New method

Abstract

In particular, the present invention provides an in vitro method for obtaining tolerogenic antigen-presenting cells (APCs) having the ability to induce tolerance to an antigen in the immune system, the method comprising (a) isolating monocytes from a sample obtained from a mammal; and (b) culturing the isolated monocytes in a cell culture to induce the monocytes to differentiate into antigen-presenting cells having a tolerogenic phenotype, wherein the cell culture comprises (i) retinoic acid and TGFβ, (ii) retinoic acid, TGFβ and an AhR agonist, or (iii) retinoic acid and an AhR agonist.

Description

Field of the Invention

[0001] The present invention particularly relates to an ex vivo method for obtaining tolerogenic antigen-presenting cells (APCs) having antigen tolerance ability, tolerogenic APCs obtained by the method of the present invention, tolerogenic APCs themselves, and their uses and methods for treating unwanted immune responses against antigens and preventing immune rejection of allogeneic transplants. Background of the Invention

[0003] Immunotherapy based on tolerogenic antigen-presenting cells (APCs) that utilize the antigen presentation mechanism in a tolerogenic manner represents a promising non-toxic method for treating immune disorders or preventing rejection of transplants (such as allogeneic transplants). They can be used as a sole therapy or as a supplement to other types of therapies, such as in combination with immunosuppressive drugs or other immunomodulatory therapies. This strategy is based on ex vivo manipulation and introduction of cell therapy products to avoid immune disorders, with the aim of inducing antigen-specific tolerance. Thus, the ultimate goal of this APC-based immunotherapy is to induce tolerance in the form of inhibitory signals delivered to effector cells and to induce and expand regulatory T cells (Tregs) in vivo. For example, patients suffering from autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis may benefit from treatment based on such tolerogenic APC-based therapies.

[0004] Inducing an antigen-specific immune response requires the engagement of professional APCs that express major histocompatibility complex (MHC) molecules as well as membrane-bound co-stimulatory and secreted pro-inflammatory molecules. In addition, such APCs must be able to uptake, process, and present antigens associated with MHC molecules.

[0005] Similarly, the induction of antigen-specific immune tolerance also requires the presentation of antigens in the context of MHC. However, unlike the situation of initiating an immune response, the induction of tolerance requires a combination of low expression of membrane-bound co-stimulatory and secreted pro-inflammatory molecules and high cell surface expression of tolerogenic molecules and secreted anti-inflammatory mediators.

[0006] The main types of professional APCs of the immune system are dendritic cells (DCs), macrophages, certain B cells, and certain activated epithelial cells. In their immature stage, APCs uptake extracellular antigens by phagocytosis or pinocytosis and process the antigens into peptides in endocytic compartments such as endosomes and phagosomes, where the peptides bind to MHC class II molecules. They also have the unique ability to load peptides from exogenous proteins into the class I MHC presentation pathway, a process called "cross-presentation". Given appropriate differentiation signals, APCs can develop into tolerogenic or non-tolerogenic APCs. Tolerogenic APCs are able to mediate the downregulation or prevention of immune responses and are thought to play a key role in maintaining peripheral tolerance.

[0007] Hemophilia A (HA) is an X-linked bleeding disorder caused by mutations in the F8 gene that encodes factor VIII (FVIII) and that interfere with the expression of the translated protein or its procoagulant function. FVIII is mainly expressed in the liver and endothelial vascular bed. Patients with HA lack sufficient procoagulant activity and are prone to bleeding events and their sequelae, including increased morbidity and mortality. The FVIII database currently identifies 2,015 unique FVIII variants based on 5,472 individual case reports. This large number of point mutations (66.5%), deletions (23.2%), and others (duplications, polymorphisms, insertions, indels, and compound) result in a variety of clinical outcomes. Patients can be treated acutely (on demand) or prophylactically with plasma-derived or recombinant FVIII.

[0008] A large number of patients develop neutralizing antibodies against FVIII, called "inhibitors" or anti-drug antibodies (ADA), which block the activity of administered FVIII because their immune system has not fully tolerated certain sequences of normal FVIII. Inhibitor development is currently the most severe and prominent treatment complication seen in HA patients.

[0009] Currently, once inhibitors are formed, the only proven method of eradication is immune tolerance induction (ITI) by frequent high-dose FVIII infusions, but this treatment strategy fails in 20 - 40% of patients.

[0010] The problem of ADA is not limited to hemophilia patients with defective FVIII expression. ADA can develop in subjects treated with other biopharmaceuticals. ADA production is a T cell-dependent process caused by a lack of central T cell tolerance. Tolerance to self-proteins is an important part of immune system development, and proteins encountered later in life are generally considered foreign, depending on the context in which they are presented, ultimately leading to the development of an antibody response.

[0011] The first step in the immune response against FVIII is thought to be the uptake of FVIII by APCs. After APCs endocytose FVIII, FVIII is processed into small peptides, which are loaded onto class II MHC molecules, and then the class II MHC-peptide complex is transported to the cell surface for presentation to FVIII-specific CD4 +T cells. Activation of these T cells requires additional activation signals provided by APCs. These activation signals are membrane-associated interactions between co-stimulatory molecules (such as CD40, CD80, and CD86) on the plasma membrane of APCs and, for example, CD28, CD154, and CTLA-4 on T cells. In addition to these receptor / ligand interactions, APCs signal to T cells through secreted cytokines (such as IL-12 or IL-10). The combination of signals determines the direction of differentiation of the activated T cells. T helper 1 (Th1) cells typically induce cytotoxic immune responses, Th2 cells induce B cell-mediated antibody responses, while Tregs are capable of inducing immunosuppression / tolerance by inhibiting activated B and T cells and through other mechanisms. Ultimately, activated FVIII-specific T cells are able to activate FVIII-specific B cells and induce affinity maturation and class switching of immunoglobulin genes in B cells. As a result, plasma cells secreting anti-FVIII antibodies and circulating FVIII-specific memory B cells are generated, which produce antibodies upon re-exposure to FVIII.

[0012] Autoimmune diseases occur when the specific adaptive immune response is wrongly established against self-antigens. The result is that the effector pathways of the immune system cause chronic inflammatory damage to tissues, which can prove to be fatal. Autoimmunity can be triggered by the activation of self-antigen-specific T cells and by the production of autoantibodies. Specific genes found within the MHC and at other immune regulatory loci play a key role in determining an individual's susceptibility to developing autoimmune diseases, probably because of their ability to regulate the adaptive T and B cell immune responses. T cell responses to self-antigens can cause tissue damage through cytotoxic T cell responses, inappropriate activation of other effector cells, and inappropriate T cell help to B cells. In addition, the immune system's failure to recognize one or more normal components of the body as "self" can lead to the production of pathogenic autoantibodies. Recognition of self-antigens as foreign by autoantibodies causes further immune responses, which result in increased T cell infiltration, production of pro-inflammatory cytokines, and extensive tissue damage. Diseases in which these roles of T cells and B cells may be important include rheumatoid arthritis, type 1 diabetes, and multiple sclerosis.

[0013] Transplantation from genetically unrelated donors of the same species is called allotransplantation. Allotransplantation is considered as the last resort for the treatment of chronic organ failure. Even with the help of advances in organ preservation and immunosuppression, the major complication after transplantation is rejection. Rejection occurs despite pre-transplant tissue typing / blood analysis and occurs to varying degrees in almost all transplant recipients. Except for hyperacute rejection which occurs due to the presence of pre-existing antibodies (caused by pregnancy, blood transfusion, and / or previous transplantation), transplant rejection can be roughly divided into two types; acute and chronic. Acute rejection is considered to be only an immune response, while chronic rejection involves both immune and non-immune mechanisms.

[0014] Alloantigen recognition is the presentation of transplant antigens (alloantigens) and is divided into two major subtypes: direct and indirect. DCs and other professional APCs migrating from the transplant initiate direct alloantigen recognition, in which recipient T cells are directly activated by allo-MHC-associated peptides. Subsequently, recipient APCs take up alloantigens shed from the graft or donor-derived dying cells and present the processed alloantigens to recipient T cells associated with self-MHC (indirect alloantigen recognition). A third subtype, semi-direct alloantigen recognition, has also been proposed, which involves recipient APCs passively acquiring donor MHC onto their cell surface during the process of inspecting the graft called trogocytosis. Naive CD4 + T helper cells are one of the first immune cells activated after transplantation and play a key role in rejection. Activated naive CD4 + T helper cells develop into Th1 (pro-inflammatory) or Th2 (anti-inflammatory) subtypes. Each subtype coordinates a characteristic spectrum of immune responses (each inhibiting the other). In the presence of transforming growth factor β (TGFβ) and IL-6, naive CD4 + T helper cells can differentiate into Th17 cells (a subset of Th cells that secrete IL-17), which are further stimulated by IL-23.

[0015] Ex vivo-generated APCs with appropriate tolerogenic functions can be implemented for the therapeutic treatment of drug responses, autoimmune diseases, and for inducing transplant tolerance. Effective inhibition of harmful immune responses involves tolerance induction in CD4 + and CD8 + T cells. Therefore, it can be expected that ex vivo-generated tolerogenic APCs should have the same characteristics for the treatment of drug responses, autoimmune diseases, and for preventing transplant rejection in vivo.

[0016] The vitamin A metabolite retinoic acid plays important roles in cell growth, differentiation, organogenesis, and reproduction, and plays a key role in mucosal immune responses. It has been reported that retinoic acid enhances Foxp3 +Induce the differentiation of inducible and IL-10-producing Treg cells, and induce gut-homing specificity in T cells (Bakdash et al., 2015). In addition, due to its regulatory activity, retinoic acid has been reported to play an important role not only in the gut but also in controlling inflammatory diseases in other tissues (Oliveira et al., 2018).

[0017] Transforming growth factor β (TGFβ) is a pleiotropic cytokine present in vertebrate and invertebrate organisms, which plays a role in many physiological and pathological processes. TGFβ affects all cells of the immune system, and among the three known TGFβ isoforms, TGFβ1 is the major isoform expressed in immune cells. TGFβ1 is known to play a key role in the function of all immune cells, particularly in the regulation of T cell development and the induction of immune tolerance in DCs. DCs regulate immune function, including immunosuppression, by secreting TGFβ (Esebanmen et al., 2017).

[0018] The aryl hydrocarbon receptor (AhR) is a transcription factor activated by several exogenous and endogenous ligands. Among several physiological roles, AhR contributes to immune homeostasis by promoting immunomodulatory effects. Activation of AhR during DC differentiation and maturation by agonist ligands such as 6-formylindolo[3,2-b]carbazole (FICZ) has been shown to result in increased expression of the enzyme IDO and reduced production of pro-inflammatory cytokines such as IL-6 and TNF-α. Studies have demonstrated that FICZ-treated DCs are able to induce naive T cell differentiation into CD4 + CD25 high FOXP3 + Treg cells, demonstrating that activation of AhR in human DCs promotes a tolerogenic phenotype (Jurado-Manzano et al., 2017).

[0019] There is a need to provide alternative methods for generating tolerogenic APCs with different tolerogenic phenotypes, which can be used to treat unwanted immune responses to biopharmaceuticals such as factor VIII, autoantigens in autoimmune diseases, and alloantigens in transplanted grafts. Summary of the Invention

[0021] The inventors of the present application have surprisingly identified a specific cell culture method, which involves using a specific combination of retinoic acid, TGFβ, and an AhR agonist, which advantageously allows the generation of tolerogenic APCs with a unique profile from a starting cell population of monocytes.

[0022] The inventors have discovered that tolerogenic APCs can be generated by a method comprising culturing monocytes in a cell culture containing a specific combination of components.

[0023] Advantageously, the tolerogenic APCs expected to be obtained by the method of the present invention can be used for targeted immunotherapy, where a mammalian subject has an undesirable immune response to an antigen or is at risk of an undesirable immune response to an antigen. The antigen can be, for example, a biopharmaceutical or an autoantigen. In addition, the tolerogenic APCs expected to be obtained by the method of the present invention can be used to prevent immune rejection of allografts in a recipient subject.

[0024] Thus, in a first aspect of the present invention, there is provided an ex vivo method for obtaining tolerogenic APCs having the ability to induce tolerance to an antigen, the method comprising,

[0025] (a) isolating monocytes from a sample obtained from a mammal; and

[0026] (b) culturing the isolated monocytes in a cell culture to induce the monocytes to differentiate into APCs having a tolerogenic phenotype,

[0027] wherein the cell culture comprises (i) retinoic acid and TGFβ, (ii) retinoic acid, TGFβ and an AhR agonist or (iii) retinoic acid and an AhR agonist.

[0028] In another aspect of the present invention, there is provided tolerogenic APCs or a population thereof obtainable or obtained by the method of the present invention.

[0029] In yet another aspect of the present invention, there is provided tolerogenic APCs or a population thereof, wherein the cells or population express CD103 and have high expression of CD141, GARP and ILT3 and low expression of CD83 and CD86 when unstimulated or when stimulated, for example, with an immunogenic stimulant such as LPS.

[0030] In a further aspect of the present invention, there is provided a method for treating a mammalian subject having an immune response to an antigen or at risk of an immune response to an antigen, comprising administering tolerogenic APCs to the mammalian subject to thereby establish immune tolerance to the antigen.

[0031] In yet another aspect of the present invention, there is provided a tolerogenic APC or a population thereof for use in a method of treating a mammalian subject having an immune response to an antigen or at risk of having an immune response to an antigen, wherein the method comprises (i) obtaining a tolerogenic APC or a population thereof having the ability to induce tolerance to an antigen according to the present invention, wherein the tolerogenic APC or population thereof is obtained from a sample of isolated monocytes derived from the mammalian subject, and (ii) administering the tolerogenic APC or population thereof back to the mammalian subject, thereby establishing immune tolerance to the antigen.

[0032] In another aspect of the present invention, there is provided a tolerogenic APC or a population thereof according to the present invention for use in a method of preventing immune rejection of an allograft in a recipient subject, wherein the allograft is derived from a donor, and the method comprises administering the tolerogenic APC to the recipient subject, thereby establishing tolerance to the allograft, wherein the tolerogenic APC is obtained from monocytes isolated from a sample taken from the donor.

[0033] In yet another aspect of the present invention, there is provided a tolerogenic APC or a population thereof according to the present invention for use in a method of preventing immune rejection of an allograft in a recipient subject, wherein the allograft is derived from a donor, and the method comprises administering the tolerogenic APC to the recipient subject, thereby establishing tolerance to the allograft, wherein the tolerogenic APC is obtained from monocytes isolated from a sample taken from the recipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Expression of the maturation marker CD83 on DCs cultured under various conditions is shown (FIGS. (A) and (B)). DCs were either unstimulated (grey bars) or stimulated with lipopolysaccharide (LPS) (black bars) (see Example 1).

[0035] Figure 2 Expression of the activation marker CD86 on DCs cultured under various conditions is shown (FIGS. (A) and (B)). DCs were either unstimulated (grey bars) or stimulated with LPS (black bars) (see Example 2).

[0036] Figure 3 Expression of the tolerogenic marker ILT3 on DCs cultured under various conditions is shown (FIGS. (A) and (B)). DCs were either unstimulated (grey bars) or stimulated with LPS (black bars) (see Example 3).

[0037] Figure 4 The tolerogenic index, defined as the ILT3 / CD86 expression level (MFI) ratio on DCs cultured under various conditions, is shown. DCs were either unstimulated (FIG. (A)) or stimulated with LPS (FIG. (B)) (see Example 4).

[0038] Figure 5 shows the ability of DCs to stimulate CD4 + T cell proliferation in the mixed lymphocyte reaction (MLR). DCs were either unstimulated (gray bars) or stimulated with LPS (panel (A)) or a mixture of proinflammatory cytokines (panel (B)) before co-culture with T cells (see Example 5).

[0039] Figure 6 shows the frequency of cells co-expressing CD141 and GARP in DCs cultured under various conditions (panels (A) and (B)). DCs were either unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 6).

[0040] Figure 7 shows the production of IL-23 by DCs cultured under various conditions (panels (A) and (B)). DCs were either unstimulated (gray bars) or stimulated with LPS (panel (A)) or a mixture of proinflammatory cytokines (panel (B)) (see Example 7).

[0041] Figure 8 shows the expression of the marker CD103 on DCs cultured under various conditions (panels (A) and (B)). DCs were either unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 8).

[0042] 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).

[0043] Figure 10 shows the ability of DCs to induce CD4 + CD25 hi Foxp3 + Tregs in a time-extended MLR (panels (A) and (B)). DCs were either unstimulated (gray bars) or stimulated with LPS (black bars) before co-culture with T cells (see Example 10).

[0044] Figure 11Shows the expression of the 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 marker CD103 (Figure F), expression level of tolerogenic marker LAP (Figure G), production of IL-23 (Figure H), T cell proliferation (Figure I), and CD4 + CD49b + LAG3 + Induction of Tr1 cells (Figure J) (see Example 11).

[0045] Figure 12 Shows the viability (Figure A), expression of the maturation marker CD83 (Figure B), tolerogenic marker ILT3 (Figure C), activation marker CD86 (Figure D), tolerogenic index defined as the ILT3 / CD86 expression (MFI) ratio (Figure E), expression level of tolerogenic marker LAP (Figure F), and expression of marker CD103 (Figure G) of DCs cultured in the presence of RA + TGFβ + AhR agonist and subjected to freeze / thaw (dark gray bars). The phenotype of the freeze / thawed cells was compared with that of fresh cells (light gray bars) (see Example 12).

[0046] Figure 13 Shows the expression of the 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), expression of marker CD103 (Figure E), frequency of CD141 and GARP co-expressing cells (Figure F), T cell proliferation (Figure G), and CD4 + CD25hiFoxp3 + Generation of Treg cells (Figure H). DCs were unstimulated (gray bars) or stimulated with LPS (black bars) (see Example 13).

[0047] Figure 14 Shows T cell proliferation when DCs cultured in the presence of RA + TGFβ + AhR agonist are loaded with tetanus toxoid and subsequently cultured in the presence of T cells. DCs were unloaded (black bars) or loaded with TT (gray bars) (see Example 14).

[0048] Figure 15Shows the expression of the maturation marker CD83 (Figure A), the expression level of the tolerogenic marker LAP (Figure B), the marker CD103 (Figure C), the tolerogenic marker ILT3 (Figure D), the activation marker CD86 (Figure E) of DCs cultured in the presence of RA+TGFb+AhR agonist and then stimulated with CD40L (black bars) or unstimulated (gray bars), and the tolerogenic index defined as the ILT3 / CD86 expression (MFI) ratio of DCs (Figure F). T cell proliferation induced by unstimulated and CD40L-stimulated DCs treated with RA+TGFb+AhR agonist is shown in (Figure G) (see Example 15).

[0049] Figure 16 Shows the antigen loading of DCs (Figure A-B), the expression of CD11c in DCs cultured in the presence of RA+TGFb+AhR agonist and antigen after antigen loading (Figure C), the expression of the maturation marker CD83 after antigen loading (Figure D), the expression of the activation marker CD86 after antigen loading (Figure E), the expression of the tolerogenic marker ILT3 after antigen loading (Figure F), the expression of the marker CD103 after antigen loading (Figure G), the expression of the tolerogenic marker GARP after antigen loading (Figure H), and the expression level of the tolerogenic marker LAP after antigen loading (Figure I) (see Example 16).

[0050] Figure 17 Shows the induction of B regulatory cells (Figure A), the ability of RA+TGFβ+AhR agonist-treated DCs, B cells and T cells to stimulate T cell proliferation (Figure B), and the ability of RA+TGFβ+AhR agonist-treated DCs, B cells and T cells to induce T cell activation (Figure C) in cultures containing RA+TGFβ+AhR agonist-treated DCs, B cells and T cells.

[0051] Figure 18 Shows the ability of RA+TGFβ+AhR agonist-treated DCs to induce T cell proliferation when co-cultured with allogeneic PBMCs (Figure A), and the ability of RA+TGFβ+AhR agonist-treated DCs to induce Tregs in the mixed lymphocyte reaction (MLR) with allogeneic PBMCs (Figure B). DETAILED DESCRIPTION OF THE INVENTION

[0053] It should be understood that different applications of the disclosed products and methods can be customized according to the specific needs in the art. It should also be understood that the terms used herein are for the purpose of describing particular embodiments of the invention only and are not limiting. In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen" includes two or more such antigens and the like.

