EP4680253A1 - Dendritic cell populations that inhibit gvhd - Google Patents

Abstract

Provided herein are compositions and methods to prevent, treat or inhibit graft vs host disease (GvHD) comprising exopolysaccharide (EPS) exposed dendritic cells (DCs).

Description

DENDRITIC CELL POPULATIONS THAT INHIBIT GVHD

PRIORITY

This application claims the benefit of U.S. Provisional Application No. 63/489,839, filed March 13, 2023, the content of which is herein incorporated by reference in its entirety. GOVERNMENT GRANT SUPPORT

This invention was made with government support under Al 155281 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Graft versus host disease (GvHD) is a severe, often lethal, complication of hematopoietic stem cell transplantation (HSCT), and although prophylactic regimens are given as standard pre-transplantation therapy, up to 50% of these patients develop aGVHD, and require additional immunosuppressive intervention.

SUMMARY OF THE INVENTION

Using a mouse GvHD model, it was previously shown that injecting mice with exopolysaccharide (EPS) from Bacillus subtilis prior to GvHD induction significantly increased 80-day survival after transplantation of complete allogeneic major histocompatibility complex-mismatched cells. To ask whether EPS might also inhibit GvHD in humans, GvHD was induced in humanized NSG-HLA-A2 mice by i.v. injection of A2^neg human peripheral blood mononuclear cells (PBMCs). As one cannot inject human donors with EPS, EPS- pretreated dendritic cells (DCs) were transferred to inhibit aGvHD. These DCs were derived from CD34⁺ human cord blood cells, treated with EPS, and then the cells were injected together with PBMCs into the NSG-HLA-A2 mice. It was found that all mice that received untreated DCs were dead by day 35, whereas 25% of mice receiving EPS-treated DCs (EPS-DCs) survived.

Provided herein are novel compositions of combined EPS and DCs and methods of use thereof, including novel methods of preventing graft vs. host disease and prolonging survival.

One embodiment provides a method to prevent, treat or inhibit graft vs host disease (GvHD) comprising administering exopolysaccharide (EPS) exposed dendritic cells (DCs) to a subject in need thereof so as to prevent, treat or inhibit GvHD in said subject.

One embodiment provides a method to decrease at least one symptom of graft vs host disease (GvHD) comprising administering to a subject in need thereof exopolysaccharide (EPS) exposed dendritic cells (DCs) so as to decrease at least one symptom in said subject. One embodiment provides a method to prolong the survival of a subject with graft vs host disease (GvHD) comprising administering exopolysaccharide (EPS) exposed dendritic cells (DCs) to a subject in need thereof so as to prolong the survival of said subject.

One embodiment provides a method to downregulate expression of activation markers (e.g., CD80 and/or CD86), upregulate expression inhibitory molecules (e.g., PD-L1 and/or PD- L2), inhibit activation of alloreactive T cells or a combination thereof comprising administering exopolysaccharide (EPS) exposed dendritic cells (DCs) to a subject in need thereof so as to downregulate expression of activation markers (e.g., CD80 and/or CD86), upregulate expression of inhibitory molecules (e.g., PD-L1 and/or PD-L2), inhibit activation of alloreactive T cells or a combination thereof.

In one embodiment, the GvHD is acute GvHD (aGvHD). In another embodiment, the GvHD is chronic GvHD (cGvHD). In one embodiment, the EPS exposed DCs are administered more than once (e.g., over a period of days, weeks, months, years). In one embodiment, the EPS exposed DCs are administered with hematopoietic stem cells (HSCs). In one embodiment, at least one other therapeutic agent to treat GvHD is administered to said subject. In one embodiment, the subject is human.

One embodiment provides a method to generate tolerogenic dendritic cells (DCs) comprising: a) providing CD34⁺ hematopoietic cells (HSCs), b) culturing the CD34⁺ cells of a) with a combination of SCF, Flt3L, TPO, IL6, StemRegenin 1 (SRI) so as to expand the CD34⁺ cells, c) culturing the cells of b) with GM-CS and IL4 so as to further expand the cells and generate DCs in vitro, d) contacting the cells of c) with exopolysaccharide (EPS), and e) optionally cryopreserving the cells of d), wherein the EPS is optionally removed from the cells of d) prior to cryopreserving. In one embodiment, the 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 of e) prior to cryopreserving or administering.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIGS. 1A-1D. EPS-DCs derived from CD34⁺ cord blood cells inhibit proliferation of alloreactive CD4 T cells in MLR. Diagrams showing DC derivation from CD34⁺ cord blood cells (A) and human monocytes (B). (C) Representative flow cytometry of CD4 T cell proliferation in MLR cultures with CD34-DCs: not treated (NT; left panel), DEPS-treated (negative control; middle 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 with CD34-DCs and EPS present throughout the culture period (D), with moDCs and EPS present throughout the culture period (E), and with CD34-DCs pretreated with EPS for 16 hours and washed out prior to addition to MLR cultures (F). Data are from >3 independent experiments. **P < .01; ***P < .001; ****P < .0001, unpaired t test.

FIGS. 2A-2D. Injection of EPS-DCs derived from CD34+ cord blood cells extends survival of humanized GVHD mice. (A) Schematic of GVHD induction and treatment. (B) Kaplan-Meier survival analysis of GVHD mice that received PBMCs alone (PBMCs; gray line), PBMCs plus PBS-treated CD34-DCs (NT DCs; dashed line), or PBMCs plus EPS-treated DCs (EPS DCs; black line). The log-rank test was performed between EPS-DCs and NT-DCs (EPS-DCs and PBMCs) (**P < .01) and NT-DCs and PBMCs (P = not significant). Data are from >3 independent experiments. (C) Body weight changes after the GvHD induction in mice that received NT-DCs (gray line) and EPS-DCs (black line). (D) GvHD clinical scores. *P < .05; **P < .01; ***P < .001, unpaired t test. Data in C and D are from a single experiment with male and female mice (total n = 15) in each group. In these experiments, we used CD34+ cells from 3 donors, all of which were HLA-A2-negative.

