significantly between tolerogenic and non-tolerogenic cells. However, immunogenic substances such as LPS or inflammatory cytokine cocktails may be present. APCs, particularly DCs, upon stimulation with stimuli may exhibit increased levels of IL-23 production. is typically increased to a much greater extent in non-tolerogenic cells than in tolerogenic cells. Desirable tolerogenic APCs, particularly DCs, are capable of responding to immunogenic stimuli such as LPS or inflammatory cytokines. When stimulated by a cocktail of cytokines, they produce low levels of IL-23, which is desirable for immune tolerance. Acute APCs, particularly DCs, respond to immunogenic stimuli, such as LPS or inflammatory sites, for IL-23 production. It is as resistant as possible to stimulation by the cain cocktail.

[0089] The tolerogenic APCs of the present invention (e.g., DCs of the present invention) are, in at least some embodiments, Upon stimulation with an immunogenic stimulus, such as LPS or a cocktail of inflammatory cytokines, e.g. For example, control cells upon stimulation with an immunogenic stimulus such as LPS or an inflammatory cytokine cocktail. They also have the advantage of showing low production of IL-23 in the absence of stimulation. The control cells were from the same subject who had not been exposed to the tolerogenic compound. It's the same type of APC.

[0090] Suitably, the tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated are 100% or less, e.g., 90% or less, e.g., 80% or less, e.g., 70% or less, of that of control cells , for example, has a production level of IL-23 that is 60% or less, for example, 50% or less.

[0091] Suitably, stimulation with an immunogenic stimulus such as, for example, LPS or a pro-inflammatory cytokine cocktail. At this time, the APCs of the present invention (e.g., DCs of the present invention) may be treated with, for example, LPS or an inflammatory cytokine cocktail. 50% or less, e.g., 40% or less, e.g., of control cells upon stimulation with an immunogenic stimulus such as As described in more detail in the Examples section, As shown in Figure 7, the tolerogenic compounds used were retinoic acid and AhR. agonist, retinoic acid and TGF-beta, and retinoic acid, TGF-beta and AhR agonist. When the APCs of the present invention are produced by the method of the present invention, stimulated control cells Compared with IL-23 production, the level of IL-23 production in stimulated APCs was reduced.

[0092] The APCs of the present invention (e.g., DCs of the present invention) may, at least in some embodiments, be used as control cells. than after stimulation with immunogenic stimuli such as LPS or an inflammatory cytokine cocktail. Merriam-Webster's analysis of IL-23 levels that are more resistant to further upregulation of IL-23 production It also has a

[0093] CD103 is a cell surface marker associated with intestinal tolerogenic DCs. Tolerogenic APCs induce Tregs and CD8 + Cross-presentation of foreign antigens to T cells It has been shown that tolerance can be maintained by protecting against tissue infection (Scott et al., 2012). 011). CD103 expression can be upregulated on the cell surface of tolerogenic APCs, particularly DCs. CD103 expression and upregulation may be independent of immunogenic stimuli, such as LPS. Non-tolerogenic and tolerogenic APCs, especially DCs, are capable of activating immune cells without stimulation (e.g., immune stimuli such as LPS). The expression levels of CD103 did not differ significantly between stimulation conditions (in the case of primordial stimulation). Levels are typically much greater in tolerogenic cells than in non-tolerogenic cells. Desirable tolerogenic APCs, particularly DCs, are either unstimulated or stimulated with an immunogenic stimulus, such as LPS. These cells express high levels of CD103 upon stimulation with .

[0094] Tolerogenic APCs of the present invention (e.g., upon stimulation with an immunogenic stimulus such as LPS) can be used to For example, DCs of the present invention may, at least in some embodiments, be either unstimulated or, e.g., , which have high CD103 expression compared to control cells upon stimulation with immunogenic stimuli such as LPS. Control cells are obtained from the same subject who has not been exposed to the tolerogenic compound. It is the same type of APC.

[0095] Suitably, the APCs of the present invention (eg, DCs of the present invention) when unstimulated are those of control cells when unstimulated. At least 150%, for example, at least 200%, for example, at least 250%, for example, have an expression level of CD103 that is at least 300%. Described in more detail in the Examples section As shown in FIG. 8, the tolerogenic compounds used were retinoic acid, TGF-β, and erythrocyte proliferation. beta and AhR agonists, the APCs of the present invention are produced by the methods of the present invention The maximum increase in CD103 expression levels in unstimulated APCs compared to unstimulated control cells was observed in The combined use of retinoic acid, TGF-beta and AhR agonists resulted in increased retinoic acid production. The use of retinoic acid and AhR agonists is superior to that of retinoic acid and AhR agonists. The use of retinoic acid and TGF-beta was superior to the use of retinoic acid and TGF-beta. The use of the agonist combination was superior to the use of retinoic acid and TGF-beta.

[0096] Suitably, the APCs of the invention (e.g., the APCs of the invention) upon stimulation with an immunogenic stimulus such as LPS. DCs) have a cell density at least 200 times that of control cells upon stimulation with an immunogenic stimulus, such as LPS. %, e.g., at least 250%, e.g., at least 300%. As described in more detail in the Examples section and shown in FIG. When the tolerogenic compounds are retinoic acid, TGF-beta and AhR agonists, When bright APCs are produced by the methods of the present invention, they are stimulated to produce more APCs than stimulated control cells. The greatest increase in CD103 expression levels was observed in stimulated APCs. The use of a combination of retinoic acid and an AhR agonist is superior to the use of retinoic acid and TGF-beta. there was.

[0097] MERTK, BTLA, LAP, HLA-G, and CD49b are cell surface markers of tolerogenic APCs, especially DCs. MERTK, BTLA, LAP, HLA-G, and CD49b are expressed on the cell surface of tolerogenic APCs, especially DCs. The levels of MERTK, BTLA, LAP, HLA-G, and CD49b expression were typically In fact, it is much higher in tolerogenic cells than in non-tolerogenic cells. Tolerogenic APCs, especially DCs, express high levels of MERTK, BTLA, LAP, and HLA-G in the unstimulated state. Desirable tolerogenic APCs, especially DCs, have MERTK, BTLA, LAP, HLA-G, and CD40 phenotypes when not stimulated. 49b has high levels of expression.

[0098] The tolerogenic APCs of the present invention (e.g., DCs of the present invention) when unstimulated have at least some In one embodiment, the cells exhibit high MERTK, BTLA, LAP, and HLA-G expression compared to unstimulated control cells. The unstimulated tolerogenic APCs of the present invention (e.g., DCs of the present invention) have the advantage of being ) in at least some embodiments, exhibits elevated MERTK, as compared to unstimulated control cells. The control cells have the advantage of expressing BTLA, LAP, HLA-G, and CD49b. APCs of the same type from the same subject who has not been exposed to the tolerogenic compound.

[0099] Suitably, the APCs of the present invention (eg, DCs of the present invention) when unstimulated are those of control cells when unstimulated. MERTK that is at least 120%, for example, at least 150%, for example, at least 175% of the As described in more detail in the Examples section and shown in Figure 9(A), The tolerogenic compounds used are retinoic acid, TGF-beta, and AhR agonists. When the APCs of the present invention are produced by the method of the present invention, The greatest increase in MERTK expression levels was observed in unstimulated APCs compared to cells. The use of a combination of retinoic acid, TGF-beta and AhR agonists has been shown to be effective in reducing the use of retinoic acid and TGF-beta. It was better than I expected.

[0100] Suitably, the APCs of the present invention (eg, DCs of the present invention) when unstimulated are those of control cells when unstimulated. BTLA that is at least 120%, for example, at least 150%, for example, at least 200% of the As described in more detail in the Examples section and shown in Figure 9(B), The tolerogenic compounds used are retinoic acid, TGF-beta, and AhR agonists. When the APCs of the present invention are produced by the method of the present invention, The greatest increase in BTLA expression levels was observed in unstimulated APCs compared to cells. The use of a combination of retinoic acid, TGF-beta and AhR agonists has been shown to be effective in reducing the use of retinoic acid and TGF-beta. It was better than I expected.

[0101] Suitably, the APCs of the present invention (eg, DCs of the present invention) when unstimulated are those of control cells when unstimulated. At least 120%, for example, at least 150%, for example, at least 200%, for example, have an expression level of LAP that is at least 250%. As described in more detail in the Examples section, As shown in Figure 9(C), the tolerogenic compounds used were retinoic acid, TG When the APC of the present invention is produced by the method of the present invention, it is an F beta and AhR agonist. The maximum increase in LAP expression levels in unstimulated APCs compared to unstimulated control cells was observed when The combined use of retinoic acid, TGF-beta and AhR agonists resulted in increased retinoic acid production. was superior to the use of acetylcholinesterase inhibitors and TGF-beta.

[0102] Suitably, the APCs of the present invention (eg, DCs of the present invention) when unstimulated are those of control cells when unstimulated. At least 120%, for example, at least 150%, for example, at least 200%, for example, have an expression level of HLA-G that is at least 250%, for example, at least 300%. As described in more detail in Section 1 and shown in Figure 9(D), the immune tolerance When the prophylactic compounds are retinoic acid, TGF beta, and AhR agonists, the APC of the present invention When produced by the methods of the present invention, the The greatest increase in HLA-G expression levels was obtained in the control group.

[0103] The APCs (e.g., DCs) of the present invention when unstimulated suitably have a higher IL than control APCs when unstimulated. The expression of T3 is high, and the expression of CD83 and CD86 is low. The APCs (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as LPS, Suitably, the expression of ILT3 is high and the expression of CD83 and CD86 is low compared to control APCs. is the same but from a subject not treated with a tolerogenic compound according to the invention. It is a type of APC.

[0104] The expression level of ILT3 in the APCs (e.g. DCs) of the present invention when unstimulated is suitably at least 150%, e.g., at least 175%, e.g., at least 200%, for example, at least 250%, for example, at least 300%. The expression level of CD83 in APCs (e.g., DCs) is suitably compared to that of unstimulated control cells. The C in the APCs (e.g., DCs) of the present invention without stimulation is 110% or less, for example, 100% or less. The expression level of D86 is suitably 110% or less, for example 100%, of that of unstimulated control cells. The following is the result.

[0105] For example, expression of ILT3 in APCs (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as LPS. The expression level is suitably compared to that of control cells upon stimulation with an immunogenic stimulus such as, for example, LPS. at least 150%, for example at least 175%, for example at least 200%, for example at least For example, at least 250%, e.g., at least 300%. The expression level of CD83 in APCs (e.g. DCs) of the present invention upon stimulation is suitably determined, for example, by stimulation with LPS. 70% or less, e.g., 60% or less, e.g., of control cells upon stimulation with an immunogenic stimulus such as For example, the expression of IL-1 in APCs (e.g., DCs) upon stimulation with an immunogenic stimulus such as LPS is 50% or less. The expression level of CD86 in the cells is suitably measured relative to that in the control cells upon stimulation with an immunogenic stimulus such as LPS. It is 80% or less, such as 70% or less, such as 60% or less, such as 50% or less, of that of the cells.

[0106] The APCs (e.g., DCs) of the present invention, either unstimulated or stimulated with an immunogenic stimulus such as LPS, Suitably, they express CD103, and most suitably, they are either unstimulated or stimulated with an immunogen such as LPS, respectively. The control cells showed higher expression of CD103 compared to control APCs upon stimulation with the immunization method according to the present invention. The same type of antigen-presenting cells from the same subject not treated with the tolerogenic compound. In APCs (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as, for example, LPS. The expression level of CD103 is suitably measured in the absence of stimulation or in the presence of an immunogenic stimulus, such as LPS, respectively. at least 200%, e.g., at least 250%, e.g., at least At most 300%.

[0107] The APCs (e.g., DCs) of the present invention when unstimulated suitably express CD103 (most suitably CD103). The expression of CD141, GARP, and ILT3 was higher than that of unstimulated control APCs, and the expression of CD83 and Furthermore, the APCs of the present invention have low expression of CD86 and CD87 upon stimulation with an immunogenic stimulus such as LPS. DCs) suitably express CD103 (most suitably have high CD103 expression) and are sensitive to, for example, LPS The expression of CD141, GARP, and ILT3 was higher than that of control APCs when stimulated with immunogenic stimuli such as The control cells were treated with the tolerogenic compound of the present invention and showed low expression of CD83 and CD86. For example, when stimulated with an immunogenic stimulus such as LPS, The expression level of CD103 in the APCs (e.g., DCs) of the present invention is suitably determined by, for example, stimulation with LPS or the like. At least 200% of that of control cells upon stimulation with any immunogenic stimulus, e.g., at least 2 50%, e.g., at least 300%. For example, upon stimulation with an immunogenic stimulus such as LPS, The level of co-expression of CD141 and GARP in APCs (e.g. DCs) of the invention may suitably be determined, for example, by: At least 200% of that of control cells upon stimulation with an immunogenic stimulus such as LPS, e.g., at least at least 250%, e.g., at least 300%, at least 400%, e.g., at least 450%, e.g., For example, at least 500%. For example, the AP of the present invention upon stimulation with an immunogenic stimulus such as LPS. The expression level of ILT3 in C (e.g. DC) is suitably determined in response to an immunogenic stimulus such as LPS. at least 150%, e.g., at least 175%, e.g., at least 150% of that of control cells upon stimulation with At least 200%, for example at least 250%, for example at least 300%. For example, L Expression levels of CD83 in APCs (e.g., DCs) of the present invention upon stimulation with immunogenic stimuli such as PS is suitably 70% or less than that of control cells upon stimulation with an immunogenic stimulus such as, for example, LPS. For example, 60% or less, for example, 50% or less. The expression level of CD86 in the APCs (e.g., DCs) of the present invention upon stimulation is suitably determined by, for example, stimulation with LPS. 80% or less, e.g., 70% or less, e.g., ... It is 60% or less, for example, 50% or less.

[0108] For example, the APCs (e.g., DCs) of the present invention when unstimulated are suitably compared to control APCs when unstimulated. and expressing one or more (e.g., two, three, or all four) of MERTK, BTLA, LAP, and HLA-G. For example, the APCs (e.g., DCs) of the present invention when unstimulated and suitably one of MERTK, BTLA, LAP, HLA-G and CD49b compared to unstimulated control APCs. express (and suitably have high expression of) more than one (e.g., two, three, or all four) of the genes. The irradiated cells were the same type from the same patient who had not been treated with the tolerogenic compound according to the invention. The expression level of MERTK in the APC (for example, DC) of the present invention without stimulation is appropriately The expression level of the serotonin-dependent ATPase inhibitor is at least 120%, e.g., at least 150%, e.g., at least 120% of that of unstimulated control cells, e.g., at least 150% of that of unstimulated control cells. The expression level of BTLA in the APCs (e.g., DCs) of the present invention without stimulation is at least 175%. Suitably, the cell number is at least 120% of that of unstimulated control cells, for example at least 150%. %, or at least 200%. The level is suitably at least 150% of that of unstimulated control cells, e.g. At least 200%, for example, at least 250%. ) is suitably 120% of that of unstimulated control cells, e.g. , at least 150%, for example, at least 200%, for example, at least 250%, for example, The expression level of CD49b in the APCs (e.g., DCs) of the present invention without stimulation is at least 300%. is suitably 120%, for example at least 150%, e.g., 150% of that of unstimulated control cells. At least 200%, such as at least 250%, for example at least 300%.

