Dendritic cell population that suppresses GVHD

Abstract

This specification provides compositions and methods for preventing, treating, or suppressing graft-versus-host disease (GvHD), comprising dendritic cells (DCs) exposed to exopolysaccharides (EPS).

Description

Technical Field

[0001] The present invention relates to dendritic cell populations that suppress graft-versus-host disease (GVHD).

Background Art

[0002] Graft-versus-host disease (GvHD) is a serious and often lethal complication of hematopoietic stem cell transplantation (HSCT). Despite prophylactic regimens being given as standard pre-transplant therapies, up to 50% of these patients develop acute GvHD (aGVHD) and require additional immunosuppressive interventions.

Summary of the Invention

Means for Solving the Problems

[0003] In previous studies using a murine GvHD model, it was shown that injecting mice with exopolysaccharide (EPS) derived from Bacillus subtilis prior to GvHD induction significantly increased the 80-day survival rate after transplantation of fully allogeneic major histocompatibility complex mismatched cells. To verify whether EPS can also suppress GvHD in humans, A2 neg GvHD was induced in humanized NSG-HLA-A2 mice by intravenous injection of human peripheral blood mononuclear cells (PBMC). Since EPS cannot be injected into human donors, dendritic cells (DC) pretreated with EPS were transplanted to suppress aGvHD. These DC were + derived from human umbilical cord blood cells, treated with EPS, and then the cells were injected into NSG-HLA-A2 mice together with PBMC. All mice that received untreated DC died by day 35, but 25% of the mice that received EPS-treated DC (EPS-DC) were found to survive.

[0004] This specification provides a novel composition combining EPS and DC, and methods of use thereof, including a novel method of preventing graft-versus-host disease and extending the survival period. One embodiment provides a method for preventing, treating, or suppressing graft-versus-host disease (GvHD), comprising administering exopolysaccharide (EPS)-exposed dendritic cells (DCs) to a subject in need, thereby preventing, treating, or suppressing GvHD in the subject.

[0005] One embodiment provides a method for reducing at least one symptom of graft-versus-host disease (GvHD), comprising administering dendritic cells (DCs) exposed to an exopolysaccharide (EPS) to a subject in need of such reduction of at least one symptom in the subject.

[0006] One embodiment provides a method for extending the survival of a subject having graft-versus-host disease (GvHD), comprising administering dendritic cells (DCs) exposed to an exopolysaccharide (EPS) to a subject in need, in such a manner that the survival of the subject is extended.

[0007] One embodiment provides a method for downregulating the expression of an activating marker (e.g., CD80 and / or CD86), upregulating the expression of an inhibitory molecule (e.g., PD-L1 and / or PD-L2), suppressing the activation of alloreactive T cells, or a combination thereof, comprising administering dendritic cells (DCs) exposed to an exopolysaccharide (EPS) to a subject in need thereof.

[0008] In one embodiment, GvHD is acute GvHD (aGvHD). In another embodiment, GvHD is chronic GvHD (cGvHD). In one embodiment, ESP-exposed DCs are administered two or more times (e.g., over a period of several days, weeks, months, or years). In one embodiment, ESP-exposed DCs are administered together with hematopoietic stem cells (HSCs). In one embodiment, at least one other therapeutic agent for treating GvHD is administered to the subject. In one embodiment, the subject is human.

[0009] One embodiment is a) CD34 + The steps of providing hematopoietic cells (HSCs) and the CD34 of b)a) + Cells were cultured with a combination of SCF, Flt3L, TPO, IL6, and StemRegenin 1 (SR1) to obtain CD34 + A method for generating tolerogenic dendritic cells (DCs) is provided, comprising the steps of: expanding cells; culturing the cells from c) and b) with GM-CS and IL4 to further expand the cells and generate DCs in vitro; contacting the cells from c) with exopolysaccharide (EPS); and cryopreserving the cells from d) optionally, wherein the EPS is optionally removed from the cells from d) before cryopreservation. In one embodiment, HSCs are obtained from umbilical cord blood. In one embodiment, the cells are cryopreserved before c), after c), and / or after d). One embodiment further comprises removing the EPS from the cells from e) before cryopreservation or administration. [Brief explanation of the drawing]

[0010] [Figure 1]Figures 1A-1D: EPS-derived dendritic cells (DCs) from CD34+ umbilical cord blood cells suppress the proliferation of alloreactive CD4 T cells in MLR cultures. Figures showing DC induction from CD34+ umbilical cord blood cells (A) and human monocytes (B). (C) Representative flow cytometry of CD4 T cell proliferation in MLR cultures using CD34-DCs: untreated (NT; left panel), DEPS-treated (negative control; center panel), and EPS-treated (right panel; EPS present throughout the culture period). (D-F) Quantification of relative proliferation of alloreactive CD4 T cells in MLR cultures when CD34-DCs and EPS are present throughout the culture period (D), when moDCs and EPS are present throughout the culture period (E), and when CD34-DCs pretreated with EPS for 16 hours and washed before being added to MLR cultures (F). Data are from three or more independent experiments. **P<.01;***P<.001;****P<.0001, Independent t-test. [Figure 2] Figures 2A-2D: Injection of EPS-DCs derived from CD34+ umbilical cord blood cells extends the survival of humanized GVHD mice. (A) Schematic diagram of GVHD induction and treatment. (B) Kaplan-Meier survival analysis of GVHD mice treated with PBMC alone (PBMC; gray line), PBMC + PBS-treated CD34-DC (NT DC; dashed line), or PBMC + EPS-treated DC (EPS DC; black line). Log-rank tests were performed between EPS-DC and NT-DC (EPS-DC and PBMC) (**P<.01) and NT-DC and PBMC (P=no significant difference). Data are from three or more independent experiments. (C) Body weight change after GvHD induction in mice treated with NT-DC (gray line) and EPS-DC (black line). (D) GvHD clinical score. *P<.05; **P<.01; ***P<.001, unpaired t-test. The data in C and D are from a single experiment using male and female mice (total n=15) in each group. In these experiments, the inventors used CD34+ cells from three donors, all of which were HLA-A2 negative. [Figure 3-1]Figures 3A-3C: DC composition, EPS uptake, and phenotypic changes induced by EPS. (A) Percentage of HLA-DR+CD11c+ DCs in initial cultures of amplified CD34+ cells (left panel), and percentage of HLA-DR+CD11c+ DCs after 5 and 12 days of culture with hGM-CSF and hIL-4 (center panel), and percentage of cDC1 (CD141+) and cDC2 (CD1c) in 12-day DC cultures (right panel). (B) EPS uptake by DCs, non-DCs, and cDC1, cDC2, and double-negative (non-cDC1 / cDC2) cells. (C) Expression of repressive PD-L1 and PL-L2 by untreated (NT) and EPS-treated DCs. Data collected from three independent experiments. *P<.05; ***P<.001, unpaired t-test. All experiments were repeated at least three times; the plots shown are representative flow plots from >3 experiments. [Figure 3-2] Same as above. [Figure 4] An example protocol for generating EPS-DCs and using them as a treatment for GVHD patients. [Modes for carrying out the invention]

[0011] A better understanding of the features and advantages of the present invention can be obtained by referring to the following detailed description, which describes exemplary embodiments in which the principles of the present invention are utilized, and to the accompanying drawings. Disclosed herein is a combination of exopolysaccharide (EPS), a bacterial probiotic molecule, and dendritic cells for reducing graft-versus-host disease (GvHD), for example, after allogeneic bone marrow transplantation. Intraperitoneal injection of EPS into donor and recipient mice has been previously demonstrated to extend the lifespan of allogeneic transplanted mice (Kalinina et al., J Immunol, 2021, 206:2101-2108). To test whether EPS can prevent GvHD in humans, we first determined whether EPS could suppress the mixed lymphocyte reaction (MLR) that occurs when peripheral blood cells (PBMCs) from two allogeneic donors are cultured together in the presence of antigen-presenting dendritic cells (DCs). In MLR, DCs present allogeneic antigens to T cells, thereby activating them; in vivo, such activation can lead to GvHD. When EPS was added to human MLR cultures, it was shown that EPS partially suppressed the proliferation of alloreactive T cells (Figure 1), demonstrating that EPS can suppress alloreactive T cells in vitro and therefore has a high potential to suppress GvHD.