FIGS. 3A-3C. DC composition, EPS uptake, and phenotypic changes induced by EPS. (A) Percentage of HLA-DR⁺CDllc⁺ DCs in the initial culture of expanded CD34⁺ cells (left panel) and after 5 and 12 days of culture with hGM-CSF and hIL-4 (middle panels), and percentages of cDCl (CD141⁺) and cDC2 (CDlc) in the 12-day DC cultures (right panel). (B) EPS uptake by DCs, non-DCs, and cDCl, cDC2, and double-negative (non-cDCl/cDC2) cells. (C) Expression of inhibitory PD-L1 and PL-L2 by untreated (NT) and EPS-treated DCs. Data compiled from 3 independent experiments. *P < .05; ***P < .001, unpaired t test. All experiments were repeated >3 times; shown are representative flow plots from >3 experiments.

FIG. 4. An example of a protocol for generation of EPS-DCs and using them as a treatment for GVHD patients.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is the combination of a bacterial probiotic molecule, exopolysaccharide (EPS), with dendritic cells for decreasing graft versus host disease (GvHD) which occurs, for example, following allogeneic bone marrow transplantation. It was previously demonstrated that intraperitoneal injection of EPS into donor and recipient mice prolongs the life of mice that received an allogeneic transplant (Kalinina et al. J Immunol 2021, 206:2101-2108). To test if EPS could prevent GvHD in humans, it was first determined whether EPS could inhibit a mixed lymphocyte reaction (MLR), which occurs when peripheral blood cells (PBMC) from two allogeneic donors are cultured together in the presence of antigen presenting dendritic cells (DCs). In the MLR, DCs present alloantigens to the T cells, thereby activating them; in vivo, such activation can lead to a GvHD. EPS was added to human MLR cultures, and it was shown that EPS partially inhibited the proliferation of alloreactive T cells (Figure 1), demonstrating that EPS can inhibit alloreactive T cells in vitro and thus likely inhibit GvHD.

To test if EPS can also ameliorate GvHD in humans, humanized NSG-HLA-A2 mice were used and GvHD was induced by i.v. injection of A2^neg human PBMCs. For this model, dendritic cell (DC) transfer was used instead of EPS injection. DCs derived from CD34⁺ cord blood cells (CD34-DC) pre-treated with EPS or PBS were used with PMBCs and it was found that injection of EPS-treated DCs prolonged the survival of GvHD mice. This DC cell therapy is a novel treatment and, as we shown here, large numbers of EPS-DCs can be generated and used as, for example, an “off the shelf’ treatment.

For the purposes of clarity and a concise description, features can be described herein as part of the same or separate embodiments; however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

Definitions

The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 14th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

References in the specification to "one embodiment," "an embodiment," etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only, " and the like, in connection with any element described herein, and/or the recitation of claim elements or use of "negative" limitations.

The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is di-substituted.

As used herein, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating a listing of items, “and/or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (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.”

As used herein, the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof, are intended to be inclusive similar to the term “comprising.”

The term "about" can refer to a variation of ± 5%, + 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment. The term about can also modify the endpoints of a recited range as discuss above in this paragraph.

As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more," and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group.

Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

As used herein, the term “comparing” refers to making an assessment of how the proportion, level or cellular localization of one or more biomarkers in a sample from a patient relates to the proportion, level or cellular localization of the corresponding one or more biomarkers in a standard or control sample. For example, “comparing” may refer to assessing whether the proportion, level, or cellular localization of one or more biomarkers in a sample from a patient is the same as, more or 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 refer to assessing whether the proportion, level, or cellular localization of one or more biomarkers in a sample from a patient is the same as, more or less than, different from or otherwise corresponds (or not) to the proportion, level, or cellular localization of predefined biomarker levels that correspond to, for example, a patient.

As used herein, the terms “indicates” or “correlates” (or “indicating” or “correlating,” or “indication” or “correlation,” depending on the context) in reference to a parameter, e.g., a modulated proportion, level, or cellular localization in a sample from a patient, may mean that the patient has or at risk of having GvHD.

As used herein, the term “subject” or refers to any animal (e.g., mammals, birds, reptiles, amphibians, fish), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” may be used interchangeably herein in reference to a subject.

Various methodologies of the instant invention include a step that involves comparing a value, level, feature, characteristic, property, etc. to a “suitable control,” referred to interchangeably herein as an “appropriate control” or a “control sample.” A “suitable control,” “appropriate control” or a “control sample” is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc., determined in a cell, organ, or patient, e.g., a control or normal cell, organ, or patient, exhibiting, for example, normal traits. For example, the biomarkers of the present invention may be assayed in a sample from an unaffected individual (UI) or a normal control individual (NC) (both terms are used interchangeably herein) or other affected individual. In another embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined prior to performing a therapy on a patient. In yet another embodiment, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to, during, or after administering a therapy into a cell, organ, or patient. In a further embodiment, a “suitable control” or “appropriate control” is a predefined value, level, feature, characteristic, property, etc.

As used herein, the term “administering” refers to providing a therapeutically effective amount of a chemical or biological compound/cell (e.g., EPS treated DCs) or pharmaceutical composition to a subject. The chemical or biological compound/cell of the present invention can be administered alone, but may be administered with other compounds, excipients, fillers, binders, carriers or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice. Administration may be by way of carriers or vehicles, such as injectable solutions, 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; and the like.

The chemical, biological compound, pharmaceutical composition or cells of the present invention may also be included, or packaged, with other non-toxic compounds, such as pharmaceutically acceptable carriers, excipients, binders and fillers including, but not limited to, glucose, lactose, gum acacia, 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, dextrans, dextrins, and the like. Moreover, the packaging material may be biologically inert or lack bioactivity, such as plastic polymers, silicone, etc. and may be processed internally by the subject without affecting the effectiveness of the agent packaged and/or delivered therewith.

The term “effective amount,” as applied to the compound(s), biologies and pharmaceutical compositions described herein, means the quantity necessary to render the desired therapeutic result. For example, an effective amount is a level effective to treat, cure, or alleviate the symptoms of a disorder for which the therapeutic compound, biologic or composition is being administered. Amounts effective for the particular therapeutic goal sought will depend upon a variety of factors including the disorder being treated and its severity and/or stage of development/progression; the bioavailability, and activity of the specific compound, biologic or pharmaceutical composition used; the route or method of administration and introduction site on the subject; the rate of clearance of the specific compound or biologic and other pharmacokinetic properties; the duration of treatment; inoculation regimen; drugs used in combination or coincident with the specific compound, biologic or composition; the age, body weight, sex, diet, physiology and general health of the subject being treated; and like factors well known to one of skill in the relevant scientific art. Some variation in dosage can occur depending upon the condition of the subject being treated, and the physician or other individual administering treatment will, in any event, determine the appropriate dose for an individual patient.