[0054] Definitions

[0055] "Monocyte" is a large mononuclear phagocyte of peripheral blood. The size of monocytes is typically in the range of 10 to 30 μm in diameter. The nucleus-to-cytoplasm ratio is typically in the range of 2:1 to 1:1. The nucleus is typically band-shaped (horseshoe-shaped) or kidney-shaped (kidney-shaped). It can fold on top of itself, thereby showing brain-like convolutions. No nucleolus is visible. The chromatin pattern is fine and arranged in a skein-like chain. The cytoplasm is abundant, presenting a blue-gray color, with many fine azurophilic granules, showing a ground-glass appearance in Giemsa staining. Vacuoles may be present. Often, the expression of specific surface markers is used to determine whether a cell is a monocyte. For example, monocytes express CD14 (monocyte marker) and do not express CD1c (DC marker), CD56 (NK cell marker), CD19 (B cell marker), CD3 (T cell marker), CD16b (neutrophil marker), or CD66b (neutrophil marker).

[0056] "Antigen-presenting cell" (APC) is part of a heterogeneous group of immune cells that mediate cellular immune responses by processing and presenting antigens for recognition by certain lymphocytes, such as T cells. APCs display antigens complexed with MHC on their surfaces. T cells can recognize these complexes using their T cell receptors (TCRs). Classical APCs include DCs, macrophages, Langerhans cells, and B cells.

[0057] "Dendritic cell" (DC) is an APC that exists in vivo, in vitro, ex vivo, or can be derived from hematopoietic stem cells, hematopoietic progenitor cells, or monocytes. DCs and their precursors can be isolated from a variety of lymphoid organs (such as the spleen, lymph nodes), as well as bone marrow and peripheral blood. DCs have a characteristic morphology of sheets (lamellipodia) that extend away from the DC body in multiple directions. DCs constitutively express class I and class II MHC molecules, which present peptide antigens to CD8 + and CD4 +T cells. In addition, human skin and mucosal DCs also express genes of the CD1 family, MHC class I-related molecules, which present microbial lipid or glycolipid antigens. The DC membrane is also rich in molecules that allow T cell adhesion (such as intercellular adhesion molecule 1 or CD54) or molecules that co-stimulate T cell activation, such as B7-1 and B7-2 (also known as CD80 and CD86, respectively).

[0058] "Tolerance" is the result of a tolerogenic mechanism of the immune response to a specific antigen, leading to the prevention, silencing, downmodulation, or downregulation of the adaptive immune response to one or more antigens, either directly or indirectly. A substance that can induce tolerance, i.e., a tolerogenic substance, is a substance that regulates APCs (such as DCs) to become tolerogenic, and the resulting tolerogenic DCs can mediate tolerance induction.

[0059] "Tolerogenic" means the ability of a cell or substance to mediate tolerance induction through mechanisms such as deletion, induction of unresponsiveness or anergy, and active inhibition of antigen-specific T cell responses by inducing Tregs. "Tolerization" refers to the induction of tolerance to an antigen in the adaptive immune system, which means that tolerogenic mechanisms have been mediated to induce an immunosuppressive effect.

[0060] "Immunogenic" means a cell or substance capable of directly or indirectly activating an adaptive immune response to an antigen.

[0061] "Tolerogenic APC" means an APC that has acquired the ability to induce tolerance due to exposure to tolerogenic stimuli, which can be of microbial origin, components of mammalian cells, combinations of cytokines, hormones, vitamins, and other biological or pharmaceutical agents. Tolerogenic APCs have a low ability to induce immunogenic responses but have a high ability to induce active Tregs and other tolerance-inducing responses.

[0062] "Tolerogenic DC" means a DC that has acquired the ability to induce tolerance due to exposure to tolerogenic stimuli, which can be of microbial origin, components of mammalian cells, combinations of cytokines, hormones, vitamins, and other biological or pharmaceutical agents. Tolerogenic DCs have a low ability to induce immunogenic responses but have a high ability to induce active Tregs and other tolerance-inducing responses.

[0063] "Autoimmune disease" means a pathological condition in which the adaptive immune system targets self-antigens in a destructive manner.

[0064] "Self - antigen" (or "autoantigen") means any molecule or chemical group of a mammal (such as a human) that acts as an antigen in an unwanted immune response (such as an antibody or effector T - cell response) in the induction of a disease state, but to which the healthy immune system of the mammal is tolerant. The terms "self - antigen" and "autoantigen" are considered synonymous and may be used interchangeably herein.

[0065] "Stimulated" means exposed to any artificial or natural compound that causes cell signaling.

[0066] Method

[0067] The present invention provides a method for obtaining tolerogenic APCs that have the ability to render the immune system tolerant to an antigen, the method comprising culturing isolated monocytes under specific culture conditions. Monocytes can be isolated from many 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 can be bone marrow DCs. The method is generally carried out under ex vivo or in vitro conditions, preferably under ex vivo conditions. Isolated monocytes are usually present in a sample taken from a mammal. The mammal from which the sample is taken is usually a human (Homo sapiens). The sample is usually a blood sample, more preferably a peripheral blood mononuclear cell (PBMC) sample prepared from blood. The sample usually contains mononuclear cells, preferably monocytes, particularly contains monocytes. The method generally includes isolating a population of monocytes from the sample (such as a PBMC sample). Preferably, the isolated cells are CD14 + monocytes. The method also includes culturing the isolated monocytes in a cell culture.

[0068] In one embodiment, the cell culture of the method of the present invention contains retinoic acid and TGFβ. In another embodiment of the present invention, the cell culture of the method of the present invention contains retinoic acid, TGFβ, and an AhR agonist. In another embodiment of the present invention, the cell culture of the method of the present invention contains retinoic acid and an AhR agonist. The cell culture preferably contains retinoic acid and TGFβ, and even more preferably contains retinoic acid, TGFβ, and an AhR agonist.

[0069] Retinoic acid is a metabolite of vitamin A1. The cell culture contains retinoic acid. Retinoic acid can exist in the cell culture in any form, such as all - trans retinoic acid, 13 - cis retinoic acid, and / or 9 - cis retinoic acid, particularly all - trans retinoic acid. Retinoic acid can be provided as a retinoic acid precursor, such as retinol and / or a variant form of vitamin A, such as retinaldehyde, which generates retinoic acid in situ.

[0070] The AhR agonist used in the cell cultures of the present invention can be any AhR agonist, such as any AhR agonist disclosed in WO2012 / 050500, the content of which is incorporated herein by reference in its entirety. The AhR agonist can be a full agonist or a partial agonist. The AhR agonist used in the method of the present invention is preferably N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-quinoline-3-carboxamide (IMA-06201) (Mahiout et al., 2017), which is referred to herein as "C1". Alternatively, the AhR agonist can be 6-formylindolo[3,2-b]carbazole (FICZ), methyl 2-(1H-indol-3-ylcarbonyl)-4-thiazolecarboxylate (ITE), N-acetyl-N-phenyl-4-acetoxy-5-chloro-1,2-dihydro-1-methyl-2-oxo-quinoline-3-carboxamide ("C2"), N-(4-trifluoromethylphenyl)-1,2-dihydro-4-hydroxy-5-methoxy-1-methyl-2-oxo-quinoline-3-carboxamide ("C3") or N-acetyl-N-(4-trifluoromethylphenyl)-4-acetoxy-1,2-dihydro-5-methoxy-1-methyl-2-oxo-quinoline-3-carboxamide ("C4"). Other potentially useful AhR agonists are described in Denison and Nagy, Ann. Rev. Pharmacol. Toxicol., 43:309-34, 2003 and the references cited therein, which are incorporated herein by reference in their entirety.

[0071] The TGFβ used in the cell cultures of the present invention can be any TGFβ isoform, such as TGFβ1, TGFβ2 or TGFβ3, preferably TGFβ1. Any TGFβ isoform can be used alone or in combination with any other TGFβ isoform.

[0072] The cell cultures generally contain GM-CSF and IL-4. Alternatively, the cell cultures can contain only GM-CSF, such as GM-CSF without IL-4, or M-CSF. The cell cultures preferably contain GM-CSF and IL-4. GM-CSF and IL-4 are added to the cell cultures to induce the differentiation of isolated monocytes into APCs, preferably DCs. M-CSF is added to the cell cultures to induce the differentiation of isolated monocytes into APCs, preferably macrophages.

[0073] Typically, GM-CSF and IL-4 or GM-CSF or M-CSF are added to the cell culture before or simultaneously with the addition of any one of TGFβ, an AhR agonist, and retinoic acid. Preferably, GM-CSF and IL-4 are added to the cell culture before or simultaneously with the addition of any one of TGFβ, an AhR agonist, and retinoic acid. Typically, an AhR agonist is added to the cell culture at a first dose while GM-CSF or M-CSF and IL-4 are first added to the cell culture. Typically, after the addition of the first dose of the AhR agonist to the cell culture, TGFβ and a second dose of the AhR agonist are added to the cell culture. Further typically, after the addition of TGFβ and the second dose of the AhR agonist to the cell culture, retinoic acid is added to the cell culture. Typically, GM-CSF or M-CSF and IL-4 are added to the cell culture at a second dose. Preferably, the second dose of GM-CSF or M-CSF and IL-4 are added to the cell culture simultaneously with the addition of TGFβ and the second dose of the AhR agonist.

[0074] GM-CSF or M-CSF can be added to the cell culture before or simultaneously with the addition of any one of TGFβ, an AhR agonist, and retinoic acid, and IL-4 is added to the cell culture after the addition of the first dose of the AhR agonist. Typically, after the addition of the first dose of the AhR agonist to the cell culture, TGFβ and a second dose of the AhR agonist are added to the cell culture. Further typically, after the addition of TGFβ and the second dose of the AhR agonist to the cell culture, retinoic acid is added to the cell culture. GM-CSF or M-CSF can be added to the cell culture at a second dose. Preferably, the second dose of GM-CSF or M-CSF is added to the cell culture simultaneously with the addition of TGFβ and the second dose of the AhR agonist.

[0075] The cell culture can further comprise an antigen or an epitope containing an antigen fragment. The antigen or the epitope containing its fragment can be associated with or expressed on monocytes isolated from the culture. The antigen or the epitope containing its fragment can be expressed on the cell surface of monocytes or APCs. The antigen or the epitope containing its fragment can be intracellular. The antigen or the epitope containing its fragment can be an antigen or an epitope containing a fragment that is not native to the monocytes, for example, the monocytes may have taken up the antigen or the epitope containing its fragment from other cells before isolation. Monocytes or APCs can be transfected with mRNA encoding the antigen to express the antigen or the epitope containing its fragment. Methods for transfecting cells with mRNA encoding an antigen are known in the art.

[0076] In the context of methods for inducing tolerance to allografts, the antigen or epitopes containing fragments thereof can be associated with or expressed on monocytes isolated from a sample taken from the graft donor, or can be expressed by APCs during or after culturing using the methods of the invention. Isolated monocytes from a sample taken from the donor, cultured using the methods of the invention, allow the production of tolerogenic APCs. Such tolerogenic APCs can be administered to a recipient to prevent immune rejection of an allograft from the donor.

[0077] In the case of isolated monocytes from a sample taken from the recipient, the monocytes can be cultured using the methods of the invention that allow the production of tolerogenic APCs. The tolerogenic APCs can be loaded with donor antigens such that they present donor-derived antigens. Alternatively, the tolerogenic APCs can be not loaded with donor antigens ex vivo. The tolerogenic APCs presenting (or not presenting) donor-derived antigens can be administered back to the recipient to prevent immune rejection of an allograft from the donor.

[0078] Alternatively, the antigen or epitopes containing fragments thereof are components added to the cell culture. Thus, the cell culture also contains the antigen or epitopes containing fragments thereof. Preferably, the antigen or epitopes containing fragments thereof are added to the cell culture before, simultaneously with, or after adding retinoic acid. The antigen or epitopes containing fragments thereof can be added as part of an antigen mixture, for example, cells, tissues, or samples containing one or more antigens can be added to the culture.

[0079] The antigen or epitopes containing fragments thereof can be a single antigen or epitopes containing fragments thereof (e.g., in purified form), or a library of antigens and / or epitopes containing fragments thereof. The antigen or epitopes containing fragments thereof can be cells, blood, tissue samples, or extracts thereof. In the context of methods for inducing tolerance to allografts, the antigen or epitopes containing fragments thereof can be a sample of the graft, or can be derived from a sample of the graft or other donor-derived tissue, cells, or blood, and the antigen or epitopes containing fragments thereof can be added directly to the cell culture.

[0080] Optionally, the antigen or epitopes containing fragments thereof can not be added directly to the cell culture. Instead, the antigen or epitopes containing fragments thereof can be present in vivo. Thus, optionally, the tolerogenic APCs can be not loaded with the antigen or epitopes containing fragments thereof ex vivo, but have the ability to load the antigen or epitopes containing fragments thereof in vivo when the tolerogenic APCs are administered. The tolerogenic APCs thus recognize and process the antigen or epitopes containing fragments thereof in vivo.

[0081] The antigen or epitopes containing fragments thereof can be or be derived from a biopharmaceutical. Thus, the biopharmaceutical can be used in whole or in part (i.e., containing partial epitopes) as an antigen.

[0082] The antigen or a fragment containing its epitope can be or be derived from a self - antigen.

[0083] Generation of regulatory T cells

[0084] The method of the present invention can further comprise culturing the obtained tolerogenic APCs ex vivo with T cells to induce the generation of Tregs. Thus, when cultured ex vivo with T cells, the tolerogenic APCs of the present invention induce Tregs. The induced Tregs can be any Tregs, such as CD4 + CD25 + FOXP3 + Tregs and / or type 1 regulatory T (Tr1) CD4 + CD49b + LAG3 + cells, preferably CD4 + CD25 + FOXP3 + Treg. A Treg or a population thereof can be obtained or obtained by the method of the present invention.

[0085] A Treg or a population thereof can be used in a method for treating a mammalian subject or a method for treating a mammalian subject that has an immune response to an antigen or is at risk of having an immune response to an antigen.

[0086] Such as Figure 5 As shown in (A) and 5(B), when unstimulated or stimulated, for example, with an immunogenic stimulant (such as LPS or a mixture of pro - inflammatory cytokines), the tolerogenic DCs of the present invention show a low ability to induce T cell proliferation. The combination of retinoic acid, TGFβ, and an AhR agonist is superior to the combinations of retinoic acid and TGFβ and retinoic acid and an AhR agonist in reducing the ability to induce T cell proliferation.

[0087] Therefore, suitably, the tolerogenic APCs of the present invention (such as the DCs of the present invention) have no more than 100%, for example, no more than 90%, for example, no more than 80%, for example, no more than 70% of the ability to induce T cell proliferation of control cells when unstimulated. In addition, suitably, when stimulated, for example, with an immunogenic stimulant (such as LPS or a mixture of pro - inflammatory cytokines), the tolerogenic APCs of the present invention (such as the DCs of the present invention) have, for example, no more than 70%, for example, no more than 60%, for example, no more than 50% of the ability of control cells to induce T cell proliferation when stimulated with an immunogenic stimulant (such as LPS). The control cells are the same type of APCs from the same subject not exposed to the tolerogenic compound.

[0088] Such as Figure 10As shown in (A) and 10 (B), the tolerogenic DCs of the present invention have high Treg induction ability. The combination of retinoic acid, TGFβ and AhR agonist is superior to the combination of retinoic acid and TGFβ or retinoic acid and AhR agonist in inducing Tregs.

[0089] Thus, suitably, the tolerogenic APCs (such as the DCs of the present invention) of the present invention have a Treg induction ability that is at least 130%, such as at least 150%, such as at least 175%, such as at least 200% of that of control cells when unstimulated. In addition, suitably, when stimulated with an immunogenic stimulant (such as LPS), the tolerogenic APCs (such as the DCs of the present invention) of the present invention suitably have a Treg induction ability that is at least 130%, such as at least 150%, such as at least 200% of that of control cells when stimulated with an immunogenic stimulant (such as LPS). Control cells are the same type of APCs from the same subject not exposed to the tolerogenic compound.

[0090] As Figure 11 As shown in (J), when CD14+ monocytes are isolated from the blood of hemophilia patients, the tolerogenic DCs of the present invention have high Treg induction ability.

[0091] Thus, suitably, the tolerogenic APCs (such as the DCs of the present invention) of the present invention have a Treg induction ability that is at least 130%, such as at least 150%, such as at least 175%, such as at least 200% of that of control cells when unstimulated. In addition, suitably, when stimulated with an immunogenic stimulant (such as LPS), the tolerogenic APCs (such as the DCs of the present invention) of the present invention suitably have a Treg induction ability that is at least 130%, such as at least 150%, such as at least 200% of that of control cells when stimulated with an immunogenic stimulant (such as LPS). Control cells are the same type of APCs from the same subject not exposed to the tolerogenic compound.

[0092] Tolerogenic antigen-presenting cells

[0093] Monocytes differentiate into DCs. During differentiation, a part of the DCs may be polarized into a tolerogenic phenotype. The method of the present invention generally allows the generation of tolerogenic APCs, such as tolerogenic DCs, with tolerogenic cell surface marker expression.

[0094] CD83 is an integral membrane protein and is an activation / maturation marker. Typically, unstimulated APCs, particularly DCs, show CD83 expression levels that do not differ significantly between tolerogenic and non-tolerogenic cells. However, when stimulated, for example, with an immunogenic stimulant such as LPS, APCs, particularly DCs, may show increased CD83 expression levels. Thus, the level of CD83 expression is generally increased to a greater extent in stimulated non-tolerogenic cells than in stimulated tolerogenic cells. Desired tolerogenic APCs, particularly DCs, have low levels of CD83 expression even upon stimulation. With respect to upregulation of CD83 expression, desired tolerogenic APCs (particularly DCs) are as resistant as possible to stimulation with immunogenic stimulants (such as LPS).

[0095] In at least some embodiments, the tolerogenic APCs of the present invention (such as the DCs of the present invention) have the advantage of having low expression of CD83 when stimulated, for example, with an immunogenic stimulant such as LPS, compared to corresponding stimulated control cells. The control cells are the same type of APCs from the same subject not exposed to the tolerogenic compound.

[0096] Suitably, the tolerogenic APCs of the present invention (such as the DCs of the present invention) have a CD83 expression level not exceeding 100%, such as not exceeding 90%, such as not exceeding 80%, such as not exceeding 70%, such as not exceeding 60%, such as not exceeding 50% of the CD83 expression level of control cells when unstimulated.

[0097] Suitably, when stimulated, for example, with an immunogenic stimulant such as LPS, the tolerogenic APCs of the present invention (such as the DCs of the present invention) have a CD83 expression level not exceeding 70%, such as not exceeding 60%, such as not exceeding 50% of the CD83 expression level of control cells when stimulated with an immunogenic stimulant such as LPS. As described in more detail in the Examples section and as Figure 1 shown, when the tolerogenic compounds used are retinoic acid, TGFβ, and AhR agonists, when generating the APCs of the present invention by the method of the present invention, the greatest reduction in the CD83 expression level in the stimulated APCs is obtained compared to the corresponding control cells. The combination of retinoic acid, TGFβ, and AhR agonists is superior to using retinoic acid and TGFβ, and using retinoic acid and TGFβ is superior to using retinoic acid and AhR agonists.

[0098] In at least some embodiments, the tolerogenic APCs of the present invention (such as the DCs of the present invention) also have the advantage of having a CD83 expression level that resists further upregulation after stimulation.

[0099] Suitably, when stimulated, for example, with an immunogenic stimulant such as LPS, the tolerogenic APCs of the present invention (such as the DCs of the present invention) have a CD83 expression level that is no more than 200%, such as no more than 175%, such as no more than 150%, such as no more than 125% of the corresponding cells when unstimulated. As described in more detail in the Examples section and as Figure 1 shown, when the tolerogenic compounds used are retinoic acid, TGFβ, and an AhR agonist, when generating the APCs of the present invention by the method of the present invention, the maximum reduction in the CD83 expression level in the stimulated APCs is obtained compared to the corresponding unstimulated cells. The combination of retinoic acid, TGFβ, and an AhR agonist is superior to the use of retinoic acid and TGFβ, and the use of retinoic acid and TGFβ is superior to the use of retinoic acid and an AhR agonist.

[0100] CD86 is an integral membrane protein and is an activation / maturation marker and a co-stimulatory molecule. Generally, unstimulated APCs, especially DCs, show a CD86 expression level with no significant difference between tolerogenic cells and non-tolerogenic cells. However, when stimulated, for example, with an immunogenic stimulant such as LPS, APCs, especially DCs, may show an increased CD86 expression level. The level of CD86 expression generally increases to a higher extent in non-tolerogenic cells than in tolerogenic cells. Desirable tolerogenic APCs (especially DCs) have a low level of CD86 expression when stimulated. In terms of the upregulation of CD86 expression, desirable tolerogenic APCs (especially DCs) are as resistant as possible to the stimulation by an immunogenic stimulant (such as LPS).