[0012] To further investigate whether EPS can improve GvHD in humans, humanized NSG-HLA-A2 mice were used, and GvHD was improved by A2 neg Human PBMCs were induced by intravenous injection. In this model, dendritic cell (DC) transfer was used instead of EPS injection. CD34 cells were pre-treated with EPS or PBS. + Using umbilical cord blood cell-derived DCs (CD34-DCs) together with PMBCs, it was found that injection of EPS-treated DCs extended the survival of GvHD mice. This DC cell therapy is a novel treatment and, as the inventors of this application demonstrate herein, can generate a large number of EPS-DCs that can be used, for example, as an "off-the-shelf" treatment.

[0013] For clarity and conciseness, features may be described herein as part of the same or distinct embodiments, but it will be understood that the scope of the invention may include embodiments having all or some combinations of the described features.

[0014] definition The following definitions are included to provide a clear and consistent understanding of this specification and the claims. Where used herein, the terms listed have the following meanings. All other terms and phrases used herein have their ordinary meanings as understood by those skilled in the art. Such ordinary meanings can be obtained by referring to specialized dictionaries such as Hawley's Condensed Chemical Dictionary, 14th edition, by R.J. Lewis, John Wiley & Sons, New York, NY, 2001.

[0015] References in this specification to “one embodiment,” “an embodiment,” etc., indicate that the embodiments described may include certain aspects, features, structures, parts, or characteristics, but not all embodiments necessarily include such aspects, features, structures, parts, or characteristics. Furthermore, such phrases may, though not necessarily, refer to the same embodiments referred to in other parts of this specification. Moreover, where certain aspects, features, structures, parts, or characteristics are described in relation to an embodiment, it is within the knowledge of those skilled in the art to know, whether explicitly stated or not, how such aspects, features, structures, parts, or characteristics affect or link to other embodiments.

[0016] The singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Therefore, for example, a reference to “a compound” includes multiple such compounds, and consequently, compound X includes multiple compounds X. It should be further noted that claims may be drafted to exclude optional elements. Therefore, when using exclusive terms such as “solely” or “only” in relation to any element described herein, and / or when describing or “negative” limitations of elements in the claims, this statement is intended to serve as a priori basis.

[0017] The term "and / or" means any one of the items to which the term relates, any combination of items, or all of the items. The phrase "one or more" is easily understood by those skilled in the art, especially when read in the context of its usage. For example, one or more substituents on a phenyl ring means 1 to 5 or 1 to 4 substituents, for example, if the phenyl ring is disubstituted.

[0018] Where used herein, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating a list of items, “and / or” or “or” shall be interpreted as inclusive, e.g., including at least one of several items, but also including two or more, and optionally including further unlisted items. “Only one of,” “exactly one of,” or, as used in claims, “consisting of,” refers to including exactly one element of a number or list of multiple elements, only when the term is clearly indicated. In general, where used herein, the term “or” shall be interpreted only as indicating an exclusive substitution (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0019] As used herein, the terms “including,” “includes,” “having,” “has,” “with,” or variations thereof are intended to be as comprehensive as the term “comprising.”

[0020] The term “approximately” can refer to a variation of ±5%, ±10%, ±20%, or ±25% of a given value. For example, “approximately 50” percent may have a variation of 45–55 percent in some embodiments. With respect to integer ranges, the term “approximately” can include one or two integers greater than and / or less than the integers enumerated at each end of the range. Unless otherwise indicated herein, the term “approximately” is intended to include a value (e.g., weight %) close to the enumerated range that is equivalent with respect to the functionality of the individual components, compositions, or embodiments. The term “approximately” can also modify the endpoints of the cited ranges, as discussed above in this paragraph.

[0021] As will be understood by those skilled in the art, all numbers, including those representing the quantity of components, molecular weight and other properties, reaction conditions, etc., are approximations and, in all cases, are to be arbitrarily modified by the term "approximately." These values ​​may vary depending on the desired properties that those skilled in the art seek to obtain using the teachings described herein. It will also be understood that such values ​​inherently include variability that inevitably arises from the standard deviation found in each of their test measurements.

[0022] As will be understood by those skilled in the art, for any and all purposes, particularly with regard to providing written explanations, all ranges enumerated herein also include any and all possible subranges and combinations thereof, as well as the individual values ​​constituting the ranges, in particular integer values. Enumerated ranges (e.g., weight percentages or carbon groups) include each specific value, integer, decimal, or identity within that range. Any enumerated range can be readily recognized as fully explaining and enabling that the same range can be divided into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range described herein can be readily decomposed into lower thirds, middle thirds, upper thirds, and so on. Also, as will be understood by those skilled in the art, all language such as “at most,” “at least,” “greater than,” “less than,” “more,” “or more,” etc., includes the numbers enumerated, and such terms subsequently refer to ranges that can be decomposed into subranges as discussed above. Similarly, all ratios enumerated herein also include all sub-ratios that fall within the range of the broader ratio. Therefore, the specific values ​​listed for radicals, substituents, and ranges are for illustrative purposes only; they do not exclude other defined values ​​or other values ​​within the defined ranges for radicals and substituents.

[0023] Those skilled in the art will also readily recognize that, when members are grouped together in a common manner such as a Markush group, the present invention encompasses not only the entire group enumerated as a whole, but also each individual member of the group and any possible subgroups of the principal group.

[0024] Furthermore, for all purposes, the present invention encompasses not only the main groups but also main groups lacking one or more of their constituent elements. Accordingly, the present invention assumes the explicit exclusion of any one or more constituent elements of the enumerated groups. Thus, conditions may apply to any of the disclosed categories or embodiments, thereby excluding one or more of the enumerated elements, species, or embodiments from such categories or embodiments, for example, for use in an explicit negative limitation.

[0025] As used herein, the term “compare” means to assess how the proportion, level, or cellular localization of one or more biomarkers in a patient sample relates to the proportion, level, or cellular localization of the corresponding one or more biomarkers in a standard or control sample. For example, “compare” may mean to assess whether the proportion, level, or cellular localization of one or more biomarkers in a patient sample is the same as, greater than, less than, or different from the proportion, level, or cellular localization of the corresponding one or more biomarkers in a standard or control sample or another patient sample. More specifically, the term may mean to assess whether the proportion, level, or cellular localization of one or more biomarkers in a patient sample is the same as, greater than, less than, different from, or otherwise corresponding to (or not corresponding to), for example, a given biomarker level corresponding to a patient.

[0026] As used herein, when referring to parameters, for example, adjusted proportions, levels, or cellular localization in a patient's sample, the terms “indicate” or “correlate” (or, depending on the context, “indicate” or “correlate,” or “indicator” or “correlate”) may mean that a patient has or is at risk of having GvHD.

[0027] As used herein, the term “subject” refers to any animal (e.g., mammals, birds, reptiles, amphibians, fish, etc.) that is a recipient of a particular treatment, including but not limited to humans, non-human primates, and rodents. Typically, the terms “subject” and “patient” may be used interchangeably herein when referring to a subject.