As used herein, “disorder” refers to a disorder, disease or condition, or other departure from healthy or normal biological activity, and the terms can be used interchangeably. The terms would refer to any condition that impairs normal function.

As used herein, “treatment” or “treating” refers to arresting or inhibiting, or attempting to arrest or inhibit, the development or progression of a disorder and/or causing, or attempting to cause, the reduction, suppression, regression, or remission of a disorder and/or a symptom thereof. As would be understood by those skilled in the art, various clinical and scientific methodologies and assays may be used to assess the development 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 a disorder or its symptoms. Additionally, treatment can be applied to a subject or to a cell culture.

Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Exopolysaccharides (EPS)

Exopolysaccharides are high-molecular-weight polymers that are composed of sugar residues and are secreted by a microorganism into the surrounding environment. Exopolysaccharide can be isolated from bacterial cultures by the method described below. Microorganisms synthesize a wide spectrum 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 Owing to the wide diversity in composition, exopolysaccharides have found multifarious applications in various food and pharmaceutical industries.

The exopolysaccharides provided herein are bacterial (e.g., Bacillus subtilis) exopolysaccharide. In one embodiment, the bacterial strain used for sourcing cxopolysaccharidcs is a probiotic strain. In another embodiment, the bacterial exopolysaccharide is from Bacillus subtilis, Bacteroides fragilis, Bifidobacterium breve, or a combination thereof.

In one embodiment, exopolysaccharides were isolated from B. subtilis DS991 (sinRtasA mutant), a strain that produces and secretes large amounts of EPS; material from this strain is designated EPS+. EPS can be isolated by methods available to an artworker, such as those described previously (Guttenplan, S. B., et al. 2010. PLoS Genet. 6: e!001243). Briefly, stationary phase supernatants or supernatants of PBS-resuspended bacteria grown on agar plates were mixed with 3-4 X volume of 100% EtOH at 4°C for > 4 hours to precipitate the EPS. The precipitant was pelleted (15,000 x 3 g, 4°C, 20 min), washed in PBS, and resupended in 0. 1 M Tris. Samples were digested with DNase (67 mg/ml) and RNase (330 pg/ml) at 37°C; after 1 h, proteinase K (40 mg/ml) was added, and samples were incubated at 55°C for 1 h. EPS was EtOH precipitated, resuspended in 0.1 M Tris (pH 8), and 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 sulfuric acid assay using serial dilutions of fructose as standard (17). Sample purity was assessed by OD26o/28o to ensure no protein or nucleic acid contamination, and by ELISA for absence of lipid A.

Dendritic Cells

Dendritic cells (DCs) are antigen-presenting cells (also known as accessory cells) of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and the adaptive immune systems.

Dendritic cells are present in those tissues that are in contact with the external environment, such as the skin (where there is a specialized dendritic cell type called the Langerhans cell) and 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 shape the adaptive immune response. At certain development stages they grow branched projections, the dendrites.

The dendritic cells for use in the method provides provided herein can be dendritic cells isolated from the patient, an immune matched donor or any donor. They can also be derived from stem cells, such as human stem cells, including, but not limited to, embryonic stem cells, iPSCs or stem cells isolated from umbilical cord blood (including HSC including CD34⁺ cells, such as those one can purchase them from Lonza or StemCell Technology or isolated by methods available to an art worker, such as isolating from umbilical cord blood (UCB) by isolating mononuclear cells from UCB and then labeling them with biotinylated anti-CD34 antibodies, followed by binding with streptavidin coated beads and isolating them out with a magnet).

In order to expand CD34⁺ to increase their numbers, the cells can be grown in serum free medium with about lOng/ml to about 100 ng/ml, including about 50 ng/ml of each of SCF, Flt3L, TPO, IL6 and/or 0.01 pM to about 1.5 pM, including about 0.75 pM, of StemRegenin 1 (SRI), an aryl hydrocarbon receptor antagonist, for about 2 weeks.

After expansion, the cells can be optionally cryopreserved (e.g., in cryopreservation media available to an art worker, such as Cryostor media (StemCell Technology) at 10⁶/ml), and then after thawing treated with GM-CSF and IL4 to further expand and generate DCs in vitro. These DCs (including as many cells as needed, such as about 1 million cells/mouse or about 10⁷-10^s cells per human undergoing HSCT) can then be contacted with EPS (such as about 5 pg/ml to about 100 pg/ml, including about 50-60 pg/ml for about 5xl0⁶ cells/ml; such as about 0.5-3.0 mg of EPS per patient; for about 10 to about 48 hours, including about 16 to about 24 hours) prior to administration to a subject in need thereof. In one embodiment, the EPS is removed prior to administering the DCs to a subject.

Thus, one can 1) expand and cry opreserve CD34⁺ cells from umbilical cord blood; 2) further expand CD34⁺ cells and differentiate them into dendritic cells using GM-CSF (lOOng/pL) and IL4 (50ng/pL); 3) treat the DCs with EPS (such as about 30 pg/mL to about 100 pg/mL, including about 60 pg/mL) and optionally freeze again prior to administration to a subject (in one embodiment administration includes 10⁷- 10^s cells per one patient undergoing HSCT (administration can be one time or can be repeated as needed)).

Graft versus host disease (GvHD)

Graft versus host disease (GvHD) is a complication that can occur after an allogeneic transplant. During an allogeneic transplant, a healthcare provider transplants hematopoietic stem cells (immature blood cells) from a donor into a host. The donated stem cells eventually become mature blood cells.

A host may need donor stem cells if the host has an issue with their blood cells, as with cancers like leukemia or lymphoma, or other bone marrow failure diseases like aplastic anemia, and also with stem cell transplantation for hemoglobinopathies such as Sickle Cell Disease and beta-thalassemia.

In GvHD, the donated stem cells (graft) view 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.”

What are the types of GvHD?