[0109] For example, antigen-presenting cells (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as LPS: Suitably, upon stimulation with an immunogenic stimulus such as, for example, LPS or a pro-inflammatory cytokine cocktail. The control cells produced less IL-23 than the control APCs. The same type of APCs from the same subject not treated with LPS or inflammatory sites. IL-23 in APCs (e.g., DCs) of the present invention upon stimulation with an immunogenic stimulus such as a kine cocktail The level of production of the antibody is suitably determined by comparing it with that of control cells upon stimulation with an immunogenic stimulus such as, for example, LPS. For example, the amount of an immunogen such as LPS is 50% or less, for example, 40% or less, for example, 30% or less. The expression level of CD86 in APCs (e.g. DCs) of the present invention upon stimulation with a sexual stimulus is suitably For example, 80% or less, e.g., 70% of that of control cells upon stimulation with an immunogenic stimulus such as LPS. For example, 60% or less, for example, 50% or less.

[0110] Suitably, the tolerogenic APCs (e.g., DCs) of the present invention are capable of expressing, for example, their cell surface markers Car presentation, high Treg induction potential, low T cell proliferation induction potential and other advantageous properties described herein The phenotype is stable with respect to

[0111] Expression of cell surface markers can be measured using methods widely used and known in the art, e.g. For example, it can be evaluated using flow cytometry analysis.

[0112] Dosage regimen The compounds in the cell culture of the present invention can be added to the culture at an appropriate dose. The actual dosage level of the compound in the culture is determined to be the level that induces the desired immune tolerance to the cells without causing toxicity. The dosage may be varied to obtain an effective amount of the compound to achieve a sexual effect.

[0113] When retinoic acid is added to the cell culture, a suitable dose of retinoic acid is, for example, For example, suitable dosages may range from about 0.5 μM to about 8 μM, 0.5 μM to about 10 μM. The concentration may be 0.5 μM to about 6 μM, 0.5 μM to about 5 μM, 0.5 μM to about 4 μM, and preferably 0.5 μM to about 3 μM. .

[0114] If an AhR agonist is added to the cell culture, an appropriate dose for the AhR agonist may be determined, e.g. , about 1 nM to about 10 μM, typically about 5 nM to about 2 μM, for example, about 5 nM to about 750 nM. For example, a suitable dosage is about 5 nM to about 500 nM, 5 nM to about 250 nM, 5 nM to about 100 nM, preferably The dose can be 5 nM to about 50 nM. If no AhR agonist is present in the cell culture, a suitable dose is 0nM.

[0115] When TGF beta is added to the cell culture, a suitable dose of TGF beta is, for example, about 1 ng / mL. ml to about 200 ng / ml, for example, about 5 ng / ml to about 200 ng / ml, typically about 5 ng / ml to about 150 ng / ml For example, suitable dosages may range from about 5 ng / ml to about 125 ng / ml, from 5 ng / ml to about 100 ng / ml, The concentration may be 5 ng / ml to about 75 ng / ml, 5 ng / ml to about 50 ng / ml, and preferably 5 ng / ml to about 30 ng / ml. A suitable dose of TGF-beta when beta is not present in the cell culture is 0 ng / ml.

[0116] When retinoic acid, TGF-beta and AhR agonists are added to cell cultures, retinoic A suitable dose of phosphate may be, for example, in the range of about 0.5 μM to about 10 μM. The dosage is preferably about 0.5 μM to about 8 μM, 0.5 μM to about 6 μM, 0.5 μM to about 5 μM, or 0.5 μM to about 4 μM. The appropriate dose of the AhR agonist may be, for example, about 1 nM to about 10 μM. M, typically in the range of about 5 nM to about 2 μM, for example, in the range of about 5 nM to about 750 nM. A suitable dosage is about 5 nM to about 500 nM, 5 nM to about 250 nM, 5 nM to about 100 nM, preferably 5 nM to about 5 A suitable dose of TGF-beta may be, for example, about 1 ng / ml to about 200 ng / ml, for example, about It may be in the range of 5 ng / ml to about 200 ng / ml, typically in the range of about 5 ng / ml to about 150 ng / ml. For example, Suitable dosages include about 5ng / ml to about 125ng / ml, 5ng / ml to about 100ng / ml, 5ng / ml to about 75ng / ml, and 5 The concentration may be from 5 ng / ml to about 50 ng / ml, preferably from 5 ng / ml to about 30 ng / ml.

[0117] When retinoic acid, TGF-beta and AhR agonists are added to cell cultures, retinoic Suitable doses of phosphates may range, for example, from about 0.5 μM to about 3 μM. An appropriate dose may be, for example, in the range of about 5 nM to about 50 nM, preferably about 20 nM. A suitable dose may be, for example, in the range of about 5 ng / ml to about 30 ng / ml, preferably about 20 ng / ml.

[0118] When retinoic acid, TGF-beta and AhR agonists are added to cell cultures, retinoic An appropriate dose of phosphate may be, for example, in the range of about 0.5 μM to about 3 μM, preferably about 2 μM. A suitable dose of the AhR agonist is, for example, in the range of about 5 nM to about 50 nM, preferably about 10 nM. A suitable dose of TGF-beta is, for example, in the range of about 5 ng / ml to about 30 ng / ml, preferably about 10 It can be ng / ml.

[0119] Dosage regimens may be adjusted to provide the optimum desired response. For example, a single dose may be administered in a single dose, or several divided doses may be administered over time, or as needed depending on the urgency of the therapeutic situation. As indicated by acute symptoms, the dose may be proportionally reduced or increased. The dosage unit form used is a physical form suitable as a single dosage for the culture conditions used. each unit contains a predetermined amount of active ingredient calculated to produce a desired therapeutic effect. Contains a compound.

[0120] The compounds used in the cell culture of the present invention may be used alone or in combination with one or more other compounds. One compound may be co-administered with one or more other compounds. Two compounds may be co-administered with one or more other compounds. It may be co-administered with other compounds listed above.

[0121] The administration of two or more compounds in combination can be achieved in a number of different ways. They may be administered together in a single composition, or in separate compositions as part of a combined administration. For example, one or more compounds may be administered before or separately from one or more other compounds. administered later or sequentially, or concurrently or simultaneously. can be given.

[0122] Treatment and prevention of immune reactions to antigens and prevention of immune rejection reactions The present invention relates to a method for treating mammals that have or are at risk of having an unwanted immune response to an antigen. The present invention provides a tolerogenic APC or population thereof for use in treating a subject. can be used with any antigen discussed herein, e.g., FVIII, FIX, antibodies, antigens that bind to grafts (e.g., For example, it may be an allograft), or an autoantigen.

[0123] The tolerogenic APCs generated by the culture method of the present invention are autologous tolerogenic APCs. The autologous tolerogenic APCs used in the treatment may be derived from the same subject. The monocytes may be obtained from autologous monocytes isolated from the collected sample. The cells may be cultured with the antigen or an epitope-containing fragment thereof, after which autologous tolerogenic APCs are obtained. The autologous tolerogenic APCs then react with the antigen when administered back to the subject. It has the ability to induce tolerance.

[0124] The present invention further provides a method for preventing allograft rejection in a recipient subject. The present invention provides tolerogenic APCs or populations thereof for use in allogeneic transplantation, including kidney, pancreas, It may be a liver, lung, heart, skin transplant, blood cell transplant, e.g., stem cell transplant, etc. The present invention provides a treatment for transplant rejection. The tolerogenic APCs used in the treatment are derived from donor The antibody may be obtained from monocytes isolated from a sample taken from a donor. The tolerogenic APCs are cultured and stimulated to react with antigens present in the allograft from the same donor. It can be obtained so as to have the ability to induce tolerance.

[0125] The tolerogenic APCs used in the treatment are isolated from a sample taken from the recipient. Monocytes collected from the recipient can be co-cultured with an antigen or a pool of antigens. The tolerogenic APCs can then be obtained. , which has the ability to induce tolerance to antigens present in the donor-derived allograft. The method may be used when obtaining monocytes from a donor is not possible, e.g., when the donor is not a living donor. It may be useful in cases where a single antibody isolated from a sample collected from a recipient according to the present invention Tolerogenic APCs derived from erythrocytes may be used to treat or prevent immune rejection of xenografts. The present invention may be used in a method for preventing

[0126] The tolerogenic APCs of the present invention may be used in manufacturing methods. in the manufacture of a medicament for treating a mammalian subject having or at risk of having a The method involves administering to a mammalian subject a tolerogenic APC or a population thereof. administering a PC or a population thereof, thereby establishing immune tolerance to the antigen. The present invention also provides a method for the immune rejection of an allograft in a recipient subject where the graft is derived from a donor. The present invention also encompasses the use of a tolerogenic APC or population thereof in the manufacture of a medicament for treating a reaction. The method includes administering to a recipient subject a tolerogenic APC or population thereof. and (b) administering to the patient a tolerogenic APC, thereby establishing tolerance to the allograft. Alternatively, the population may be derived from monocytes isolated from a sample taken from a donor or recipient. Such a production method also allows for the production of tolerogenic APCs or populations thereof from the recipient. It may be applied to xenografts when derived from monocytes isolated from samples taken from the xenograft.

[0127] mammal Mammals from which samples according to the method of the invention are obtained and mammals treated by the method of the invention Mammals that can be treated by the methods of the present invention include, in particular, humans (Homo sapiens). The mammal that can be used is preferably a human (Homo sapiens).

[0128] sample The methods of the present invention may involve isolating monocytes from any suitable sample from a subject. The sample may be a blood sample, a fractionated buffy coat sample, a leukapheresis material sample, or a PBMC sample. The sample used in the method of the present invention is preferably a PBMC sample. The sample may be autologous, allogeneic or xenogeneic, preferably the sample is autologous or allogeneic.

[0129] Obtaining APCs from mammals According to the present invention, monocytes are first obtained from a mammal. One suitable method is to The first step is to collect PBMCs by apheresis. PBMCs collected by apheresis are The mixture may be kept under temperature controlled conditions, for example, at ambient temperature, for example, 18°C ​​to 25°C, or PBMCs collected by pheresis may be frozen at temperatures below -4°C, -20°C, or -80°C. Alternatively, PBMCs can be isolated by density centrifugation. Monocytes are then separated from the solid phase. For example, by plastic adhesion or using beads, e.g., anti-CD14 They can be positively selected from PBMCs by magnetic bead isolation.

[0130] Administration of cells to a subject The cells can be administered to a mammal by a variety of routes, for example, intravenously, subcutaneously or intradermally, or intranodally. or directly back into the lesion site where it is accessible for injection. The antibody suitably contains human albumin as a cytoprotective protein. Typically, the antibody has a cytoprotective effect of 1 to 100%. x10 7 The dose of cells / dose is administered weekly to biweekly in 1 to 10 doses. The dosage regimen can be modified until the desired tolerance is achieved.

[0131] antigen Antigens or epitope-containing fragments thereof can be used in biological drugs, e.g., protein drugs (especially Both are proteins containing 50, at least 200, or at least 1000 amino acid residues. The biopharmaceutical may comprise a polysaccharide component.

[0132] Examples of biologics include blood factors (including, for example, FVIII or factor IX), hormones (insulin and EPO), growth factors (including EGF, IGF, KGF, HGF, and FGF), cytokines (e.g., interleukins), enzymes (e.g., imiglucerase, rasburicase, imiglucerase algalase, agalsidase beta, alglucosidase alpha, laronidase, idur Further examples include GCSF and Analogs such as filgrastim and their pegylated versions (pegfilgrastim) These include interferons (e.g., interferon beta-1a) and steroids (e.g., steroids). In one preferred embodiment of the invention, the drug is FVIII. In another embodiment of the invention, the drug is For FVIII, commercially available products include ReFacto AF, Helixate NexGen, Kogenate Bayer, Kovaltry, Advate, NovoEight, Esperoct, Nuwiq, Beriate, Beriate P, Feiba, Haemoct Various combinations including phenanthrene, Hemofil, Monoclate-P, Octanate [LV], Optivate and Recombinant These products contain antigens and antibodies that can be derived from any of these products. and epitope-containing fragments can be used.

[0133] Further example biopharmaceuticals include engineered proteins (such as fusion and chimeric proteins) and In some embodiments, the drug may be an antibody. can be, for example, a monoclonal antibody, such as a humanized or fully human monoclonal antibody. Drugs can also be protein constructs that contain fragments of immunoglobulins. The terms include bispecific antibodies, antibody drug conjugates and antibody nanoparticle conjugates, as well as and pegylated or otherwise extended analogs. The antibody may be a single light chain antibody, a domain antibody or an antibody comprising a VHH, scFv, Fab, F(ab')2 or BiTE. The polypeptides may be fragments as well as pegylated or otherwise extended analogs thereof.

[0134] Exemplary antibodies include infliximab (chimeric antibody, anti-TNF alpha), adalimumab (human antibody, anti-TNF alpha), basiliximab (chimeric antibody, anti-IL-2), abciximab (chimeric antibody, anti-IL-2) RA antibody, anti-GpIIb / IIIa), daclizumab (humanized antibody, anti-IL-2), gemtuzumab (humanized antibody , anti-CD33), alemtuzumab (humanized antibody, anti-CD52), edrecolomab (murine Ig2a, anti-EpCA M), rituximab (chimeric antibody, anti-CD20), palivizumab (humanized antibody, anti-respiratory syncytial cell virus), trastuzumab (humanized antibody, anti-HER2 / neu (erbB2) receptor), bevacizumab (human Cetuximab (chimeric antibody, anti-EGFR), Eculizumab (humanized antibody, anti-VEGF) CD5), efalizumab (humanized antibody, anti-CD1Ia), ibritumomab (mouse anti muromonab-CD3 (mouse antibody, anti-T cell CD3 receptor), natalizumab (humanized antibody IgG1, anti-α4 integrin), nimotuzumab (humanized IgG1, anti-EGF receptor), omalizumab (human panitumumab (human antibody, anti-EGFR), ranibizumab (human antibody, anti-VEGF) , I-131 tositumomab (humanized antibody, anti-CD20), ofatumumab (human antibody, anti-CD20), cell Tolizumab (humanized antibody, anti-TNF alpha), golimumab (human antibody, anti-TNF alpha), emi Shigella (humanized bispecific antibody, anti-FIXa / FX), denosumab (human antibody, anti-RANK ligand) and concizumab (humanized antibody, anti-tissue factor pathway inhibitor).

[0135] Examples of biologic drugs that are fusion proteins include etanercept.

[0136] An extensive list of biological protein drugs in clinical development and approved products is available at: A 2013 PhARMA report, "Biolog," gives details of 907 biologics targeting over 100 diseases. ics” - https: / / web.archive.org / web / 20161011093352 / http: / / www.phrma.org / sites / def ault / files / pdf / biologics2013.pdf, which is incorporated by reference in its entirety. The present invention incorporates these and other biological responses that result in the development of an immune response in a subject. It is believed that the compounds may be used for biological therapeutic agents.