[0028] Various methodologies of the present invention involve comparing values, levels, features, properties, etc., with a "suitable control," which is interchangeably referred to herein as "appropriate control" or "control sample." A "suitable control," "appropriate control," or "control sample" is any control or standard well known to those skilled in the art and useful for comparison purposes. In one embodiment, the "suitable control" or "appropriate control" is a value, level, feature, property, etc., determined in a cell, organ, or patient exhibiting normal traits, e.g., a control or normal cell, organ, or patient. For example, the biomarkers of the present invention may be assayed in samples from unaffected individuals (UI) or normal control individuals (NC) (both terms are used interchangeably herein) or other affected individuals. In another embodiment, the "suitable control" or "appropriate control" is a value, level, feature, property, etc., determined before treatment is administered to a patient. In another embodiment, transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristics or properties, genotype, phenotype, etc., may be determined before, during, or after administration of treatment to cells, organs, or patients. In a further embodiment, a “preferred control” or “appropriate control” is a predetermined value, level, characteristic, property, etc.

[0029] As used herein, the term “administer” refers to providing a therapeutically effective amount of a chemical or biological compound / cell (e.g., EPS-treated DC) or pharmaceutical composition. The chemical or biological compounds / cells of the present invention may be administered alone, but may also be administered with other compounds, excipients, fillers, binders, carriers or other vehicles selected based on a chosen route of administration and standard pharmaceutical practice. Administration may be by injection, including sterile aqueous or non-aqueous solutions or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants including microimplants; eye drops; ear drops; sprays including nasal sprays; other proteins and peptides; synthetic polymers; microspheres; nanoparticles or other carriers or vehicles.

[0030] The chemical, biological compounds, pharmaceutical compositions, or cells of the present invention may also be included with or packaged with other non-toxic compounds (e.g., pharmaceutically acceptable carriers, excipients, binders, and fillers (including, but not limited to, glucose, lactose, acacia gum, gelatin, mannitol, xanthan gum, locust bean gum, galactose, oligosaccharides and / or polysaccharides, starch paste, magnesium trisilicate, talc, corn starch, starch fragments, keratin, colloidal silica, potato starch, urea, dextran, dextrin, etc.)). Furthermore, the packaging material may be biologically inert or lacking in biological activity (e.g., plastic polymers, silicones, etc.) and may be internally processed by the subject without affecting the efficacy of the drug packaged and / or delivered with it.

[0031] The term “effective dose” as applied herein to the compounds, biologics, and pharmaceutical compositions means the amount required to produce the desired therapeutic outcome. For example, an effective dose is the level at which the therapeutic compound, biologic, or composition is effective in treating, curing, or alleviating the symptoms of the disorder to which it is administered. The effective dose for a particular therapeutic goal depends on a variety of factors, including the disorder being treated and its severity and / or stage of onset / progression; the bioavailability and activity of the particular compound, biologic, or pharmaceutical composition used; the route or method of administration and the site of introduction on the target; the rate of clearance of the particular compound or biologic and other pharmacokinetic properties; the duration of treatment; the inoculation regimen; drugs used in combination with or concurrently with the particular compound, biologic, or composition; the age, weight, sex, diet, physiological function, and overall health status of the subject being treated; and similar factors known to those skilled in the art of the relevant scientific field. Some variation in dosage may occur depending on the condition of the subject being treated, and the physician or other individual administering the treatment will, in any case, determine the appropriate dose for the individual patient.

[0032] As used herein, “disorder” means a disorder, disease or condition, or any other deviation from health or normal biological activity, and these terms may be used interchangeably. These terms refer to any condition that impairs normal function.

[0033] As used herein, “treatment” or “to treat” means stopping or inhibiting the onset or progression of a disorder, and / or causing or attempting to cause a reduction, suppression, regression, or remission of the disorder and / or its symptoms. As will be understood by those skilled in the art, various clinical and scientific methodologies and assays may be used to assess the onset or progression of a disorder, and similarly, various clinical and scientific methodologies and assays may be used to assess the reduction, regression, or remission of the disorder or its symptoms. Furthermore, treatments may be applied to subjects or cell cultures.

[0034] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the field and are described in methodological manuals (e.g., Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0035] Exopolysaccharide (EPS) Exopolysaccharides are high molecular weight polymers composed of sugar residues that are secreted into the surrounding environment by microorganisms. Exopolysaccharides can be isolated from bacterial cultures by the methods described below. Microorganisms synthesize a wide range of multifunctional polysaccharides, including intracellular polysaccharides, structural polysaccharides, and extracellular polysaccharides or exopolysaccharides (EPS). Exopolysaccharides generally consist of numerous different monosaccharides, such as glucose, mannose, and galactose. Due to the wide diversity of their compositions, exopolysaccharides have found a wide variety of applications in various food and pharmaceutical industries.

[0036] The exopolysaccharides provided herein are bacterial (e.g., Bacillus subtilis) exopolysaccharides. In one embodiment, the bacterial strain used to supply the exopolysaccharide is a probiotic strain. In another embodiment, the bacterial exopolysaccharide is derived from Bacillus subtilis, Bacteroides fragilis, Bifidobacterium breve, or a combination thereof.

[0037] In one embodiment, the exopolysaccharide was isolated from B. subtilis DS991 (sinRtasA mutant), a strain that produces and secretes large amounts of EPS; the substance derived from this strain is named EPS+. EPS can be isolated by methods available to those skilled in the art, such as those previously described (Guttenplan, SB et al., 2010, PLoS Genet., 6:e1001243). Briefly, stationary-stage supernatant grown on agar plates or supernatant of bacteria resuspended in PBS was mixed with 3-4 times the volume of 100% EtOH at 4°C for at least 4 hours to precipitate EPS. The precipitate was pelletized (15,000 × 3 g, 4°C, 20 min), washed in PBS, and resuspended in 0.1 M Tris. The sample was digested with DNase (67 mg / ml) and RNase (330 μg / ml) at 37°C, and after 1 hour, proteinase K (40 mg / ml) was added, and the sample was incubated at 55°C for 1 hour. EPS was precipitated with EtOH, resuspended in 0.1 M Tris (pH 8), further purified by gel filtration on an S1000 column in 0.1 M Tris (pH 8), and then desalted by dialysis. EPS was quantified by a colorimetric phenol-sulfate assay using serial dilutions of fructose as a standard (17). The purity of the sample was measured by OD. 260 / 280 The samples were evaluated to confirm the absence of protein or nucleic acid contamination, and ELISA confirmed the absence of lipid A.

[0038] Dendritic cells Dendritic cells (DCs) are antigen-presenting cells (also known as accessory cells) of the mammalian immune system. Their primary function is to process antigens and present them on their cell surface to T cells of the immune system. They act as messengers between the innate and adaptive immune systems.

[0039] Dendritic cells are present in the skin (where there is a specialized dendritic cell type called Langerhans cells) and in tissues that come into contact with the external environment, such as the inner lining of the nose, lungs, stomach, and intestines. They can also be found in an immature state in the blood. Once activated, they migrate to the lymph nodes, where they interact with T cells and B cells to initiate and form an adaptive immune response. At a certain developmental stage, they grow branched projections, dendrites.