There are two main types of GvHD. Previously, healthcare providers classified GvHD based on when symptoms started. More recently, providers diagnose the specific type of GvHD based on symptoms and clinical signs (e.g., results of tests or imaging) in addition to the timing of symptom onset.

• Acute graft versus host disease (aGvHD): occurs shortly after transplant, usually within the first 100 days. But symptoms of aGvHD may also start later. aGvHD most often affects skin, gastrointestinal (GI) tract or liver.

• Chronic graft versus host disease (cGvHD): can appear any time after an allogenic transplant, but most cases start within two years. Chronic GvHD can affect skin, mouth, liver, lungs, GI tract, muscles, joints or genitals.

As an allogeneic transplant recipient, one might experience either form of GvHD, both forms or neither.

What are the symptoms of GvHD?

Symptoms of GvHD range from mild to moderate to severe (potentially fatal).

Acute GvHD

Symptoms of aGvHD most often affect skin, gastrointestinal (GI) tract or liver.

The most common symptom of skin aGvHD is a rash or reddened areas on skin (similar to a sunburn). The rash may feel painful or itchy. These rashes usually start on the neck, shoulders, ears and the palms of hands and soles of feet. The rash can spread to other parts of the body.

The most common symptoms of GI aGvHD are nausea, vomiting and diarrhea. Symptoms can be mild or severe. Symptoms can include: rash and/or itching, diarrhea, nausea and vomiting, abdominal cramping, and/or jaundice (yellow discoloration of skin and/or eyes).

Chronic GvHD cGvHD most commonly affects skin, liver, GI tract and lungs, but it can affect any body part. Symptoms can include: rash and/or itching, skin tightness and swelling, hair on head and body, dry mouth, mouth sores, gum disease, dry or gritty feeling in eyes, vision changes, diarrhea, nausea and vomiting, yellow discoloration of skin and/or eyes (jaundice), shortness of breath (dyspnea), dry, persistent cough, fatigue, muscle weakness, cramping or pain, decreased range of motion in joints, vaginal dryness, itchiness or pain with intercourse and/or itchy penis or scrotum or pain with intercourse.

How is graft versus host disease diagnosed?

A healthcare provider can diagnose GvHD during a physical exam by observing certain symptoms and evaluating lab tests and biopsy results.

What is the treatment for GvHD?

Currently, one is likely to receive preventive (prophylactic) medicines to suppress the host immune system after transplant. These immunosuppressive medicines decrease donor cells’ ability to start an immune response (attack) against host tissues.

If these medicines don’t prevent the development of GvHD, there are other treatments commercially available.

Treating acute GvHD

Healthcare providers successfully treat many people with aGvHD by increasing immunosuppressive medicines in the form of oral (given by mouth), intravenous (given through a vein) or topical (applied to skin) corticosteroids. If steroids don’t help, a provider may prescribe Ruxolitinib (Jakafi®). One may also be eligible for clinical trials.

Treating chronic GvHD

Providers usually prescribe long-term immunosuppressive medicines to treat cGvHD. If these medicines don’t improve the condition, these may also be prescribed: Ruxolitinib, (Jakafi®), Belumosudil (Rezurock™), Ibrutinib (Imbruvica®) and/or Photopheresis.

Immunosuppressive medicines weaken the immune system, putting one at risk for fungal, bacterial and viral infections. A healthcare provider can prescribe several medicines to help prevent potentially life-threatening infections from occurring.

Treating or Preventing GvHD with EPS-DC

Disclosed herein are methods of treating or preventing GvHD comprising administering an effective amount of EPS exposed DCs to said subject.

The appropriate dosage of EPS-DCs will depend, for example, on the condition to be treated, the severity and course of the condition, whether the EPS-DCs are administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to EPS-DCs, and the discretion of the attending physician. EPS-DCs are suitably administered to the patient at one time or over a series of treatments and may be administered to the patient at any time as necessary for treatment or prevention of GvHD. EPS-DCs may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating GvHD.

Routes of administration

EPS-DCs can be administered systemically or locally. In various embodiments, the route of administration can be auricular (otic), buccal, conjunctival, cutaneous, dental, electroosmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intraarterial, intra- articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronal, dental, intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, not applicable, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanicureteral, urethral, vaginal, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

Formulations and Dosage Forms

EPS-DCs can be provided in a pharmaceutical composition (e.g., for the first administration the cells can be injected along with the HSCT cells in PBS; for additional administrations, the cells can be injected i.v. in, for example, PBS). The pharmaceutical composition can comprise pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). The pharmaceutical compositions can include other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and/or buffers. Methods well known in the art for making formulations are to be found in, for example, Remington: The Science and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A R., 2000, Lippencott Williams & Wilkins. Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors. Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.

The number of EPS-DCs in the formulations can vary depending upon a number of issues, including the dosage to be administered, and the route of administration.

The EPS-DCs and HSCs may be administered simultaneously or subsequently. The EPS- DCs and HSCs may be administered in a single dose or multiple doses, singly or in combination. The EPS-DCs and HSCs may be administered by the same mode of administration or by different modes of administration, and may be administered once, twice or multiple times, one or more in combination or individually. Thus, EPS-DCs may be administered in an initial dose followed by a subsequent dose or doses, particularly depending on the response, EXAMPLES

Example 1

Introduction

Hematologic malignancies are often treated with allogeneic hematopoietic stem cell transplantation (HSCT) with curative intent. However, HSCT is complicated by acute graft- vs-host disease (aGvHD), a severe, potentially lethal complication initiated when donor alloreactive T cells attack host cells and organs (1,2). Furthermore, aGvHD is risk factor for developing chronic (cGvHD) (3), thus preventing aGvHD dramatically decreases the likelihood of developing cGVHD. Although prophylactic regimens for aGvHD are standard pre-transplantation therapy, up to 50% of these patients treated with calcineurin-based regimens develop grade II or higher aGVHD (grade 0-IV) (1) and require additional immunosuppressive intervention (4). GvHD is caused by activation of donor allogeneic T cells in response to molecules on host cells. This T cell activation results in the recognition and destruction of the recipient tissues and organs by the donor immune effector cells, primarily affecting the skin, liver, and gastrointestinal tract (5,6). T cell activation also causes inflammation and a cytokine storm that further exacerbates disease (7). Two standard regimens used clinically to prevent this complication include a calcineurin inhibitor with short-course methotrexate and posttransplantation cyclophosphamide (PTCy) with a calcineurin inhibitor and mycophenolate (1,6,8). Although prophylaxis with calcineurin inhibitors combined with methotrexate has been the customary approach for decades (1,6), high-dose PTCy given on days +3 and +4 post-transplantation has been shown to decrease the rates of both aGVHD and cGVHD in a phase 3 comparison trial versus tacrolimus and methotrexate (8). However, in this trial, there was no difference in disease-free survival or overall survival for the PTCy group, as higher rates of organ failure, primary graft failure, and hemorrhage likely directly related to cyclophosphamide toxicity off-set the benefits of lower mortality from aGvHD and cGvHD. Thus, a new method of aGvHD amelioration without chemotherapy toxicity, such as the novel immunosuppressive method described here, can improve not only aGvHD rates, but also overall survival.