[0137] Therefore, antigens or epitope-containing antigens thereof for use in the methods described herein are The fragment may be, for example, FVIII or a derivative or fragment thereof, or factor IX or a derivative thereof. The antibody may be a conductor or fragment thereof, or an antibody or antibody fragment thereof.

[0138] The antigen or epitope-containing fragment thereof may bind to the allograft. Antigen-presenting tolerogenic APCs can be used to treat allograft rejection .

[0139] The antigen or epitope-containing fragment thereof may be or be derived from an autoantigen. Tolerogenic APCs that present self-antigens can be used to treat autoimmune diseases. The range of autoantigens involved in autoimmune diseases includes desmoglein 3, BP180, BP230, (pemphigus) ), dystonin and / or type XVII collagen (pemphigoid), myelin (multiple sclerosis), pancreatic vein Cellular protein (type 1 diabetes mellitus), nicotinic acetylcholine receptor (myasthenia gravis) , neuronal surface proteins (autoimmune epilepsy and encephalitis), 2-hydrolase (autoimmune Addison's disease), FceRI (chronic autoimmune urticaria) and acetylcholine receptors (myasthenia gravis) , fibrillarin (scleroderma) and cardiolipin (systemic lupus erythematosus). These autoantigens, or epitope-containing fragments thereof, can be used as antigens in the methods of the present invention. It can be used.

[0140] The antigen or epitope-containing fragment may be unknown. Therefore, the unknown antigen may be cultured using the method of the present invention. The unknown antigen may be bound to a tissue sample or extract thereof taken from the subject. possible.

[0141] Antigens are delivered from the exogenous medium across the cell membrane by any suitable antigen delivery method or vehicle. For example, antigen delivery methods or vehicles may include nanoparticles. Therefore, the antigen can be a nanoparticle, i.e., an antigen-nanoparticle conjugate. The antigen-nanoparticle may be a FVIII-nanoparticle conjugate. The lipid nanoparticles may be lipid nanoparticles such as vesicles, micelles, or micelles.

[0142] Alternatively, the antigen delivery vehicle may be a cell-penetrating peptide. Cell permeability, which allows transport across lipid bilayers from the external environment to intracellular compartments The cell-penetrating peptide may be fused to a peptide called penetratin or polyarginine. Transactivation of HIV-1 is mediated by the third helix of the homeodomain of Antennapedia. The cell-penetrating peptide may be a Tat peptide from the protein Tat. obtain.

[0143] Immune Responses and Uses of Tolerogenic APCs of the Invention The tolerogenic APCs of the present invention are capable of suppressing unwanted immune responses in a subject receiving the cells. The undesired immune response may include an antibody response and / or a cellular response. obtain.

[0144] The tolerogenic APCs of the present invention can be used, for example, in the treatment of mammalian subjects in which an immune response is generated. , any drug capable of producing said reaction, e.g., any biological drug, e.g., protein It is suitable for treatments involving the production of ADA to protein drugs.

[0145] The tolerogenic APCs of the present invention are directed to treatment of bleeding disorders (hemophilia A and B; FVIII and factor IX, respectively). deficiency), growth factor deficiency (deficiency of EGF, IGF, KGF, HGF, FGF, etc.), hormone deficiency (EPO deficiency), enzyme replacement therapy (e.g., imiglucerase (e.g., CEREZYME (commercially available)) α-galactosidase A (α-gal A) (e.g., agalsidase beta, Fabriza FABRYZYME), acid α-glucosidase (GAA) (e.g., alglucosidase alfa, LUMIZYME™, MYOZYME™, and arylsulfataben enzyme B (e.g., laronidase, ALDURAZYME™, idursulfa , ELAPRASE (trademark), galsulfase, NAGLAZYME (trademark) Anti-inflammatory and autoimmune disorders (e.g., anti-TNF alpha monoclonal antibodies) In many drug treatment situations, immune responses to drugs, including the production of ADAs, such as monoclonal antibodies, Suitable for use in treating mammalian subjects who develop a reaction.

[0146] The tolerogenic APCs of the present invention are capable of causing an autoimmune disease and producing the response. The present invention is suitable for use in treating mammalian subjects who have developed an immune response to a specific autoantigen.

[0147] The tolerogenic APCs of the present invention can be used to treat patients suffering from graft, e.g., allograft, rejection. or at risk of developing a reaction to antigens associated with the graft that can cause such a reaction. Suitable for use in the treatment of mammalian subjects in which an immune response has occurred or may occur There are.

[0148] autoimmune disease or disorder The tolerogenic APCs according to the present invention may be useful in the treatment of autoimmune diseases. Autoimmune diseases or disorders include achlorhydria, acquired hemophilia, acute hemorrhagic leukoencephalitis, and acquired thrombocytopenia. Hypoplastic purpura, Addison's disease, alopecia areata, anemia, ankylosing spondylitis, anti-glomerular basement membrane disease, anti-liver syndrome Lipid syndrome, aplastic anemia, atopic allergy, autoimmune atrophic gastritis, autoimmune Infectious hearing loss, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hypoparathyroidism, autoimmune Autoimmune hypophysitis, autoimmune lymphoproliferative, autoimmune myocarditis, autoimmune oophoritis, autoimmune Autoimmune orchitis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, autoimmune Autoimmune polyendocrinopathy, autoimmune sensory deafness, autoimmune syndrome type II, autoimmune ulcerative colitis Uveitis, Behcet's syndrome / disease, celiac disease, Chagas disease, chronic active hepatitis, Chronic inflammatory demyelinating polyneuropathy, chronic lymphocytic thyroiditis, Churg-Strauss syndrome , Crohn's disease, cryoglobulinemia, Cushing's syndrome, dermatitis herpetiformis, dermatomyositis, 1 Type 2 diabetes mellitus, Schilder's diffuse cerebral sclerosis, epidermolysis bullosa acquisita, erythroderma, Felty's disease syndrome, glomerulonephritis, membranous glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hammann-Rich syndrome, idiopathic thrombocytopenic purpura, inflammation Inflammatory bowel disease, insulin resistance type B, Lambert-Eaton myasthenic syndrome, lens-induced Uveitis, lichen sclerosus et atrophicus, lymphopenia, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, carnivorous corneal ulcer, mucocutaneous lymph node syndrome, multifocal motor neuropathy, Sclerosis, myasthenia gravis, transverse myelitis, myocarditis, narcolepsy, neuromyelitis optica, ophthalmopathy Cicatricial pemphigoid, oculo-vestibulo-auditory syndrome, sympathetic ophthalmia, opsoclonus-myoclonus Symptoms: pancreatitis, bullous pemphigoid, pemphigus foliaceus, pemphigus vulgaris, polyarteritis nodosa, rheumatoid arthritis Polymyalgia, polyradiculopathy, primary biliary cholangitis, primary biliary cirrhosis, psoriasis, Raynaud's disease, Reiter's disease, relapsing polychondritis, rheumatic fever, rheumatoid arthritis, sarcoid sclerosing cholangitis, scleroderma, sclerosing cholangitis, Sjögren's syndrome, stiff-person syndrome, adults Onset Still's disease, Takayasu's arteritis, temporal arteritis, thyrotoxicosis, type B insulin resistance, ulcer urticaria, uveomeningoencephalitis syndrome, vitiligo, and Wegener's granulomatosis. can be.

[0149] Suitably, the autoimmune disease is diabetes mellitus type 1, rheumatoid arthritis, chronic lymphocytic thyroiditis , multiple sclerosis and ulcerative colitis.

[0150] In addition, diseases that may be partially related to autoimmune reactions include arteriosclerosis, Parkinson's disease, and and Alzheimer's disease.

[0151] Cryopreservation, storage and thawing The tolerogenic APCs of the present invention may be produced manually, semi-automatically, or by a fully automated closed system. The cells of the present invention (for example, DCs of the present invention) can be produced in a medium containing 2 to 10% DMSO (dimethyl sulfoxide). The cells may then be cryopreserved as a single cell suspension in a cryopreservation medium containing the desired Preferably, the cells are washed with a physiological sodium chloride buffer solution containing human serum albumin, and then cryopreserved in a medium. , preferably 0.5 to 20 × 10 6 May be resuspended in the range of cells / ml and transferred to cryovials The cells may then be placed in a freezing container in a -80°C freezer. After the injection, the cryovials are transferred to a -150°C freezer or liquid nitrogen tank for long-term storage. Preferably, the cells of the present invention (e.g., DCs of the present invention) are cryopreserved at temperatures below -120°C. can be.

[0152] The cells of the invention (e.g., DCs of the invention) are cryopreserved until only a small chunk of ice remains before use. It may be thawed in the vial.

[0153] Storage and transportation The cells of the invention (e.g., DCs of the invention) can be stored as single cells or in multicellular aggregates. Single cells or multicellular aggregates may be entrapped or encapsulated in the hydrogel. The capture or encapsulation of cells within the membrane allows them to maintain their functionality at hypothermia and provides mechanistic protection. Maintain and effectively form multicellular aggregates while preserving cell morphology, integrity, viability, and function. It can be used for storage and / or transportation.

[0154] Single cells or multicellular aggregates may be encapsulated in hydrogels in vitro. Typically, the cells are viable or living cells with structurally intact cell membranes, and the cells of the present invention The hydrogel has a cell morphology representative of a cell (e.g., DC of the present invention). To confine the cells or aggregates, at least the majority of the single cells or multicellular aggregates must be completely The hydrogel coating may be a coating that completely or incompletely covers / surrounds the surface. Formed separately from single cells or multicellular aggregates and then placed on the single cells or multicellular aggregates. The hydrogel coating may be formed separately from the aggregates (i.e., spatially separated). Alternatively, the hydrogel coating may comprise a layer of cross-linked alginate (separated from the surface). They may be formed in situ (ie, in the presence of single cells or multicellular aggregates).

[0155] Single cells or multicellular aggregates can be cultured in a reversibly crosslinked hydrogel, e.g., alginate hydrogel. The hydrogel may be entrapped or encapsulated in a "reversibly crosslinked hydrogel." Refers to hydrogels formed by bridging (i.e., crosslinking so that the hydrogel returns to solution). The reversal of crosslinking can be achieved by (e.g., at the time of their use / transport or storage). After completion of the process, the captured or encapsulated multicellular aggregate(s) are released from the hydrogel. Examples of reversibly crosslinked hydrogels are well known in the art. and suitable hydrogels can be readily identified by one of ordinary skill in the art.

[0156] The hydrogel may be a hydrogel having a crosslinked or network structure or matrix. The hydrogel may comprise a polymeric polymer; and interstitial fluid. The hydrogel is semi-permeable and can inhibit or prevent cell differentiation in the aggregates. The hydrogel can be crosslinked under appropriate conditions to form a network structure or matrix. and the interstitial fluid and multicellular aggregates. The hydrogel may contain internal pores. That's fine.

[0157] The hydrogel-forming polymer can be alginic acid or a metal ion alginate. Preferably, the metal is a Group 1 metal (e.g., lithium, sodium, or calcium alginate). sodium) or Group 2 metals (e.g., calcium, magnesium, barium or is strontium). Preferably, the polymer is calcium alginate or alginate. The hydrogel-forming polymer is a cross-linked polymer, typically sodium phosphate or strontium alginate. It can be an acrylic acid-based (e.g., polyacrylamide) polymer. crosslinkable cellulose derivatives, hydroxyl ether polymers (e.g., poloxamer), The sugar may be cellulose, pectin or a natural gum.

[0158] In the case of multicellular aggregates, the cells are directly or indirectly connected to one another in a manner that forms the aggregate of cells. Matrices, substrates, or scaffolds are collectively referred to as "structures." The structure may be a synthetic or natural polymer. Preferably, the structure is biodegradable. The structure may be made of, for example, polylactic acid, collagen, Nylon, e.g., nylon mesh, collagen, gelatin, alginate, cellulose The membrane may be glass, glass or a polymer, including Matrigel.

[0159] Cells are cultured in an extracellular matrix (ECM), e.g., Alvatex polystyrene scaffolds for 3D cell culture. Alternatively, the multicellular aggregates may be structure-free.

[0160] Single cells or multicellular aggregates encapsulated in hydrogels can be preserved from a first location to a second location. The cells can be packaged and sealed in a tube or container for transport. The multicellular aggregates are stable for at least 1 hour, at least 2 hours, or at least 5 hours. The hydrogel may have a time period of at least 12 hours, at least 24 hours, etc. Single cells or multicellular aggregates are grown in the hydrogel (and sealed container) at temperatures ranging from -80°C to 45°C. and preferably at 4 to 45°C, or at ambient temperature, for example, 10 to 25°C, preferably 15 to 20°C. It can be stored or transported.

[0161] The hydrogel-encapsulated single cells or multicellular aggregates can be cultured under cell culture conditions (e.g., at about 37°C). °C, about 5% CO2 and about 95% humidity, or under chilled conditions, for example, at 4 to 6 °C, preferably about 4 °C. The single cells or multicellular aggregates encapsulated in the hydrogel can be stored or transported, e.g. It may be stored at 2-8°C or 8-15°C and refrigerated during transport. Single cells or multicellular aggregates should be stored or transported at controlled room temperature (CRT), defined as 15–25°C. They may be stored or transported refrigerated or at CRT (i.e., 8-25°C). Single cells or multicellular aggregates encapsulated in the rogel are grown at low body temperatures (i.e., below about 35°C, typically It can be stored or transported at temperatures ranging from 0 to 32°C.

[0162] The hydrogel containing the multicellular aggregates may be frozen prior to storage and / or transportation. Extending the time that cells in multicellular aggregates remain viable after thawing and / or increasing the effective transport time Therefore, hydrogels can be used as post-cryoprotectants. For example, the temperature of the hydrogel containing the aggregates may be less than 0°C, less than -15°C, or less than -80°C. The hydrogel containing the multicellular aggregates may be reduced to , preferably at a gradual rate of temperature increase, controlled or uncontrolled defrosting. ) or thaw, i.e., raise its temperature above 0°C, or In other instances, the hydrogels of the present invention are not cooled or frozen.

[0163] Single cells or multicellular aggregates encapsulated in hydrogels can be stored and stored for up to 10 or 20 weeks. Preferably, the single cells or multicellular aggregates are released from the hydrogel. The gel is stored in the hydrogel for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks before being removed. Preferably, the single cells or multicellular aggregates are allowed to stand for up to 1, 2, 3 minutes before being released from the hydrogel. The cells are stored in the hydrogel for 4, 5, 6, 7, 8, 9, or 10 days.

[0164] Single cells or multicellular aggregates are prepared by suitable cell-compatible conditions, i.e., cell and / or cell membrane can be released from the hydrogel by conditions that are not detrimental or significantly detrimental to the integrity of the For example, the hydrogel can be released by chemical degradation or by, e.g. Dissolve using an appropriate alginate dissolution buffer.

[0165] Storage and / or transportation for multicellular aggregation is described herein in its entirety, the contents of which are incorporated by reference. and WO2019142004, which is incorporated herein by reference.