[0040] The dendritic cells for use in the methods provided herein can be dendritic cells isolated from a patient, an immunocompatible donor, or any donor. They can also be stem cells, e.g., but not limited to, embryonic stem cells, iPSCs, or umbilical cord blood (HSCs containing CD34 + cells, such as those that can be purchased from Lonza or StemCell Technology, or isolated from umbilical cord blood (UCB) by isolating mononuclear cells therefrom, then labeling them with biotinylated anti-CD34 antibody, subsequently binding them to streptavidin-coated beads, and isolating them with a magnet, including those isolated by methods available to those skilled in the art such as isolation from UCB) and can be derived from human stem cells containing stem cells isolated therefrom.

[0041] CD34 + To amplify and increase their number, the cells can be grown for about 2 weeks in a serum-free medium containing about 10 ng / ml to about 100 ng / ml (each containing about 50 ng / ml of SCF, Flt3L, TPO, IL6) and / or 0.01 μM to about 1.5 μM (including about 0.75 μM) of StemRegenin 1 (SR1) (aryl hydrocarbon receptor antagonist).

[0042] After amplification, the cells can optionally be cryopreserved (e.g., 10 6The cells can then be further amplified and produced in vitro by treating them with GM-CSF and IL4 after thawing in a cryopreservation medium readily available to those skilled in the art, such as Cryostor medium (StemCell Technology) at a concentration of / ml. These DCs (in any number of cells required, e.g., about 1 million cells per mouse or about 10 per human undergoing HSCT) are then used to produce the DCs. 7 ~10 8 Before administering the cells (including EPS) to the target that requires it, 6 Approximately 5 μg / ml to 100 μg / ml of EPS (e.g., approximately 0.5 to 3.0 mg per patient; approximately 10 to 48 hours, including approximately 16 to 24 hours) can be used for contact. In one embodiment, the EPS is removed before administration to the DCs.

[0043] Therefore, 1) CD34 derived from umbilical cord blood + 1) Amplifying and cryopreserving cells; 2) Using GM-CSF (100 ng / μL) and IL4 (50 ng / μL), CD34 + Further amplification of cells and differentiation of them into dendritic cells; 3) Treatment of DCs with EPS (approximately 30 μg / mL to approximately 100 μg / mL, including approximately 60 μg / mL) and optionally refreezing before administration to the subject (in one embodiment, administration is 10 per patient undergoing HSCT) 7 ~10 8 Contains individual cells (administration may be a single dose or may be repeated as needed).

[0044] Graft-versus-host disease (GvHD) Graft-versus-host disease (GvHD) is a complication that can occur after allogeneic transplantation. During allogeneic transplantation, healthcare providers transplant hematopoietic stem cells (immature blood cells) from a donor into a host. The donated stem cells eventually develop into mature blood cells.

[0045] A host may need donor stem cells if they have problems with their blood cells, such as in stem cell transplantation for cancers like leukemia or lymphoma, other bone marrow failure disorders like aplastic anemia, and abnormal hemoglobin disorders like sickle cell disease and beta-thalassemia.

[0046] In GvHD, the donated stem cells (graft) perceive the recipient's cells (host) as an unfamiliar threat. As a result, the donated cells attack the recipient's cells. This is why the disease is called "graft" versus "host."

[0047] What are the different types of GvHD? There are two main types of GvHD. Previously, healthcare providers classified GvHD based on when symptoms began. More recently, providers diagnose specific types of GvHD based on symptoms and clinical signs (e.g., test or imaging results), in addition to the timing of symptom onset.

[0048] • Acute graft-versus-host disease (aGvHD): Occurs immediately after transplantation, usually within the first 100 days. However, symptoms of aGvHD can also begin later. aGvHD often affects the skin, gastrointestinal (GI) tract, or liver.

[0049] • Chronic graft-versus-host disease (cGvHD): Can appear at any point after allogeneic transplantation, but most cases begin within two years. Chronic GvHD can affect the skin, mouth, liver, lungs, GI ducts, muscles, joints, or genitals.

[0050] As an allogeneic transplant recipient, you may experience one or both forms of GvHD, or neither. What are the symptoms of GvHD? GvHD symptoms range from mild to moderate to severe (potentially fatal).

[0051] Acute GvHD Symptoms of aGvHD most commonly affect the skin, gastrointestinal (GI) tract, or liver. The most common symptom of cutaneous aGvHD is a rash or reddened area of ​​skin (similar to a sunburn). The rash may also be painful or itchy. These rashes usually begin on the neck, shoulders, ears, as well as the palms of the hands and soles of the feet. The rash may spread to other parts of the body.

[0052] The most common symptoms of GI aGvHD are nausea, vomiting, and diarrhea. Symptoms can be mild or severe. Symptoms may include rash and / or itching, diarrhea, vomiting, abdominal cramps, and / or jaundice (yellowing of the skin and / or eyes).

[0053] Chronic GvHD cGvHD most commonly affects the skin, liver, GI ducts, and lungs, but can affect any part of the body. Symptoms may include rash and / or itching, skin tightness and swelling, hair loss on the head and body, dry mouth, sores in the mouth, gum disease, dry or gritty eyes, vision changes, diarrhea, nausea and vomiting, yellowing of the skin and / or eyes (jaundice), shortness of breath (dyspnea), dryness, persistent cough, fatigue, muscle weakness, cramps or pain, decreased range of motion in the joints, vaginal dryness, itching or dyspareunia, and / or itching or dyspareunia of the penis or scrotum.

[0054] How is graft-versus-host disease diagnosed? Healthcare providers can diagnose GvHD during a physical examination by observing specific symptoms and evaluating clinical tests and biopsy results.

[0055] What are the treatment options for GvHD? Currently, transplant recipients are likely to receive prophylactic (preventive) medication to suppress the host immune system. These immunosuppressants reduce the ability of donor cells to initiate an immune response (attack) against host tissue.

[0056] If these medications do not prevent the onset of GvHD, other over-the-counter treatments are available. Treatment of acute GvHD Healthcare providers successfully treat many people with aGvHD by increasing the use of immunosuppressants in the form of oral (administered by mouth), intravenous (administered through a vein), or topical (applied to the skin) corticosteroids. If steroids are ineffective, providers may prescribe ruxolitinib (Jakafi®). ​​Clinical trials may also be available.

[0057] Treatment of chronic GvHD Providers typically prescribe long-term immunosuppressants to treat cGvHD. If these medications do not improve the condition, these may also be prescribed: ruxolitinib (Jakafi®), belumosudil (Rezurock®), ibrutinib (Imbruvica®), and / or photopheresis.

[0058] Immunosuppressants weaken the immune system, increasing the risk of fungal, bacterial, and viral infections. Healthcare providers may prescribe certain medications to help prevent potentially life-threatening infections.

[0059] Treatment or prevention of GvHD with EPS-DC A method for treating or preventing GvHD is disclosed herein, comprising administering an effective dose of DCs exposed to EPS to the subject.

[0060] The appropriate dosage of EPS-DC depends, for example, on the condition being treated, its severity and course, whether EPS-DC is administered for preventive or therapeutic purposes, previous treatments, the patient's medical history and response to EPS-DC, and the discretion of the attending physician. EPS-DC may be administered to the patient as a single dose or over a series of procedures, and may be administered to the patient at any time necessary for the treatment or prevention of GvHD. EPS-DC may be administered as a monotherapy or in combination with other drugs or treatments useful in treating GvHD.