[0101] In at least some embodiments, the tolerogenic APCs of the present invention (such as the DCs of the present invention) have the advantage of low expression of CD86 when stimulated, for example, with an immunogenic stimulant such as LPS, compared to control cells when stimulated. The control cells are the same type of APCs from the same subject not exposed to the tolerogenic compound.

[0102] Suitably, the tolerogenic APCs of the present invention (such as the DCs of the present invention) have a CD86 expression level that is no more than 100%, no more than 90%, such as no more than 80%, such as no more than 70%, such as no more than 60%, such as no more than 50% of the control cells when unstimulated when unstimulated.

[0103] Suitably, when stimulated, for example, with an immunogenic stimulant such as LPS, the tolerogenic APCs of the present invention (such as the DCs of the present invention) have a CD86 expression level that is no more than 80%, such as no more than 70%, such as no more than 60%, such as no more than 50% of the CD86 expression level of the control cells when stimulated, for example, with an immunogenic stimulant such as LPS. As described in more detail in the Examples section and as Figure 2As shown, when the tolerance-inducing compounds used are retinoic acid, TGFβ, and an AhR agonist, when generating the APCs of the present invention by the method of the present invention, the maximum reduction in the CD86 expression level in the stimulated APCs is obtained compared to the corresponding control cells at the time of stimulation. The combination of retinoic acid, TGFβ, and an AhR agonist is superior to using retinoic acid and TGFβ, and using retinoic acid and TGFβ is superior to using retinoic acid and an AhR agonist.

[0104] The tolerogenic state of DCs is characterized by low co-stimulatory potential and high expression of inhibitory molecules. ILT3 is an inhibitory cell surface receptor that can be expressed by tolerogenic APCs (preferably DCs). DCs overexpressing ILT3 show lower levels of NF-κB phosphorylation and cannot stimulate the complete program of Th proliferation and maturation, triggering alternative Treg cell differentiation (Vlad et al. 2009). ILT3 expression and upregulation are generally independent of immunogenic stimuli such as LPS. Typically, non-tolerogenic and tolerogenic APCs (especially DCs) show that there is not much difference in the ILT3 expression level between unstimulated and stimulated (e.g., with an immunogenic stimulus such as LPS) conditions. The ILT3 expression level is generally much higher in tolerogenic cells than in non-tolerogenic cells. Desired tolerogenic APCs (especially DCs) have a high level of ILT3 expression that is not downregulated upon stimulation with an immunogenic stimulus (e.g., LPS).

[0105] In at least some embodiments, the tolerogenic APCs of the present invention (e.g., the DCs of the present invention) have the advantage of having high expression of ILT3 when unstimulated or stimulated (e.g., with an immunogenic stimulus such as LPS), respectively, compared to control cells when unstimulated or stimulated. The control cells are of the same type of antigen-presenting cells from the same subject not exposed to the tolerance-inducing compound.

[0106] Suitably, the tolerogenic APCs of the present invention (e.g., the DCs of the present invention) have at least 150%, e.g., at least 175%, e.g., at least 200%, e.g., at least 250%, e.g., at least 300% of the ILT3 expression level of the control cells when unstimulated and when stimulated (e.g., with an immunogenic stimulus such as LPS), respectively. As described in more detail in the Examples section and as Figure 3 As shown, when the tolerance-inducing compounds used are retinoic acid, TGFβ, and an AhR agonist, when generating the APCs of the present invention by the method of the present invention, the maximum increase in the ILT3 expression level in the unstimulated and stimulated APCs is obtained compared to the control cells. The combination of retinoic acid, TGFβ, and an AhR agonist is superior to using retinoic acid and TGFβ, and using retinoic acid and TGFβ is superior to using retinoic acid and an AhR agonist.

[0107] The tolerogenic capacity of APCs (especially DCs) can be determined by the ratio between the ILT3 and CD86 expression levels. The ILT3 / CD86 ratio is generally much higher in tolerogenic cells than in non-tolerogenic cells. Desired tolerogenic APCs (especially DCs) have a high ILT3 / CD86 ratio, independent of whether they are unstimulated or stimulated with immunogenic stimulants (such as LPS).

[0108] In at least some embodiments, the tolerogenic APCs (such as the DCs of the present invention) of the present invention have the following advantages: they have a high ILT3 / CD86 ratio when unstimulated or stimulated (such as with an immunogenic stimulant, such as LPS), as compared to control cells when unstimulated or stimulated, respectively. The control cells are the same type of APCs from the same subject not exposed to the tolerogenic compound.

[0109] Suitably, the APCs (such as the DCs of the present invention) of the present invention have an ILT3 / CD86 ratio that is at least 150%, such as at least 200%, such as at least 250%, such as at least 300%, such as at least 350%, such as at least 400%, such as at least 500% of the ILT3 / CD86 ratio of control cells when unstimulated. As described in more detail in the Examples section and as Figure 4 shown, when generating the APCs of the present invention by the method of the present invention, the maximum increase in the ILT3 / CD86 ratio in unstimulated APCs compared to control cells is obtained. The compounds used are retinoic acid, TGFβ, and an AhR agonist. Using a combination of retinoic acid, TGFβ, and an AhR agonist is superior to using retinoic acid and TGFβ, and using retinoic acid and TGFβ is superior to using retinoic acid and an AhR agonist.

[0110] Suitably, when stimulated with an immunogenic stimulant (such as LPS), for example, the APCs (such as the DCs of the present invention) of the present invention have an ILT3 / CD86 ratio that is at least 150%, such as at least 200%, such as at least 250%, such as at least 300%, such as at least 350%, such as at least 400%, such as at least 500% of the ILT3 / CD86 ratio of control cells when stimulated with an immunogenic stimulant (such as LPS). As described in more detail in the Examples section and as Figure 4 shown, when the tolerogenic compounds used are retinoic acid, TGFβ, and an AhR agonist, when generating the APCs of the present invention by the method of the present invention, the maximum increase in the ILT3 / CD86 ratio in stimulated APCs compared to stimulated control cells is obtained. Using a combination of retinoic acid, TGFβ, and an AhR agonist is superior to using retinoic acid and TGFβ, and using retinoic acid and TGFβ is superior to using retinoic acid and an AhR agonist.

[0111] CD141 and GARP are tolerogenic APC markers when co-expressed.

[0112] Tolerogenic APCs co-expressing CD141 and GARP have been shown to be associated with an enhanced ability to induce Tregs (Agrawal et al., 2016). CD141 and GARP can be co-expressed on the cell surface of unstimulated and stimulated APCs, particularly DCs. Co-expression and upregulation of CD141 and GARP on tolerogenic APCs may be independent of immunogenic stimulants such as LPS. The frequency of CD141 and GARP co-expressing cells is generally much higher in tolerogenic cells than in non-tolerogenic cells. Desired tolerogenic APCs (particularly DCs) have a high frequency of CD141 and GARP co-expressing cells when unstimulated or stimulated by immunogenic stimulants such as LPS.

[0113] In at least some embodiments, the tolerogenic APCs of the invention (such as the DCs of the invention) have the advantage of having a high degree of co-expression of CD141 and GARP when unstimulated or stimulated, compared to control cells when unstimulated or stimulated, respectively. Control cells are the same type of APCs from the same subject not exposed to the tolerogenic compound.

[0114] Suitably, the APCs of the invention (such as the DCs of the invention) contain a high frequency of cells co-expressing CD141 and GARP when unstimulated, which is at least 400%, such as at least 450%, such as at least 500%, such as at least 600%, such as at least 700%, such as at least 800%, such as at least 900%, such as at least 1000% of the control cells when unstimulated. As described in more detail in the Examples section and as Figure 6 shown, when the tolerogenic compounds used are retinoic acid, TGFβ and AhR agonists, when generating the APCs of the invention by the method of the invention, a maximum increase in the frequency of CD141 and GARP co-expressing cells in unstimulated APCs is obtained compared to unstimulated control cells. The combination of retinoic acid, TGFβ and AhR agonists is superior to the use of retinoic acid and AhR agonists, while the use of retinoic acid and AhR agonists is superior to the use of retinoic acid and TGFβ. The combination of retinoic acid, TGFβ and AhR agonists is superior to the use of retinoic acid and TGFβ.

[0115] Suitably, when stimulated with an immunogenic stimulant such as LPS, for example, the APCs of the invention (such as the DCs of the invention) have a frequency of CD141 and GARP co-expressing cells that is at least 200%, such as at least 250%, such as at least 300%, at least 400%, such as at least 450%, such as at least 500% of the control cells when stimulated. As described in more detail in the Examples section and asFigure 6 As shown, when the tolerogenic compounds used are retinoic acid, TGFβ, and an AhR agonist, the method of the present invention produces the APCs of the present invention, and the maximum increase in the frequency of CD141- and GARP-coexpressing cells in the stimulated APCs is obtained compared to stimulated control cells. The combination of retinoic acid, TGFβ, and an AhR agonist is superior to the use of retinoic acid and TGFβ.

[0116] IL-23 is a pro-inflammatory cytokine released from APCs (especially DCs) during inflammation. This cytokine, produced by DCs and macrophages, promotes host protection against mucosal pathogens by inducing IL-17 and related cytokines in lymphocytes. IL-23 is usually only released from APCs (especially DCs) when the cells are activated. Generally, unstimulated APCs, especially DCs, show IL-23 production levels that do not differ significantly between tolerogenic and non-tolerogenic cells. However, when stimulated with an immunogenic stimulant such as LPS or a mixture of pro-inflammatory cytokines, APCs, especially DCs, may show increased levels of IL-23 production. The level of IL-23 production generally increases much more in non-tolerogenic cells than in tolerogenic cells. When stimulated with an immunogenic stimulant (such as LPS or a mixture of pro-inflammatory cytokines), the desired tolerogenic APCs (especially DCs) produce low levels of IL-23. The desired tolerogenic APCs, especially DCs, are as resistant as possible to stimulation by immunogenic stimulants (such as LPS or a mixture of pro-inflammatory cytokines) in terms of IL-23 production.

[0117] In at least some embodiments, the tolerogenic APCs of the present invention (such as the DCs of the present invention) have the advantage of exhibiting low IL-23 production when stimulated, for example, with an immunogenic stimulant (such as LPS or a mixture of pro-inflammatory cytokines), compared to control cells when stimulated, for example, with an immunogenic stimulant (such as LPS or a mixture of pro-inflammatory cytokines). When unstimulated, they also exhibit low IL-23 production. The control cells are the same type of APCs from the same subject not exposed to the tolerogenic compound.

[0118] Suitably, the tolerogenic APCs of the present invention (such as the DCs of the present invention) have an IL-23 production level that is no more than 100%, for example, no more than 90%, for example, no more than 80%, for example, no more than 70%, for example, no more than 60%, for example, no more than 50% of the IL-23 production level of control cells when unstimulated.

[0119] Suitably, when stimulated, for example, with an immunogenic stimulant (such as LPS or a mixture of pro-inflammatory cytokines), the APCs of the present invention (such as the DCs of the present invention) have an IL-23 production level that is no more than 50%, such as no more than 40%, such as no more than 30% of that of control cells when stimulated with an immunogenic stimulant (such as LPS or a mixture of pro-inflammatory cytokines). As described in more detail in the Examples section and as Figure 7 shown, when the tolerogenic compounds used are retinoic acid and an AhR agonist, retinoic acid and TGFβ, and retinoic acid, TGFβ, and an AhR agonist, the production level of IL-23 in the stimulated APCs of the present invention produced by the method of the present invention is reduced compared to that of the stimulated control cells.

[0120] In at least some embodiments, the APCs of the present invention (such as the DCs of the present invention) also have the advantage that the production level of IL-23 is more resistant to further upregulation compared to control cells, for example, after stimulation with an immunogenic stimulant (such as LPS or a mixture of pro-inflammatory cytokines).

[0121] CD103 is a cell surface marker associated with intestinal tolerogenic DCs. It has been shown that tolerogenic APCs expressing CD103 can maintain tolerance by inducing Tregs and by cross-presenting foreign antigens to CD8+ T cells to protect against tissue infection (Scott et al. 2011). CD103 expression can be upregulated on the cell surface of tolerogenic APCs (especially DCs). CD103 expression and upregulation may not depend on an immunogenic stimulant such as LPS. Generally, non-tolerogenic and tolerogenic APCs, especially DCs, show CD103 expression levels that do not differ greatly between unstimulated and stimulated (such as with an immunogenic stimulant like LPS) conditions. CD103 expression levels are generally increased much more in tolerogenic cells than in non-tolerogenic cells. When unstimulated or stimulated with an immunogenic stimulant (such as LPS), the desired tolerogenic APCs, especially DCs, have a high level of CD103 expression.

[0122] In at least some embodiments, the tolerogenic APCs of the present invention (such as the DCs of the present invention) have the advantage of high CD103 expression when unstimulated or stimulated (such as with an immunogenic stimulant like LPS), compared to control cells when unstimulated or stimulated (such as with an immunogenic stimulant like LPS), respectively. The control cells are the same type of APCs from the same subject not exposed to the tolerogenic compound.

[0123] Suitably, the APCs of the present invention (e.g., the DCs of the present invention) have an expression level of CD103 that is at least 150%, such as at least 200%, such as at least 250%, such as at least 300% of that of control cells when unstimulated. As described in more detail in the Examples section and as Figure 8 shown, when the tolerogenic compounds used are retinoic acid, TGFβ, and an AhR agonist, when generating the APCs of the present invention by the method of the present invention, the maximum increase in the expression level of CD103 in the unstimulated APCs is obtained compared to unstimulated control cells. The combination of retinoic acid, TGFβ, and an AhR agonist is superior to the use of retinoic acid and an AhR agonist, and the use of retinoic acid and an AhR agonist is superior to the use of retinoic acid and TGFβ. The combination of retinoic acid, TGFβ, and an AhR agonist is superior to the use of retinoic acid and TGFβ.

[0124] Suitably, when stimulated with an immunogenic stimulant (such as LPS), the APCs of the present invention (e.g., the DCs of the present invention) have an expression level of CD103 that is at least 200%, such as at least 250%, such as at least 300% of that of control cells when stimulated with an immunogenic stimulant (such as LPS). As described in more detail in the Examples section and as Figure 8 shown, when the tolerogenic compounds used are retinoic acid, TGFβ, and an AhR agonist, when generating the APCs of the present invention by the method of the present invention, the maximum increase in the expression level of CD103 in the stimulated APCs is obtained compared to stimulated control cells. The combination of retinoic acid, TGFβ, and an AhR agonist is superior to the use of retinoic acid and TGFβ.

[0125] MERTK, BTLA, LAP, HLA-G, and CD49b are cell surface markers of tolerogenic APCs (especially DCs). MERTK, BTLA, LAP, HLA-G, and CD49b can be expressed on the cell surface of tolerogenic APCs (especially DCs). The levels of expression of MERTK, BTLA, LAP, HLA-G, and CD49b are generally much higher in tolerogenic cells than in non-tolerogenic cells. Desired tolerogenic APCs, especially DCs, have a high level of expression of MERTK, BTLA, LAP, and HLA-G when unstimulated. Desired tolerogenic APCs, especially DCs, have a high level of expression of MERTK, BTLA, LAP, HLA-G, and CD49b when unstimulated.

[0126] In at least some embodiments, the tolerogenic APCs of the present invention (e.g., the DCs of the present invention) have the advantage of having high MERTK, BTLA, LAP, and HLA-G expression when unstimulated, compared to control cells when unstimulated. In at least some embodiments, the tolerogenic APCs of the present invention (e.g., the DCs of the present invention) have the advantage of having high MERTK, BTLA, LAP, HLA-G, and CD49b expression when unstimulated, compared to control cells when unstimulated. Control cells are the same type of APCs from the same subject not exposed to tolerogenic compounds.

[0127] Suitably, the APCs of the present invention (e.g., the DCs of the present invention) have an MERTK expression level when unstimulated that is at least 120%, such as at least 150%, such as at least 175% of the MERTK expression level of control cells when unstimulated. As described in more detail in the Examples section and as Figure 9 (A) shows, when the tolerogenic compounds used are retinoic acid, TGFβ, and an AhR agonist, when generating the APCs of the present invention by the method of the present invention, the greatest increase in the MERTK expression level in unstimulated APCs is obtained compared to unstimulated control cells. The combination of retinoic acid, TGFβ, and an AhR agonist is superior to the use of retinoic acid and TGFβ.

[0128] Suitably, the APCs of the present invention (e.g., the DCs of the present invention) have a BTLA expression level when unstimulated that is at least 120%, such as at least 150%, such as at least 200% of the BTLA expression level of control cells when unstimulated. As described in more detail in the Examples section and as Figure 9 (B) shows, when the tolerogenic compounds used are retinoic acid, TGFβ, and an AhR agonist, when generating the APCs of the present invention by the method of the present invention, the greatest increase in the BTLA expression level in unstimulated APCs is obtained compared to unstimulated control cells. The combination of retinoic acid, TGFβ, and an AhR agonist is superior to the use of retinoic acid and TGFβ.

[0129] Suitably, the APCs of the present invention (e.g., the DCs of the present invention) have an LAP expression level when unstimulated that is at least 120%, such as at least 150%, such as at least 200%, such as at least 250% of the LAP expression level of control cells when unstimulated. As described in more detail in the Examples section and as Figure 9 (C) shows, when the tolerogenic compounds used are retinoic acid, TGFβ, and an AhR agonist, when generating the APCs of the present invention by the method of the present invention, the greatest increase in the LAP expression level in unstimulated APCs is obtained compared to unstimulated control cells. The combination of retinoic acid, TGFβ, and an AhR agonist is superior to the use of retinoic acid and TGFβ.

[0130] Suitably, when not stimulated, the APCs of the present invention (such as the DCs of the present invention) have an HLA-G expression level that is at least 120%, such as at least 150%, such as at least 200%, such as at least 250%, such as at least 300% of the HLA-G expression level of control cells when not stimulated. As described in more detail in the Examples section and as Figure 9 shown in (D), when the tolerogenic compounds used are retinoic acid, TGFβ, and AhR agonists, when generating the APCs of the present invention by the method of the present invention, a maximum increase in the HLA-G expression level in the unstimulated APCs is obtained compared to unstimulated control cells.

[0131] Compared to control APCs when not stimulated, the APCs (such as DCs) of the present invention suitably have a high expression of ILT3 and a low expression of CD83 and CD86 when not stimulated. In addition, compared to control APCs when stimulated with an immunogenic stimulant (such as LPS stimulation), for example, when the APCs (such as DCs) of the present invention are stimulated with an immunogenic stimulant (such as LPS), they suitably have a high expression of ILT3 and a low expression of CD83 and CD86. The control cells are the same type of APCs from the same subject, however, they have not been treated with the tolerogenic compounds according to the present invention.

[0132] When not stimulated, the ILT3 expression level in the APCs (such as DCs) of the present invention is suitably at least 150%, such as at least 175%, such as at least 200%, such as at least 250%, such as at least 300% of the control cells when not stimulated. When not stimulated, the CD83 expression level in the APCs (such as DCs) of the present invention suitably does not exceed 110%, such as does not exceed 100% of the control cells when not stimulated. When not stimulated, the CD86 expression level in the APCs (such as DCs) of the present invention suitably does not exceed 110%, such as does not exceed 100% of the control cells when not stimulated.

[0133] When stimulated with an immunogenic stimulant (such as LPS) for example, the ILT3 expression level in the APCs (such as DCs) of the present invention is suitably at least 150%, such as at least 175%, such as at least 200%, such as at least 250%, such as at least 300% of the control cells when stimulated with an immunogenic stimulant (such as LPS) for example. When stimulated with an immunogenic stimulant (such as LPS) for example, the CD83 expression level in the APCs (such as DCs) of the present invention suitably does not exceed 70%, such as does not exceed 60%, such as does not exceed 50% of the control cells when stimulated with an immunogenic stimulant (such as LPS) for example. When stimulated with an immunogenic stimulant (such as LPS) for example, the CD86 expression level in the APCs (such as DCs) of the present invention suitably does not exceed 80%, such as does not exceed 70%, such as does not exceed 60%, such as does not exceed 50% of the control cells when stimulated with an immunogenic stimulant (such as LPS) for example.

[0134] When unstimulated or stimulated (e.g., with an immunogenic stimulant such as LPS), the APCs (e.g., DCs) of the present invention appropriately express CD103 and, most appropriately, have a high expression of CD103 as compared to control APCs when unstimulated or stimulated (e.g., with an immunogenic stimulant such as LPS), respectively. The control cells are the same type of antigen-presenting cells from the same subject not treated with the tolerogenic compound according to the present invention. When unstimulated or stimulated (e.g., with an immunogenic stimulant such as LPS), the expression level of CD103 in the APCs (e.g., DCs) of the present invention is appropriately at least 200%, e.g., at least 250%, e.g., at least 300% of that of the control cells when unstimulated or stimulated (e.g., with an immunogenic stimulant such as LPS).