[0166] QC (Quality Control) Release Assay RA + TGF-beta + AhR agonist-treated DCs showed a significant increase in the expression of several different markers and in the MLR. The quality of a given antibody can be tested based on its ability to reduce T cell proliferation in a given patient. For example, RA + TGF beta + Ah R agonist-treated DCs express the markers CD11c, HLA-DR, CD80, CD83, CD86, ILT3, GARP, and CD141 RA + TGFbeta + AhR agonist-treated DCs can express LAP, CD103, BTLA, HLA-G, and CD49b. preferably at least CD11c, HLA-DR, CD80, CD83, CD86 and ILT3, more preferably Preferably at least CD11c, HLA-DR, CD80, CD83, CD86 and ILT3, and even more preferably at least Both can express CD11c, HLA-DR, CD80, CD83, CD86, ILT3, GARP, CD141, and LAP. The proliferation is 3 T cell proliferation can be assessed by H-thymidine incorporation, CFDA, CD25 expression, and KI-67. The cell proliferation is preferably 3 H-thymidine incorporation, more preferably CFDA, and CD25 expression 3 H- Thymidine incorporation, and even more preferably CFDA, CD25 expression, KI-67 and 3 H-thymidine uptake It can be evaluated by the inclusion. [Example]

[0167] (Example) Materials and Methods for Examples 1-18 and 20-25 Isolation of human monocytes and T cells PBMCs were cultured on Lymphoprep™ (StemCell Technologies, Vancouver, Canada) or The cells were separated by density centrifugation using stainless steel tubes (BD Bioscience, San Jose, United States). Monocytes were isolated using anti-CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). ) were positively selected from PBMCs. + T cells were isolated using EasySep Human CD4 + T Cell Isolation Kit (StemCell Technologies).

[0168] Generation of dendritic cells (DCs) CD14 + Monocytes were cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 ng / ml; PeproTech , London, UK) and interleukin 4 (IL-4; 100 ng / ml; PeproTech) in the presence of HEPES, G containing GlutaMAX and penicillin-streptomycin solution (Thermo Fisher, Waltham, MA) 1.25 × 10 in MP DC medium (CellGenix, Freiburg, Germany) for 7 days. 6 The cells were cultured at 3 cells / ml. On day 1, cells were replenished with fresh medium and cytokines. Control DCs were not added with additional compounds. The cells were differentiated in GM-CSF and IL-4 without treatment and, optionally, on day 6, were further treated with lipopolysaccharide (LPS; 0.5 μg / ml; S igma-Aldrich, Saint Louise, MO) or TNF-alpha (10 ng / ml; PeproTech), IL-1 beta (1 inflammatory agent consisting of prostaglandin E2 (PGE2; 1 μg / ml; Sigma) and prostaglandin B (PGE2; 1 μg / ml; PeproTech). Immunogenic DCs were generated by treatment and stimulation with a cytokine cocktail. Compound: On days 0 and 3, AhR agonist "C1" (C1; 20 nM; IMA-06201; Immunahr AB, Lund, SS) was administered. weden), on day 3 TGF-beta 1 (10 or 20 ng / ml; PeproTech), and on day 6 retinoic acid (2 μ Tolerogenic DCs (tolDCs) were generated by treatment with IgG (IgG; Sigma-Aldrich). Illuminated DCs were treated with dexamethasone (Dex; 100 nM; Sigma-Aldrich) and vitamin D3 (VitD) on day 3. The phenotype of TolDCs was established by treatment with 100 nM of erythrocyte-derived leukocyte antigen (ESA) (STEMCell Technologies). Qualitative analysis was performed in the last 24 hours of culture with LPS (0.5 μg / ml), TNF alpha (10 ng / ml), IL-1 beta (10 ng / ml), PGE2 (1 μg / ml), and an inflammatory cytokine cocktail consisting of IL-1 in Example 24. On day 7, DCs were cultured in a 24-well platelet-free medium containing 100 mg / ml of erythropoietin (10 ng / ml) of erythropoietin (10 ng / ml). They were harvested and washed extensively before undergoing phenotyping and functional assays.

[0169] DC phenotyping Cell surface expression was measured using the following fluorescently conjugated antibodies: BD Biosciences (Franklin Lakes, NJ), BioLe Gend (San Diego, CA) and CD11c (B-Ly6), CD83 (HB15e), and CD86 (BU) from Thermo Fisher. 63), ILT3 (ZM4.1), CD141 (M80), GARP (7B11), CD103 (B-Ly7), MERTK (590H11G1E3), BTLA (J168-540), CD49b (P1E6-C5), HLA-G (87G), and LAP (FNLAP) were used for the study. Wash and resuspend in staining buffer (phosphate-buffered saline supplemented with 0.5% BSA and 2 mM EDTA). After 20 minutes of incubation with antibody, the cells were washed and analyzed using a MACSQuant 10 flow cytometer (Miller et al., 2011). Biopsies were acquired using a microscope (Tenyi Biotec) and analyzed using FlowJo software (BD Biosciences). Cells were distinguished from dead cells using Fixable Viability dye (FVD) from Thermo Fisher.

[0170] DC cytokine production The Luminex platform (Invitrogen) was used to measure the activity of inflammatory cytokines in the presence or absence of LPS or inflammatory cytokine stimulation. en, Carlsbad, USA) was used to determine IL-23 production in supernatants from DC cultures.

[0171] DC / T cell culture: MLR and Treg induction To analyze the T cell stimulatory capacity of the generated DC population, an allogeneic MLR was performed. Complete medium: fetal bovine serum (FBS; 10%; Thermo Fisher), HEPES, GlutaMAX, and penicillin The treatment was carried out in RPMI-1640 (Thermo Fisher) containing lecithin-streptomycin solution. T cells (10 5 Thin T cells / well) were cultured with DCs at a T cell:DC ratio of 10:1 for 5–7 days. 3 H-Chimi Proliferation was determined by CD4 uptake. + CD49b + LAG3 + For the determination of Tr1 cells, The cells were phenotyped as follows: CD25 高 Foxp3 + For determination of Treg induction, MLR cultures The derived T cells were rested for an additional 7 days in complete medium containing IL-2 (20 IU / ml; PeproTech) and then and subjected to phenotyping as described below.

[0172] Treg phenotyping Cell surface and intracellular marker expression was measured using the following fluorescently labeled antibodies: BD Biosciences and BioLeg CD4 (A161A1), CD25 (M-A251), CD49b (P1E6-C5), LAG3 (11C3C65), and Foxp3 ( T cells were washed, resuspended in staining buffer, and incubated with antibody for 20 minutes. After incubation, the cells were washed, fixed, permeabilized, and stained with Foxp3 / transcription factor staining buffer set (eBi Intracellular staining of Foxp3 was performed using a Thermo Fisher Scientific (Tetrascience, Thermo Fisher Scientific). Fixable Viability Dye (FVD) was used to distinguish dead cells. was carried out as described above.

[0173] Freezing and thawing of DCs Wash DCs once and then add 10-20 x 10 6 Resuspend in freezing medium at a cell concentration of 100 cells / ml and cryovialectically freeze. These vials were placed in a freezing container and placed in a -80°C freezer. The freezing rate was controlled at approximately -1°C / min. After 24 hours in a -80°C freezer, the cells were frozen for long-term storage. The cryovials were transferred to a -150°C freezer for freezing. Two different freezing media were used: 10% DMSO (C CryoStor (CS, Biolife solution) with 50% human serum albumin (S10), 10% DMSO, and 50% human serum albumin. The DCs were frozen in a vial in a water bath at 37°C. Thaw and carefully transfer the cell suspension into a 15 ml tube containing cold GMP DC medium (CellGenix) for 1 After washing, the cells were stained according to the DC phenotyping method described above.

[0174] Example 1 - Effect of various compounds on DC expression of CD83 The low expression level of the DC maturation marker CD83 is one of several factors that contribute to the In addition, it should be as resistant as possible to upregulation by immunogenic stimuli. Various compounds were tested for their ability to induce a tolerogenic phenotype in certain DCs. Irradiated DCs were cultured with GM-CSF and IL-4 for 7 days to differentiate into CD14 + Control DCs were differentiated from monocytes. Immunogenic DCs were generated by either no stimulation or stimulation with LPS added on day 6. TolDCs, or The less optimal variants were cultured with GM-CSF and IL-4 as described above, either alone or in combination. Various tolerogenic compounds were administered as a control: 20 nM AhR agonist C1 (AhR agonist C1) on days 0 and 3; ag), 20 ng / ml TGF-beta on day 3, and 2 μM retinoic acid on day 6 (sections 1 and 2 of this example). Treatment with RA (abbreviated as "RA" in the figure) resulted in CD14 + They were differentiated from monocytes and shown in Figure 1(A) and (B). As shown, the synergistic effect of the claimed combination was demonstrated. The inflammatory response was established by treating the eyes with 100 nM Dex and 100 nM VitD3 (see section of this example). On day 6, some samples were treated with LPS to assess phenotypic stability. On day 7, all cells were collected and fluorescently labeled (Figure 1(A) and (B); black bars). The cells were stained for CD83 cell surface expression using an anti-CD83 antibody, followed by flow cytometry. Analysis was conducted using the following method.

[0175] The mean fluorescence intensity (MFI) values ​​in live cells are shown as mean ± SD in Figures 1(A) and (B).

[0176] The results in Figure 1(A) show that DCs treated with RA+TGF-beta and RA+TGF-beta+AhR agonist were significantly more effective in inhibiting LPS Matched control DCs and Dex / VitD3-treated comparator tolDCs, whether stimulated with or without RA + TGF-beta + AhR expressed lower levels of CD83 compared to RA. Treatment with rhesus monkeys was superior to treatment with RA+TGF-beta in reducing CD83 expression in DCs. was.

[0177] The results in Figure 1(B) show that RA + TGF-beta, RA + TGF-beta + AhR agonist, and RA + AhR agonist Treated DCs expressed lower levels of CD83 when stimulated with LPS compared with matched control DCs. Treatment with RA + TGF beta + AhR agonist and RA + TGF beta Superior to RA plus AhR agonist treatment, and even single agent, in reducing CD83 expression in DCs was superior to treatment with RA.

[0178] Example 2 - Effect of various compounds on DC expression of CD86 One of the factors is the low expression level of the costimulatory molecule CD86, and in addition, Immune tolerance in DCs, which should be as resistant as possible to upregulation by pathogenic stimuli Various compounds were tested for their ability to induce a proinflammatory phenotype. Control DCs were treated with GM-CSF and IL-4 By culturing for 7 days at 4°C, CD14 + Control DCs were differentiated from monocytes. TolDCs, or their less optimal counterparts, were stimulated with LPS added on day 1 to generate immunogenic control DCs. The mutants were cultured with GM-CSF and IL-4 as described above and were then incubated with various IL-4 inhibitors either alone or in combination. Tolerogenic compounds: 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3; 20 nM AhR agonist C1 (AhR ag) on ​​day 3; On day 6, treatment with 2 μM RA increased the CD14 + Differentiated from monocytes As shown in Figures 2(A) and (B), the synergistic effect of the claimed combination was demonstrated. TolDCs for the study were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some of the tolDCs were treated with LPS and tested for phenotypic stability (Fig. 2(A) and (B; black bars). On day 7, all cells were collected and analyzed for CD86 cells using a fluorescently labeled anti-CD86 antibody. Staining for surface expression was followed by analysis by flow cytometry.

[0179] The MFI values ​​of live cells are shown as mean ± SD in Figures 2(A) and (B).

[0180] The results in Figure 2(A) show that DCs treated with RA+TGF-beta and RA+TGF-beta+AhR agonist were significantly more effective against LPS Matched control DCs and Dex / VitD3-treated comparator tolDCs, whether stimulated with or without RA + TGF-beta + AhR expressed lower levels of CD86 compared to RA. Treatment with rhesus monkeys was superior to treatment with RA+TGF-beta in reducing CD86 expression in DCs. was.

[0181] The results in Figure 2(B) show that DCs treated with RA+TGF-beta and RA+TGF-beta+AhR agonist were significantly more effective against LPS expressed lower levels of CD86 compared with corresponding control DCs, regardless of whether they were stimulated with RA+AhR agonist-treated DCs showed RA+TGF-mediated expression when stimulated with LPS. DCs treated with RA, TGF-beta, and AhR agonist expressed higher levels of CD86 compared to DCs treated with RA, TGF-beta, and AhR agonist. RA-treated DCs were resistant to CD86 upregulation upon LPS stimulation compared with stimulated control DCs. Treatment with RA + TGFbeta + AhR agonist did not show any significant difference from treatment with RA + TGFbeta or RA + AhR agonist. Furthermore, treatment with all three combinations was superior to treatment with LPS alone. When stimulated with RA, it was superior to RA as a single agent in reducing CD86 expression in DCs.

[0182] Example 3 - Effect of various compounds on DC expression of ILT3 One of the factors is the high expression of the tolerogenic marker ILT3. In addition, immune tolerance in DCs should be resistant to downregulation by immunogenic stimuli. Various compounds were tested for their ability to induce a proinflammatory phenotype. Control DCs were treated with GM-CSF and IL-4 By culturing for 7 days at 4°C, CD14 + Control DCs were differentiated from monocytes. TolDCs, or their less optimal counterparts, were stimulated with LPS added on day 1 to generate immunogenic control DCs. The mutants were cultured with GM-CSF and IL-4 as described above and were then incubated with various IL-4 inhibitors either alone or in combination. Tolerogenic compounds: 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3; 20 nM AhR agonist C1 (AhR ag) on ​​day 3; On day 6, treatment with 2 μM RA increased the CD14 + Differentiated from monocytes As shown in Figures 3(A) and (B), the synergistic effect of the claimed combination was demonstrated. TolDCs for the study were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some of the tolDCs were treated with LPS and tested for phenotypic stability (Fig. 3(A) and (B; black bars). On day 7, all cells were collected and analyzed for ILT3 cells using a fluorescently labeled anti-ILT3 antibody. Staining for surface expression was followed by analysis by flow cytometry.

[0183] The MFI values ​​of live cells are shown as mean ± SD in Figures 3(A) and (B).

[0184] The results in Figure 3(A) show that DCs treated with RA+TGF-beta and RA+TGF-beta+AhR agonist were able to inhibit LPS Matched control DCs and Dex / VitD3-treated comparator tolDCs, whether stimulated with or without These results indicate that RA + TGF-beta + AhR expressed higher levels of ILT3 compared to RA + TGF-beta + AhR. Treatment with RA was superior to treatment with RA+TGF-beta in inducing ILT3 expression in DCs. was.