Cellular therapy is an attractive treatment for aGvHD (4). However, cellular therapy in animal models using CD34⁺ monocytes (9), regulatory T cells (Tregs) (10), mesenchymal stem cells (11), DCs (12-14), or myeloid-derived suppressor cells (15) has had limited success thus far (16). DCs treated with vitamin D can delay GvHD in humanized NSG mice, indicating that DCs can protect from GvHD (12).

It was discovered that a probiotic exopolysaccharide (EPS) from Bacillus subtilis, a harmless probiotic soil bacterium, has potent cellular immunosuppressive activities in a variety of disorders, including allergies (17), colitis (18-20], sepsis (21,22), and GvHD (23). EPS induces tolerogenic DCs and anti-inflammatory macrophages, which inhibit proliferation of activated T cells (17-20,23). In mice, transfer of in vitro EPS-treated bone marrow-derived DCs ameliorates allergic eosinophilia (17), and peritoneal macrophages isolated from EPS-treated mice and transferred to recipient mice protect from disease caused by an enteric pathogen (17,20). In a mouse GvHD model, injection of EPS prior to GvHD induction increased 80-day survival from 10% (in control, PBS-treated mice) to 70% (in EPS-treated mice) after transplantation of complete allogeneic major histocompatibility complex (MHC)-mismatched cells (23). Here it shown that human EPS-treated DCs affect human alloreactive T cells and can ameliorate GvHD in humanized mice. A protocol for generation of large quantities of EPS- DCs was also developed and suggests usage of them as “off the shelf’ treatment for GVHD patients.

Materials and Methods

Mice and Reagents

All mice were housed in pathogen-free conditions in the animal facility at Loyola University Chicago.NSG-HLA-A2 (strain009617) mice were purchased from The Jackson Laboratory or bred in-house. Age-and sex-matched 8-to 12-week-old mice were used for all experiments and all experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Loyola University Chicago. Base media and supplements were purchased from Life Technologies; StemSpan SFEM II media, human AB serum, and Stem regeninl (SRI), from STEMCELL Technologies); bacterial media, from BD; all antibodies (Ab) from BioLegend or eBioscience; and all cytokines from PeproTech (Thermo Fisher Scientific).

Preparation of B.siibtilis-Derived EPS

EPS was prepared from B.subtilis DS991 (sinR tas ), which over produces and secretes EPS. AEPS is carbohydrate prepared from B.subtilis DS5187 (sinRtasAepsH mutant), which does not produce EPS (19,24). The bacteria were plated at ODeoo .6 to .7on Luria-Bertani agar plates (150mm) for 4 hours, bacterial lawns were harvested, and supernatants were treated with DNase (67mg/mL), RNase (330mg/mL), and protease K (40mg/mL). Carbohydrates were treated with 75% EtOH, and after resuspension, EPS was purified by gel filtration on Sephacryl S-500. Carbohydrate-positive fractions identified by a modified phenol sulfuric acid assay (25,26) were pooled, dialyzed extensively, and air-dried. EPS purity was assessed by the lack of detectable protein or nucleic acid (OD260/280) and the absence of cytotoxicity in vitro. All EPS preparations were sterile filtered and undetectable levels of endotoxin (lipopolysaccharide) was confirmed by ELISA. Each preparation was assessed for its capacity to induce peritoneal M2 macrophages, as described previously (20), or to inhibit a mixed lymphocyte reaction (MLR), as described below.

CD34 Cell Expansion and Derivation of DCs

Umbilical cord blood CD34+ cells (LonzaGroup) were expanded for 7 days in expansion medium (StemSpan SFEMII, 0.75 uM Stemregenerin, Ixpenicillin-streptomycin, 50 ng/mL of Flt3L, SCF, TPO, and IL-6). After 1 week, the cells (10⁶/mL) in Cryostor media (StemCell Technology). 1-week-expanded CD34⁺ cells were thawed and expanded for another week in expansion medium. The 2-week-expanded CD34⁺ cells (100,000/mL) were plated in DC-derivation medium consisting of a 1: 1:1 mixture of aMEM:IMDM:RPMI with 10% FBS, 2% human AB serum with lOOng/mL human GM-CSF (hGM-CSF), and 50 ng/mL hIL-4. One- half of the medium was exchanged every 2 days. On day 12, the cells were pretreated with EPS or PBS overnight before using them in aGvHD experiments or in MLR testing. Monocyte- derived DCs were generated by isolating CD14⁺ cells from human peripheral blood mononuclear cells (hPBMCs) using the MojoSort Human CD14⁺ Monocytes Isolation Kit (BioLegend) according to the manufacturer’s instructions, then cultured in RPMI with 10% FBS with 50ng/m LhGM-CSF and 25ng/mL hIL-4 for 10 days. One-half of the medium was exchanged every other day.