[0061] Route of administration EPS-DC can be administered systemically or topically. In various embodiments, the route of administration is via the auricle (ear), buccal mucosa, conjunctiva, skin, teeth, electroosmosis, intracervical, intranasal sinuses, intratracheal, intestinal, dura mater, extraamniotic, extracorporeal, hemodialysis, infiltration, interstitium, intraperitoneal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilage, intracavitary, intracavernosus, intracavitary, intracerebral, intrasacral, intracorneal, and intracoronal regions. Tooth, coronal tissue, intracorpus cavernosum, intradermal tissue, intervertebral disc, intratubular tissue, duodenum, dura mater, epidermal tissue, esophagus, stomach, gingiva, ileum, lesion, lumen, lymphatic vessel, medullary cavity, meninges, muscle, eye, ovary, pericardium, abdominal cavity, pleura, prostate, lung, paranasal sinuses, spinal cord, synovial membrane, tendon, testis, medullary cavity , intrathoracic, intratubular, intratumoral, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous infusion, intraventricular, intrabladder, intravitreous, iontophoresis, lavage, larynx, nasal cavity, nasogastrostomy, not applicable, closed dressing method, eye, oral cavity, oropharynx, other, parenteral, percutaneous, periarticular, epidural, perineurial, periodontal, rectum, respiratory tract (inhalation), posterior ocular, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, local, percutaneous, transmucosal, transplacental, transtracheal, transtympanic, urethra, vagina, or by other methods known to those skilled in the art or any combination thereof (see, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, incorporated herein by reference).

[0062] Formulations and Dosage Forms EPS-DCs may be provided in a pharmaceutical composition (for example, for the first dose, cells may be injected together with HSCT cells in PBS; for further doses, cells may be IV injected in PBS, for example). The pharmaceutical composition may contain pharmaceutically acceptable diluents, excipients, or carriers. The pharmaceutical composition may also contain other medical or pharmaceutical agents, carriers, adjuvants (e.g., preservatives, stabilizers, wetting agents, or emulsifiers), solubilizers, salts for adjusting osmotic pressure, and / or buffers.

[0063] Well-known methods in the art for preparing formulations can be found, for example, in Remington: The Science and Practice of Pharmacy, (20th edition), ARGennaro A R., 2000, Lippencott Williams & Wilkins. Formulations for parenteral administration may include, for example, sterile water or saline, polyalkylene glycol (e.g., polyethylene glycol), plant-derived oil, or hydrogenated naphthalene as excipients, and biocompatible, biodegradable lactide polymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the factors of the present invention. Other potentially useful parenteral delivery systems for these factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable injection systems, and liposomes. The formulation for inhalation may contain, for example, lactose as an excipient, or may be an aqueous solution containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be an oily solution for administration in the form of a nasal spray, or may be a gel applied into the nasal cavity.

[0064] The number of EPS-DCs in the formulation may vary depending on several factors, including the dosage and route of administration. EPS-DC and HSC may be administered simultaneously or sequentially. EPS-DC and HSC may be administered in single or multiple doses, individually or in combination. EPS-DC and HSC may be administered in the same or different dosing modes, and may be administered once, twice, or multiple times, in combination of one or more, or individually. Thus, EPS-DC may be administered in an initial dose, followed by subsequent doses (one or more), particularly depending on the response. [Examples]

[0065] Example 1 Introduction Hematological malignancies are often treated with allogeneic hematopoietic stem cell transplantation (HSCT) aimed at cure. However, HSCT is contraindicated by acute graft-versus-host disease (aGvHD), a serious and potentially fatal complication initiated when donor alloreactive T cells attack host cells and organs (1, 2). Furthermore, aGvHD is a risk factor for developing chronic aGvHD (cGvHD) (3), and therefore, preventing aGvHD dramatically reduces the likelihood of developing cGvHD. While prophylactic regimens for aGvHD are standard pre-transplant therapy, up to 50% of these patients treated with calcineurin-based regimens develop grade II or higher aGvHD (grades 0-IV) (1), requiring further immunosuppressive intervention (4).

[0066] GvHD is caused by the activation of donor allogeneic T cells in response to molecules on host cells. This T cell activation leads to the recognition and destruction of recipient tissues and organs by donor immune effector cells, primarily affecting the skin, liver, and gastrointestinal tract (5,6). T cell activation also triggers inflammation and cytokine storms that further exacerbate the disease (7). Two standard regimens clinically used to prevent this complication include calcineurin inhibitors with short-term methotrexate, and post-transplant cyclophosphamide (PTCy) with calcineurin inhibitors and mycophenolates (1,6,8). Although prophylaxis with calcineurin inhibitors in combination with methotrexate has been the conventional approach for decades (1,6), high doses of PTCy administered on +3 and +4 post-transplant days have been shown to reduce the rates of both aGVHD and cGVHD in a phase 3 comparative trial against tacrolimus and methotrexate (8). However, in this trial, the increased incidence of organ failure, primary transplant failure, and bleeding, which were thought to be directly attributable to cyclophosphamide toxicity, offset the benefits of reduced mortality due to aGvHD and cGvHD, and therefore no difference was observed in disease-free survival or overall survival in the PTCy group. Thus, new methods for improving aGvHD without chemotherapy toxicity, such as the novel immunosuppressive methods described herein, can improve not only the aGvHD rate but also overall survival.

[0067] Cell therapy is an attractive treatment for aGvHD (4). However, CD34 + Cell therapy in animal models using monocytes (9), regulatory T cells (Tregs) (10), mesenchymal stem cells (11), dendritic cells (DCs) (12-14), or bone marrow-derived suppressor cells (15) has so far achieved only limited success (16). Vitamin D-treated DCs can delay GvHD in humanized NSG mice, demonstrating that DCs can protect against GvHD (12).

[0068] Probiotic exopolysaccharide (EPS) derived from the harmless probiotic soil bacterium Bacillus subtilis has been found to possess potent cellular immunosuppressive activity in various disorders, including allergies (17), colitis (18-20), sepsis (21,22), and GvHD (23). EPS induces tolerogenic DCs and anti-inflammatory macrophages that suppress the proliferation of activated T cells (17-20,23). In mice, in vitro transfer of EPS-treated bone marrow-derived DCs improved allergic eosinophilia (17), and peritoneal macrophages isolated from EPS-treated mice and transferred to recipient mice protected against diseases caused by enteropathogenic bacteria (17,20). In a mouse GvHD model, injection of EPS before GvHD induction increased the 80-day survival rate from 10% (control, PBS-treated mice) to 70% (EPS-treated mice) after transplantation of complete allogeneic major histocompatibility complex (MHC) mismatch cells (23).

[0069] Here, it was demonstrated that human EPS-treated dendritic cells (DCs) can influence human alloreactive T cells and improve GvHD in humanized mice. A protocol for generating large quantities of EPS-DCs was also developed, suggesting their use as an "off-the-shelf" treatment for GVHD patients.

[0070] material and method Mice and reagents All mice were housed in a sterile environment at the Loyola University Chicago animal facility. NSG-HLA-A2 (strain 009617) mice were purchased from Jackson Laboratory or bred in-house. Age- and sex-matched 8- to 12-week-old mice were used in all experiments, and all experiments were conducted according to protocols approved by the Loyola University Chicago Institutional Animal Care and Use Committee. Basic media and supplements were purchased from Life Technologies, StemSpan SFEM II medium, human AB serum, and Stem regenin 1 (SR1) were purchased from STEMCELL Technologies, bacterial media were purchased from BD, all antibodies (Ab) were purchased from BioLegend or eBioscience, and all cytokines were purchased from PeproTech (Thermo Fisher Scientific).