[0185] The results in Figure 3(B) show that RA + AhR agonist, RA + TGF-beta, and RA + TGF-beta + AhR agonist The treated DCs, whether stimulated with LPS or not, showed more cytotoxicity compared with the corresponding control DCs. Treatment with RA + TGF-beta + AhR agonist resulted in high levels of ILT3 expression. , superior to treatment with RA+TGF-beta or RA+AhR agonist, and even better than treatment with RA+TGF-beta. Treatment was superior to treatment with RA plus AhR agonist in inducing ILT3 expression in DCs Treatment with RA + TGF-beta + AhR agonist and RA + TGF-beta did not affect the response to LPS stimulation. However, it was superior to treatment with RA as a single agent in inducing ILT3 expression in DCs. Treatment with RA plus AhR agonist significantly reduced ILT3 expression in DCs upon stimulation with LPS. was superior to treatment with RA as a single agent.

[0186] Example 4 - Effect of various compounds on DC tolerogenic index ILT3 / CD86 Optimal tolDCs express both low levels of costimulatory molecules and high levels of tolerogenic molecules. Therefore, to take this combinatorial effect into account, representative immune tolerance genes The ratio between the expression levels of stimulatory and costimulatory molecules determines the tolerogenic potential of specific subsets of tolDCs. Figure 4 (A) and (B) show the ILT3 / CD86 expression ratio (MFI) as a proxy for the ILT3 / CD86 expression. and under various conditions in the absence (Fig. 4(A); grey bars) or presence (Fig. 4(B); black bars) of LPS. 1 shows such tolerogenic index in DCs cultured with .

[0187] The MFI values ​​of live cells are shown as mean ± SD in Figures 4(A) and (B).

[0188] The results in Figure 4(A) show that DCs treated with RA+TGF-beta and RA+TGF-beta+AhR agonist showed a significantly higher response rate than the control DCs. DC and Dex / VitD3-treated comparator tolDC had a higher tolerogenic index, and furthermore, +TGF-beta + AhR agonist-treated DCs had a higher tolerogenic index than RA + TGF-beta-treated DCs. Indicates that it has.

[0189] The results in Figure 4(B) show the same pattern as the results in Figure 4(A). In particular, Figure 4(B) shows that the RA+AhR agonist DCs treated with RA, RA + TGF beta, and RA + TGF beta + AhR agonist showed significantly higher proliferation rates than stimulated control DCs. These results indicate that the treatment with RA, TGF-beta, and AhR agonists has a higher immune tolerance index than the treatment with RA, TGF-beta, and AhR agonists. TolDCs treated with RA+TGF-beta had a higher tolerogenic index than those treated with RA+Ah The RA + TGF-beta + AhR agonist treatment has a higher immune tolerogenic index than those treated with the AhR agonist. DCs treated with RA agonist, RA + TGF-beta, and RA + AhR agonist were all treated with RA as a single agent. They have a higher tolerogenic index than DCs treated with IgG.

[0190] Example 5 - Effect of various compounds on DCs and their ability to induce T cell proliferation This translates into a reduced ability to induce T cell proliferation and is upregulated by immunogenic stimuli Induce a tolerogenic phenotype in DCs that should be as resistant as possible to being infected Various compounds were tested for their ability to inhibit DC proliferation. Control DCs were cultured with GM-CSF and IL-4 for 7 days. CD14 + Control DCs were either unstimulated or stimulated with LPS added on day 6. TolDCs, or their less optimal variants, were stimulated with the above-mentioned immunization protocol to generate immunogenic control DCs. The cells were cultured with GM-CSF and IL-4 and various tolerogenic compounds, either alone or in combination, as follows: On days 0 and 3, 20 nM of AhR agonist C1 (AhR ag) was administered, and on day 3, 10 ng / ml of TGF-beta (Figure 5(B) )) or 20 ng / ml TGF-beta (Figure 5(A)), and on day 6, treatment with 2 μM RA increased the CD14 + The synergistic effect of the claimed combination was observed as shown in Figures 5(A) and (B) after differentiation from monocytes. For comparison, TolDCs were treated with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some of the DCs were treated with LPS (Fig. 5(A); black bars) or TNF-alpha, IL-1. and treated with a pro-inflammatory cytokine cocktail consisting of PGE2 (Figure 5(B); black bars). Phenotypic stability was tested under various conditions. On day 7, all cells were harvested and CD4 + T thin Co-cultures with DCs were set up in an MLR setting at a T cell:DC ratio of 10:1. At the end of 7 days of culture, , T cell proliferation, 3 Determined by 3H-thymidine incorporation.

[0191] Counts per minute (CPM) values ​​from triplicate or more samples are shown in Figures 5(A) and (B) as mean ± SD. show.

[0192] The results in Figure 5(A) show that RA+AhR agonist, RA+TGF-beta, and RA+TGF-beta+AhR agonist The treated DCs showed a lower ability to induce T cell proliferation than the corresponding control DCs, including after LPS challenge. Treatment with RA + TGF-beta + AhR agonist resulted in a decreased ability to induce T cell proliferation. This was superior to treatment with RA plus AhR agonist or RA plus TGF-beta in reducing the inflammatory response.

[0193] The results in Figure 5(B) show the same pattern as the results in Figure 5(A). In particular, Figure 5(B) shows that the RA+AhR agonist DCs treated with RA, RA + TGF beta, and RA + TGF beta + AhR agonist suppressed inflammatory cytokines. The corresponding control DCs, Dex / VitD3-treated comparator tolDCs, and Lower ability to induce T cell proliferation than DCs treated with RA, TGF-beta, or AhR agonist as single agents Treatment with RA + TGF-beta + AhR agonist resulted in a reduced ability to induce T cell proliferation. This was superior to treatment with RA plus AhR agonist or RA plus TGF-beta in reducing the inflammatory response.

[0194] Example 6 - CD141 + GARP + (Effect of various compounds on DC frequency in One of several factors that contribute to the immune tolerance of DCs is the co-expression of CD141 and GARP. Various compounds were tested for their ability to induce a sexual phenotype. Control DCs were treated with GM-CSF and IL-4. By culturing for 7 days, CD14 + Control DCs were differentiated from monocytes without stimulation or at day 6. TolDCs, or their less optimal counterparts, were stimulated with LPS added to the IL-16 / IL-16 cells to generate immunogenic control DCs. The mutants were cultured with GM-CSF and IL-4 as described above and subjected to various immunizations, either alone or in combination. Tolerogenic compound: AhR agonist C1 (AhR ag) at 20 nM on days 0 and 3, and 10 ng / ml on day 3 Treatment with 0.05% TGF-beta (Fig. 6(A)) or 20 ng / ml TGF-beta (Fig. 6(B)), and 2 μM RA on day 6 By doing so, CD14 + The monocytes were differentiated to produce the claimed compounds, as shown in Figures 6(A) and 6(B). For comparison, TolDCs were treated with 100 nM Dex and 100 nM V on day 3. On day 6, some of the tolDCs were treated with LPS and expressed The stability of the cells was tested (Fig. 6(B); black bars). On day 7, all cells were collected and fluorescently labeled. Cell surface expression was stained using anti-CD141 and anti-GARP antibodies, followed by flow cytometry. Analysis by chromatometry was performed.

[0195] CD141 in live cells + GARP + The frequencies (%) of these are shown as mean ± SD in Figures 6(A) and (B).

[0196] The results in Figure 6(A) show that RA+AhR agonist, RA+TGF-beta, and RA+TGF-beta+AhR agonist The treated DCs were compared with control DCs, Dex / VitD3-treated comparator tolDCs, and DCs treated with RA, TGF-beta, and AhR agonists. DCs treated with sucrose and TGFbeta + AhR agonist showed higher frequencies of CD141 than DCs treated with sucrose and TGFbeta + AhR agonist. + GARP + Contains cells Treatment with RA + TGF beta + AhR agonist was significantly superior to treatment with RA + AhR agonist or RA + TGF beta. Treatment with RA + AhR agonist, RA + TGF-beta, and RA + TGF-beta were superior to those with RA + AhR agonist, RA + TGF-beta, and RA + TGF-beta. DCs treated with AhR agonists were treated with TGF-beta or AhR agonists as single agents. DCs treated with Dex / VitD3 or Dex / VitD3 expressed a higher frequency of CD141 + GARP + Contains RA+AhR cells DCs treated with RA, TGF beta, and AhR agonist showed a higher frequency than DCs treated with RA. CD141 + GARP + Contains cells.

[0197] The results in Figure 6(B) show that DCs treated with RA+TGF-beta and RA+TGF-beta+AhR agonist were significantly more potent than DCs treated with LPS Higher frequencies of CD than matched control DCs and Dex / VitD3-treated comparator tolDCs, including after stimulation. 141 + GARP + Treatment with RA + TGF-beta + AhR agonist resulted in a decrease in the number of RA + TGF-beta cells. It was better than the F-Beta handling.

[0198] Example 7 - Effect of various compounds on DC generation of IL23 One of several factors is the low production of the cytokine IL-23 in response to immunogenic stimuli. Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs, including Control DCs were cultured with GM-CSF and IL-4 for 7 days to differentiate into CD14 + Differentiated from monocytes. DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. C, or its less optimal variants, was cultured with GM-CSF and IL-4 as described above, either alone or in combination with IL-4. various tolerogenic compounds in combination: 20 nM AhR agonist on days 0 and 3; C1 (AhR ag), 10 ng / ml TGF beta (Fig. 7(B)) or 20 ng / ml TGF beta (Fig. 7(A)) on day 3; and CD14 by treatment with 2 μM RA on day 6. + They were differentiated from monocytes, and the results shown in Figure 7(A) and (B) As shown, the synergistic effect of the claimed combination was demonstrated. The tolDCs were established by treating them with 100 nM Dex and 100 nM VitD3. Some were treated with LPS (Fig. 7(A); black bars) or TNF-alpha, IL-1 beta, and PGE2 (Fig. 7(B); black bars). The supernatant was collected from DC cultures on day 7. and IL-23 levels were measured using the Luminex platform.

[0199] IL-23 concentration values ​​are shown as mean ± SD in Figures 7(A) and (B).

[0200] The results in Figure 7(A) show that RA+AhR agonist, RA+TGF-beta, and RA+TGF-beta+AhR agonist DCs treated with IL-23 produced less IL-23 upon LPS activation compared to stimulated control DCs. RA + TGF-beta was superior to RA + TGF-beta + AhR agonist and RA + TGF-beta + AhR agonist in reducing IL-23 production. and RA+AhR agonists.

[0201] The results in Figure 7(B) show a similar pattern to Figure 7(A). In particular, Figure 7(B) shows the effect of RA + AhR agonist DCs treated with RA+TGF beta and RA+TGF beta+AhR agonist showed significantly higher IL-1 expression than the corresponding stimulated control DCs. DCs treated with TGFbeta or AhR agonists as single agents, or TGFbeta + AhR agonists IL-23 production upon stimulation with an inflammatory cytokine cocktail compared with DCs treated with nitin This indicates that there was little

[0202] Example 8 - Effect of various compounds on DC expression of CD103 One of several factors that contribute to immune tolerance in DCs is the highly expressed marker CD103. Various compounds were tested for their ability to induce a proinflammatory phenotype. Control DCs were treated with GM-CSF and IL-4 By culturing for 7 days at 4°C, CD14 + Control DCs were differentiated from monocytes. TolDCs, or their less optimal counterparts, were stimulated with LPS added to the eye to generate immunogenic control DCs. The mutants were cultured with GM-CSF and IL-4 as described above and treated with various immunizing agents, either alone or in combination. Tolerogenic compounds: 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 10 ng / m on day 3 1 ng / ml TGF-beta (Fig. 8(A)) or 20 ng / ml TGF-beta (Fig. 8(B)), and on day 6, treatment with 2 μM RA. By doing so, CD14 + The monocytes were differentiated to produce the claimed compounds, as shown in Figures 8(A) and (B). For comparison, TolDCs were treated with 100 nM Dex and 100 nM V on day 3. On day 6, some of the tolDCs were treated with LPS and expressed On day 7, all cells were harvested and fluorescently labeled. Cell surface expression was stained using an anti-CD103 antibody, followed by flow cytometry. An analysis was conducted.

[0203] The MFI values ​​of live cells are shown as mean ± SD in Figures 8(A) and (B).

[0204] The results in Figure 8(A) show that DCs treated with RA + AhR agonist and RA + TGF beta + AhR agonist Higher than RA, TGFbeta, RA+TGFbeta, control DC and Dex / VitD3 treated comparator tolDC Treatment with RA + TGF-beta + AhR agonist resulted in C It was superior to treatment with RA plus AhR agonist in inducing D103 expression.

[0205] The results in Figure 8(B) show that the RA + TGF-beta + AhR agonist treatment, regardless of whether they were stimulated with LPS, DCs treated with Dex / VitD3 expressed higher levels of CD103 than control DCs and Dex / VitD3-treated comparator tolDCs. Treatment with RA, TGF-beta, and AhR agonist induced CD103 expression. was superior to treatment with RA plus TGF-beta in

[0206] Example 9 - Effect of various compounds on DC expression of MERTK, BTLA, LAP, HLA-G and CD49b Induce a tolerogenic phenotype in DCs consisting of the expression of various tolerogenic markers Various compounds were tested for their ability to induce inflammatory responses in DCs. Control DCs were cultured with GM-CSF and IL-4 for 7 days. From CD14 + Control DCs were either unstimulated or stimulated with LPS added on day 6. TolDCs were cultured with GM-CSF and IL-4 as described above to generate immunogenic control DCs. )–(E) various tolerogenic compounds: 20 nM AhR agonist C1 (AhR agonist C1) on days 0 and 3; ag), 10 ng / ml TGF-beta on day 3 (for MERTK and LAP, Figures 9(A), (C), and (E)) or 20 n g / ml TGF-beta (for BTLA and HLA-G; Figures 9(B) and (D)), and treatment with 2 μM RA on day 6. By doing so, CD14 + TolDCs were differentiated from monocytes. For comparison, TolDCs were treated with 100 nM Dex on day 3. On day 7, all cells were harvested and fluorescently analyzed. Cell surface expression was examined using labeled anti-MERTK, anti-BTLA, anti-LAP, anti-HLA-G, and anti-CD49b antibodies. The cells were stained with HCl and subsequently analyzed by flow cytometry.

[0207] The MFI values ​​of live cells are shown as mean ± SD in Figures 9(A) to (D).

[0208] The results showed that DCs treated with RA+TGF beta and RA+TGF beta+AhR agonist showed significantly higher levels of erythrocyte proliferation than control DCs and De x / VitD3-treated comparator tolDC showed higher levels of MERTK (Fig. 9(A)), BTLA (Fig. 9(B)), and LAP In all cases, RA + TGF-beta + AhR agonist expression was observed (Figure 9(C)). Treatment with RA+TGF-beta was superior to treatment with RA+TGF-beta. Dex / VitD3-treated DCs showed significantly higher inflammatory responses than control DCs and Dex / VitD3-treated DCs, as shown in Figures 9(D) and (E), respectively. Compared with rat DCs, they expressed higher levels of HLA-G and CD49b.