GvHD and MLR hPBMCs isolated from heathy HLA-A2 negative donors were purchased from STEMCELL Technologies. To induce GvHD in 8- to 12- week-old NSG-HLA-A2 mice, they were conditioned with 2.5 Gy from an X-ray source (RS 2000Biological Research Irradiator) and 4 hours later i.v. injected them with 4 x 10⁶ PBMCs alone or a mixture of 4 x 10⁶ PBMCs with 10⁶ cord blood CD34⁺ hematopoietic stem cells (CD34-DCs) from a different donor. The CD34-DCs were pretreated with EPS (60ug/mL) or PBS for 16 hours. For MLR, PBMCs were labelled with 5uM Cell Trace Violet (CTV) (Thermo Fisher Scientific) according to the manufacturer’s instructions and cultured either alone (2.5x I (Feel Is, negative control) or with CD34-derived DCs (7.5xl0⁴ in 96-well U-bottom plates) with EPS (60ug/mL) or AEPS (60ug/mL). In some experiments, third-party irradiated PBMCs were added to the cultures as initiators. Four days later, T cell proliferation and activation were measured by flow cytometry. Two different CD34⁺ cell donors were evaluated long with multiple (>8) hPBMC donors.

Flow Cytometry

For flow cytometry, cells were treated with anti-CD 16/32 Fc block, then stained with Abs specific for CD4, CD8, CD25, andCD44. Cells were analyzed using a FACSCanto II or LSRFortessa flow cytometer (BD Biosciences). Proliferation was measured based on the dilution of the CTV dye, as determined by flow cytometry, and the data was analyzed using FlowJo software (BD Biosciences).

Statistical Analysis

The unpaired 2-tailed Student t test was used for statistical analyses unless noted otherwise, with P < .05 considered to indicate statistical significance. The survival curves were modeled using Kaplan-Meier methods and a log-rank test was applied to make comparisons between groups. All analyses were performed using Prism 5.0(GraphPad Software).

Results

EPS effect on human dendritic cells (DCs)

EPS inhibits T cell proliferation in murine MLR cultures by converting bone marrow derived DCs into inhibitory DCs (23). To evaluate whether EPS also affects human cells, its effects in MLR on DCs derived from CD34-DCs was investigated (FigurelA) or from CD14⁺ peripheral blood monocytes (moDCs; FigurelB). These DCs were used as initiators in MLR cultures, along with CTV-labeled PBMCs from allogeneic donors. To determine whether EPS converts DCs into inhibitory cells, 60ug/mL of EPS or the negative control AEPS was added to the cultures, and T cell activation and proliferation were measured 4 days later by flow cytometry. It was found that CD34-DCs inhibited alloreactive T cell proliferation by > 50% compared to the same cultures without EPS added or with AEPS added as a negative control (Figurel C,D). Cultures with CD14⁺-derived moDCs and EPS did not inhibit T cell proliferation (FigurelE).

To ask whether the observed inhibitory effects of CD34-DCs were due to DC only and if EPS-treated DCs retain their inhibitory properties after EPS is removed from cultures, CD34- DCs were pretreated with EPS overnight and added to MLR cultures after washing away EPS. It was determined that the DCs still inhibited proliferation of alloreactive T cells in MLR cultures (Figure IF) to a similar extent as that observed when EPS was present throughout the culture period (Figure ID). These results indicate that EPS converts human CD34-derived DCs into inhibitory DCs, and that these cells significantly inhibit proliferation of alloreactive T cells in MLR cultures. It was also shown that human CD34-DCs retain their inhibitory properties after EPS is removed, leading to the hypothesis that EPS-treated CD34-DCs (EPS-DCs) can be used as a cell-based therapy to ameliorate human GvHD.

EPS-treated CD34-DCs prolong survival of NSG-HLA-A2 mice experiencing GvHD

To investigate whether EPS-DCs can ameliorate GvHD, a humanized mouse model of induced GvHD was used, where human peripheral blood mononuclear cells (hPBMCs) were transferred to lightly irradiated NSG-HLA-A2 mice (27). To determine whether EPS-DCs can reduce GvHD-related mortality, GvHD was induced with 4xl0⁶ hPBMCs and separate cohorts of mice were simultaneously injected with CD34-DCs pretreated for 16 hours with EPS, or with PBS as a negative control, at a 4: 1 hPBMC:DC ratio (Figure2A). All control mice that received PBS-treated CD34-DCs died between 10 and 36 days after disease induction (Figure2B). Mice that received EPS-DCs had significantly improved survival. In the control cohort that received only hPBMCs (4xl0⁶), the median survival was 23 days, which was not significantly different from that in the control group that received PBS-treated CD34-DCs and hPBMCs (21 days). The similar survival dynamic (Figure2B), as well as similar median days of lethal induction (21 and 23 days) in mice that received hPBMCs with untreated DCs (NT- DCs) and hPBMCs alone, respectively, indicate that adding HLA-mis -matched DCs derived from cord blood CD34⁺ cells does not exacerbate GvHD in humanized mice.

In a separate GvHD experiment, 2xl0⁶ hPBMCs and .5xl0⁶untreated or EPS-treated CD34-DCs were transferred, and mice were analyzed for weight loss and clinical score. It was found that mice that received EPS-DCs lost significantly less weight than those receiving untreated DCs (Fig. 2C). Similarly, mice with EPS-DCs showed essentially no evidence of disease in the first 30 days as measured by clinical score, whereas mice receiving untreated DCs had evidence of disease based on clinical score (Figure2D). After 30 days, however, the clinical score of the EPS-DC-treated mice increased, suggesting that in this model, another dose of EPS-DCs could provide further protection from disease. It was concluded that EPS- DCs significantly increased survival and decreased GvHD symptoms.

Generating EPS-DCs from Human Cord Blood CD34⁺ stem cells

To meet clinical applications for using EPS-DCs to prevent GvHD, large numbers of DCs need to be easily generated. It is well established that CD34⁺ hematopoietic cells (HSCs) can give rise to DCs in vitro, however, the number of CD34⁺ HSCs in circulation is low (<0.1% of PBMC). Although this number is higher in umbilical cord blood, the volumes of clinically obtain cord blood are small. Human cord blood CD34⁺ cells are commercially available but are expensive ($1500-2000/ 10⁶ cells; StemCell Technologies). A protocol was used to expand the cord blood CD34⁺ cells in serum-free medium using a titrated cocktail of SCF, Flt3L, TPO, IL- 6, and StemRegenin 1 (an aryl hydrocarbon receptor antagonist). Using CD34⁺ cells from 4 different donors, it was found that after 3 weeks in culture with the cytokines, the number of CD34⁺ cells expanded by more than 2 x 10⁴-fold (Table 1). These data show that one can produce >2 x 10⁹ CD34⁺ cells from 10⁵ CD34⁺ cells. Table 1