[0135] When compared to control APCs when unstimulated, the APCs (e.g., DCs) of the present invention appropriately express CD103 (most appropriately have a high expression of CD103) when unstimulated and have a high expression of CD141, GARP, and ILT3 and a low expression of CD83 and CD86. Further, when stimulated (e.g., with an immunogenic stimulant such as LPS), the APCs (e.g., DCs) of the present invention appropriately express CD103 (most appropriately have a high expression of CD103) and have a high expression of CD141, GARP, and ILT3 and a low expression of CD83 and CD86 as compared to control APCs when stimulated (e.g., with an immunogenic stimulant such as LPS). The control cells are the same type of APCs from the same subject not treated with the tolerogenic compound according to the present invention.

[0136] When stimulated (e.g., with an immunogenic stimulant such as LPS), the expression level of CD103 in the APCs (e.g., DCs) of the present invention is appropriately at least 200%, e.g., at least 250%, e.g., at least 300% of that of the control cells when stimulated (e.g., with an immunogenic stimulant such as LPS). When stimulated (e.g., with an immunogenic stimulant such as LPS), the co-expression level of CD141 and GARP in the APCs (e.g., DCs) of the present invention is appropriately at least 200%, e.g., at least 250%, e.g., at least 300%, at least 400%, e.g., at least 450%, e.g., at least 500% of that of the control cells when stimulated (e.g., with an immunogenic stimulant such as LPS).

[0137] When stimulated with an immunogenic stimulant (such as LPS), the expression level of ILT3 in the APCs (such as DCs) of the present invention is suitably at least 150%, such as at least 175%, such as at least 200%, such as at least 250%, such as at least 300% of that of the control cells when stimulated with an immunogenic stimulant (such as LPS). When stimulated with an immunogenic stimulant (such as LPS), the expression level of CD83 in the APCs (such as DCs) of the present invention does not exceed 70%, such as does not exceed 60%, such as does not exceed 50% of that of the control cells when stimulated with an immunogenic stimulant (such as LPS). When stimulated with an immunogenic stimulant (such as LPS), the expression level of CD86 in the APCs (such as DCs) of the present invention does not exceed 80%, such as does not exceed 70%, such as does not exceed 60%, such as does not exceed 50% of that of the control cells when stimulated with an immunogenic stimulant (such as LPS).

[0138] Compared with the control APCs when unstimulated, the APCs (such as DCs) of the present invention suitably express one or more (such as two, three or all four) of MERTK, BTLA, LAP and HLA-G (and suitably have high expression) when unstimulated. Compared with the control APCs when unstimulated, the APCs (such as DCs) of the present invention suitably express one or more (such as two, three or all four) of MERTK, BTLA, LAP, HLA-G and CD49b (and suitably have high expression) when unstimulated. The control cells are the same type of APCs from the same patient who has not been treated with the tolerogenic compound according to the present invention. The expression level of MERTK in the APCs (such as DCs) of the present invention when unstimulated is suitably at least 120%, such as at least 150%, such as at least 175% of that of the control cells when unstimulated. The expression level of BTLA in the APCs (such as DCs) of the present invention when unstimulated is suitably at least 120%, such as at least 150%, such as at least 200% of that of the control cells when unstimulated. The expression level of LAP in the APCs (such as DCs) of the present invention when unstimulated is suitably at least 150%, such as at least 200%, such as at least 250% of that of the control cells when unstimulated. The expression level of HLA-G in the APCs (such as DCs) of the present invention when unstimulated is suitably 120%, such as at least 150%, such as at least 200%, such as at least 250%, such as at least 350% of that of the control cells when unstimulated. The expression level of CD49b in the APCs (such as DCs) of the present invention when unstimulated is suitably 120%, such as at least 150%, such as at least 200%, such as at least 250%, such as at least 300% of that of the control cells when unstimulated.

[0139] Compared to control APCs when stimulated with, for example, an immunogenic stimulant (such as LPS or a mixture of pro-inflammatory cytokines), when stimulated with, for example, an immunogenic stimulant (such as LPS), the antigen-presenting cells (such as DCs) of the present invention suitably have low IL-23 production. The control cells are the same type of APCs from the same subject not treated with the tolerogenic compound according to the present invention. When stimulated with, for example, an immunogenic stimulant (such as LPS or a mixture of pro-inflammatory cytokines), the level of IL-23 production in the APCs (such as DCs) of the present invention suitably does not exceed 50%, for example, does not exceed 40%, for example, does not exceed 30% of the production level of control cells when stimulated with, for example, an immunogenic stimulant (such as LPS). When stimulated with, for example, an immunogenic stimulant (such as LPS), the level of CD86 expression in the APCs (such as DCs) of the present invention suitably does not exceed 80%, for example, does not exceed 70%, for example, does not exceed 60%, for example, does not exceed 50% of control cells when stimulated with, for example, an immunogenic stimulant (such as LPS).

[0140] Suitably, the tolerogenic APCs (such as DCs) of the present invention are phenotypically stable, for example, in terms of their cell surface marker presentation, high Treg induction ability, low T cell proliferation induction ability, and other advantageous properties described herein.

[0141] The expression of cell surface markers can be evaluated using methods widely used and known in the art, such as flow cytometry analysis and the like.

[0142] Dose regimen

[0143] The compounds in the cell cultures of the present invention can be added to the cultures at suitable doses. The actual dose levels of the compounds in the cell cultures of the present invention can be varied in order to obtain an amount of the compound that effectively achieves the desired tolerogenic effect on the cells without toxicity.

[0144] When retinoic acid is added to the cell culture, a suitable dose of retinoic acid can be, for example, in the range of about 0.5 uM to about 10 uM. For example, a suitable dose can be about 0.5 uM to about 8 uM, 0.5 uM to about 6 uM, 0.5 uM to about 5 uM, 0.5 uM to about 4 uM, preferably 0.5 uM to about 3 uM.

[0145] When an AhR agonist is added to the cell culture, a suitable dose of the AhR agonist can be, for example, in the range of about 1 nM to about 10 uM, typically about 5 nM to about 2 uM, for example, about 5 nM to about 750 nM. For example, a suitable dose can be about 5 nM to about 500 nM, 5 nM to about 250 nM, 5 nM to about 100 nM, preferably 5 nM to about 50 nM. When not present in the cell culture, the suitable dose of the AhR agonist is 0 nM.

[0146] When TGFβ is added to the cell culture, a suitable dose of TGFβ can be, for example, in the range of about 1 ng / ml to about 200 ng / ml, such as about 5 ng / ml to about 200 ng / ml, typically about 5 ng / ml to about 150 ng / ml. For example, suitable doses can be about 5 ng / ml to about 125 ng / ml, 5 ng / ml to about 100 ng / ml, 5 ng / ml to about 75 ng / ml, 5 ng / ml to about 50 ng / ml, preferably 5 ng / ml to about 30 ng / ml. When not present in the cell culture, the suitable dose of TGFβ is 0 ng / ml.

[0147] When retinoic acid, TGFβ, and an AhR agonist are added to the cell culture, a suitable dose of retinoic acid can be, for example, in the range of about 0.5 μM to about 10 μM. For example, suitable doses can be about 0.5 μM to about 8 μM, 0.5 μM to about 6 μM, 0.5 μM to about 5 μM, 0.5 μM to about 4 μM, preferably 0.5 μM to about 3 μM. A suitable dose of the AhR agonist can be, for example, in the range of about 1 nM to about 10 μM, typically about 5 nM to about 2 μM, such as about 5 nM to about 750 nM. For example, suitable doses can be about 5 nM to about 500 nM, 5 nM to about 250 nM, 5 nM to about 100 nM, preferably 5 nM to about 50 nM. A suitable dose of TGFβ can be, for example, in the range of about 1 ng / ml to about 200 ng / ml, such as about 5 ng / ml to about 200 ng / ml, typically about 5 ng / ml to about 150 ng / ml. For example, suitable doses can be about 5 ng / ml to about 125 ng / ml, 5 ng / ml to about 100 ng / ml, 5 ng / ml to about 75 ng / ml, 5 ng / ml to about 50 ng / ml, preferably 5 ng / ml to about 30 ng / ml.

[0148] When retinoic acid, TGFβ, and an AhR agonist are added to the cell culture, a suitable dose of retinoic acid can be, for example, in the range of about 0.5 μM to about 3 μM. A suitable dose of the AhR agonist can be, for example, in the range of about 5 nM to about 50 nM, preferably about 20 nM. A suitable dose of TGFβ can be, for example, in the range of about 5 ng / ml to about 30 ng / ml, preferably about 20 ng / ml.

[0149] When retinoic acid, TGFβ, and an AhR agonist are added to the cell culture, a suitable dose of retinoic acid can be, for example, about 0.5 μM to about 3 μM, preferably about 2 μM. A suitable dose of the AhR agonist can be, for example, in the range of about 5 nM to about 50 nM, preferably about 10 nM. A suitable dose of TGFβ can be, for example, in the range of about 5 ng / ml to about 30 ng / ml, preferably about 10 ng / ml.

[0150] The dosage regimen can be adjusted to provide the optimal desired response. For example, a single dose can be administered, several separate doses can be administered over time, or the dose can be proportionally decreased or increased depending on the urgency of the treatment situation. As used herein, the form of the dosage unit refers to a physically discrete unit suitable as a unit dose for the culture conditions employed; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect.

[0151] The compounds used in the cell cultures of the present invention can be administered alone or in combination with one or more compounds. One compound can be co-administered with one or more other compounds. Two compounds can be co-administered with one or more other compounds.

[0152] The co-administration of two or more compounds can be achieved in many different ways. Both can be administered together in a single composition, or they can be administered in separate compositions as part of a co-administration. For example, one or more, or two or more compounds can be administered before, separately from, after, or sequentially to, or in parallel or simultaneously with one or more other compounds.

[0153] Treatment and prevention of antigen immune responses and prevention of immune rejection

[0154] The present invention provides tolerogenic APCs or populations thereof for treating mammalian subjects having an unwanted immune response against an antigen or at risk of an unwanted immune response against an antigen. The antigen can be any antigen discussed in this specification, such as FVIII, FIX, an antibody, an antigen associated with a graft (e.g., an allograft), or an autoantigen.

[0155] The tolerogenic APCs produced by the culturing method of the present invention can produce autologous tolerogenic APCs or populations thereof. The autologous tolerogenic APCs used in treatment can be obtained from autologous monocytes isolated from a sample taken from the same subject. The monocytes taken from the subject can be cultured with an antigen or an epitope containing a fragment thereof, and then autologous tolerogenic APCs are obtained. Then, the autologous tolerogenic APCs have the ability to induce tolerance to the antigen when administered back to the subject.

[0156] The present invention further provides tolerogenic APCs or populations thereof for use in methods for preventing allograft rejection in a recipient subject. The allograft can be a kidney, pancreas, liver, lung, heart, skin graft, blood cell graft, such as a stem cell graft, etc. Thus, the present invention provides a treatment for transplant rejection. The tolerogenic APCs used in the treatment can be obtained from monocytes isolated from a sample taken from a donor. The monocytes taken from the donor can be cultured to obtain tolerogenic APCs such that the tolerogenic APCs have the ability to induce tolerance to antigens present in an allograft derived from the same donor.

[0157] The tolerogenic APCs used in the treatment can be obtained from monocytes isolated from a sample taken from the recipient. The monocytes taken from the recipient can be cultured with an antigen or antigen pool and then tolerogenic APCs are obtained. Then, the tolerogenic APCs have the ability to induce tolerance to antigens present in an allograft derived from the donor. This method can be used in situations where it is not possible to derive monocytes from the donor, such as when the donor is not a living donor. The tolerogenic APCs sourced from monocytes isolated from a sample taken from the recipient according to the present invention can be used in methods for treating immune rejection or for preventing immune rejection of a xenograft.

[0158] The tolerogenic APCs of the present invention can be used in manufacturing methods. The present invention includes the use of tolerogenic APCs or populations thereof in the preparation of a medicament for treating a mammalian subject having an unwanted immune response or at risk of an unwanted immune response. The method can include administering the tolerogenic APCs or populations thereof to a mammalian recipient so as to establish immune tolerance to an antigen. The present invention also includes the use of tolerogenic APCs or populations thereof in the preparation of a medicament for treating immune rejection of an allograft in a recipient subject, wherein the graft is derived from a donor. The method can include administering the tolerogenic APCs or populations thereof to the recipient subject so as to establish tolerance to the allograft, wherein the tolerogenic APCs or populations thereof are obtained from monocytes isolated from a sample taken from the donor or the recipient. When the tolerogenic APCs or populations thereof are sourced from monocytes isolated from a sample taken from the recipient, such manufacturing methods can also be applied to xenografts.

[0159] Mammal

[0160] The mammals from which the samples for the methods according to the present invention are obtained and the mammals that can be treated by the methods according to the present invention particularly include humans (Homo sapiens). The mammals that can be treated by the methods of the present invention are preferably humans (Homo sapiens).

[0161] Sample

[0162] The method of the present invention may include isolating monocytes from any suitable sample from an object. The sample may be a blood sample, a 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, and preferably the sample is autologous or allogeneic.

[0163] Obtaining APCs from a mammal

[0164] According to the present invention, monocytes are first obtained from a mammal. A suitable method is to collect PBMCs by apheresis. The PMBCs collected by apheresis can be maintained under temperature-controlled conditions, such as at ambient temperature, such as at 18°C - 25°C, or the PBMCs collected by apheresis can be frozen, such as at -4°C, -20°C, or -80°C or lower. Alternatively, PBMCs can be separated by density centrifugation. Thereafter, monocytes can be positively selected from PBMCs using a solid phase, such as by plastic adhesion or by using beads, such as anti-CD14 magnetic beads for separation.

[0165] Administering the cells to an object

[0166] The cells can be administered back to the mammal by various routes, such as intravenously, subcutaneously or intradermally, intranodally or directly at the site of a lesion suitable for injection. The cell-containing medium suitably contains human albumin as a cell-protecting protein. Generally, 1 - 100×10 7 cells / dose are administered in 1 - 10 doses at intervals of weekly to bi-weekly. The treatment can be extended, and the dosing regimen can be altered until the desired tolerance is achieved.

[0167] Antigen

[0168] The antigen or an epitope containing a fragment thereof may be or be derived from a biopharmaceutical, such as a protein drug (especially a drug containing a protein of at least 50 or at least 200 or at least 1000 amino acid residues). The biopharmaceutical may contain a polysaccharide component.

[0169] Exemplary biopharmaceuticals include blood factors (including, for example, FVIII or factor IX), hormones (including insulin and EPO), growth factors (including EGF, IGF, KGF, HGF, and FGF), cytokines (such as interleukins), enzymes (such as imiglucerase, rasburicase, imiglucerase, beta-galactosidase, alglucosidase alfa, rilonacept, idursulfase, and sulfamidase), and the like. Other examples include GCSF and analogs such as filgrastim and its pegylated forms (such as pegylated filgrastim) and interferons (such as interferon beta-1a). In a preferred aspect of the invention, the drug is FVIII. In another aspect of the invention, the drug is factor IX. In the case of FVIII, various recombinant drugs are available, including the commercial products ReFacto AF, Helixate NexGen, Kogenate Bayer, Kovaltry, Advate, NovoEight, Esperoct, Nuwiq, Beriate, Beriate P, Feiba, Haemoctin, Hemofil, Monoclate-P, Octanate[LV], Optivate, and Recombinate. These products can be used as antigens, and epitopes containing fragments can be derived from any of these products.

[0170] Additional exemplary biopharmaceuticals include engineered proteins (such as fusion and chimeric proteins) and recombinant proteins. In some embodiments, the drug can be an antibody. The drug can be, for example, a monoclonal antibody, such as a humanized or fully human monoclonal antibody. The drug can also be a protein construct containing immunoglobulin fragments. The term antibody also includes bispecific antibodies, antibody-drug conjugates, and antibody-nanoparticle conjugates, as well as pegylated or otherwise extended analogs. In some embodiments, the antibody can be a domain antibody or an antibody fragment, including single light chain antibodies, VHHs, scFvs, Fabs, F(ab')2s, or BiTEs, and their pegylated or otherwise extended analogs.

[0171] Exemplary antibodies include infliximab (chimeric antibody, anti-TNFα), adalimumab (human antibody, anti-TNFα), basiliximab (chimeric antibody, anti-IL-2), abciximab (chimeric antibody, anti-GpIIb / IIIa), daclizumab (humanized antibody, anti-IL-2), gemtuzumab (humanized antibody, anti-CD33), alemtuzumab (humanized antibody, anti-CD52), edrecolomab (murine Ig2a, anti-EpCAM), rituximab (chimeric antibody, anti-CD20), palivizumab (humanized antibody, anti-respiratory syncytial virus), trastuzumab (humanized antibody, anti-HER2 / neu (ErbB2) receptor), bevacizumab (humanized antibody, anti-VEGF), cetuximab (chimeric antibody, anti-EGFR), eculizumab (humanized antibody, anti-complement system protein C5), efalizumab (humanized antibody, anti-CD11a), ibritumomab tiuxetan (murine antibody, anti-CD20), muromonab-CD3 (murine antibody, anti-T cell CD3 receptor), natalizumab (humanized antibody, anti-α4 integrin), nimotuzumab (humanized IgG1, anti-EGF receptor), omalizumab (humanized antibody, anti-IgE), panitumumab (human antibody, anti-EGFR), ranibizumab (humanized antibody, anti-VEGF), 1-131 tositumomab (humanized antibody, anti-CD20), ofatumumab (human antibody, anti-CD-20), certolizumab pegol (humanized antibody, anti-TNFα), golimumab (human antibody, anti-TNFα), emicizumab (humanized bispecific antibody, anti-FIXa / FX), denosumab (human antibody, anti-RANK ligand), and concizumab (humanized antibody, anti-tissue factor pathway inhibitor).

[0172] Exemplary biopharmaceuticals that are fusion proteins include etanercept.

[0173] A comprehensive list of biologic protein drugs in clinical development and approved products was published in the 2013 PhARMA report “Biologics” - https: / / web.archive.org / web / 20161011093352 / http: / / www.phrma.org / sites / default / files / pdf / biologics2013.pdf - which gives details of 907 biologics for over 100 diseases. This document is incorporated herein by reference in its entirety. It is contemplated that the present invention can be used against these and other biotherapeutic agents that produce an immune response in the subject being treated.

[0174] Thus, the antigen or epitope containing a fragment thereof for use in the methods described herein can be, for example, FVIII or a derivative or fragment thereof, or factor IX or a derivative or fragment thereof, or an antibody or an antibody fragment.

[0175] The antigen or epitope containing a fragment thereof can be associated with an allograft. Tolerogenic APCs presenting antigens associated with an allograft can be used to treat allograft rejection.

[0176] The antigen or epitope containing a fragment thereof can be or be derived from an autoantigen. Tolerogenic APCs presenting autoantigens 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 (bullous pemphigoid), myelin (multiple sclerosis), pancreatic β-cell proteins (type 1 diabetes), nicotinic acetylcholine receptor (myasthenia gravis), neuronal surface proteins (autoimmune epilepsy and encephalitis), 2-hydrolyase (autoimmune Addison's disease), FcεRI (chronic autoimmune urticaria) and acetylcholine receptor (myasthenia gravis), fibrillin (scleroderma), and cardiolipin (systemic lupus erythematosus). These autoantigens or epitopes containing fragments thereof can be used as antigens in the methods of the present invention.

[0177] The antigen or epitope containing a fragment can be unknown. Thus, unknown antigens can be used in the culturing methods of the present invention. The unknown antigen can be associated with a tissue sample taken from a subject or an extract thereof.

[0178] The antigen can be delivered from an exogenous medium through the cell membrane into the intracellular compartment by any suitable antigen delivery method or carrier. For example, the antigen delivery method or carrier can be nanoparticles. Thus, the antigen can be associated with the nanoparticles, i.e., an antigen-nanoparticle conjugate. The antigen-nanoparticle can be an FVIII-nanoparticle conjugate. The nanoparticles can be lipid nanoparticles such as vesicles or micelles.

[0179] Alternatively, the antigen delivery method of the carrier can be a cell-penetrating peptide. The antigen can be fused with the cell-penetrating peptide, thereby allowing transport from the extracellular environment across the lipid bilayer membrane into the intracellular compartment. The cell-penetrating peptide can be the Tat peptide from the transactivator protein Tat of HIV1, called penetratin or the third helix of the antennapedia homeodomain of polyarginine. The cell-penetrating peptide can be cell-selective.