[0071] Preparation of EPS derived from B. subtilis EPS was prepared from B. subtilis DS991 (sinR:tasA), which overproduces and secretes EPS. ΔEPS is a carbohydrate prepared from B. subtilis DS5187 (sinRtasAepsH mutant), which does not produce EPS (19,24). 600 The bacteria were spread on Luria Bertani agar (150 mm) at a pH of 0.6-0.7 for 4 hours, and a homogeneous layer of bacteria was collected. The supernatant was treated with DNase (67 mg / mL), RNase (330 mg / mL), and protease K (40 mg / mL). Carbohydrates were treated with 75% EtOH, and after resuspending, the EPS was purified by gel filtration with Sephacryl® S-500. Carbohydrate-positive fractions identified by modified phenol-sulfuric acid assay (25,26) were pooled, broadly dialyzed, and air-dried. EPS purity was measured for detectable proteins or nucleic acids (OD). 260 / 280EPS preparations were evaluated by the absence of ) and the absence of cytotoxicity in vitro. All EPS preparations were sterile filtered and confirmed by ELISA for undetectable levels of endotoxins (lipopolysaccharides). Each preparation was evaluated for its ability to induce peritoneal M2 macrophages, as previously described (20), or for its ability to suppress the mixed lymphocyte reaction (MLR), as described below.

[0072] CD34 cell expansion and dendritic cell induction Umbilical cord blood CD34+ cells (Lonza Group) were amplified for 7 days in amplification medium (StemSpan SFEMII, 0.75 μM Stemregenerin, 1 × penicillin-streptomycin, 50 ng / mL Flt3L, SCF, TPO, and IL-6). After 1 week, the cells (10 6 CD34 ( / mL) was added to Cryostor medium (StemCell Technology). CD34 was amplified for one week. + The cells were thawed and amplified for another week in amplification medium. CD34 was amplified for two weeks. + Cells (100,000 / mL) were plated in DC induction medium consisting of a 1:1:1 mixture of aMEM:IMDM:RPMI containing 10% FBS, 2% human AB serum with 100 ng / mL human GM-CSF (hGM-CSF), and 50 ng / mL hIL-4. Half of the medium was replaced every two days. On day 12, the cells were pre-treated overnight with EPS or PBS before use in aGvHD experiments or MLR tests. MojoSort Human CD14 + Using the Monocytes Isolation Kit (BioLegend) according to the manufacturer's instructions, CD14 was extracted from human peripheral blood mononuclear cells (hPBMCs). + Monocyte-derived dendritic cells (DCs) were generated by isolating cells and then cultured for 10 days in RPMI containing 10% FBS, 50 ng / mL hGM-CSF, and 25 ng / mL hIL-4. Half of the culture medium was replaced every other day.

[0073] GvHD and MLR hPBMCs isolated from healthy HLA-A2-negative donors were purchased from STEMCELL Technologies. To induce GvHD in 8-12 week old NSG-HLA-A2 mice, they were prepared with 2.5 Gy from an X-ray source (RS 2000 Biological Research Irradiator), and after 4 hours, they were given 4 × 10⁶ cells. 6 Individual PBMCs, or 4 x 10 6 10 individual PBMCs from different donors 6 Individual umbilical cord blood CD34 + A mixture with hematopoietic stem cells (CD34-DC) was administered intravenously. CD34-DC was pre-treated with EPS (60 μg / mL) or PBS for 16 hours. For MLR, PBMCs were labeled with 5 μM Cell Trace Violet (CTV) (Thermo Fisher Scientific) according to the manufacturer's instructions and individually (2.5 × 10⁻⁶). 5 Cells, negative control, or CD34-derived DCs containing EPS (60 ug / mL) or ΔEPS (60 ug / mL) (7.5 × 10 in a 96-well U-bottom plate) 4 They were cultured together with PBMCs. In some experiments, third-party irradiated PBMCs were added to the culture as initiators. After 4 days, T cell proliferation and activation were measured by flow cytometry. Two different CD34 + Cell donors were evaluated over the long term along with multiple (>8) hPBMC donors.

[0074] Flow cytometry For flow cytometry, cells were treated with anti-CD16 / 32Fc block and then stained with Ab specific for CD4, CD8, CD25, and CD44. Cells were analyzed using FACSCanto® II or LSRFortessa® flow cytometers (BD Biosciences). Proliferation was measured based on the dilution of CTV dyes determined by flow cytometry, and the data were analyzed using FlowJo® software (BD Biosciences).

[0075] statistical analysis Unless otherwise specified, an independent two-tailed Student's t-test was used for statistical analysis, and P<.05 was considered statistically significant. Survival curves were modeled using the Kaplan-Meier method, and log-rank tests were applied to compare groups. All analyses were performed using Prism 5.0 (GraphPad Software).

[0076] result EPS effect on human dendritic cells (DCs) EPS suppresses T cell proliferation in mouse MLR cultures by converting bone marrow-derived DCs into suppressor DCs (23). To evaluate whether EPS also affects human cells, we used DCs derived from CD34-DCs (Figure 1A) or CD14-DCs. + The effects of dendritic cells (DCs) derived from peripheral blood monocytes (moDCs; Figure 1B) on MLR were investigated. These DCs, along with CTV-labeled PBMCs derived from allogeneic donors, were used as initiators in MLR cultures. To determine whether EPS converts DCs into suppressor cells, 60 μg / mL of EPS or a negative control ΔEPS was added to the cultures, and T cell activation and proliferation were measured by flow cytometry after 4 days. CD34-DCs were found to suppress alloreactive T cell proliferation by >50% compared to the same culture without EPS or with ΔEPS added as a negative control (Figure 1C, D). CD14 + Cultures containing derived moDCs and EPS did not suppress T cell proliferation (Figure 1E).

[0077] To investigate whether the observed inhibitory effect of CD34-DCs was solely due to the DCs themselves, and whether EPS-treated DCs retained their inhibitory properties after EPS was removed from the culture, CD34-DCs were pre-treated with EPS overnight, and then added to MLR cultures after washing off the EPS. The DCs were found to still inhibit the proliferation of alloreactive T cells in the MLR cultures to a similar degree to that observed when EPS was present throughout the culture period (Figure 1D) (Figure 1F). These results indicate that EPS converts human CD34-derived DCs into inhibitory DCs, and that these cells significantly inhibit the proliferation of alloreactive T cells in MLR cultures. It was also shown that human CD34-DCs retained their inhibitory properties after EPS removal, leading to the hypothesis that EPS-treated CD34-DCs (EPS-DCs) could be used as a cell-based therapy to improve human GvHD.

[0078] EPS-treated CD34-DC extends the survival of NSG-HLA-A2 mice experiencing GvHD. To investigate whether EPS-DCs can improve GvHD, we used a humanized mouse model of induced GvHD, in which human peripheral blood mononuclear cells (hPBMCs) were transferred into lightly irradiated NSG-HLA-A2 mice (27). To determine whether EPS-DCs could reduce GvHD-related mortality, 4 × 10⁶ mice were subjected to a 4 × 10⁶ test. 6 GvHD was induced using individual hPBMCs, and separate cohorts of mice were simultaneously injected with either CD34-DC pretreated with EPS for 16 hours, or PBS as a negative control, in a 4:1 hPBMC:DC ratio (Figure 2A). All control mice treated with PBS-treated CD34-DC died between 10 and 36 days after disease induction (Figure 2B). Mice treated with EPS-DC showed significantly improved survival. 6In the control cohort that received only ) the median survival time was 23 days, which was not significantly different from the median survival time of the control group that received PBS-treated CD34-DCs and hPBMCs (21 days). Similar survival kinetics (Figure 2B), as well as similar median days to lethality (21 and 23 days), were observed in mice administered with hPBMCs together with untreated DCs (NT-DCs) and with hPBMCs alone, respectively. + This study demonstrates that the addition of cell-derived HLA-mismatched DCs does not exacerbate GvHD in humanized mice.