Expansion of 034* Celh

Characterization of CD34-Derived DCs and Their EPS Uptake

By flow cytometry, essentially none of the CD34⁺ cells had HLA-DR or CDl lc DC markers after the 3-week expansion, but after differentiating these cells to DCs, it was found that by day 5 and day 12, 50% and 72%, respectively, were HLA-DR⁺CD1 lc⁺(Figure3A). The 12-day DCs essentially were all conventional DCs (eDCs), with ~ 20% having cDCl markers and >70% having cDC2 markers (Figure2A). Using fluorescently labeled EPS, it was tested which cells take up EPS and found that essentially all DCs takeup EPS, but cDC2 cells takeup more compared to cDCl cells (Figure3B). The identity of the contaminating -20% HLA-DR" CD1 Ic cells that were also CD34 , CD 14 , and CD3, is not known, but they did not takeup EPS and likely did not contribute to protection by EPS. EPS-DCs were also analyzed for upregulated expression of the inhibitory molecules PD-LlandPD-L2 that are upregulated on mouse DCs after treatment with EPS and found increased expression of both PD-L1 andPL-L2 in EPS-DCs (Figure3C), as expected.

A protocol for generating and using EPS-DCs to prevent and/or treat aGvHD in humans

One embodiment of the protocol is illustrated in Figure 4, showing 1 ) Expanding and cryopreserving CD34⁺ cells from umbilical cord blood; and 2) further expanding CD34⁺ cells and differentiating them into dendritic cells using GM-CSF ( I OOng/ L) and IL4 (50ng/pL), treated with EPS (60 g/mLj and freezing or using right away. Using a xenograft (x) GvHD model, donor hPBMCs are injected into the NSG-A2 mouse; timing and dosing of EPS-DCs are optimized to give maximum protection (for example, hPBMC 4 x 10⁶, EPS-DC 10⁶, mixed and injected i.v. once on day 0 - initiation of GVHD). Cell-based GvHD prophylaxis is advantageous because EPS-DCs are non-toxic, can be easily generated and cryopreserved (e.g., in Cryostor media, Stem Cell Technology at about 3 x 10⁶/ml), and do not require HLA- matching to donor or recipient. The approach guarantees one can generate therapeutic doses of EPS-DCs for infusion. DISCUSSION

HSCT is often the sole curative option to treat life-threatening hematologic diseases, including leukemias, hemoglobinopathies, and some forms of anemia, including sickle cell anemia. However, GvHD is a main cause of morbidity and mortality after HSCT, severely limiting its curative potential. The high incidence of GvHD, with >10% of patients dying from this complication (28) despite current prophylactic treatments, underscores the unmet need for new therapies to prevent and/or treat GVHD. It was discovered that EPS from a harmless probiotic soil bacterium, B. subtilis, induces tolerogenic DCs (EPS-DCs) that prevent activated T cells from proliferating in MLR cultures. A humanized NSG-HLA-A2 transgenic mouse model was used, and it was found that injecting EPS-DCs when transferring hPBMCs significantly increased survival, from a median of 22 days with NT-DCs to 31 days with EPS- DCs. Two of the 32 EPS-DC-treated mice survived at least 100 days. In addition, the EPS-DC- treated mice showed significantly less weight loss and clinical symptoms compared to the NT- DC-treated mice. The decrease in clinical score was most apparent in the first month after transplantation, suggesting that more than Idose of EPS-DCs may be needed to optimize treatment.

Cellular therapy is an attractive approach to treating GvHD (9,10,12-16,29,30). The use of mesenchymal stem cells has shown variable success in treating patients with steroid refractory GvHD (29,30); however, in a recent phase III trial, significant improvement was observed only in pediatric patients and not in adult patients (30). Tregs also have shown promise in treating GvHD in humans, but no large-scale clinical trial results are available to date. Studies in humans have been performed primarily in patients with established GvHD, and it seems likely that more success will come by preventing GvHD instead of treating it (31). DCs — specifically, tolerogenic DCs — have the can prevent GvHD by inhibiting alloreactive T cell activation and proliferation. 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 here are tolerogenic DCs that inhibit alloreactive T cells (Fig. 1 C,D,F) and prolong survival in a humanized GvHD model (Figure 2B). Unique DC subsets have been described, including conventional DCs (eDCs), moDCs, and plasmacytoid DCs, each with different phenotypes and functions. Tolerogenic EPS-DCs are derived from human cord blood CD34⁺ stem cells and are mostly eDCs, especially cDC2 (Figure3 B and (32)), whereas VitD-DCs and other tolerogenic DCs used in the clinic are moDCs derived from peripheral blood monocytes (33-36). EPS does not induce tolerogenic functions in moDCs (Figure 1C), underscoring the novel mechanisms that EPS uses to tolerize a specific subset of DCs.

Activated donor allogeneic T cells cause GvHD as they attack recipient organs. In a mouse GvHD model using biosensor mice, it is shown that injecting EPS inhibited alloreactive T cell proliferation in vivo (23). In the clinic, the current treatment of choice to prevent GvHD is PTCy. Its use was shown to reduce the incidence of severe aGVHD to <10% and to apparently reduce the incidence of cGVHD as well (37-40). Wacshmuth et al. (41) demonstrated that the protective effect of PTCy in an MHC-haploidentical HSCT model resulted from a dampening of alloreactive donor T cell proliferation, primarily through Treg enhancement. Here it is shown that human EPS-treated DCs that inhibit alloreactive T cell proliferation in vitro in MLR cultures have increased expression of inhibitory molecules PD-LlandPD-L2. These molecules may contribute to the mechanism by which EPS -DCs ameliorate GvHD in humanized mice. In this case, EPS -DCs could be interacting directly with donor T cells to inhibit their activation and proliferation, as occurs in MLR cultures. Moreover, EPS-DCs could decrease systemic inflammation in GvHD mice, leading to decreased donor T cell activation. Recent studies have demonstrated that extracellular factors, including the tissue microenvironment, which can be altered by host microbiota or microbiota-derived metabolites such as short-chain fatty acids, affect allogeneic immune responses (42,43). Provided herein, EPS-DCs can function by altering the tissue microenvironment and/or by making T cells tolerant to proinflammatory microenvironments. Thus, EPS-DCs may exert a similar effect as PTCy on alloreactive T cells, as they directly inhibit the proliferation of activated T cells, including alloreactive T cells (23). In contrast to PTCy, an alkylating agent that alters nucleic acid and can be toxic at higher concentrations, it does not appear that EPS-DCs are toxic. It is further expected that EPS-DC treatments would be short-term and that they can serve as a nontoxic alternative, or in addition, to PTCy to prevent or treat GvHD.