[0180] Immune responses and uses of the tolerogenic APCs of the present invention

[0181] The tolerogenic APCs of the present invention can be used to inhibit undesired immune responses in a subject receiving the cells. The undesired immune responses can include antibody responses and / or cellular responses.

[0182] The tolerogenic APCs of the present invention are useful, for example, in treating mammalian subjects that develop an immune response, including the production of ADA against any drug capable of eliciting such a response (e.g., any biologic drug, such as a protein drug).

[0183] The tolerogenic APCs of the present invention are useful in treating mammalian subjects that have an immune response to a drug, including the production of ADA in many drug treatment scenarios, such as in the treatment of bleeding disorders (hemophilia A and B; deficiencies in FVIII and factor IX, respectively), growth factor deficiencies (deficiencies in EGF, IGF, KGF, HGF, FGF, etc.), hormone deficiencies (deficiency in EPO), enzyme replacement therapy (e.g., for imiglucerase (e.g., Cerezyme TM ), alpha-galactosidase A (α-Gal A) (e.g., galactosidase β, FABRYZYME), acid alpha-glucosidase (GAA) (e.g., against aglucerase alfa, LUMIZYME TM , MYOZYME TM ), and arylsulfatase B (e.g., laronidase, ALDURAZYME TM , idursulfase, ELAPRASE TM , galsulfase, NAGLAZYME TM )-produced ADA), and inflammatory and autoimmune disorders (e.g., anti-TNFα monoclonal antibodies).

[0184] The tolerogenic APCs of the present invention are useful in treating mammalian subjects with an autoimmune disease who have developed an immune response to an autoantigen capable of eliciting such a response.

[0185] The tolerogenic APCs of the present invention are useful in treating mammalian subjects with graft (e.g., allograft) rejection or at risk of graft rejection who have developed or may develop an immune response to an antigen associated with the graft capable of eliciting such a response.

[0186] Autoimmune disease or disorder

[0187] The tolerogenic APCs according to the present invention can be used to treat autoimmune diseases. Suitably, the autoimmune disease or disorder is selected from achlorhydria, acquired hemophilia, acute hemorrhagic leukoencephalitis, acquired thrombocytopenic purpura, Addison's disease, alopecia areata, anemia, ankylosing spondylitis, anti-glomerular basement membrane disease, antiphospholipid syndrome, aplastic anemia, atopic allergy, autoimmune atrophic gastritis, autoimmune hearing loss, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune lymphoproliferative, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, autoimmune polyendocrinopathy, autoimmune sensorineural hearing loss, autoimmune type II syndrome, autoimmune uveitis, Behcet's syndrome / disease, celiac disease, Chagas disease, chronic active hepatitis, chronic inflammatory demyelinating disease, chronic lymphocytic thyroiditis, Churg-Strauss syndrome, Crohn's disease, cryoglobulinemia, Cushing's syndrome, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, Diffuse cerebral sclerosis of Schilder, epidermolysis bullosa acquisita, erythema disease, Felty's syndrome, glomerulonephritis, membranous glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hamman-Rich syndrome, idiopathic thrombocytopenic purpura, inflammatory bowel disease, insulin resistance type B, Lambert-Eaton myasthenic syndrome, lens-induced uveitis, lichen sclerosus et atrophicus, lymphocytopenia, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease, Mullen's ulcer, mucocutaneous lymph node syndrome, multifocal motor neuropathy, multiple sclerosis, myasthenia gravis, transverse myelitis, myocarditis, narcolepsy, neuromyelitis optica, ocular cicatricial pemphigoid, oculovestibular auditory syndrome, sympathetic ophthalmia, opsoclonus myoclonus syndrome, pancreatitis, bullous pemphigoid, pemphigus foliaceus, pemphigus vulgaris, polyarteritis nodosa, polymyalgia rheumatica, polyradiculoneuropathy, primary biliary cholangitis, primary biliary cirrhosis, psoriasis, Raynaud's disease, Reiter's disease, relapsing polychondritis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, sclerosing colitis, Sjogren's syndrome, stiff-person syndrome, adult Still's disease, Takayasu arteritis, temporal arteritis, thyrotoxicosis, insulin resistance type B, ulcerative colitis, uveomeningoencephalitis syndrome, vitiligo, and Wegener's granulomatosis.

[0188] Suitably, the autoimmune disease is type 1 diabetes, rheumatoid arthritis, chronic lymphocytic thyroiditis, multiple sclerosis, and ulcerative colitis.

[0189] In addition, diseases that may be partially involved in autoimmune responses are arteriosclerosis, Parkinson's disease, and Alzheimer's disease.

[0190] Cryopreservation, storage, and thawing

[0191] The tolerogenic APCs of the present invention can be manufactured by manual, semi - automatic, or fully automatic closed systems. The cells of the present invention (e.g., the DCs of the present invention) can be stored as a single - cell suspension in a cryopreservation medium containing 2 - 10% DMSO (dimethyl sulfoxide). The cells can then be washed, preferably with a physiological sodium chloride buffer containing human serum albumin, and resuspended in the cryopreservation medium, preferably in the range of 0.5 - 20×10 6 cells / ml and transferred to cryovials. The cells can then be placed in a freezing container in an - 80°C freezer. After 24 hours in the - 80°C freezer, the cryovials can be transferred to a - 150°C freezer or a liquid nitrogen tank for long - term storage. Preferably, the cells of the present invention (e.g., the DCs of the present invention) are cryopreserved below - 120°C.

[0192] The cells of the present invention (e.g., the DCs of the present invention) can be thawed in the cryovial until only a small piece of ice remains before use.

[0193] Storage and transportation

[0194] The cells of the present invention (e.g., the DCs of the present invention) can be stored as single cells or multicellular aggregates. The single cells or multicellular aggregates can be embedded or encapsulated in a hydrogel. Entrapping or encapsulating the cells in a hydrogel can maintain their function at low temperatures and can be used for effectively storing and / or transporting the multicellular aggregates while providing mechanical protection and maintaining cell morphology, integrity, viability, and function.

[0195] The single cells or multicellular aggregates can be encapsulated in a hydrogel in vitro. The cells generally have a structurally intact cell membrane, are living cells or viable cells, and have the cell morphology representative of the cells of the present invention (e.g., the DCs of the present invention). The hydrogel can be a coating that completely or incompletely covers / encloses at least most of the single cells or multicellular aggregates so as to trap the cells or aggregates in the hydrogel. The hydrogel coating can be formed separately from the single cells or multicellular aggregates and then placed on the single cells or multicellular aggregates. The hydrogel coating can comprise a cross - linked alginate layer formed separately (i.e., spatially separated) from the aggregate. Alternatively, the hydrogel coating can be formed in situ (i.e., in the presence of the single cells or multicellular aggregates).

[0196] Single - cell or multicellular aggregates can be embedded or encapsulated in a reversibly cross - linked hydrogel (e.g., alginate hydrogel). A "reversibly cross - linked hydrogel" refers to a hydrogel formed by reversible cross - linking (i.e., the cross - linking can be reversed such that the hydrogel reverts to a solution). Reversal of the cross - linking enables the release of the embedded or encapsulated multicellular aggregates from the hydrogel (e.g., upon its use / after completion of transportation or storage). Examples of reversibly cross - linked hydrogels are well known in the art, and those skilled in the art can readily identify suitable hydrogels.

[0197] The hydrogel can comprise a hydrogel - forming polymer having a cross - linked or network structure or matrix; and interstitial fluid. The hydrogel is capable of inhibiting or preventing cell differentiation in the aggregates encapsulated or embedded therein. The hydrogel can be semi - permeable. The hydrogel can be a "hydrogel - forming polymer" that is capable of forming a cross - linked or network structure or matrix under appropriate conditions, within which the interstitial fluid and the multicellular aggregates can be retained. The hydrogel can comprise internal pores.

[0198] The polymer forming the hydrogel can be alginic acid or an alginate of a metal ion. Preferably, the metal is a Group 1 metal (e.g., lithium alginate, sodium alginate or potassium alginate) or a Group 2 metal (e.g., calcium alginate, magnesium alginate, barium alginate or strontium alginate). Preferably, the polymer is calcium alginate or sodium alginate or strontium alginate. The hydrogel - forming polymer can be a polymer based on cross - linked acrylic acid (e.g., polyacrylamide). The polymer forming the hydrogel can be a cross - linkable cellulose derivative, a hydroxy - ether polymer (e.g., poloxamer), pectin or a natural gum.

[0199] For multicellular aggregates, the cells can be directly or indirectly adjacent or connected to each other in a manner that forms a cell aggregate. A matrix, substrate or scaffold, commonly referred to as a "structure", can connect the adjacent cells into an aggregate. The structure can be a synthetic or natural polymer. Preferably, the structure is biodegradable. The structure can be, for example, a polymer comprising polylactic acid, collagen, nylon (e.g., nylon mesh), collagen, gelatin, alginate, cellulose, glass or Matrigel.

[0200] The cells can be adjacent via an extracellular matrix (ECM), such as an Alvatex polystyrene scaffold for 3D cell culture. Alternatively, the multicellular aggregate can be unstructured.

[0201] Single - cell or multi - cell aggregates encapsulated in a hydrogel can be packaged and sealed in a container for storage or transportation from a first location to a second location. The single - cell or multi - cell aggregates encapsulated in a hydrogel can have a storage stability time of at least 1 hour, at least 2 hours, at least 5 hours, at least 12 hours, at least 24 hours, etc. The single - cell or multi - cell aggregates encapsulated in a hydrogel can be stored or transported in the hydrogel (and sealed container) at a temperature of - 80°C to 45°C, preferably 4°C to 45°C, or at ambient temperature, such as 10 - 25°C, preferably 15 - 20°C.

[0202] Single - cell or multi - cell aggregates encapsulated in a hydrogel can be stored or transported under cell culture conditions (e.g., about 37°C, about 5% CO2) or under refrigeration conditions, such as 4 - 6°C, preferably about 4°C. Single - cell or multi - cell aggregates encapsulated in a hydrogel can be refrigerated during storage or transportation, such as 2 - 8°C or 8 - 15°C. Single - cell or multi - cell aggregates encapsulated in a hydrogel can be stored or transported at a controlled room temperature (CRT) (which is defined as 15 to 25°C). They can be stored or transported at a cool or CRT (i.e., 8 to 25°C). Single - cell or multi - cell aggregates encapsulated in a hydrogel can be stored or transported at a low temperature (i.e., below about 35°C, typically in the range of 0 to 32°C).

[0203] The hydrogel containing multi - cell aggregates can be frozen before storage and / or transportation. This can extend the time that the cells of the multi - cell aggregates survive after thawing and / or increase the available transportation time. Thus, the hydrogel can be used as a cryoprotectant in this way. For example, the temperature of the hydrogel containing aggregates can be reduced to below 0°C, below - 15°C or below - 80°C. The hydrogel containing multi - cell aggregates may or may not allow thawing or be thawed, i.e., raise its temperature above 0°C during storage and / or transportation, preferably at a slow, controlled or uncontrolled rate of temperature increase. In other instances, the hydrogels of the present invention are not refrigerated or frozen.

[0204] Single - cell or multi - cell aggregates encapsulated in a hydrogel can be stored and / or transported for up to 10 or 20 weeks. Preferably, the single - cell or multi - cell aggregates are stored in the hydrogel for up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks before being released from the hydrogel. More preferably, the single - cell or multi - cell aggregates are stored in the hydrogel for up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days before being released from the hydrogel.

[0205] Single - cell or multicellular aggregates can be released from the hydrogel under appropriate cytocompatible conditions, i.e., conditions that are not harmful or not significantly harmful to the integrity of the cells and / or cell membranes. For example, the hydrogel can be dissociated by chemical disintegration or dissolution, such as using a suitable alginate - dissolving buffer.

[0206] The storage and / or transportation of multicellular aggregates is described in WO2019142004, the content of which is incorporated herein by reference in its entirety. QC (Quality Control) release tests determine

[0207] Quality testing of RA + TGFβ+AhR agonist - treated DCs can be based on the expression of several different markers and the ability to reduce T - cell proliferation in the MLR. For example, RA + TGFβ+AhR agonist - treated DCs can express the markers CD11c, HLA - DR, CD80, CD83, CD86, ILT3, GARP, CD141, LAP, CD103, BTLA, HLA - G, and CD49b. RA + TGFβ+AhR agonist - treated DCs can preferably express at least CD11c, HLA - DR, CD80, CD83, CD86, and ILT3, more preferably at least CD11c, HLA - DR, CD80, CD83, CD86, and ILT3, and even more preferably at least CD11c, HLA - DR, CD80, CD83, CD86, ILT3, GARP, CD141, and LAP. T - cell proliferation can be evaluated by incorporation of 3 3H - thymidine, CFDA, CD25 expression, and KI - 67. T - cell proliferation can preferably be evaluated by incorporation of 3 3H - thymidine, more preferably by CFDA, CD25 expression, and incorporation of 3 3H - thymidine, and even more preferably by CFDA, CD25 expression, KI - 67, and incorporation of 3 3H - thymidine. Examples

[0208] Materials and methods for Examples 1–18 and 20–25

[0209] Isolation of human monocytes and T cells

[0210] Using Lymphoprep TM(StemCell Technologies, Vancouver, Canada) or PBMC were isolated by density centrifugation using a cell preparation tube (BD Bioscience, San Jose, United States). Monocytes were positively selected from PBMC using anti-CD14 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4 + T cells were purified using the EasySep human CD4 + T cell isolation kit (StemCell Technologies).

[0211] Generation of dendritic cells (DCs)

[0212] CD14 + monocytes were seeded at 1.25 × 10 6Cells / ml were cultured for 7 days in GMP DC medium (CellGenix, Freiburg, Germany) containing HEPES, GlutaMAX and penicillin-streptomycin solution (Thermo Fisher, Waltham, MA) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 ng / ml; Peprotech, London, UK) and interleukin-4 (IL-4; 100 ng / ml; PeproTech). Cells were supplemented with fresh medium and cytokines on day 3. Control DCs were differentiated in GM-CSF and IL-4 without the addition of any other compounds and optionally further treated with lipopolysaccharide (LPS, 0.5 μg / ml; Sigma-Aldrich, Saint Louise, MO) or a mixture of pro-inflammatory cytokines consisting of TNFα (10 ng / ml; PeproTech), IL-1β (10 ng / ml; PeproTech) and prostaglandin E2 (PGE2; 1 μg / ml; Sigma) on day 6 to generate stimulated immunogenic DCs. Tolerogenic DCs (tolDCs) were generated by treatment with various tolerogenic compounds: AhR agonist "C1" (C1; 20 nM; IMA-06201; Immunahr AB, Lund, Sweden) on days 0 and 3, TGFβ1 (10 or 20 ng / ml; PeproTech) on day 3 and retinoic acid (2 μM; Sigma-Aldrich) on day 6. Control tolDCs for comparison were established by treatment with dexamethasone (Dex; 100 nM; Sigma-Aldrich) and vitamin D3 (VitD3; 100 nM, StemCell Technologies) on day 3. The phenotypic stability of TolDCs was investigated by adding LPS (0.5 μg / ml) or a mixture of pro-inflammatory cytokines consisting of TNFα (10 ng / ml), IL-1β (10 ng / ml) and PGE2 (1 μg / ml) in the last 24 hours of culture, and for Example 24 also IL-6 (10 ng / ml). On day 7, DCs were harvested and washed thoroughly, and then subjected to phenotypic analysis and functional assays.

[0213] DC Phenotypic Analysis

[0214] Cell surface expression was studied using the following fluorescently labeled antibodies: CD11c (B-Ly6), CD83 (HB15e), CD86 (BU63), ILT3 (ZM4.1), CD141 (M80), GARP (7B11), CD103 (B-Ly7), MERTK (590H11G1E3), BTLA (J168-540), CD49b (P1E6-C5), HLA-G (87G), LAP (FNLAP) from BD Biosciences (Franklin Lakes, NJ), BioLegend (San Diego, CA) and Thermo Fisher. DCs were washed and resuspended in staining buffer (phosphate buffered saline supplemented with 0.5% BSA and 2 mM EDTA) and incubated with the antibodies for 20 min, then washed and harvested on a MACSQuant 10 flow cytometer (Miltenyi Biotec) and analyzed using FlowJo software (BD Biosciences). Fixable viability dye (FVD) from Thermo Fisher was used to distinguish live cells from dead cells.

[0215] DC cytokine production

[0216] IL-23 production was measured in the supernatants of DC cultures with or without LPS or pro-inflammatory cytokine stimulation using the Luminex platform (Invitrogen, Carlsbad, USA).

[0217] DC / T cell cultures: MLR and Treg induction

[0218] To analyze the T cell stimulatory capacity of the generated DC populations, allogeneic MLRs were performed. DC / T cell cultures were carried out in complete medium: RPMI-1640 containing fetal bovine serum (FBS; 10%; Thermo Fisher), HEPES, GlutaMAX and penicillin-streptomycin solution. T cells were cultured at a T cell:DC ratio of 10:1 (10 5 cells / well) for 5 - 7 days. Proliferation was measured by incorporation of 3 H-thymidine during the last 18 h of culture. To phenotype the cells for CD4 + CD49b + LAG3 + Tr1 cells, the cells were phenotyped as described below. To phenotype the cells for CD25 hi Foxp3 +Induction of Tregs: T cells from MLR cultures were additionally rested for 7 days in complete medium containing IL-2 (20 IU / ml; PeproTech), and then phenotypic analysis was performed as described below.

[0219] Treg Phenotypic Analysis

[0220] The following fluorescently labeled antibodies from BD Biosciences and BioLegend were used to study cell surface and intracellular marker expression: CD4 (A161A1), CD25 (M-A251), CD49b (P1E6-C5), LAG3 (11C3C65), and Foxp3 (259D / C7). T cells were washed and resuspended in staining buffer and incubated with the antibodies for 20 min, and then washed, fixed, permeabilized, and intracellular staining for Foxp3 was performed using the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience, Thermo Fisher). Fixable viability dye (FVD) from Thermo Fisher was used to distinguish live cells from dead cells. Flow cytometry analysis was performed as described above.

[0221] Freezing and Thawing of DCs

[0222] DCs were washed once and resuspended in freezing medium at a cell concentration of 10 - 20×10 6 cells / ml and transferred to cryovials. These vials were placed in a freezing container and put into an -80°C refrigerator. The freezing container allows a controlled freezing rate of approximately -1°C / min. After 24 hours in an -80°C freezer, the cryovials were transferred to an -150°C freezer for long-term storage. Two different freezing media were tested; CryoStor (CS, BioLifeSolutions) containing 10% DMSO (CS10) and a conventional freezing medium containing 10% DMSO, 50% human serum albumin, and 40% cell culture medium. DCs were thawed in a 37°C water bath in the vials, and the cell suspension was carefully transferred to a 15 mL tube containing cold GMP DC medium (CellGenix) and washed once. Then the cells were stained according to the DC phenotypic typing method described above.

[0223] Example 1 - Effects of Various Compounds on DC Expression of CD83

[0224] The ability of various compounds to induce a tolerogenic phenotype in DCs was tested, where, as one of several factors, it is indicated by a low expression level of the DC maturation marker CD83 and it should additionally be as resistant as possible to upregulation by immunogenic stimulants. By culturing for 7 days in GM-CSF and IL-4, from CD14 +Monocyte - differentiated control DC. Control DC was either unstimulated or stimulated with LPS added on day 6 to generate immunogenic DC. Monocyte - differentiated tolDC or its sub - optimal variants were generated by culturing as above in GM - CSF and IL - 4 and treating with various tolerogenic compounds used either as a single reagent or in combination, starting from CD14 + to demonstrate the synergistic effect of the combination, as shown in Figure 1 (A) and (B): An AhR agonist C1 (AhR ag) at 20 nM on days 0 and 3, TGFβ at 20 ng / mL on day 3, and retinoic acid (abbreviated as 'RA' in this example section and figures) at 2 μM on day 6. TolDC for comparison was established by treating with 100 nM Dex and 100 nM VitD3 on day 3 (abbreviated as 'Dex / VitD3' in this example section and figures). On day 6, some samples were treated with LPS to test phenotypic stability ( Figure 1 (A) and (B), black bars). All cells were harvested on day 7 and stained for CD83 cell - surface expression using a fluorescent - labeled anti - CD83 antibody, followed by flow - cytometry analysis.