[0209] Example 10 - Effect of various compounds on DCs and their ability to induce Tregs A tolerogenic phenotype in DCs that can translate into enhanced induction of Treg cells during T cell / DC coculture Various compounds were tested for their ability to induce inflammatory cytokines. Control DCs were cultured with GM-CSF and IL-4 for 7 days. By CD14 + Control DCs were either unstimulated or added on day 6. TolDCs, or their less optimal variants, were stimulated with LPS to generate immunogenic control DCs. Cultured with GM-CSF and IL-4 as described above, the cells were treated with various tolerogenic compounds as shown in Figures 10(A) and 10(B). 20 nM AhR agonist C1 (AhR ag) on ​​days 0 and 3, 20 ng / ml TGF-beta on day 3, and and on day 6, treatment with 2 μM RA + Differentiated from monocytes. TolD for comparison. C was established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. Some tolDCs were then treated with LPS to test for phenotypic stability (Fig. 10(A); black bars). Cells were collected on day 7 and CD4 + Co-cultures with T cells were set up in an MLR setting at a T cell / DC ratio of 10:1. At the end of the first 7 days of culture, T cells were washed and incubated in medium containing IL-2 for an additional 7 days. Then, all the cells were collected and analyzed using fluorescently labeled anti-CD4 and anti-CD25 antibodies. Cell surface expression and intracellular expression of Foxp3 were stained using a fluorescently labeled anti-Foxp3 antibody. staining and subsequent analysis by flow cytometry.

[0210] CD4 + CD25 in live T cells 高 Foxp3 + The frequencies (%) of these are shown in Figures 10(A) and (B) as mean ± SD.

[0211] The results in Figure 10(A) show that the effect of RA + TGF-beta + AhR agonist on the proliferation of RA, regardless of whether it was stimulated with LPS, was not observed. and the enhanced ability of RA+TGFbeta-treated DCs to promote Treg induction compared with matched control DCs. Treatment with RA, TGF-beta, and AhR agonist resulted in the induction of Tregs. was superior to treatment with RA plus TGF-beta in

[0212] The results in Figure 10(B) show that RA+TGFbeta+AhR agonist-treated DCs were significantly superior to control DCs and Dex / VitD3-treated DCs. Enhanced Treg induction compared with para- and TGFbeta+AhR agonist-treated DCs. Furthermore, RA+AhR agonist-treated DCs were significantly more potent than control DCs and had an enhanced ability to induce Tregs compared with TGFbeta + AhR agonist-treated DCs Treatment with RA + TGF-beta + AhR agonist was significantly superior to treatment with RA + AhR agonist in the induction of Tregs. It was better than logic.

[0213] Example 11 - CD83, CD86, ILT3, ILT3 / CD86, GARP+CD141+, CD103, LAP, IL-23 and CD4 RA+TGF-beta+AhR agonism on DCs derived from the blood of hemophilia A patients in association with 9b+LAG3+Tr1 cells (impact of strike) The AhR agonist mentioned in the examples is C1. High expression levels of the costimulatory molecule CD86, high expression levels of the immune tolerogenic marker ILT3, and ILT3 / CD86 expression Tolerogenicity index, defined as the expression of CD141 and GARP, CD103 expression, and LAP expression. , low spontaneous production of the cytokine IL-23, reduced ability to induce T cell proliferation and Treg cell generation The ability to induce a tolerogenic phenotype in DCs is depicted herein by enhanced induction of For this, the combination of RA + TGF beta + AhR agonist was tested. Control DCs were treated with GM-CSF and CD14 from healthy donors or subjects with hemophilia was cultured in IL-4 for 7 days. + From monocytes TolDCs, or their less optimal variants, were differentiated in GM-CSF and IL-4 as described above. and treated with a combination of RA, TGF-beta, and AhR agonist, as shown in Figure 11(A)-(J). By doing so, CD14 + Differentiated from monocytes.

[0214] The MFI values ​​of live cells are shown as mean ± SD in Figures 11(A) to (G).

[0215] IL-23 concentration values ​​are shown as mean±SD in FIG. 11(H).

[0216] T cell proliferation, assessed as counts per minute (CPM) values ​​from triplicate or more samples, was measured as the mean. The values ​​are shown in Figure 11(I) as ±SD.

[0217] CD4 + CD49b + LAG3 + The frequency (%) of Tr1 cells is shown as mean ± SD in Figure 11(J).

[0218] These results suggest that RA+TGF-beta+ cells generated from CD14+ monocytes isolated from the blood of hemophilia patients AhR agonist-treated DCs induced immune responses similar to those obtained with DCs generated from healthy donor blood. These results further demonstrate that hemophilia patients acquire a tolerogenic phenotype and function. RA+TGFbeta+AhR agonist-treated DCs generated from CD14+ monocytes isolated from the blood of healthy subjects DCs generated from normal blood-derived cells showed the same reduced T cell proliferation induction ability and increased regulatory T cell generation. Demonstrate that this shows addition.

[0219] Example 12 - Viability experiments of DCs treated with RA + TGFbeta + AhR agonist The AhR agonist mentioned in the examples is C1. RA + TGF beta + AhR agonist-treated DCs were generated. The DCs were then analyzed for their viability before freezing / thawing, the expression level of the DC maturation marker CD83, and the tolerogenic marker The expression level of ILT3 and the expression level of the costimulatory molecule CD86 are defined as ILT3 / CD86 expression. The tolerogenic index, LAP expression, and CD103 expression were tested. DCs were cultured in conventional freezing medium. Control DCs were frozen in either GM-CSF or CryoStor10 (CS, Biolife solutions). and IL-4 for 7 days. + Differentiated from monocytes.

[0220] The survival rates before and after freezing are shown as mean ± SD in Figure 12(A).

[0221] MFI values ​​in live cells for CD83, ILT3, CD86, LAP, and CD103 are shown as mean ± SD. This is shown in Figures 12(B) to (D) and (F) to (G).

[0222] The tolerogenic index, defined as the ILT3 / CD86 expression (MFI) ratio, is shown in Figure 12(E).

[0223] RA+TGFbeta+AhR agonist-treated DCs frozen in CS10 compared to conventional freezing medium (conv) 1 is a table showing the survival rate and recovery rate of the 100 cells / ml ... [Table 1]

[0224] The results presented in the table above show that cryopreservation and thawing of RA+TGF beta+AhR agonist-treated DCs DCs treated with RA + TGFbeta + AhR agonist resulted in significant cell survival compared to control DCs. These results also demonstrate that RA + TGF-beta + AhR induces a higher survival rate. Cryopreservation and thawing of agonist-treated DCs resulted in similar expression of surface markers as before freezing. Using CS10, cells were cryopreserved in a RA+TGF-vector medium compared to those cryopreserved in standard freezing medium. These results demonstrate that AhR agonist-treated DCs are better able to recover and survive. .

[0225] Example 13 - CD83, CD86, ILT3, ILT3 / CD86, CD141+GARP+, CD103, T cell proliferation and CD25 高 Effects of various compounds on DCs associated with Foxp3+ Treg cells Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs. C, or its less optimal variants, was cultured with GM-CSF and IL-4 as described above, either alone or in combination with IL-4. Various tolerogenic compounds in combination: 10 nM AhR agonist C on days 0 and 3; 1 (AhR ag), 10 ng / ml TGF-beta on day 3, and 2 μM RA on day 6. CD14 + The synergistic effect of the claimed combination was demonstrated by differentiation from monocytes, as shown in Figure 13. Control DCs were cultured with GM-CSF and IL-4 for 7 days to induce CD14 + Differentiated from monocytes DCs were either unstimulated (gray bars) or stimulated with LPS added 2 hours after RA addition on day 6 (gray bars). (black bars), immunogenic control DCs were generated and tested for phenotypic stability. Cells were collected and stained for cell surface expression using fluorescently labeled antibodies, followed by flow cytometry. Analysis was performed using a tree.

[0226] The MFI values ​​of live cells for CD83, CD86, ILT3, and CD103 are shown in Figure 13 (A) as mean ± SD. ) to (C) and (E).

[0227] The tolerogenic index, defined as the ILT3 / CD86 expression (MFI) ratio, is shown in Figure 13(D).

[0228] CD141 + GARP + The frequency (%) of double-positive cells is shown as the mean ± SD in Figure 13(F).

[0229] T cell proliferation, assessed as counts per minute (CPM) values ​​from triplicate or more samples, was measured as the mean. The values ​​are shown in Figure 13(G) as ±SD.

[0230] CD4 + CD25 + Foxp3+ The frequency (%) of Tregs is shown as mean±SD in FIG. 13(H).

[0231] These results, as seen in Figure 13C, show that RA-treated DCs exhibited higher ILT compared with control DCs. 3 expression and higher frequency of GARP + CD141 + RA-treated DCs were significantly more potent than control DCs. Induce less T cell proliferation and more Tregs compared to IgG, Figures 13G and H.

[0232] AhR agonist-treated DCs showed significantly increased IL-1 expression compared to control DCs, as seen in Figures 13A, B, and E, respectively. AhR agonist-treated DCs have lower CD83 and CD86 expression and higher CD103 expression. Compared to DCs, without stimulation with LPS, there was slightly more T cell proliferation and slightly more Tregs. Induce (Figures 13(G) and (H)).

[0233] TGF-beta-treated DCs had a slightly higher tolerogenic ratio (ILT3 / CD86) and Furthermore, TGF-beta-treated DCs showed less CD103 expression compared to control DCs (Fig. 13(D-E)). Induce no T cell proliferation and more Tregs (Figures 13(G)-(H)).

[0234] RA+TGFbeta-treated DCs showed lower CD83 and CD86 expression and higher CD86 expression compared to control DCs. RA+TGFbeta-treated DCs also showed ILT3 expression compared to control DCs (Figures 13A, B, and C, respectively). and higher frequency GARP + CD141 + RA+TGF-beta treated DCs were found to have the same phenotype as the combined DCs (Fig. 13(F)). When used in combination, they did not induce T cell proliferation as much as when compared individually (Figure 13(G)). In summary, TGF-beta potentiates the tolerogenic effects of RA.

[0235] RA+AhR agonist-treated DCs showed lower CD83 and CD86 expression and more CD86-dependent markers than control DCs. RA+AhR agonist treatment D shows higher ILT3 and CD103 expression, respectively (Fig. 13A, B, C, and E). DC also showed higher GARP activity compared with control DC. + CD141 + The frequency of cells is high, but RA+TGF-beta or is not as high as that of RA+TGF-beta+AhR agonist-treated DCs (Fig. 13(F)). C, when used in combination, do not induce T cell proliferation as significantly as compared to each other (Figure 13(G)). In summary, AhR agonists enhance the tolerogenic effects of RA.

[0236] The use of AhR agonists in combination with RA and TGF-beta for the generation of tolerogenic DCs is more This results in a high tolerogenicity ratio and higher CD103 expression (Figures 13(D) and (E)). When A + TGF beta + AhR agonist was used, Treg induction was slight compared to RA + TGF beta. very high (Figure 13(H)).

[0237] Example 14 - RA+TG vs DC and their ability to suppress tetanus toxoid T cell proliferation (effect of Fbeta + AhR agonist) The AhR agonist referred to in the examples is C1.

[0238] Additional Materials and Methods Generation of TT-loaded dendritic cells (DCs) CD14 + Monocytes were cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 ng / ml; PeproTec h, London, UK) and interleukin-4 (IL-4; 100 ng / ml; PeproTech) in the presence of HEP ES, GlutaMAX, and penicillin-streptomycin solution (Thermo Fisher, Waltham, MA) 1.25 × 10 6 cells / ml Cells were replenished with fresh medium and cytokines on day 3. Control DCs were treated with additional compounds. On day 6, the cells were differentiated with GM-CSF and IL-4 without any addition of TNF-α. pha (10 ng / ml; PeproTech), IL-1 beta (10 ng / ml; PeproTech), and prostaglandins. 4 hours before treatment with a proinflammatory cytokine cocktail consisting of Prostaglandin E2 (PGE2; 1 ug / ml; Sigma) TT (30 nM) was added to the cultures to generate immunogenic DCs. TolDCs were generated as previously described. , generated by treatment with the tolerogenic compound AhR agonist TGF beta 1 and RA. For loaded cells, TT (30 nM) was added to the cultures on day 6, 2 hours after RA addition. On day 7, DCs were collected, washed extensively, and subjected to phenotyping and functional assays.

[0239] Freezing and thawing of PBMCs PBMCs were resuspended in freezing medium containing 10% DMSO and transferred to cryovials. The vials were placed in a freezing container, which was then placed in a -80°C freezer. The freezing container was frozen at a rate of approximately -1°C / min. After 24 hours in a -80°C freezer, the cells were transferred to a cryovial for long-term storage. The vials were transferred to a -150°C freezer. PBMCs were thawed in a vial in a 37°C water bath and diluted with 10% FCS. Transfer the cell suspension carefully into a 15 ml tube containing cold RPMI supplemented with DNase A and rinse 1 min before use. Washed twice.

[0240] Autologous DC / T cell culture To analyze TT-specific T cell responses, DCs were loaded with 30 nM TT and treated with TNF-α, IL-1β, and PGE2. and IL-6-matured DCs (mDCs), TT-loaded DCs, and RA+TGF beta+AhR agonist-treated DCs were mixed at 10:1 mDCs were cultured with autologous T cells for 6 days at a T cell:DC ratio of 0.01 to 0.01. DC / T cell cultures were incubated in complete medium: OpTmizer™ T Cell Expansion Medium along with OpTmizer™ T Cell Expansion Medium, which was used as a control. Supplement (Thermo Fisher) and CTS with penicillin-streptomycin solution (commercial). The cells were grown in OpTmizer™ T cell proliferation medium. Expansion was continued for the final 18 hours of culture. 3 H-Chimiji The uptake of ATP was determined by the

[0241] RA+TGF-beta+AhR agonist-treated DCs were loaded with TT and co-cultured with autologous TT-loaded mDCs and T cells. Furthermore, TT-specific effects in T cell proliferation induced by RA + TGF-beta + AhR agonist-treated DCs were observed. The specific reduction of RA, TGF-beta, and AhR agonist-treated DCs was investigated. The other was used as a control.

[0242] T cell proliferation assessed as counts per minute (CPM) values ​​from triplicate samples is shown as mean ± SD. This is shown in Figure 14.

[0243] These results suggest that TT-loaded RA+TGFβ+AhR agonist-treated DCs induce TT-specific ATP-dependent responses in mDCs. We demonstrate that IL-16 inhibits target T cell proliferation.

[0244] Example 15 - CD83, LAP, CD103, ILT3, CD86, ILT3 / CD86-associated DCs after CD40L treatment Effects of RA + TGF-beta + AhR agonist on T cell proliferation and T cell proliferation Additional Materials and Methods Phenotypic stability and sustained tolerogenicity after CD40L stimulation On day 7, the addition of CD40L (100 ng / ml) after harvesting enhanced the phenotypic stability and proliferation of TolDCs. The DCs were stimulated for 24 hours, after which they were harvested and thoroughly washed. The cells were purified and subjected to phenotyping and functional assays.