[0079] In another GvHD experiment, 2 × 10 6 Individual hPBMC and 0.5 × 10 6 Mice were transfused with either untreated or EPS-treated CD34-DCs and analyzed for weight loss and clinical scores. Mice treated with EPS-DCs showed significantly less weight loss compared to mice treated with untreated DCs (Figure 2C). Similarly, mice treated with EPS-DCs did not essentially show evidence of disease during the first 30 days, as measured by clinical scores, while mice treated with untreated DCs did show evidence of disease based on clinical scores (Figure 2D). However, after 30 days, clinical scores increased in EPS-DC-treated mice, suggesting that in this model, higher doses of EPS-DCs may provide further protection from disease. It was concluded that EPS-DCs significantly increased survival and reduced GvHD symptoms.

[0080] Human umbilical cord blood CD34 + Generation of EPS-DCs from stem cells To meet the clinical requirements for using EPS-DCs to prevent GvHD, it is necessary that a large number of DCs be readily generated. CD34 + It is well established that hematopoietic cells (HSCs) can generate DCs in vitro, but circulating CD34 + The number of HSCs is small (<0.1% of PBMCs). While this number is higher in umbilical cord blood, the amount of umbilical cord blood clinically available is small. Human umbilical cord blood CD34 +Cells are commercially available, but they are expensive ($1500-$2000 / 10 6 Cells (StemCell Technologies). Using the protocol, umbilical cord blood CD34 was extracted in serum-free medium using a titration cocktail of SCF, Flt3L, TPO, IL-6, and StemRegenin 1 (aryl hydrocarbon receptor antagonist). + The cells were amplified. CD34 from four different donors. + Using cells, CD34 was detected after 3 weeks of culture with cytokines. + The number of cells is 2 × 10 4 It was found that the amplification was more than double (Table 1). These data were 10 5 individual CD34 + From cells >2 × 10 9 individual CD34 + This demonstrates the ability to produce cells.

[0081] [Table 1]

[0082] Characterization of CD34-derived DCs and their EPS acquisitions Flow cytometry essentially reveals any CD34 + The cells also lacked the HLA-DR or CD11cDC marker after 3 weeks of amplification. However, after differentiating these cells into DCs, 50% and 72% of them developed the HLA-DR marker by day 5 and day 12, respectively. + CD11c + It was found that (Figure 3A). On day 12, all DCs were essentially conventional DCs (cDCs), with approximately 20% possessing the cDC1 marker and >70% possessing the cDC2 marker (Figure 2A). Using fluorescently labeled EPS, we tested which cells took up EPS and found that essentially all DCs took up EPS, but cDC2 cells took up more than cDC1 cells (Figure 3B). Approximately 20% of the HLA-DR cells were contaminated. - CD11c - cells (CD34 - CD14- The identity of (and CD3) remains unknown, but it is highly likely that they did not take up EPS and did not contribute to EPS-mediated defense. We also analyzed the upregulated expression of inhibitory molecules PD-L1 and PD-L2, which are upregulated on mouse DCs after EPS treatment, and, as predicted, we found increased expression of both PD-L1 and PD-L2 in EPS-DCs (Figure 3C).

[0083] Protocol for generating and using EPS-DCs to prevent and / or treat aGvHD in humans Figure 4 shows one embodiment of the protocol, 1) CD34 derived from umbilical cord blood + 1) Amplifying and cryopreserving cells; and 2) Using GM-CSF (100 ng / μL) and IL4 (50 ng / μL), CD34 + This demonstrates further cell amplification, differentiation into dendritic cells, treatment with EPS (60 μg / mL), and freezing or immediate use. Using a xenograft (×) GvHD model, donor hPBMCs are injected into NSG-A2 mice; the timing and dosing of EPS-DCs are optimized to obtain maximum protection (e.g., 4 × 10 hPBMCs). 6 EPS-DC10 6 Mix the two and administer as an intravenous injection once on day 0 of the start of GvHD. EPS-DCs are nontoxic and can be easily prepared and cryopreserved (for example, in Cryostor medium (StemCell Technology), approximately 3 × 10⁻¹⁶). 6 Cell-based GvHD prophylaxis is advantageous because it does not require HLA matching for the donor or recipient (at / ml). This approach ensures that therapeutic doses of EPS-DCs for infusion can be generated.

[0084] Consideration HSCT is often the only curative option for treating life-threatening blood disorders, including leukemia, hemoglobinopathy, and several forms of anemia, including sickle cell anemia. However, GvHD is the leading cause of morbidity and mortality after HSCT, significantly limiting its cure potential. Despite current prophylactic treatments, the high incidence of GvHD, with >10% of patients dying from this complication (28), highlights the still unmet need for new therapies to prevent and / or treat GvHD. EPS derived from the harmless probiotic soil bacterium B. subtilis was found to induce tolerogenic DCs (EPS-DCs) that prevent the proliferation of activated T cells in MLR cultures. Using a humanized NSG-HLA-A2 transgenic mouse model, injection of EPS-DCs during hPBMC transfer was found to significantly increase survival from a median of 22 days in NT-DCs to 31 days in EPS-DCs. Two of the 32 EPS-DC treated mice survived for at least 100 days. In addition, EPS-DC treated mice showed significantly less weight loss and clinical symptoms compared to NT-DC treated mice. The reduction in clinical scores was most evident in the first month after transplantation, suggesting that more than one dose of EPS-DC may be required to optimize the treatment.

[0085] Cell therapy is an attractive approach to treating GvHD (9,10,12-16,29,30). The use of mesenchymal stem cells has shown varying successes in treating patients with steroid-refractory GvHD (29,30), although recent Phase III trials have shown significant improvement only in pediatric patients and not in adult patients (30). Tregs have also shown promise for treating GvHD in humans, but results from large-scale clinical trials are not yet available. Human studies have been conducted primarily in patients with established GvHD and appear to be more successful by preventing GvHD rather than treating it (31). DCs, specifically tolerogenic DCs, can prevent GvHD by suppressing the activation and proliferation of alloreactive T cells. In a preclinical model, vitamin D-treated DCs delayed GvHD in humanized NSG mice but did not enhance survival (12). In contrast, the EPS-DCs described herein are tolerogenic DCs that suppress alloreactive T cells (Figure 1C, D, F) and extend survival in a humanized GvHD model (Figure 2B). A distinct set of DC subsets is described, including conventional DCs (cDCs), moDCs, and plasmacytoid DCs, each with different phenotypes and functions. Tolerogenic EPS-DCs are derived from human umbilical cord blood CD34 + These are stem cell-derived, mostly cDCs, particularly cDC2s (Figures 3B and (32)), while the VitD-DCs and other tolerogenic DCs used clinically are moDCs derived from peripheral blood monocytes (33-36). EPS does not induce tolerogenic function in moDCs (Figure 1C), highlighting a novel mechanism that EPS uses to tolerate specific subsets of DCs.