[0225] The mean fluorescence intensity (MFI) values on live cells are shown as mean ± SD in Figure 1 (A) and (B).

[0226] Figure 1 (A) The results show that, with or without LPS stimulation, DCs treated with RA + TGFβ and RA + TGFβ+AhR agonist express lower levels of CD83 compared to the corresponding control DCs and Dex / VitD3 - treated tolDCs for comparison. Treatment with RA + TGFβ+AhR agonist is superior to treatment with RA + TGFβ in reducing CD83 expression in DCs.

[0227] Figure 1 (B) The results show that when stimulated with LPS, DCs treated with RA + TGFβ, RA + TGFβ+AhR agonist, and RA + AhR agonist express lower levels of CD83 compared to the corresponding control DCs. Treatment with RA + TGFβ+AhR agonist or RA + TGFβ is superior to treatment with RA + AhR in reducing CD83 expression in DCs, and furthermore is superior to treatment with RA as a single reagent.

[0228] Example 2 - Effects of various compounds on DC expression of CD86

[0229] The ability of various compounds to induce a tolerogenic phenotype in DCs was tested, including low expression levels of the costimulatory molecule CD86 as one of several factors and which should additionally be as resistant as possible to upregulation by immunogenic stimuli. Control DCs were differentiated from CD14 + monocytes by culturing for 7 days in GM-CSF and IL-4. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic DCs. TolDCs or their suboptimal variants were differentiated from CD14 + monocytes by culturing as above in GM-CSF and IL-4 and by treatment with various tolerogenic compounds used either as a single reagent or in combination, to demonstrate the synergistic effect of the combination, as Figure 2 (A) and (B) show: the AhR agonist C1 (AhR ag) at 20 nM on days 0 and 3, TGFβ at 20 ng / mL on day 3, and RA at 2 uM on day 6. TolDCs for comparison were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some samples were treated with LPS to test phenotypic stability ( Figure 2 (A) and (B), black bars). All cells were harvested on day 7 and stained for CD86 cell surface expression using a fluorescently labeled anti-CD86 antibody, followed by analysis by flow cytometry.

[0230] The MFI values on live cells are shown as mean ± SD in Figure 2 (A) and (B).

[0231] Figure 2 (A) The results show that, with or without LPS stimulation, DCs treated with RA + TGFβ and RA + TGFβ + AhR agonist expressed lower levels of CD86 compared to the corresponding control DCs and the comparison tolDCs treated with Dex / VitD3. Treatment with RA + TGFβ + AhR agonist was superior to treatment with RA + TGFβ in reducing CD86 expression in DCs.

[0232] Figure 2The results of (B) showed that, with or without LPS stimulation, DCs treated with RA+TGFβ and RA+TGFβ+AhR agonist expressed lower levels of CD86 compared to the corresponding control DCs. When stimulated with LPS, DCs treated with RA+AhR agonist expressed higher levels of CD86 compared to DCs treated with RA+TGFβ and RA+TGFβ+AhR agonist. DCs treated with RA did not show resistance to upregulation of CD86 after LPS stimulation compared to stimulated control DCs. Treatment with RA+TGFβ+AhR agonist was superior to treatment with RA+TGFβ or RA+AhR agonist in reducing CD86 expression in DCs when stimulated with LPS, and in addition, treatment with all three combinations was superior to RA as a single reagent.

[0233] Example 3 - Effects of various compounds on DC expression of ILT3

[0234] The ability of various compounds to induce a tolerogenic phenotype in DCs was tested, including high expression levels of the tolerogenic marker ILT3 as one of several factors, and it should additionally be as resistant as possible to downregulation by immunogenic stimulants. Control DCs were differentiated from CD14 + monocytes by culturing in GM-CSF and IL-4 for 7 days. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs or their suboptimal variants were differentiated from CD14 + monocytes by culturing as above in GM-CSF and IL-4 and treating with various tolerogenic compounds as a single reagent or in combination to demonstrate the synergistic effect of the combination, as Figure 3 (A) and (B) show: an AhR agonist C1 (AhR ag) at 20 nM on days 0 and 3, TGFβ at 20 ng / mL on day 3, and RA at 2 μM on day 6. TolDCs for comparison were established by treating with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some samples were treated with LPS to test phenotypic stability ( Figure 3 (A) and (B), black bars). All cells were harvested on day 7 and stained for cell surface expression of ILT3 using a fluorescently labeled anti-ILT3 antibody, followed by flow cytometry analysis.

[0235] MFI values on live cells are shown as mean ± SD in Figure 3 (A) and (B).

[0236] Figure 3The results in (A) showed that, with or without LPS stimulation, DCs treated with RA+TGFβ and RA+TGFβ+AhR agonist expressed higher levels of ILT3 compared to the corresponding control DCs and Dex / VitD3-treated tolDCs. In terms of inducing ILT3 expression in DCs, treatment with RA+TGFβ+AhR agonist was superior to treatment with RA+TGFβ.

[0237] Figure 3 The results in (B) showed that, with or without LPS stimulation, DCs treated with RA+AhR agonist, RA+TGFβ, and RA+TGFβ+AhR agonist expressed higher levels of ILT3 compared to the corresponding control DCs. In terms of inducing ILT3 expression in DCs, treatment with RA+TGFβ+AhR agonist was superior to treatment with RA+TGFβ or RA+AhR agonist, and furthermore treatment with RA+TGFβ was superior to treatment with RA+AhR agonist. With or without LPS stimulation, in terms of inducing ILT3 expression in DCs, treatment with RA+TGFβ+AhR agonist and RA+TGFβ was superior to treatment with RA as a single reagent. In terms of inducing ILT3 expression in DCs when stimulated with LPS, treatment with RA+AhR agonist was superior to treatment with RA as a single reagent.

[0238] Example 4 - Effects of various compounds on the tolerogenic index ILT3 / CD86 of DCs

[0239] Optimal tolDCs should express low levels of co-stimulatory molecules and high levels of tolerogenic molecules. Thus, to account for this combinatorial effect, the ratio between the expression levels of representative tolerogenic and co-stimulatory molecules can be depicted as representative of the tolerogenic capacity of a subset of tolDCs. Figure 4 (A) and (B) showed that in the absence of LPS ( Figure 4 (A); grey bars) or in the presence of LPS ( Figure 4 (B); black bars), this tolerogenic index defined as the ILT3 / CD86 expression (MFI) ratio on DCs cultured under various conditions.

[0240] The MFI values on live cells are shown as mean ± SD in Figure 4 (A) and (B).

[0241] Figure 4 The results in (A) showed that DCs treated with RA+TGFβ and RA+TGFβ+AhR agonist had a higher tolerogenic index than control DCs and Dex / VitD3-treated tolDCs, and furthermore DCs treated with RA+TGFβ+AhR agonist had a higher tolerogenic index than DCs treated with RA+TGFβ.

[0242] Figure 4 (B)'s results showed the same pattern as Figure 4 (A). In particular, Figure 4 (B) showed that DCs treated with RA+AhR agonist, RA+TGFβ, and RA+TGFβ+AhR agonist had higher tolerogenic indices than stimulated control DCs. TolDCs treated with RA+TGFβ+AhR agonist had higher tolerogenic indices than tolDCs treated with RA+TGFβ, and tolDCs treated with RA+TGFβ had higher tolerogenic indices than tolDCs treated with RA+AhR agonist. DCs treated with RA+TGFβ+AhR agonist, RA+TGFβ, and RA+AhR agonist all had higher tolerogenic indices than DCs treated with RA as a single reagent.

[0243] Example 5 - Effects of various compounds on DCs and their ability to induce T cell proliferation

[0244] Various compounds were tested for their ability to induce a tolerogenic phenotype in DCs, which should be translated as a reduced ability to induce T cell proliferation and which should additionally be as resistant as possible to upregulation by immunogenic stimulants. Control DCs were differentiated from CD14 + monocytes by culturing in GM-CSF and IL-4 for 7 days. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs or their suboptimal variants were differentiated from CD14 + monocytes by culturing as above in GM-CSF and IL-4 and by treatment with various tolerogenic compounds either as single reagents or in combination to demonstrate the synergistic effect of the combination, as Figure 5 (A) and (B) show: an AhR agonist C1 (AhR ag) at 20 nM on days 0 and 3, TGFβ at 10 ng / ml ( Figure 5 (B)) or 20 ng / ml ( Figure 5 (A)) on day 3, and RA at 2 μM on day 6. TolDCs for comparison were established by treatment with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some samples were treated with LPS ( Figure 5 (A), black bars) or a mixture of proinflammatory cytokines consisting of TNFα, IL-1β, and PGE2 ( Figure 5 (B); black bars) to test phenotypic stability under various conditions. All cells were harvested on day 7 and co-cultured with CD4 + T cells at a T cell:DC ratio of 10:1 in an MLR setup. At the end of the 7-day culture, by 3H-thymidine incorporation was used to assay T cell proliferation.

[0245] Counts per minute (CPM) values from triplicate or more samples are shown as mean ± SD in Figure 5 (A) and (B).

[0246] Figure 5 (A) Results showed that DCs treated with RA+AhR agonist, RA+TGFβ, and RA+TGFβ+AhR agonist exhibited a lower ability to induce T cell proliferation compared to the corresponding control DCs, including after LPS challenge. In terms of reducing the ability to induce T cell proliferation, treatment with RA+TGFβ+AhR agonist was superior to treatment with RA+AhR agonist or RA+TGFβ.

[0247] Figure 5 (B) Results showed the same pattern as Figure 5 (A). Specifically, Figure 5 (B) showed that DCs treated with RA+AhR agonist, RA+TGFβ, and RA+TGFβ+AhR agonist exhibited a lower ability to induce T cell proliferation compared to the corresponding control DCs, compared tolDCs treated with Dex / VitD3, and DCs treated with RA, TGFβ, or AhR agonist as single reagents, including after stimulation with a mixture of pro-inflammatory cytokines. In terms of reducing the ability to induce T cell proliferation, treatment with RA+TGFβ+AhR agonist was superior to treatment with RA+AhR agonist or RA+TGFβ.

[0248] Example 6 - Effects of various compounds on the frequency of CD141 + GARP + DCs

[0249] The ability of various compounds to induce a tolerogenic phenotype in DCs was tested, including co-expression of CD141 and GARP as one of several factors. Control DCs were differentiated from CD14 + monocytes by culturing in GM-CSF and IL-4 for 7 days. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs or their sub-optimal variants were differentiated from CD14 + monocytes by culturing as above and treating with various tolerogenic compounds as single reagents or in combination to demonstrate the synergistic effect of the combination, as shown in Figure 6 (A) and (B): AhR agonist C1 (AhR ag) at 20 nM on days 0 and 3, 10 ng / mL on day 3 ( Figure 6 (A)) or 20 ng / ml ( Figure 6TGFβ of (B), and 2 μM of RA on day 6. TolDC for comparison was established by treating with 100 nM of Dex and 100 nM of VitD3 on day 3. On day 6, some samples were treated with LPS to test phenotypic stability ( Figure 6 (B), black bars). All cells were harvested on day 7 and stained for cell surface expression with fluorescently labeled anti-CD141 and anti-GARP antibodies, followed by flow cytometry analysis.

[0250] Frequency (%) of CD141 in live cells + GARP + is shown as mean ± SD in Figure 6 (A) and (B).

[0251] Figure 6 (A) Results showed that compared with control DC, comparison tolDC treated with Dex / VitD3, and DC treated with RA, TGFβ, AhR agonist, and TGFβ + AhR agonist, DC treated with RA + AhR agonist, RA + TGFβ, and RA + TGFβ + AhR agonist contained a higher frequency of CD141 + GARP + cells. Treatment with RA + TGFβ + AhR agonist was superior to treatment with RA + AhR agonist or RA + TGFβ. Compared with DC treated with TGFβ or AhR agonist as a single reagent or treated with Dex / VitD3, DC treated with Ra + AhR agonist, RA + TGFβ, and RA + TGFβ + AhR agonist contained a higher frequency of CD141 + GARP + cells. DC treated with RA + AhR agonist and RA + TGFβ + AhR agonist contained a higher frequency of CD141 + GARP + cells than DC treated with RA.

[0252] Figure 6 (B) Results showed that compared with the corresponding control DC and comparison tolDC treated with Dex / VitD3, DC treated with RA + TGFβ and RA + TGFβ + AhR agonist contained a higher frequency of CD141 + GARP + cells, including after LPS stimulation. Treatment with RA + TGFβ + AhR agonist was superior to treatment with RA + TGFβ.

[0253] Example 7 - Effects of various compounds on DC production of IL23

[0254] The ability of various compounds to induce a tolerogenic phenotype in DCs was tested, including low production of the cytokine IL-23 in response to immunogenic stimuli as one of several factors. Control DCs were differentiated from CD14 + monocytes by culturing in GM-CSF and IL-4 for 7 days. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. tolDCs or their suboptimal variants were differentiated from CD14 + monocytes by culturing as above in GM-CSF and IL-4 and treating with various tolerogenic compounds either as a single reagent or in combination, to demonstrate the synergistic effect of the combination, as shown in Figure 7 (A) and (B): the AhR agonist C1 (AhR ag) at 20 nM on days 0 and 3, TGFβ at 10 ng / mL ( Figure 7 (B)) or 20 ng / ml ( Figure 7 (A)) on day 3, and RA at 2 uM on day 6. tolDCs for comparison were established by treating with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some tolDCs were treated with LPS ( Figure 7 (A), black bars) or a mixture of pro-inflammatory cytokines consisting of TNFα, IL-1β, and PGE2 ( Figure 7 (B); black bars). Supernatants were collected from the DC cultures on day 7, and IL-23 levels were measured using the Luminex platform.

[0255] IL-23 concentration values are shown as mean ± SD in Figure 7 (A) and (B).

[0256] Figure 7 (A) The results showed that DCs treated with RA+AhR agonist, RA+TGFβ, and RA+TGFβ+AhR agonist produced less IL-23 after LPS activation compared to stimulated control DCs. In terms of reducing IL-23 production, RA+TGFβ was superior to RA+TGFβ+AhR agonist and RA+AhR agonist.

[0257] Figure 7 (B) The results showed a pattern similar to Figure 7 (A). Specifically, Figure 7 (B) showed that DCs treated with RA+AhR agonist, RA+TGFβ, and RA+TGFβ+AhR agonist produced less IL-23 after stimulation with the pro-inflammatory cytokine mixture compared to the corresponding stimulated control DCs and DCs treated with TGFβ or AhR agonist as a single reagent or with TGFβ+AhR agonist.

[0258] Example 8 - Effects of Various Compounds on DC Expression of CD103

[0259] The ability of various compounds to induce a tolerogenic phenotype in DCs was tested, including high expression of the marker CD103 as one of several factors. Control DCs were differentiated from CD14 monocytes by culturing in GM - CSF and IL - 4 for 7 days. The control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. TolDCs or their sub - optimal variants were differentiated from CD14 monocytes by culturing as above in GM - CSF and IL - 4 and treating with various tolerogenic compounds as a single reagent or in combination to demonstrate the synergistic effect of the combination, as shown in + (A) and (B): An AhR agonist C1 (AhR ag) at 20 nM on days 0 and 3, TGFβ at 10 ng / mL( + (A)) or 20 ng / ml( Figure 8 (B)) on day 3, and RA at 2 uM on day 6. TolDCs for comparison were established by treating with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some tolDCs were treated with LPS to test phenotypic stability( Figure 8 (A)); black bars). All cells were harvested on day 7 and stained for cell - surface expression using a fluorescent - labeled anti - CD103 antibody, followed by flow - cytometry analysis. Figure 8 (B)). The MFI values on live cells are shown as mean ± SD in ​ (A) and (B).

[0260] The results in ​ (A) showed that DCs treated with RA + AhR agonist and RA + TGFβ + AhR agonist expressed higher levels of CD103 than RA, TGFβ, RA + TGFβ, control DCs, and comparison tolDCs treated with Dex / VitD3. In terms of inducing CD103 expression, treatment with RA + TGFβ + AhR agonist was superior to treatment with RA + AhR agonist.

[0261] ​ (B) showed that DCs treated with RA + TGFβ + AhR agonist expressed higher levels of CD103 than control DCs and comparison tolDCs treated with Dex / VitD3, whether or not stimulated with LPS. In terms of inducing CD103 expression, treatment with RA + TGFβ + AhR agonist was superior to treatment with RA + TGFβ.

[0262] ​ (B) showed that DCs treated with RA + TGFβ + AhR agonist expressed higher levels of CD103 than control DCs and comparison tolDCs treated with Dex / VitD3, whether or not stimulated with LPS. In terms of inducing CD103 expression, treatment with RA + TGFβ + AhR agonist was superior to treatment with RA + TGFβ.

[0263] Example 9 - Effects of various compounds on the DC expression of MERTK, BTLA, LAP, HLA-G, and CD49b

[0264] The ability of various compounds to induce a tolerogenic phenotype in DCs, which consists of the expression of various tolerogenic markers, was tested. Control DCs were differentiated from CD14 + monocytes by culturing for 7 days in GM-CSF and IL-4. Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. tolDCs were differentiated from CD14 ​ (A) to (E) monocytes by culturing as above in GM-CSF and IL-4 and treating with various tolerogenic compounds as described in + : 20 nM AhR agonist C1 (AhR ag) on days 0 and 3, 10 ng / mL (for MERTK and LAP, ​ (A), (C), and (E)) or 20 ng / ml (for BTLA and HLA-G; ​ (B) and (D)) TGFβ on day 3, and 2 μM RA on day 6. tolDCs for comparison were established by treating with 100 nM Dex and 100 nM VitD3 on day 3. All cells were harvested on day 7 and stained for cell surface expression using fluorescently labeled anti-MERTK, anti-BTLA, anti-LAP, anti-HLA-G, and anti-CD49b antibodies, followed by flow cytometry analysis.

[0265] MFI values on live cells are shown as mean ± SD in ​ (A) to (D).

[0266] Results showed that DCs treated with RA + TGFβ and RA + TGFβ + AhR agonist expressed higher levels of MERTK( ​ (A)), BTLA( ​ (B)), and LAP( ​ (C)) compared to control DCs and Dex / VitD3-treated comparison tolDCs. In all cases, treatment with RA + TGFβ + AhR agonist was superior to treatment with RA + TGFβ. In addition, DCs treated with RA + TGFβ + AhR agonist expressed higher levels of HLA-G and CD49b compared to control DCs and Dex / VitD3-treated comparison tolDCs, shown in ​ (D) and (E), respectively.

[0267] Example 10 - Effects of various compounds on DCs and their ability to induce Tregs

[0268] The ability of various compounds to induce a tolerogenic phenotype in DCs, which can be interpreted as enhanced Treg cell induction on T cell / DC co-cultures, was tested. Control DCs were differentiated from CD14 monocytes by culturing in GM-CSF and IL-4 for 7 days. The control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. tolDCs and their suboptimal variants were differentiated from CD14 monocytes by culturing as above and treating with various tolerogenic compounds as depicted in (A) and (B): the AhR agonist C1 (AhR ag) at 20 nM on days 0 and 3, TGFβ at 20 ng / ml on day 3, and RA at 2 uM on day 6. tolDCs for comparison were established by treating with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some tolDCs were treated with LPS to test phenotypic stability ((A); black bars). All cells were harvested on day 7 and co-cultured with CD4 T cells at a T cell / DC ratio of 10:1 in an MLR setting. At the end of the initial 7-day culture, the T cells were washed and re-incubated in medium containing IL-2 for an additional 7 days. Then all cells were harvested and stained for cell surface expression using fluorescently labeled anti-CD4 and anti-CD25 antibodies and for intracellular expression of Foxp3 using a fluorescently labeled anti-Foxp3 antibody, and then analyzed by flow cytometry. + Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. tolDCs and their suboptimal variants were differentiated from CD14 monocytes by culturing as above and treating with various tolerogenic compounds as depicted in (A) and (B). ​ (A) and (B) + Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. tolDCs and their suboptimal variants were differentiated from CD14 monocytes by culturing as above and treating with various tolerogenic compounds as depicted in (A) and (B): the AhR agonist C1 (AhR ag) at 20 nM on days 0 and 3, TGFβ at 20 ng / ml on day 3, and RA at 2 uM on day 6. tolDCs for comparison were established by treating with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some tolDCs were treated with LPS to test phenotypic stability ((A); black bars). ​ (A); black bars + Control DCs were either unstimulated or stimulated with LPS added on day 6 to generate immunogenic control DCs. tolDCs and their suboptimal variants were differentiated from CD14 monocytes by culturing as above and treating with various tolerogenic compounds as depicted in (A) and (B): the AhR agonist C1 (AhR ag) at 20 nM on days 0 and 3, TGFβ at 20 ng / ml on day 3, and RA at 2 uM on day 6. tolDCs for comparison were established by treating with 100 nM Dex and 100 nM VitD3 on day 3. On day 6, some tolDCs were treated with LPS to test phenotypic stability ((A); black bars). At the end of the initial 7-day culture, the T cells were washed and re-incubated in medium containing IL-2 for an additional 7 days. Then all cells were harvested and stained for cell surface expression using fluorescently labeled anti-CD4 and anti-CD25 antibodies and for intracellular expression of Foxp3 using a fluorescently labeled anti-Foxp3 antibody, and then analyzed by flow cytometry.