[0245] These results suggest that RA+TGFβ+AhR agonist-treated DCs were stable after stimulation with CD40L. These results demonstrate that DC:T cell contact mimics the phenotype (Figures 15(A)-(F)). Furthermore, RA+TGFbeta+AhR agonist-treated DCs after stimulation with CD40L were significantly more potent than unstimulated RA+TGF demonstrate that beta + AhR agonist-treated DCs have the ability to induce T cell proliferation equivalent to that of AhR agonist-treated DCs ( In conclusion, these results suggest that RA+TGFbeta+AhR agonist-treated DCs express CD40L. We demonstrate that the cells exhibit a sustained phenotype and tolerogenicity after stimulation with .

[0246] Example 16 - Incorporation of antigens of various sizes and maintaining a stable tolerogenic phenotype Effect of RA + TGF-beta + AhR agonist on DCs for The AhR agonist referred to in the examples is C1. Additional Materials and Methods Antigen loading of DCs On the 3rd, 6th, or 7th day of culture, 100 nM FITC-labeled dextran, 1-10 μg / mL AF488 labeled dextran, and 10 μg / mL AF488 labeled dextran were added. KLH, 100 nM AF488-labeled tetanus toxoid (TT), or 10–100 nM FVIII were used for antigen challenge. To assess antigen uptake, DCs were added to cultures of RA+TGFbeta+AhR agonist-treated DCs. For FVIII detection, cells were incubated with FVII after fixation and permeabilization. DC phenotype analysis was performed by incubating the cells with 60 nM FVII for 2, 4, 8, or 20 hours. The percentage of antigen-positive cells was compared with that of DCs cultured without antigen, RA+TGFbeta+Ah. R agonist-treated DCs were compared.

[0247] FITC-dextran, AF488-KLH on day 7 after incubation with fluorescently labeled antigen from day 3 to day 7 or AF488-TT-positive CD11c+RA+TGFbeta+AhR agonist-treated DCs (Fig. 16(A)). For the incorporation, RA+TGFβ+AhR agonist-treated DCs were cultured for 10, 30, or 4 days from day 3 to day 7. On day 7, the cells were stained for CD11c and then cultured in the presence of 100 nM FVIII (Fig. 16(B)). After permeabilization and fixation, the cells were analyzed by FITC-labeled FVIII-specific antibody (Sanquin, the Netherlands). This makes it possible to perform intracellular staining.

[0248] RA+TGFbeta+AhR agonist-treated DCs were cultured in the presence of 60 nM FVIII for 2, 4, 8 or 20 days on day 7. The cells were cultured for 1 hour, and the phenotype of the cells in the presence of FVIII was determined (FIGS. 16(C) to (I)).

[0249] These results suggest that RA+TGFbeta+AhR agonist-treated DCs are able to differentiate into various sizes and chemical classes. These results also demonstrate that RA+T has the ability to incorporate a diverse range of antigens. GFβ + AhR agonist-treated DCs have the ability to take up antigen at different time points in culture and We demonstrate that the cells have a stable phenotype after FVIII challenge at a given time point.

[0250] Example 17 - DCs affect Bregs, B and T cell levels, T cell proliferation and activation Effect of RA + TGF-beta + AhR agonist on The AhR agonist referred to in the examples is C1.

[0251] Additional Materials and Methods B cell / T cell / DC culture: Breg induction On day 7, autologous B cells were isolated by negative selection using a human B cell isolation kit (StemCell technologies). Cells were isolated from PBMCs. 40,000 B cells were cultured with 40,000 autologous T cells and treated as described above. CD4+ T cells were isolated using the EasySep Human CD4+ T Cell Isolation Kit (StemCell technologies) as described above. 10,000 RA+TGFbeta+AhR agonist-treated or control DCs were generated in 1000 cells. The cells were stimulated with 0.25 μM CpG-OGN to enhance the response. Breg induction was assessed by intracellular IL-10 staining, KI67 T cell proliferation was determined by staining, and T cell activation was assessed by CD154 expression. Staining for IL-10 (JES3-10) from Biolegend, KI67, and CD154 was performed using the following fluorescently labeled antibodies: IL-10 (JES3-10) from Biolegend, KI67, and CD154. 9D7) and KI67 (B56) and CD154 (TRAP1) from BD as described above.

[0252] These data suggest that IL-10 expression after co-culture of autologous DCs, B cells, and T cells + CD19 + Determined as a cell The frequency of Bregs was significantly increased in the RA+TGF-treated group compared to the control DC co-culture, as shown in Figure 17(A). We demonstrate that autologous DCs, B cells, and T cells are increased in the presence of DCs treated with AhR agonists. After co-culture with KI67 (Fig. 17(B)) and CD154 (Fig. 17(C)), T cell proliferation and activation were observed, respectively. It was determined by evaluation.

[0253] Bregs are immunoregulatory cells that have the ability to suppress the activity of other immune cells. Induction of Bregs activates another level of immune suppression, Brems can function in synergy with Tregs. These results suggest that B cells and T cells interact synergistically. RA+TGFβ+AhR agonist-treated DCs cultured together showed significantly higher cellular proliferation compared to control DCs. Furthermore, these results demonstrate that B cells are more likely to produce IL-10 than Bregs. RA+TGFbeta+AhR agonist-treated DCs cultured with blastocysts and T cells showed increased cytotoxicity compared with control DCs. Demonstration that it does not significantly induce T cell proliferation and activation. indicates induction of regulatory cells, Bregs, Tregs, or both.

[0254] Example 18 - RA+TGFbeta+AhR on DCs derived from donor blood when cultured with allogeneic PBMCs agonist effects) DCs generated from monocytes isolated from four healthy donors (designated AD) were incubated with RA, TGF-beta, and Ah. RA + TGFbeta + AhR agonist-treated DCs expressed low levels of CD83 and and CD86, high expression of ILT3, CD103, and LAP, and high ILT3 / CD86 expression The expression profile was the same as that described in the previous example, which was a virulence index.

[0255] RA+TGF-beta+AhR agonist-treated DCs from donors A to D were cultured with allogeneic PBMCs. For example, DCs from donor A were cultured with PBMCs from donors B, C, and D in an MLR. R agonist-treated DCs have different abilities to induce T cell proliferation when co-cultured with allogeneic PBMCs. Furthermore, RA+TGFbeta+AhR agonist-treated DCs inhibited MHC with allogeneic PBMCs, Figure 18(A). They had different abilities to induce Tregs in the LR, FIG. 18(B).

[0256] Evaluated as the frequency of KI67+ cells for RA+TGF-beta+AhR agonist-treated DCs and control DCs The resulting T cell proliferation is shown in Figure 18(A).

[0257] Tregs (CD4 + CD25 + Foxp3 + )of The frequency (%) is shown in Figure 18(B).

[0258] These results indicate that the majority of donor RA+TGFbeta+AhR agonist-treated DCs co-expressed with allogeneic PBMCs. We demonstrate that co-culture of IgG1 with IgG1 reduced T cell proliferation and increased Treg induction. Furthermore, these results suggest that the level of mismatch, which can be assessed by HLA typing, Affects the outcome of the tolerogenic response induced by A+TGF beta+AhR agonist-treated DCs This shows that it can have an impact.

[0259] Example 19 - Clinical Trial Study The study will include two periods: an initial dose escalation portion (Part 1) and an expansion study portion (Part 2).

[0260] First Study Part (Group 1): The first part of the trial will evaluate the safety of escalating doses. Additional subjects may be included during safety reviews by the Internal Monitoring Board (IMB). Patient 1 will receive three doses of cells, spaced two weeks apart. IMB will be available to administer the final dose to Patient 1. Patient 2 will receive the final dose after Patient 1 has received the final dose. The patient will receive the first dose of cells at least two weeks after the initial administration, followed by two more doses at two-week intervals. Patient 3 will receive their first dose at least 2 weeks after the second patient's last dose, and will receive their first dose at least 2 weeks after the second patient's last dose. Patient 4 will receive two more doses at intervals of at least 2 weeks after the last dose of patient 3. The patient receives an initial dose, followed by two more doses at two-week intervals. If this is not reached, the patient will receive all harvested cells and another patient will be enrolled in the study.

[0261] Expansion Part 2 (Group 2): Eight subjects. After completion of the dose-escalation part (Part 1), the expansion Part 2 of the study will begin. IMB will be administered to the group after the last dose before proceeding to Part 2 of the expanded study (Group 2). Once the safety of the increasing doses administered to study subjects in Group 1 is established, Group 1 will be evaluated. Eight patients in loop 2 (numbers 5, 6, 7, 8, 9, 10, 11, and 12) received the maximum RT-PCR determined in group 1. The highest tolerated dose obtained without exceeding the highest tolerated dose determined in Group 1 The production yield is given by intravenous injection three times every two weeks.

[0262] Procedure for generating and transporting active material to each study clinic: Peripheral blood mononuclear cells were used to generate the active material. Fresh leukapheresis material should be collected from the patient via leukapheresis. Patients should be sent to the P facility (Radboud University Medical Center, Nijmegen, Netherlands). There, monocytes are enriched and cultured with a mixture of compounds that cause the monocytes to differentiate into dendritic cells. A tolerance-inducing cocktail of idogens then converts these dendritic cells into tolerogenic dendritic cells. These are then transfected with recombinant FVIII (Kovaltry®, Octocog alpha) as a final step. The cells are then cryopreserved and shipped back to each study site.

[0263] Administration route: Intravenous administration of cells. The frozen cell suspension is thawed and administered to each clinical site. It is administered directly intravenously at the

[0264] When patients develop antibodies to FVIII, the first treatment option is ITI therapy with high doses of FVIII. However, ITI is not always successful. This is usually due to the need for daily injections. It is a very expensive treatment that requires more than a year and fails in one-third of patients (Aledort 2019, Carcao 2019, Lacroix-Desmazes 2020, Ljung 2019). This is especially burdensome for very young children, who are more likely to develop inhibitors. Increased morbidity and medical costs associated with adverse events (such as bleeding into joints and muscles) (D'Angiole lla Reference 2018, CDC (Centers for Disease Control and Prevention) tion)) June 3, 2019) highlights the urgency of identifying plans to prevent blockages from occurring. can be.

[0265] The Phase I / IIa study of Idogen involves the development of an inhibitor of FVIII, an active FVIII inhibitor, HA studies that have an immune response and are not suppressed by established treatment protocols for ITI This patient category includes patients undergoing treatment with anti-FVIII drugs to eradicate inhibitors to FVIII. No other standard treatment alternatives remain. The risks of this trial are unknown, but there is a possibility of developing autoimmune tolerance. No serious safety issues have been reported from published clinical trials with inflammatory dendritic cell therapy. (Bell 2017, Benham 2015, Dhodapkar 2001, Dhodapkar and Steinm 2002, Giannoukakis 2011, Harry 2010, Hilkens and Isaacs et al. 2013, Jauregui-Amezaga 2015, Joo 2014, Ten Brinke 2015, Thomas 2011, Willekens and Cools 2018, Willekens 2019, Zubizar Reta 2019), the cells exhibit a tolerogenic phenotype in in vitro experiments (Lee 2019). 2016, Gordon 2014, Lutz 2000, Steinbrink 1997, Bartosik-Psu Jek (2010), Huang (2001), Hussien (2001), Bellinghausen (2012), Boks 2012, Chu 2012, Raich-Regue 2012, Saito 2011). Nevertheless, serious adverse events suspected to be related to treatment may occur. It cannot be excluded. An alternative to antihistamine prophylaxis is the administration of ItolDC-028. These are also given to patients.

[0266] Potential risks in studies involving subjects with bleeding diathesis include blood collection, leukocyte apheresis, However, with experienced hands, bleeding after puncture is unlikely to occur. This is rare and should be managed by personnel experienced in managing patients with bleeding disorders. If the puncture is traumatic, digital pressure or a pressure dressing at the puncture site may be necessary. Subcutaneous, intradermal, and small intramuscular injections are administered consistently and may prevent other complications. If acupressure is maintained for at least 5 minutes, hematomas rarely occur (Powell and Ro dgers et al. 2013 ).

[0267] Example 20 - Blood samples from patients with autoimmune diseases associated with IL-10's B cell production and T cell regulatory functions Effects of RA+TGF-beta+AhR agonist on DCs derived from rhesus mast cells RA+TGFbeta+AhR agonist-treated DCs were challenged with one or several well-characterized antigens. Generated from monocytes from patients with autoimmune diseases. RA + TGF-beta + AhR agonist The treated DCs are loaded with one or several well-characterized antigens. The DCs are then transfected with autologous T cells. T cells may be co-cultured with control DCs loaded with the same antigen. Furthermore, DCs and T cells treated with RA, TGF-beta, and AhR agonist were less activated than DCs and T cells treated with RA, TGF-beta, and AhR agonist. In co-culture with DCs, a higher frequency of T cells was observed compared with T cells co-cultured with control DCs under the same conditions. A high degree of regulatory T cells will be induced.

[0268] RA+TGF-beta+AhR agonist-treated DCs generated from patients with autoimmune diseases were co-cultured with B cells. B cells will have a higher frequency of regulatory markers such as IL-10.

[0269] RA+TGF-beta+AhR agonist-treated DCs generated from patients with autoimmune diseases were used to stimulate B and T cells. T cells showed increased regulatory activity compared to B and T cells co-cultured with control DCs. It will have the function.

[0270] Tolerogenicity of both T and B cells after co-culture with RA+TGF beta+AhR agonist-treated DCs The induction of phenotype and function is important because these cells reduce T and B cell autoreactive activity. , beneficial in autoimmune diseases.

[0271] Example 21 - Involving the B cell production and T cell regulatory functions of IL-10 in patients with autoimmune diseases DCs derived from the blood of patients treated with several unknown and / or complex antigens or tissue samples Effect of RA + TGF-beta + AhR agonist on RA+TGFbeta+AhR agonist-treated DCs were used to express several unknown and / or complex antigens. DCs are generated from monocytes of patients with autoimmune diseases. RA + TGF beta + AhR agonist-treated DCs are challenged with several unknown and / or complex antigens or tissue sample extracts from the same patient, They are then co-cultured with autologous T cells. T cells are then co-cultured with control DCs loaded with the same antigen. Furthermore, RA + TGF-beta + AhR agonist treatment Co-culture of DCs with T cells resulted in higher T cell proliferation compared with T cells co-cultured with control DCs under the same conditions. A high frequency of regulatory T cells will be induced.

[0272] RA+TGF-beta+AhR agonist-treated DCs generated from patients with autoimmune diseases were co-cultured with B cells. B cells will have a higher frequency of regulatory markers such as IL-10.

[0273] RA+TGFbeta+AhR agonist-treated DCs are co-cultured with B cells and T cells. T cells are co-cultured with control DCs. These cells will have increased regulatory function compared to B and T cells co-cultured with IgG.

[0274] Tolerogenicity of both T and B cells after co-culture with RA+TGF beta+AhR agonist-treated DCs The induction of phenotype and function is thought to be a key factor in the development of autoimmune diseases, as these cells attenuate the autoreactive activity of T and B cells. Beneficial in autoimmune diseases.