[0086] Activated donor allogeneic T cells cause GvHD when they attack recipient organs. In a mouse GvHD model using biosensor mice, injection of EPS has been shown to suppress alloreactive T cell proliferation in vivo (23). In clinics, the current treatment of choice for preventing GvHD is PTCy. Its use has been shown to reduce the incidence of severe aGVHD to <10% and also significantly reduce the incidence of cGVHD (37-40). Wacshmuth et al. (41) demonstrated that the protective effect of PTCy in an MHC-haplotype-matched HSCT model arose from attenuation of alloreactive donor T cell proliferation, mainly through Treg enhancement. Herein, it is shown that human EPS-treated DCs that suppress alloreactive T cell proliferation in vitro in MLR cultures increased the expression of inhibitory molecules PD-L1 and PD-L2. These molecules may contribute to the mechanism by which EPS-DCs improve GvHD in humanized mice. In this case, EPS-DCs may directly interact with donor T cells, as occurs in MLR cultures, to suppress their activation and proliferation. Furthermore, EPS-DCs may reduce systemic inflammation in GvHD mice, leading to reduced donor T cell activation. Recent studies have demonstrated that extracellular factors, including the tissue microenvironment, which can be modified by the host microbiota or microbiota-derived metabolites such as short-chain fatty acids, influence allogeneic immune responses (42,43). The EPS-DCs provided herein may function by altering the tissue microenvironment and / or by making T cells tolerant to a pro-inflammatory microenvironment. Therefore, EPS-DCs may exert a similar effect on alloreactive T cells as PTCy, since they directly suppress the proliferation of activated T cells, including alloreactive T cells (23). In contrast to PTCy, an alkylating agent that alters nucleic acids and can be toxic at high concentrations, EPS-DCs do not appear to be toxic. Furthermore, EPS-DC treatment is short-term and is expected to act as a non-toxic alternative to PTCy, or added to culture media, to prevent or treat GvHD.

[0087] Using EPS-DC to prevent GvHD in humans is possible because of umbilical cord blood CD34 + A standardized protocol is required to generate DCs from cells (or other sources) and treat them with EPS, after which they can be cryopreserved until thawed and injected. A large number of EPS-DCs are needed to meet the clinical application for the prevention of GvHD. CD34 + HSCs can produce DCs after culture with GM-CSF and IL-4, but only a small number of CD34 + HSC can only be obtained from peripheral blood or umbilical cord blood. Provided herein is CD34 + Cells grow >2 x 10 in 2 weeks 3 This is a protocol that amplifies the signal by two. 10 7 ~10 8 It is estimated that the therapeutic dose of EPS-DC is required for a person undergoing HSCT, <10 5 individual CD34 + It was demonstrated that this number of DCs could be produced from cells. After being treated with EPS overnight, the EPS-DCs could be cryopreserved, thawed, and transferred to pre-conditioned human patients simultaneously with donor HSCs. It should be noted that in this study, only 30% of the mice survived, and these mice received only a single injection of EPS-DCs. Survival may be significantly increased with additional doses of EPS-DCs after HSCT.

[0088] This specification describes a method for generating EPS-treated dendritic cells (DCs), which have been demonstrated to improve GvHD in a humanized model of the disease. Using EPS-DCs as a cell-based therapy for GvHD prevention has clear advantages and is safe because the cells are "normal" and not genetically modified. (Umbilical cord blood CD34) + The advantage of using HSCs is that a large number of samples are readily available in cord blood banks worldwide, due to the increased availability of alternative donors for donors who do not have a matched sibling donor or an unrelated donor. These cord blood cells are >10 3It can be amplified twofold, allowing for the creation and cryopreservation of large stocks of EPS-DCs. This approach enables the use of a readily available product, which, when used in combination with standard, less toxic but less effective GvHD prophylaxis regimens other than PTCy, or in combination with PTCy, could be potentially useful for preventing lethal aGvHD. This could allow for the administration of PTCy at lower, less toxic doses.

[0089] There are several strategies for the clinical use of these EPS-DCs. One approach is to develop a ready-to-use EPS-DC product, derived from umbilical cord blood stem cells and injected within the same timeframe as cultured PTCy (i.e., immediately after donor stem cell infusion) to prevent aGVHD, as suggested by the strategy used to treat mice herein. UCB CD34 using cytokines in the presence of nicotinamide + Ex vivo amplification of stem cells has recently been observed with CD34 + Cellular amplification 130 times (median, 6.6 × 10⁻⁶) 8 individual CD34 + It was approved by the U.S. Food and Drug Administration for clinical transplantation based on a Phase 3 clinical trial, demonstrating that it shows sufficient CD34 cells and results in superior engraftment compared to non-amplified UCB grafts (44). + Cells can be obtained from these amplified stem cells and used to produce EPS-DCs for multiple patients, with broad availability for all transplantation programs. However, there are potential risks in using third-party DCs for this purpose. This is because, although third-party tolerable DCs have been tested in mouse models of GvHD and appear to be effective in extending survival time, they express low levels of MHC (5,6).

[0090] Another strategy is CD34 +The goal is to develop a specific, single-type EPS-DC derived from a donor using peripheral blood G-CSF-mobilized stem cell grafts. This technique may be used for patients receiving allogeneic grafts from matched related and unrelated donors, but likely not for recipients of incompatible or haplotype-matched grafts for the reasons mentioned above.

[0091] References

[0092] [Table 2-1]

[0093] [Table 2-2]

[0094] [Table 2-3]

[0095] [Table 2-4]

[0096] [Table 2-5]

[0097] All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference. In the event of any conflict between the definition of a term incorporated by reference and a term defined herein, this specification shall prevail.

Claims

1. A method for preventing, treating, or suppressing graft-versus-host disease (GvHD), comprising administering dendritic cells (DCs) exposed to an exopolysaccharide (EPS) to a subject in need, thereby preventing, treating, or suppressing GvHD in the subject.

2. A method for reducing at least one symptom of graft-versus-host disease (GvHD), comprising administering dendritic cells (DCs) exposed to an exopolysaccharide (EPS) to a subject in need of such reduction of at least one symptom in the subject.

3. A method for extending the survival of a subject having graft-versus-host disease (GvHD), comprising administering dendritic cells (DCs) exposed to an exopolysaccharide (EPS) to a subject in need, in such a manner that the survival of the subject is extended.

4. A method for downregulating the expression of an activating marker (e.g., CD80 and / or CD86), upregulating the expression of an inhibitory molecule (e.g., PD-L1 and / or PD-L2), suppressing the activation of alloreactive T cells, or a combination thereof, comprising administering dendritic cells (DCs) exposed to an exopolysaccharide (EPS) to a subject in need thereof.

5. The method according to any one of claims 1 to 4, wherein the GvHD is acute GvHD (aGvHD).

6. The method according to any one of claims 1 to 4, wherein the GvHD is chronic GvHD (cGvHD).

7. The method according to any one of claims 1 to 6, wherein the DC exposed to the EPS is administered two or more times (for example, over a period of several days, several weeks, several months, or several years).

8. The method according to claim 7, wherein DCs exposed to the EPS are administered together with hematopoietic stem cells (HSCs).

9. The method according to any one of claims 1 to 8, wherein at least one other therapeutic agent for treating GvHD is administered to the subject.

10. The method according to any one of claims 1 to 9, wherein the subject is a human.

11. A method for generating tolerogenic dendritic cells (DCs), a) CD34 ＋ To provide hematopoietic cells (HSCs), b) CD34 of a) ＋ Cells were cultured with a combination of SCF, Flt3L, TPO, IL6, and StemRegenin 1 (SR1) to obtain CD34 ＋ To amplify cells, c) The cells from b) are cultured with GM-CS and IL4 to further amplify the cells and generate DCs in vitro. d) and c) the cells are brought into contact with exopolysaccharide (EPS), e) Cryopreserving the cells of d) at the discretion of the user, wherein the EPS is optionally removed from the cells of d) before cryopreservation. Methods that include...

12. The method according to claim 11, wherein the HSC is obtained from umbilical cord blood.

13. The method according to claim 11 or 12, wherein the cells are cryopreserved before c), after c), and / or after d).

14. The method according to any one of claims 11 to 13, wherein the EPS is removed from the cells of e) before cryopreservation or administration.

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