[0269] CD25 hi FOXP3 + The frequency (%) of + live CD4 ​ (A) and (B) is shown as mean ± SD.

[0270] ​ (A) The results showed that compared to the corresponding control DCs, whether stimulated with LPS or not, DCs treated with RA+TGFβ+AhR agonist and RA+TGFβ had enhanced ability to promote Treg induction. In terms of Treg induction, treatment with RA+TGFβ+AhR agonist was superior to treatment with RA+TGFβ.

[0271] ​The results of (B) showed that compared with control DCs, tolDCs treated with Dex / VitD3, and DCs treated with TGFβ+AhR agonist, DCs treated with RA+TGFβ+AhR agonist had enhanced ability to induce Tregs. In addition, compared with control DCs and DCs treated with TGFβ+AhR agonist, DCs treated with RA+AhR agonist had enhanced ability to induce Tregs. In terms of inducing Tregs, treatment with RA+TGFβ+AhR agonist was superior to treatment with RA+AhR agonist.

[0272] Example 11 - Effects of RA+TGFβ+AhR agonist on DCs derived from blood of patients with hemophilia A related to CD83, CD86, ILT3, ILT3 / CD86, GARP+CD141+, CD103, LAP, IL-23, and CD49b+LAG3+Tr1 cells

[0273] The AhR agonist mentioned in the example is C1. The ability of the combination of RA+TGFβ+AhR agonist to induce a tolerogenic phenotype in DCs was tested, and the phenotype was represented here by low expression levels of the DC maturation marker CD83, low expression levels of the costimulatory molecule CD86, high expression levels of the tolerogenic marker ILT3, a tolerogenic index defined as ILT3 / CD86 expression, co-expression of CD141 and GARP, expression of CD103, expression of LAP, low spontaneous production of the cytokine IL-23, reduced ability to induce T cell proliferation, and enhanced induction of Treg cell production. Control DCs were differentiated from CD14 + monocytes from healthy donors or subjects with hemophilia by culturing in GM-CSF and IL-4 for 7 days. tolDCs or their suboptimal variants were differentiated from CD14 + monocytes by culturing as described above in GM-CSF and IL-4 and by treatment with the combination of RA+TGFβ+AhR agonist, as ​ described in (A) to (J).

[0274] MFI values on live cells are shown as mean ± SD in ​ (A) to (G).

[0275] IL-23 concentration values are shown as mean ± SD in ​ (H).

[0276] T cell proliferation, evaluated as counts per minute (CPM) values from triplicate or more samples, is shown as mean ± SD in ​ (I).

[0277] CD4 + CD49b+ LAG3 + The frequency (%) of TR1 cells is shown as mean ± SD in ​ (J).

[0278] These results demonstrate that DCs generated from CD14+ monocytes isolated from the blood of hemophilia patients and treated with RA+TGFβ+AhR agonist acquire a tolerogenic phenotype and function similar to those obtained with DCs generated from the blood of healthy donors. The results further demonstrate that RA+TGFβ+AhR agonist-treated DCs generated from CD14+ monocytes isolated from the blood of hemophilia patients show the same ability to reduce T cell proliferation induction and increase the ability to generate regulatory T cells as DCs generated from cells from healthy blood.

[0279] Example 12 - Viability experiment of DCs treated with RA+TGFβ+AhR agonist

[0280] The AhR agonist mentioned in the example is C1. DCs treated with RA+TGFβ+AhR agonist are generated and their viability, expression levels of the DC maturation marker CD83, expression levels of the tolerogenic marker ILT3, expression levels of the costimulatory molecule CD86, tolerogenic index defined as the ILT3 / CD86 expression, expression of LAP, and expression of CD103 are tested, and then they are frozen / thawed. The DCs are frozen in conventional freezing medium or Cryostor 10 (CS, Biolife solutions). Control DCs are differentiated from CD14 + monocytes by culturing for 7 days in GM-CSF and IL-4.

[0281] The viability before and after freezing is shown as mean ± SD in ​ (A).

[0282] The MFI values of CD83, ILT3, CD86, LAP, and CD103 on live cells are shown as mean ± SD in ​ (B) to (D) and (F) to (G).

[0283] The tolerogenic index (defined as the ILT3 / CD86 expression (MFI) ratio) is shown in ​ (E).

[0284] Table showing the viability and recovery of RA+TGFβ+AhR agonist-treated DCs frozen in CS10 compared to conventional freezing medium (conv).

[0285]

[0286] The results presented in the above table demonstrate that cryopreservation and thawing of RA+TGFβ+AhR agonist-treated DCs yield useful cell viability, and that RA+TGFβ+AhR agonist-treated DCs have greater viability compared to control DCs. These results also demonstrate that cryopreservation and thawing of RA+TGFβ+AhR agonist-treated DCs result in surface marker expression similar to that before freezing, and that the use of CS10 leads to better recovery and viability of RA+TGFβ+AhR agonist-treated DCs compared to when the cells have been cryopreserved with standard cryomedia.

[0287] Example 13 - Effects of various compounds on DCs related to CD83, CD86, ILT3, ILT3 / CD86, CD141+GARP+, CD103, T cell proliferation, and CD25hiFoxp3+ Treg cells

[0288] The ability of various compounds to induce a tolerogenic phenotype in DCs was tested. TolDC or its suboptimal variants were differentiated from CD14 + monocytes by culturing in GM-CSF and IL-4 as described above and treating with various tolerogenic compounds as single reagents or in combination, to demonstrate the synergistic effect of the combination, as ​ shown. AhR agonist C1 (AhR ag) at 10 nM on days 0 and 3, TGFβ at 10 ng / ml on day 3, and RA at 2 uM on day 6. Control DCs were differentiated from CD14 + monocytes by culturing in GM-CSF and IL-4 for 7 days. DCs were either unstimulated (gray bars) or stimulated with added LPS 2 hours after RA addition on day 6 (black bars) to generate immunogenic control DCs and test phenotypic stability. All cells were harvested on day 7 and stained with fluorescently labeled antibodies against cell surface expression, followed by flow cytometry analysis.

[0289] MFI values for CD83, CD86, ILT3, and CD103 on live cells are shown as mean ± SD in ​ (A) to (C) and (E).

[0290] The tolerogenic index (defined as the ILT3 / CD86 expression (MFI) ratio) is shown in ​ (D).

[0291] CD141 + GARP + The frequency (%) of double-positive cells is shown as mean ± SD in ​ (F).

[0292] T cell proliferation, assessed as counts per minute (CPM) values from triplicate or more samples, is shown as mean ± SD in ​ (G).

[0293] CD4 + CD25 + Foxp3 + The frequency (%) of Tregs is shown as mean ± SD in ​ (H).

[0294] These results demonstrate that RA-treated DCs have higher ILT3 expression and a higher frequency of GARP + CD141 + cells, as shown in ​ panel C, compared to control DCs. RA-treated DCs induce less T cell proliferation and more Tregs compared to control DCs, ​ panels G and H.

[0295] AhR agonist-treated DCs have lower CD83 and CD86 expression and higher CD103 expression compared to control DCs, as shown in ​ panels A, B, and E, respectively. When not stimulated with LPS, AhR agonist-treated DCs induce slightly more T cell proliferation and slightly more Tregs compared to control DCs ( ​ (G) and (H)).

[0296] TGFβ-treated DCs have a slightly higher tolerogenic ratio (ILT3 / CD86) and CD103 expression compared to control DCs ( ​ (D - E)). Additionally, TGFβ-treated DCs induce less T cell proliferation and more Tregs compared to control DCs ( ​ (G) through (H)).

[0297] RA + TGFβ-treated DCs have lower CD83 and CD86 expression and higher ILT3 expression compared to control DCs, as shown in ​ panels A, B, and C, respectively. RA + TGFβ-treated DCs also have a higher frequency of GARP + CD141 + cells ( ​ (F)). When used in combination, RA + TGFβ-treated DCs induce less T cell proliferation compared to when used individually ( ​ (G)). In summary, TGFβ enhances the tolerogenic effect of RA.

[0298] Compared with control DCs, DCs treated with RA+AhR agonist showed lower expression of CD83 and CD86 and higher expression of ILT3 and CD103, respectively, as ​ A, B, C, and E. DCs treated with RA+AhR agonist also had a higher frequency of GARP + CD141 + cells, but not as high as those of DCs treated with RA+TGFβ or RA+TGFβ+AhR agonist ( ​ (F)). When used in combination compared to use alone, DCs treated with Ra+AhR agonist induced less T cell proliferation( ​ (G)). In summary, the AhR agonist enhances the tolerogenic effect of RA.

[0299] Using an AhR agonist together with RA+TGFβ to generate tolerogenic DCs resulted in a higher tolerogenic ratio and higher CD103 expression( ​ (D) and (E)). In addition, compared with RA+TGFβ, when using RA+TGFβ+AhR agonist, the induction of Tregs was slightly higher( ​ (H)).

[0300] Example 14 - Effects of RA+TGFβ+AhR agonist on DCs and their ability to inhibit tetanus toxoid T cell proliferation

[0301] The AhR agonist mentioned in the example is C1.

[0302] Additional materials and methods

[0303] Generation of dendritic cells (DCs) loaded with TT

[0304] In the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 ng / ml; Peprotech, London, UK) and interleukin-4 (IL-4; 100 ng / ml; PeproTech), CD14 + monocytes were seeded at 1.25×10 6Cells / ml were cultured in GMPDC medium (CellGenix, Freiburg, Germany) containing HEPES, GlutaMAX, and penicillin-streptomycin solution (Thermo Fisher, Waltham, MA) for 7 days. Cells were supplemented with fresh medium and cytokines on day 3. Control DCs were differentiated in GM-CSF and IL-4 without adding any other compounds, and for antigen-loaded cells, TT (30 nM) was added to the culture 4 hours before treatment with a pro-inflammatory cytokine mixture consisting of TNFα (10 ng / ml; PeproTech), IL-1β (10 ng / ml; Peprotech), and prostaglandin E2 (PGE2; 1 μg / ml; Sigma) on day 6 to generate immunogenic DCs. TolDCs were generated by treatment with the tolerogenic compounds AhR agonist, TGFβ1, and RA as previously described. For antigen-loaded cells, TT (30 nM) was added to the culture 2 hours after adding RA on day 6. On day 7, DCs were harvested and washed thoroughly, and then subjected to phenotypic analysis and functional assays.

[0305] Freezing and thawing of PBMCs

[0306] PBMCs were resuspended in freezing medium containing 10% DMSO and transferred to cryovials. These vials were placed in a freezing container and put into an -80 °C freezer. The freezing container allows a controlled freezing rate of approximately -1 °C / min. After 24 hours in the -80 °C freezer, the cryovials were transferred to a -150 °C freezer for long-term storage. PBMCs were thawed in a 37 °C water bath in the vials, and the cell suspension was carefully transferred to a 15 ml tube containing cold RPMI with 10% FCS supplemented with DNase and washed once before use.

[0307] Autologous DC / T cell culture

[0308] To analyze the TT-specific T cell response, DCs loaded with 30 nM TT and stimulated with DCs matured with TNF-α, IL-1β, PGE2, and IL-6 (mDC) and DCs treated with RA + TGFβ + AhR agonist loaded with TT were cultured with autologous T cells at a T cell:DC ratio of 10:1 for 6 days. mDCs with or without TT were used as controls with autologous T cells. DC / T cell culture was performed in complete medium: CTS with OpTmizer TM T cell expansion supplement (Thermo Fisher) and penicillin-streptomycin solution TM Optmizer TMT cell expansion medium. Proliferation was determined by incorporation of 3H-thymidine during the last 18 hours of culture.

[0309] DCs treated with TT-loaded RA + TGFβ + AhR agonist were co-cultured with autologous, TT-loaded mDCs and T cells to study the TT-specific reduction in T cell proliferation induced by DCs treated with RA + TGFβ + AhR agonist. Unloaded cells, mDCs, and DCs treated with both RA + TGFβ + AhR agonist were used as controls.

[0310] T cell proliferation, evaluated as counts per minute (CPM) values from triplicate samples, is shown as mean ± SD ​ in

[0311] These results indicate that TT-loaded DCs treated with RA + TGFβ + AhR agonist inhibit mDC-induced TT-specific T cell proliferation.

[0312] Example 15 - Effects of RA + TGFβ + AhR agonist on DCs in relation to CD83, LAP, CD103, ILT3, CD86, ILT3 / CD86, and T cell proliferation after CD40L treatment

[0313] Additional materials and methods

[0314] Phenotypic stability and persistent tolerogenicity after CD40L stimulation

[0315] On day 7, the phenotypic stability and persistent tolerogenicity of TolDCs were studied by adding CD40L (100 ng / ml) after harvesting. The cells were stimulated for 24 hours, then the DCs were harvested and washed thoroughly, and then phenotypic analysis and functional assays were performed.

[0316] These results demonstrated that DCs treated with RA + TGFβ + AhR agonist showed a stable phenotype ( ​ (A) to (F)) after stimulation with CD40L to mimic DC:T cell contact. These results further demonstrated that DCs treated with RA + TGFβ + AhR agonist after stimulation with CD40L had the ability to induce T cell proliferation comparable to that of unstimulated DCs treated with RA + TGFβ + AhR agonist ( ​ (G)). In summary, these results demonstrated that DCs treated with RA + TGFβ + AhR agonist showed persistent phenotype and tolerogenicity after stimulation with CD40L.

[0317] Example 16 - Effects of RA + TGFβ + AhR agonist on DC uptake of antigens of various sizes and maintenance of a stable tolerogenic phenotype

[0318] The AhR agonist mentioned in the example is C1.

[0319] Additional materials and methods

[0320] Antigen loading of DC

[0321] On the 3rd, 6th, or 7th day of culture, 100 nM FITC-labeled dextran, 1 - 10 μg / mL AF488-labeled KLH, 100 nM AF488-labeled tetanus toxoid (TT), or 10 - 100 nM FVIII was added to the cultures of RA + TGFβ + AhR agonist-treated DCs for antigen loading. Antigen uptake was detected by flow cytometry. For FVIII detection, after fixation and permeabilization, the cells were stained with an FITC-labeled antibody specific for FVIII. DCs loaded with 60 nM FVIII were phenotyped for 2, 4, 8, or 20 hours. The percentage of antigen-positive cells was compared with RA + TGFβ + AhR agonist-treated DCs cultured without antigen.

[0322] After culturing with fluorescently labeled antigens from day 3 to day 7, the percentage of FITC-dextran, AF488-KLH, or AF488-TT-positive, CD11c+ RA + TGFβ + AhR agonist-treated DCs on day 7 ( ​ (A)). For FVIII uptake, from day 3 to day 7, RA + TGFβ + AhR agonist-treated DCs were cultured in the presence of 10, 30, or 100 nM FVIII ( ​ (B)). On day 7, the cells were stained for CD11c and then permeabilized and fixed to allow intracellular staining with an FITC-labeled FVIII-specific antibody (Sanquin, Netherlands).

[0323] On day 7, RA + TGFβ + AhR agonist-treated DCs were cultured in the presence of 60 nM FVIII for 2, 4, 8, or 20 hours to determine the phenotype of the cells in the presence of FVIII ( ​ (C) to (I)).

[0324] These results demonstrate that RA + TGFβ + AhR agonist-treated DCs have the ability to uptake a diverse range of antigens of various sizes and chemical classes. These results also demonstrate that RA + TGFβ + AhR agonist-treated DCs have the ability to uptake antigens at different time points during culture and have a stable phenotype after loading with FVIII at different time points.

[0325] Example 17 - Effects of RA + TGFβ + AhR agonist on DCs to affect the levels of Breg, B cells, and T cells, T cell proliferation, and activation

[0326] The AhR agonist mentioned in the examples is C1.

[0327] Additional materials and methods

[0328] B cell / T cell / DC cultures: Breg induction

[0329] On day 7, autologous B cells were isolated from PBMCs by negative selection using a human B cell isolation kit (StemCell technologies). 40,000 B cells were cultured together with 40,000 autologous T cells isolated as described above using the EasySep human CD4+ T cell isolation kit (StemCell technologies) and 10,000 RA+TGFβ+AhR agonist-treated DCs or control DCs generated as described above. The selected cultures were stimulated with 0.25 μM CpG-OGN to enhance the response. Breg induction was evaluated by intracellular IL-10 staining, T cell proliferation was determined by KI67 staining, and T cell activation was determined by CD154 expression. Staining for IL-10, KI67, and CD154 was performed as described above, using the following fluorescently labeled antibodies: IL-10 (JES3-9D7) from Biolegend and KI67 (B56) and CD154 (TRAP1) from BD.

[0330] These data demonstrate that, as ​ (A) shows, the frequency of Bregs, as determined by IL-10 + CD19 + cells, increased in the presence of RA+TGFβ+AhR agonist-treated DCs compared to co-culture with control DCs after co-culturing autologous DCs, B cells, and T cells. T cell proliferation and activation were determined by evaluating KI67 ( ​ (B)) and CD154 ( ​ (C)) respectively after co-culturing autologous DCs, B cells, and T cells.

[0331] Bregs are immunomodulatory cells with the ability to suppress the activity of other immune cells. Bregs are also antigen-presenting cells. By inducing Bregs, another level of immunosuppression will be activated, and Bregs can act synergistically with Tregs. These results demonstrate that RA+TGFβ+AhR agonist-treated DCs co-cultured with B cells and T cells induce more IL-10-producing Bregs compared to when co-cultured with control DCs. Additionally, these results demonstrate that RA+TGFβ+AhR agonist-treated DCs co-cultured with B cells and T cells induce less T cell proliferation and activation compared to control DCs. Reduced T cell proliferation and activation indicate the induction of regulatory cells, Bregs, Tregs, or both.

[0332] Example 18 - Effects of RA+TGFβ+AhR agonist on DCs derived from donor blood when co-cultured with allogeneic PBMCs

[0333] DCs generated from monocytes isolated from four healthy donors (named A - D) were treated with RA+TGFβ+AhR agonist. RA+TGFβ+AhR agonist-treated DCs showed the same expression profile as described in the previous example, with low expression levels of CD83 and CD86, high expression of ILT3, CD103, and LAP, and a high tolerogenic index (defined as ILT3 / CD86 expression).

[0334] RA+TGFβ+AhR agonist-treated DCs from donors A - D were co-cultured with allogeneic PBMCs. For example, in the MLR, PBMCs from donors B, C, and D were co-cultured with donor A-derived DCs. When co-cultured with allogeneic PBMCs, RA+TGFβ+AhR agonist-treated DCs had different abilities to induce T cell proliferation, ​ (A). Additionally, RA+TGFβ+AhR agonist-treated DCs had different abilities to induce Tregs in the MLR using allogeneic PBMCs, ​ (B).

[0335] T cell proliferation was evaluated as the frequency of KI67+ cells in RA+TGFβ+AhR agonist-treated DCs and control DCs, as shown in ​ (A).

[0336] For the frequency (%) of Tregs (CD4 + CD25 + Foxp3 + ) in RA+TGFβ+AhR agonist-treated DCs and control DCs, it is shown in ​ (B).

[0337] These results demonstrated that when co-cultured with allogeneic PBMCs, most donor RA+TGFβ+AhR agonist-treated DCs reduced T cell proliferation and increased Treg induction. In addition, these results indicated that the level of mismatch, which can be evaluated by HLA typing, could affect the outcome of tolerogenic responses induced by RA+TGFβ+AhR agonist-treated DCs.

[0338] Example 19 - Clinical Trial Study

[0339] This study will consist of two phases: an initial dose escalation part (Part 1) and an extended study part (Part 2).

[0340] First study part (Group 1): In the first part of the trial, the safety of escalating doses will be evaluated. There will be four subjects. Through the safety review of the Internal Monitoring Board (IMB), additional subjects may be included. Patient 1 will receive three doses of cells at two-week intervals. The IMB will decide whether dose escalation can proceed after Patient 1 receives the last dose. Patient 2 will receive the first dose of cells at least two weeks after the last dose given to Patient 1 and will receive two additional doses at two-week intervals. Patient 3 will receive the first dose at least two weeks after the last dose of the second patient and will receive two additional doses at two-week intervals. Patient 4 will receive the first dose at least two weeks after the last dose of the third patient and will then receive two additional doses at two-week intervals. In the case where the yield does not reach the predetermined dose, the patient will receive all harvested cells, and another patient will be included in the study.