[0275] Example 22 - Involvement of Breg induction after in vivo administration in patients with autoimmune disease and without antigen Effect of RA + TGF-beta + AhR agonist on DCs derived from the blood of treated patients RA+TGFbeta+AhR agonist-treated DCs were then treated with autoimmune therapy against one or several unknown antigens. DCs are generated from monocytes of patients with HIV. RA + TGF-beta + AhR agonist-treated DCs are generated from monocytes of patients with HIV. DCs are injected in situ without antigen loading or freezing, and take up disease-related antigens in situ. By presenting this antibody to T cells, disease-specific regulatory T cells are induced.

[0276] RA+TGFbeta+AhR agonist-treated DCs from patients with autoimmune diseases were used in one or several DCs were cultured with several unknown antigens. DCs were administered in situ. RA + TGF-beta + AhR agonist treatment DCs induce Bregs, promote tolerance to transplantation, and reduce harmful antibody production. This would occur if the recipient received DCs from an immunogenic donor or This is in contrast to what would happen if they received no DC at all.

[0277] Example 23 - RA+TGFbeta+AhR on FVIII-loaded DCs derived from the blood of a patient with hemophilia A Effects of agonists on T cell activation and Treg induction Hemophilia A patients who are resistant to treatment with exogenous FVIII have FVII that has the ability to activate B cells. Generate FVIII-specific antibodies (inhibitors) to upregulate II-specific effector T cells It has been found that.

[0278] DCs generated from monocytes from hemophilia A patients were treated with RA, TGF-beta, and AhR agonists. DCs were loaded with FVIII according to the method, and treated with RA, TGF-beta, and AhR agonist. In the second study, DCs were co-cultured in vitro with T cells from the same hemophilia patient. Furthermore, RA + TGF-beta + AhR cells will be less activated compared to co-cultured T cells. T cells co-cultured with agonist-treated DCs showed higher cytotoxicity than T cells co-cultured with control DCs under the same conditions. This will induce higher frequencies of regulatory T cells.

[0279] Example 24 - RA+TGFbeta+A on DCs from allogeneic donors in relation to Treg and Breg induction effects of hR agonists) RA+TGFbeta+AhR agonist-treated DCs are generated from allogeneic donor monocytes. +AhR agonist-treated DCs, when administered to transplant recipients, stimulate the production of autoregulatory T cells and transplant recipients. This will induce tolerance to DCs derived from immunogenic donors. This contrasts with what happens if you receive only a DC or no DC at all.

[0280] RA + TGF beta + AhR agonist-treated DCs from the donor are administered to the transplant recipient. GFβ + AhR agonist-treated DCs induce Bregs and further promote transplant tolerance. This will reduce the production of harmful antibodies, which is why the recipient is more likely to develop immunogenic donor-derived antibodies. This contrasts with what happens if you receive DC or no DC at all. be.

[0281] Example 25 - RA on DCs from allograft recipients in relation to Treg and Breg induction +TGF-beta +AhR agonist effects) RA+TGFbeta+AhR agonist-treated DCs were generated from monocytes from allograft recipients. RA+TGFbeta+AhR agonist-treated DCs were treated with donor-derived mAbs before administration to transplant recipients. Such a mixture of antigens can be obtained from donor blood or from a donor. It may be derived from a tissue sample, preferably from donor cells, tissues, organs to be transplanted. The sample may be a liver or other graft sample. RA + TGF beta + AhR agonist-treated DCs are used in transplantation recipes. When administered to patients, it will induce autoregulatory T cells and tolerance to transplantation. This is because the recipient is treated with RA + TGF beta + AhR agonist without donor antigens. This contrasts with what happens if you receive a DC or no DC at all. is.

[0282] RA+TGF-beta+AhR agonist-treated DCs from the recipient were loaded with antigen from the donor. RA + TGF beta + AhR agonist-treated DCs induce Bregs, It may also promote tolerance induction to transplantation and reduce harmful antibody production. If donor antigen-free RA + TGFbeta + AhR agonist-treated DCs were received, or if DCs were not This is in stark contrast to what happens if you don't receive it at all.

[0283] (References) [Table 2]

[0284] (abbreviation) [Table 3]

[0285] Throughout this specification and the claims that follow, unless the context otherwise requires: The word "comprise" and the terms "comprises" and "comprising" Any variation may include a specified integer, step, group of integers or group of steps, but may not include any other It is understood to mean that no integer, step, group of integers or group of steps is excluded. can be.

[0286] All patents and patent applications referred to herein are incorporated by reference in their entirety. There are.

Claims

1. The present invention provides an exogenous method for obtaining tolerogenic antigen-presenting cells capable of inducing tolerance to antigens.

1. An in vivo method comprising: (a) isolating monocytes from a sample obtained from a mammal; and (b) culturing the isolated monocytes in cell culture to produce antigen-presenting cells with a tolerogenic phenotype; inducing differentiation of said monocytes into cells; and wherein the cell culture comprises: (i) retinoic acid and TGF-beta; (ii) retinoic acid, TGF-beta; The method further comprises (iii) a retinoic acid and an AhR agonist or (iv) a retinoic acid and an AhR agonist.

2. The method of claim 1, wherein the cell culture comprises retinoic acid and TGF-beta.

3. 10. The method of claim 1, wherein the cell culture comprises retinoic acid, TGF beta, and an AhR agonist. How to do it.

4. The method of claim 1, wherein the cell culture comprises retinoic acid and an AhR agonist.

5. The AhR agonist is N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1- Any one of claims 1 to 4, which is methyl-2-oxoquinoline-3-carboxamide (IMA-06201). The method described in claim 1.

6. The cell culture further comprises GM-CSF and IL-4 to induce differentiation of monocytes into antigen-presenting cells. The method of any one of claims 1 to 5, further comprising:

7. Any one of TGF-beta, AhR agonist and retinoic acid was added to the cell culture.

7. The method of claim 6, wherein GM-CSF and IL-4 are added to the cell culture before or simultaneously with the addition of the GM-CSF and IL-4.

8. The GM-CSF and IL-4 are first added to the cell culture, and at the same time, the AhR agonists are 8. The method of claim 6 or 7, wherein the agent is added to the cell culture in a first dose.

9. After the first dose of AhR agonist is added to the cell culture, the TGF-beta and and a second dose of an AhR agonist is added to the cell culture. How to post.

10. After the second dose of the TGF beta and the AhR agonist is added to the cell culture, The method of any one of claims 6 to 9, wherein the retinoic acid is added to the cell culture. 。

11. 11. The method of claim 6, wherein the GM-CSF and IL-4 are added to the cell culture in a second dose. or the method described in any one of claims 1 to 5.

12. the second dose of the TGF beta and the AhR agonist is added to the cell culture; 12. The method of claim 11, wherein the second dose of GM-CSF and IL-4 is added to the cell culture at the same time. method.

13. Any one of TGF-beta, AhR agonist and retinoic acid was added to the cell culture. The GM-CSF is added to the cell culture before or at the same time as the first application of the AhR agonist.

7. The method of claim 6, wherein the amount of IL-4 is added to the cell culture after the amount of IL-4 is added to the cell culture. How to do it.

14. After the first dose of AhR agonist is added to the cell culture, the TGF-beta and 14. The method of claim 6 or 13, wherein a first dose of an AhR agonist and a second dose of an AhR agonist are added to the cell culture.

15. After the second dose of the TGF beta and the AhR agonist is added to the cell culture, 15. The method of claim 6, wherein the retinoic acid is added to the cell culture. How to post.

16. 16. The method of claim 6, wherein the GM-CSF is added to the cell culture in a second dose. The method described in claim 1.

17. the second dose of the TGF beta and the AhR agonist is added to the cell culture; 17. The method of claim 16, wherein the second dose of GM-CSF is added to the cell culture at the same time.

18. 18. The method of claim 1, wherein the tolerogenic antigen-presenting cells are dendritic cells. method.

19. The isolated monocytes are CD14 + The method of any one of claims 1 to 18, wherein the cells are monocytes.

20. The method of any one of claims 1 to 19, wherein the mammal is a human.

21. The method of any one of claims 1 to 20, wherein the sample is a sample of peripheral blood mononuclear cells.

22. 22. The method of claim 1, wherein the cell culture comprises the antigen or an epitope-containing fragment thereof. The method according to any one of claims 1 to 5.

23. 23. The method of claim 22, wherein the antigen or epitope-containing fragment thereof is bound to isolated monocytes. How to do it.

24. 23. The method of claim 22, wherein the antigen or epitope-containing fragment thereof is added to the cell culture. method.

25. The antigen or enzyme may be added to the cell culture simultaneously or after the retinoic acid is added to the cell culture.

25. The method of claim 24, wherein the pitope-containing fragment is added to the cell culture.

26. The antigens or epitope-containing fragments may be a pool of antigens and / or epitope-containing fragments thereof.

26. The method of claim 24 or claim 25, wherein the pool is a pool of:

27. 10. The method of claim 9, wherein the antigen or epitope-containing fragment thereof is or is derived from a biologic. The method according to any one of claims 1 to 22 and 24 to 26.

28. 28. The method of claim 27, wherein the biologic is Factor VIII or a derivative or fragment thereof.

29. 28. The method of claim 27, wherein the biologic is Factor IX or a derivative or fragment thereof.

30. 28. The method of claim 27, wherein the biologic is an antibody or an antibody fragment thereof.

31. Claims 1 to 22 and 2, wherein the antigen or epitope-containing fragment thereof is bound to an allograft.

27. The method of any one of claims 4 to 26.

32. The antigen or epitope-containing fragment thereof is an autoantigen or is derived from an autoantigen.

27. The method of any one of claims 1 to 22 and 24 to 26.

33. The tolerogenic antigen-presenting cells are expressed either unstimulated or stimulated by, for example, LPS or inflammatory cytokines. Upon stimulation with immunogenic stimuli such as insulin or inflammatory cytokines, high expression of ILT3 and The method of any one of claims 1 to 32, having low expression of CD83 and CD86.

34. The method of claim 33, wherein the tolerogenic antigen-presenting cells express CD103.

35. The tolerogenic antigen-presenting cells induce regulatory T cells when cultured with T cells. Item 35. The method according to any one of items 1 to 34.

36. The tolerogenic antigen-presenting cells are further cultured with T cells, thereby promoting the production of regulatory T cells. The method of any one of claims 1 to 35, wherein the synthesis is induced.

37. The regulatory T cells are CD4 + CD25 hi Foxp3 + or Tr1 regulatory T cells. The method of claim 36.

38. The tolerogenic antigen-presenting cells are expressed either unstimulated or stimulated by, for example, LPS or inflammatory cytokines. Low T cell proliferation upon stimulation with immunogenic stimuli such as insulin or inflammatory cytokine cocktails The method of any one of claims 1 to 37, which has the ability to induce proliferation.

39. The tolerogenic antigen-presenting cells are expressed either unstimulated or stimulated by, for example, LPS or inflammatory cytokines. Highly regulatable upon stimulation with immunogenic stimuli such as erythropoietin or inflammatory cytokine cocktails The method of any one of claims 1 to 38, having T cell inducibility.

40. Tolerogenicity obtainable or obtained by the method of any one of claims 1 to 35 Antigen-presenting cells or populations thereof.

41. Tolerogenicity obtainable or obtained by the method of any one of claims 1 to 23 Antigen-presenting cells or populations thereof.

42. When unstimulated or when stimulated with, for example, LPS or inflammatory cytokines or inflammatory cytokine clusters Upon stimulation with immunogenic stimuli such as steroids, they express CD103 and have high expression of CD141, GARP, and ILT3. and a tolerogenic antigen-presenting cell or population thereof having low expression of CD83 and CD86.

43. Plus: (i) HLA-G; (ii) BTLA; (iii) MERTK; (iv) LAP 43. The tolerogenic antigen presenting agent of claim 42, characterized by expressing one or more of: A cell or a population thereof.

44. When unstimulated or when stimulated with, for example, LPS or inflammatory cytokines or inflammatory cytokine clusters 42 or 43, which exhibits low production of IL-23 upon stimulation with an immunogenic stimulus such as a steroid. The antigen-presenting cell or population thereof described above.

45. Methods for treating a mammalian subject having or at risk of having an immune response to an antigen A tolerogenic antigen-presenting cell or a tolerogenic antigen-presenting cell according to any one of claims 40 to 44 for use in wherein the population comprises administering to the mammalian subject the tolerogenic antigen-presenting cells. and thereby establishing immune tolerance to said tolerogenic antigen. A presenting cell or population thereof.

46. A method for treating a mammalian subject having or at risk of having an immune response to an antigen. The tolerogenic antigen-presenting cell or population thereof according to any one of claims 40 to 44 is used in the method of the present invention. administering to a mammalian subject, thereby establishing immune tolerance to said antigen. How to write.

47. In a method of treating a mammalian subject having or at risk of having an immune response to an antigen, 1. A tolerogenic antigen presenting cell or population thereof for use in a method comprising: (i) The ability to induce tolerance to the antigen by the method of any one of claims 1 to 35. and tolerogenic antigen-presenting cells obtained from a sample of isolated monocytes from said mammalian subject. or acquiring a group thereof; and (ii) administering the tolerogenic antigen-presenting cells or population thereof back to the mammalian subject. Thus, immune tolerance to the antigen is established. The tolerogenic antigen-presenting cell or population thereof, comprising:

48. A method of treating a mammalian subject having or at risk of having an immune response to an antigen. So, (i) A method according to any one of claims 1 to 35, which has the ability to induce tolerance to the antigen. and tolerogenic antigen-presenting cells or antigen-presenting cells obtained from a sample of isolated monocytes from said mammalian subject. to acquire the group, and (ii) administering the tolerogenic antigen-presenting cells or population thereof back to the mammalian subject. Thus, immune tolerance to the antigen is established. The method comprising:

49. Methods for preventing immune rejection of a donor-derived allograft in a recipient subject - Patent Application 20070122997 42. The tolerogenic antigen-presenting cell or population thereof according to claim 41 for use in The method further comprises the step of: obtaining immune tolerance from monocytes isolated from a sample collected from the donor; and administering progenitor antigen-presenting cells to the recipient subject, thereby inducing tolerance to the allograft. and establishing a tolerogenic antigen-presenting cell or population thereof.

50. Methods for preventing immune rejection of a donor-derived allograft in a recipient subject - Patent Application 20070122997 42. The method of claim 41, wherein the method is obtained from monocytes isolated from a sample taken from the donor. Tolerogenic antigen-presenting cells are administered to the recipient subject, thereby providing immunity to the allograft. establishing tolerance to the antibody.

51. Methods for preventing immune rejection of a donor-derived allograft in a recipient subject - Patent Application 20070122997 42. The tolerogenic antigen-presenting cell or population thereof according to claim 41 for use in The method further comprises the step of: obtaining an immune response from monocytes isolated from a sample taken from the recipient; Tolerogenic antigen-presenting cells are administered to the recipient subject, thereby providing immunity to the allograft. and establishing tolerance to the tolerogenic antigen-presenting cells or population thereof.

52. A method for preventing immune rejection of a donor-derived allograft in a recipient subject.

42. The method according to claim 41, wherein the antibody is obtained from monocytes isolated from a sample taken from the recipient. to the recipient subject, thereby administering to the recipient subject to administer the tolerogenic antigen-presenting cells of establishing tolerance to the fragment.

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