Using EPS-DCs to prevent GvHD in humans will require a standardized protocol for generating DCs from cord blood CD34⁺ cells (or other sources) and treating them with EPS, after which they can be cryopreserved until they are thawed and infused. Large numbers of EPS-DCs would be needed to meet the clinical application for preventing GvHD. CD34⁺ HSCs can give rise to DCs after culture with GM-CSF and IL-4, but only small numbers of CD34⁺ HSCs can be obtained from peripheral blood or umbilical cord blood. Provided herein is a protocol to expand CD34⁺ cells by >2 x 10³-fold in 2weeks. It is estimated that a therapeutic dose of 10⁷ to 10⁸ EPS-DCs would be necessary for humans undergoing HSCT and demonstrated that one could produce this number of DCs from <10⁵ CD34⁺ cells. After overnight treatment with EPS, the EPS -DCs can be cryopreserved and, on thawing, transferred to preconditioned human patients simultaneously with donor HSCs. Although in the present study, only 30% of the mice survived, it should be noted that these mice received only a single injection of EPS-DCs. Survival can significantly increase with additional doses of EPS-DCs following HSCT.

Herein it is described how to generate EPS -treated DCs and demonstrated that they ameliorate GvHD in a humanized model of the disease. Using EPS-DCs as a cell-based therapy for GvHD prophylaxis has a distinct advantage and is safe, because the cells are “normal” and not genetically manipulated. An advantage of using cord blood CD34⁺ HSCs is the ready availability of numerous samples in cord blood banks world-wide owing to the increase in available alternative donors for those without a m matched sibling or unrelated donor. These cord blood cells can be expanded by >10³-fold, and large stocks of EPS-DCs can be generated and cryopreserved. This approach allows the use of an off-the-shelf product, making this a potentially useful product to prevent lethal aGvHD in combination with standard and less toxic, but less effective, GvHD prophylactic regimens other than PTCy or together with PTCy, potentially allowing for PTCy administration at lower and less toxic doses.

There are several strategies for the clinical use of these EPS-DCs. One approach, as suggested by the strategy used to treat the mice herein, would be to develop an off-the-shelf EPS -DC product made from umbilical cord blood stem cells and infused in the same time frame as PTCy (i.e., shortly after the infusion of donor stem cells) to prevent aGVHD. Ex vivo expansion of UCB CD34⁺ stem cells using cytokines in the presence of nicotinamide has recently been approved by the US Food and Drug Administration for clinical transplantation based on a phase 3 trial showing a 130-fold expansion of CD34⁺ cells (median, 6.6xl0⁸CD34⁺ cells) with resulting superior engraftment compared to unexpanded UCB grafts (44). Thus, sufficient CD34⁺ cells can be obtained from these expanded stem cells to manufacture EPS- DCs for multiple patients, with widespread availability to all transplantation programs. However, there are potential risks of using third-party DCs for this purpose, as they express low levels of MHC, although such third-party tolerogenic DCs have been tested in a murine model of GvHD and appear to be effective in prolonging survival (5,6).

Another strategy would be to develop specific one-of-a kind donor-derived EPS-DCs fromCD34⁺ peripheral blood G-CSF-mobilized stem cell grafts. This technique could be used for patients undergoing allografts from matched related and unrelated donors but perhaps not for recipients of mismatched or haploidentical transplants for the reasons mentioned above.

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All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the definition of a term incorporated by reference conflicts with a term defined herein, this specification shall control.

Claims

WHAT IS CLAIMED IS:

1. A method to prevent, treat or inhibit graft vs host disease (GvHD) comprising administering exopolysaccharide (EPS) exposed dendritic cells (DCs) to a subject in need thereof so as to prevent, treat or inhibit GvHD in said subject.

2. A method to decrease at least one symptom of graft vs host disease (GvHD) comprising administering to a subject in need thereof exopolysaccharide (EPS) exposed dendritic cells (DCs) so as to decrease at least one symptom in said subject.

3. A method to prolong the survival of a subject with graft vs host disease (GvHD) comprising administering exopolysaccharide (EPS) exposed dendritic cells (DCs) to a subject in need thereof so as to prolong the survival of said subject.

4. A method to downregulate expression of activation markers (e.g., CD80 and/or CD86), upregulate expression inhibitory molecules (e.g., PD-L1 and/or PD-L2), inhibit activation of alloreactive T cells or a combination thereof comprising administering exopolysaccharide (EPS) exposed dendritic cells (DCs) to a subject in need thereof so as to downregulate expression of activation markers (e.g., CD80 and/or CD86), upregulate expression of inhibitory molecules (e.g., PD-L1 and/or PD-L2), inhibit activation of alloreactive T cells or a combination thereof.

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

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

7. The method of any one of claims 1 to 6, wherein the EPS exposed DCs are administered more than once (e.g., over a period of days, weeks, months, years).

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

9. The method of any one of claims 1 to 8, wherein at least one other therapeutic agent to treat GvHD is administered to said subject.

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

11. A method to generate tolerogenic dendritic cells (DCs) comprising: a) providing CD34⁺ hematopoietic cells (HSCs), b) culturing the CD34⁺ cells of a) with a combination of SCF, Flt3L, TPO, IL6, StemRegenin 1 (SRI) so as to expand the CD34⁺ cells, c) culturing the cells of b) with GM-CS and IL4 so as to further expand the cells and generate DCs in vitro, d) contacting the cells of c) with exopolysaccharide (EPS), and e) optionally cry opreserving the cells of d), wherein the EPS is optionally removed from the cells of d) prior to cryopreserving.

12. The method of claim 11, where in the HSCs are obtained from umbilical cord blood.

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

14. The method of any of claims 11 to 13, further comprising wherein the EPS is removed from the cells of e) prior to cryopreserving or administering.