[0341] Extended second study part (Group 2): There will be eight subjects. After completion of the dose escalation trial part (Part 1), the extended Part 2 of the study will be initiated. The IMB will evaluate Group 1 after the last dose and then proceed with the extended Part 2 trial (Group 2). Once the safety of the escalating doses given to the study subjects in Group 1 is confirmed, the 8 patients (Patients 5, 6, 7, 8, 9, 10, 11, and 12) in Group 2 will be administered intravenously 3 times at the highest tolerated dose determined in Group 1 or the highest yield obtained but not exceeding the highest tolerated dose determined in Group 1 every two weeks.

[0342] Procedure for preparing the active substance and transferring it to each research clinic: Peripheral blood mononuclear cells are collected from patients by apheresis. Fresh leukapheresis material will be sent to a GMP facility (Radboud University Medical Center, Nijmegen, the Netherlands), where monocytes are enriched and cultured with a mixture of compounds that differentiate the monocytes into dendritic cells. The tolerogenic induction mixture of Idogen then converts these dendritic cells into tolerogenic dendritic cells, and then as a final step, loads recombinant FVIII( Octocog alpha). The cells are then cryopreserved and sent back to each research site.

[0343] Route of administration: The cells are administered intravenously. The cryopreserved cell suspension is thawed and administered directly intravenously at the corresponding clinical site.

[0344] When patients develop antibodies against FVIII, the first treatment option is ITI therapy with high-dose FVIII. However, ITI is not always successful. It is an extremely expensive treatment that usually lasts for more than 1 year, requires daily injections, and fails in 1 / 3 of patients (Aledort 2019, Carcao 2019, Lacroix-Desmazes 2020, Ljung 2019). It is burdensome, especially for inhibitor-producing children who are usually very young. The increased morbidity and healthcare costs associated with inhibitors (bleeding into joints and muscles, etc.) (D'Angiolella 2018, CDC (Centers for Disease Control and Prevention), June 3, 2019) highlight the urgency of identifying strategies to prevent the development of inhibitors at the root.

[0345] The Phase I / IIa trial of Idogen will include hemophilia A (HA) subjects with FVIII inhibitors who have an active immune response to FVIII and have not been immunosuppressed with established treatment regimens for immune tolerance induction (ITI). There are no remaining standard treatment alternatives for eradicating their FVIII inhibitors in this patient category. The risks of the trial are unknown, although no serious safety issues have been reported in published clinical trials of autologous tolerogenic dendritic cell therapies (Bell 2017, Benham 2015, Dhodapkar 2001, Dhodapkar and Steinman 2002, Giannoukakis 2011, Harry 2010, Hilkens and Isaacs 2013, Jauregui-Amezaga 2015, Joo 2014, Ten Brinke 2015, Thomas 2011, Willekens and Cools 2018, Willekens 2019, Zubizarreta 2019), and the cells have shown a tolerogenic phenotype in in vitro experiments (Lee 2016, Gordon 2014, Lutz 2000, Steinbrink 1997, Bartosik-Psujek 2010, Huang 2001, Hussien 2001, Bellinghausen Raich-Regue 2012, Saito 2011). Nevertheless, serious adverse events possibly related to the treatment cannot be excluded. Prophylactic coverage with antihistamines will be given to patients in conjunction with the administration of ItolDC-028.

[0346] Potential risks in trials involving subjects with a bleeding diathesis are bleeding during blood sampling, leukapheresis, or cell infusion. However, bleeding after venipuncture is rare for experienced personnel, and subjects will be handled by individuals proficient in managing patients with bleeding disorders. If venipuncture is traumatic, finger pressure or a pressure dressing over the puncture site can prevent further complications. Subcutaneous, intradermal, and small intramuscular injections rarely produce hematomas if firm finger pressure is maintained for at least 5 minutes (Powell and Rodgers 2013).

[0347] Example 20 - Effects of RA + TGFβ + AhR agonists related to B cell production and T cell regulatory function of IL-10 on DCs from the blood of patients with autoimmune diseases

[0348] DCs treated with RA+TGFβ+AhR agonist are generated from monocytes of patients suffering from autoimmune diseases with one or several well-characterized antigens. The DCs treated with RA+TGFβ+AhR agonist are loaded with one or several well-characterized antigens. The DCs can then be co-cultured with autologous T cells. The T cells will be less activated compared to T cells co-cultured with control DCs loaded with the same antigen. In addition, in the co-culture of DCs treated with RA+TGFβ+AhR agonist and T cells, a higher frequency of regulatory T cells will be induced compared to T cells co-cultured with control DCs under the same conditions.

[0349] Co-culture the RA+TGFβ+AhR agonist-treated DCs generated from patients suffering from autoimmune diseases with B cells. The B cells will have a higher frequency of regulatory markers such as IL-10.

[0350] Co-culture the DCs treated with RA+TGFβ+AhR agonist generated from patients suffering from autoimmune diseases with B cells and T cells. The T cells will have increased regulatory function compared to B cells and T cells co-cultured with control DCs.

[0351] After co-culture with DCs treated with RA+TGFβ+AhR agonist, the induction of tolerogenic phenotypes and functions in both T cells and B cells is beneficial in autoimmune diseases as these cells attenuate the autoreactive activities of T and B cells.

[0352] Example 21 - Effects of RA+TGFβ+AhR agonist on blood DCs derived from patients suffering from autoimmune diseases treated with several unknown and / or complex antigens or tissue samples in relation to B cell production of IL-10 and T cell regulatory function

[0353] DCs treated with RA+TGFβ+AhR agonist are generated from monocytes of patients suffering from autoimmune diseases with several unknown and / or complex antigens. The DCs treated with RA+TGFβ+AhR agonist are loaded with several unknown and / or complex antigens or tissue sample extracts from the same patient and then co-cultured with autologous T cells. The T cells will be less activated compared to T cells co-cultured with control DCs loaded with the same antigen. In addition, in the co-culture of DCs treated with RA+TGFβ+AhR agonist and T cells, a higher frequency of regulatory T cells is induced compared to T cells co-cultured with control DCs under the same conditions.

[0354] Co-culture the RA+TGFβ+AhR agonist-treated DCs generated from patients suffering from autoimmune diseases with B cells. The B cells will have a higher frequency of regulatory markers such as IL-10.

[0355] Co-culture DCs treated with RA + TGFβ + AhR agonist with B cells and T cells. T cells will have increased regulatory function compared to B cells and T cells co-cultured with control DCs.

[0356] After co-culture with DCs treated with RA + TGFβ + AhR agonist, the induction of tolerogenic phenotypes and functions in both T cells and B cells is beneficial in autoimmune diseases because these cells inhibit the autoreactive activities of T and B cells.

[0357] Example 22 - Effect of RA + TGFβ + AhR agonist on DCs from the blood of patients with autoimmune diseases without any antigen treatment after in vivo administration, related to Breg induction

[0358] DCs treated with RA + TGFβ + AhR agonist are generated from monocytes of patients with autoimmune diseases with one or several unknown antigens. Without prior antigen loading and cryopreservation, DCs treated with RA + TGFβ + AhR agonist are injected in situ, where the DCs uptake disease-related antigens to be presented to T cells, thereby inducing disease-specific regulatory T cells.

[0359] Co-culture DCs treated with RA + TGFβ + AhR agonist from patients with autoimmune diseases with one or several unknown antigens. Administer DCs in situ. DCs treated with RA + TGFβ + AhR agonist will induce Bregs, further promoting the induction of tolerance to the graft and reducing the production of harmful antibodies. This is contrary to what would happen if the recipient received immunogenic donor-derived DCs or no DCs at all.

[0360] Example 23 - Effect of RA + TGFβ + AhR agonist on T cell activation and Treg induction of DCs derived from the blood of patients with hemophilia A and loaded with FVIII

[0361] It has been found that patients with hemophilia A resistant to exogenous FVIII treatment upregulate FVIII-specific effector T cells, which have the ability to activate B cells, thereby producing FVIII-specific antibodies (inhibitors).

[0362] According to the method described above previously, DCs generated from monocytes from hemophilia A patients treated with RA+TGFβ+AhR agonist are loaded with FVIII, and in a recall antigen assay, the RA+TGFβ+AhR agonist-treated DCs are co-cultured in vitro with T cells from the same hemophilia patient. The T cells will be less activated compared to T cells co-cultured with control DCs. In addition, the T cells co-cultured with RA+TGFβ+AhR agonist-treated DCs will induce a higher frequency of regulatory T cells compared to T cells co-cultured with control DCs under the same conditions.

[0363] Example 24 - Effect of RA+TGFβ+AhR agonist on DCs derived from allogeneic donors related to Treg and Breg induction

[0364] RA+TGFβ+AhR agonist-treated DCs are generated from monocytes from allogeneic donors. The RA+TGFβ+AhR agonist-treated DCs will induce autologous regulatory T cells and tolerance to the graft when administered to transplant recipients. This is contrary to what would happen if the recipient received immunogenic donor-derived DCs or no DCs at all.

[0365] The RA+TGFβ+AhR agonist-treated DCs from the donor are administered to the transplant recipient. The RA+TGFβ+AhR agonist-treated DCs will induce Bregs, further promoting tolerance induction to the graft and reducing harmful antibody production. This is contrary to what would happen if the recipient received immunogenic donor-derived DCs or no DCs at all.

[0366] Example 25 - Effect of RA+TGFβ+AhR agonist on DCs derived from allogeneic graft recipients related to Treg and Breg induction

[0367] RA+TGFβ+AhR agonist-treated DCs are generated from monocytes from allogeneic graft recipients. The RA+TGFβ+AhR agonist-treated DCs are loaded with a mixture of donor-derived antigens before being administered to the transplant recipient. This antigen mixture can be derived from donor blood or from a tissue sample of the donor, preferably a sample from donor cells, tissues, organs, or other grafts to be transplanted. The RA+TGFβ+AhR agonist-treated DCs will induce autologous regulatory T cells and tolerance to the graft when administered to the transplant recipient. This is contrary to what would happen if the recipient received RA+TGFβ+AhR agonist-treated DCs without donor antigens or no DCs at all.

[0368] Donor antigen-loaded dendritic cells (DCs) from recipients treated with RA+TGFβ+AhR agonist are administered to transplant recipients. RA+TGFβ+AhR agonist-treated DCs will induce Bregs, further promoting graft tolerance induction and reducing harmful antibody production. This is contrary to what would occur if the recipient received RA+TGFβ+AhR agonist-treated DCs without donor antigen or no DCs at all.

[0369] References

[0370] Agrawal S, Ganguly S, Tran A, Sundaram P, Agrawal A. 2016. Retinoic acid-treated human dendritic cells induce T regulatory cells via the expression of CD141 and GARP which is impaired with age. Aging 8:1223-35.

[0371] Bakdash G, Vogelpoel LT, van Capel TM, Kapsenberg ML, de Jong EC. 2015. Retinoic acid primes human dendritic cells to induce gut-homing, IL-10-producing regulatory T cells. Mucosal Immunol. 8:265-78.

[0372] Denison and Nagy 2003. Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Ann. Rev. Pharmacol. Toxicol., 43:309-34.

[0373] Esebanmen GE, Langridge WHR. 2017. The role of TGF-beta signaling in dendritic cell tolerance. Immunol Res. 65:987-994.

[0374] Jurado-Manzano BB, Zavala-Reyes D, Turrubiartes-Martínez EA, Portales-Pérez DP, González-Amaro R, Layseca-Espinosa E. 2017. FICZ generates human tDCs that induce CD4 + CD25high Foxp3 + Treg-like cell differentiation. Immunol Lett. 190:84-92.

[0375] Mahiout S, Lindén J, Esteban J, Sánchez-Pérez I, Sankari S, Pettersson L, H,

[0376] Pohjanvirta R. 2017. Toxicological characterisation of two novel selective aryl hydrocarbon receptor modulators in Sprague-Dawley rats. Toxicol Appl Pharmacol. 326:54-65.

[0377] Oliveira LM, Teixeira FME, Sato MN. 2018. Impact of Retinoic Acid on Immune Cells and Inflammatory Diseases. Mediators Inflamm. 2018:3067126.

[0378] Scott CL, Aumeunier AM, Mowat AM. 2011. Intestinal CD103+ dendritic cells: master regulators of tolerance? Trends Immunol. 32:412-9.

[0379] Vlad G, Chang CC, Colovai AI, Berloco P, Cortesini R, Suciu-Foca N. 2009. Immunoglobulin-like transcript 3: A crucial regulator of dendritic cell function. Hum Immunol. 70:340-4.

[0380] Abbreviation

[0381] HA Hemophilia A

[0382] DC Dendritic cell

[0383] Dex Dexamethasone

[0384] tolDC Tolerogenic dendritic cell

[0385] APC Antigen-presenting cell

[0386] MHC Major histocompatibility complex

[0387] IFN Interferon

[0388] PBS Phosphate-buffered saline

[0389] PBMC Peripheral blood mononuclear cell

[0390] FVIII Factor VIII

[0391] s.c. Subcutaneous

[0392] TGF Transforming growth factor

[0393] AhR Aryl hydrocarbon receptor

[0394] AhR ag Aryl hydrocarbon receptor agonist

[0395] TNF Tumor necrosis factor

[0396] GM-CSF Granulocyte-macrophage colony-stimulating factor

[0397] Ig Immunoglobulin

[0398] mAb Monoclonal antibody

[0399] FACS Fluorescence-activated cell sorting

[0400] Treg Regulatory T cell

[0401] IL Interleukin

[0402] RNA Ribonucleic acid

[0403] RA all-trans retinoic acid

[0404] MFI mean fluorescence intensity

[0405] VitD3 vitamin D3

[0406] FICZ 6-formylindolo[3,2-b]carbazole

[0407] GARP glycoprotein-A repeat predominant

[0408] ILT3 immunoglobulin-like transcript 3

[0409] LPS lipopolysaccharide

[0410] MERTK MER proto-oncogene tyrosine kinase

[0411] BTLA B and T lymphocyte associated protein

[0412] LAP latency-associated peptide

[0413] HLA-G human leukocyte antigen G

[0414] uM micromole

[0415] C1 N-ethyl-N-phenyl-5-chloro-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-quinoline-3-carboxamide

[0416] Throughout the specification and the following claims, unless the context requires otherwise, the word "comprise" and its variations such as "comprises" and "comprising" shall be understood to imply the inclusion of the stated integer, step, group of integers or group of steps but not the exclusion of any other integer, step, group of integers or group of steps.

[0417] All patents and patent applications mentioned herein are incorporated by reference in their entirety.

Claims

1. An ex vivo method for obtaining tolerogenic dendritic cells capable of inducing tolerance to an antigen, the method comprising, (a) Isolate CD14 from a sample obtained from a mammal + monocytes; and (b) culturing isolated monocytes in a cell culture to induce the monocytes to differentiate into antigen-presenting cells with a tolerogenic phenotype, wherein the cell culture contains retinoic acid, TGFβ1 and an AhR agonist and further contains GM-CSF and IL-4 to induce the monocytes to differentiate into antigen-presenting cells, wherein the AhR agonist is added to the cell culture at a first dose while GM-CSF and IL-4 are first added to the cell culture, wherein after the first dose of the AhR agonist is added to the cell culture, TGFβ and a second dose of the AhR agonist are added to the cell culture, wherein after TGFβ and the second dose of the AhR agonist are added to the cell culture, retinoic acid is added to the cell culture, wherein the AhR agonist is IMA-06201.

2. The method according to claim 1, wherein GM-CSF and IL-4 are added to the cell culture at a second dose.

3. The method according to claim 2, wherein the second dose of GM-CSF and IL-4 are added to the cell culture while TGFβ and the second dose of the AhR agonist are added to the cell culture.

4. The method according to claim 1, wherein GM-CSF is added to the cell culture before or simultaneously with the addition of any one of TGFβ, the AhR agonist and retinoic acid to the cell culture, and IL-4 is added to the cell culture after the first dose of the AhR agonist is added to the cell culture.

5. The method according to claim 4, wherein after the first dose of the AhR agonist is added to the cell culture, TGFβ and a second dose of the AhR agonist are added to the cell culture.

6. The method according to claim 4 or 5, wherein after TGFβ and the second dose of the AhR agonist are added to the cell culture, retinoic acid is added to the cell culture.

7. The method according to claim 1, wherein GM-CSF is added to the cell culture at a second dose.

8. The method according to claim 7, wherein the second dose of GM-CSF is added to the cell culture while TGFβ and the second dose of the AhR agonist are added to the cell culture.

9. The method according to any one of claims 1, 3-5 and 7-8, wherein the mammal is a human.

10. The method according to any one of claims 1, 3-5 and 7-8, wherein the sample is a sample of peripheral blood mononuclear cells.

11. The method according to any one of claims 1, 3-5 and 7-8, wherein the cell culture contains an antigen or an epitope containing a fragment thereof.

12. The method according to claim 11, wherein the antigen or the epitope containing a fragment thereof is associated with the isolated monocytes.

13. The method according to claim 11, wherein the antigen or the epitope containing a fragment thereof is added to the cell culture.

14. The method according to claim 13, wherein the antigen or epitope containing a fragment is added to the cell culture before, simultaneously with, or after adding retinoic acid to the cell culture.

15. The method according to claim 13 or claim 14, wherein the antigen or epitope containing a fragment is a library of antigens and / or a library of epitopes containing fragments thereof.

16. The method according to claim 11, wherein the antigen or epitope containing a fragment thereof is a biopharmaceutical or derived from a biopharmaceutical.

17. The method according to claim 16, wherein the biopharmaceutical is factor VIII or a derivative or fragment thereof.

18. The method according to claim 16, wherein the biopharmaceutical is factor IX or a derivative or fragment thereof.

19. The method according to any one of claims 1, 3 - 5, 7 - 8, and 12 - 14, wherein the antigen or epitope containing a fragment thereof is associated with an allograft.

20. The method according to any one of claims 1, 3 - 5, 7 - 8, and 12 - 14, wherein the antigen or epitope containing a fragment thereof is an autoantigen or derived from an autoantigen.

21. The method according to any one of claims 1, 3 - 5, 7 - 8, and 12 - 14, wherein the tolerogenic dendritic cells, when unstimulated or stimulated, have high expression of ILT3 and low expression of CD83 and CD86.

22. The method according to claim 21, wherein the tolerogenic dendritic cells are unstimulated or stimulated with an immunogenic stimulant.

23. The method according to claim 22, wherein the immunogenic stimulant is LPS or a pro - inflammatory cytokine or a mixture of pro - inflammatory cytokines.

24. The method according to claim 21, wherein the tolerogenic dendritic cells express CD103.

25. The method according to any one of claims 1, 3 - 5, 7 - 8, 12 - 14, and 22 - 23, wherein the tolerogenic dendritic cells induce regulatory T cells when cultured with T cells.

26. The method according to any one of claims 1, 3 - 5, 7 - 8, 12 - 14, and 22 - 23, wherein the tolerogenic dendritic cells are further cultured with T cells, thereby inducing the generation of regulatory T cells.

27. The method according to claim 25, wherein the regulatory T cell is CD4 + CD25 hi Foxp3 + or a Tr1 regulatory T cell.

28. The method according to claim 26, wherein the regulatory T cell is CD4 + CD25 hi Foxp3 + or a Tr1 regulatory T cell.

29. The method according to any one of claims 1, 3 - 5, 7 - 8, 12 - 14, 22 - 23, and 27 - 28, wherein the tolerogenic dendritic cells, when unstimulated or stimulated, have a low ability to induce T cell proliferation.

30. The method according to claim 29, wherein the tolerogenic dendritic cells are unstimulated or stimulated with an immunogenic stimulant.

31. The method according to claim 30, wherein the immunogenic stimulant is LPS or a pro - inflammatory cytokine or a mixture of pro - inflammatory cytokines.

32. The method according to any one of claims 1, 3 - 5, 7 - 8, 12 - 14, 22 - 23, 27 - 28, and 30 - 31, wherein the tolerogenic dendritic cells, when unstimulated or stimulated, have a high ability to induce regulatory T cells.

33. The method according to claim 32, wherein the tolerogenic dendritic cells are unstimulated or stimulated with an immunogenic stimulant.

34. The method according to claim 33, wherein the immunogenic stimulant is LPS or a pro-inflammatory cytokine or a mixture of pro-inflammatory cytokines.

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