Mesenchymal progenitor or stem cells with enhanced immunosuppressive activity

Abstract

To develop a ready-to-use ex vivo expanded allogeneic mesenchymal progenitor or stem cell (MLPSC) product having improved immunosuppressive activity.SOLUTION: The present disclosure relates to cell therapy products comprising mesenchymal lineage precursor or stem cells and potency assays for these products. The present disclosure also relates to a method for treating an immune or inflammatory disease, for treating or preventing graft versus host disease (GVHD) or one or more symptoms associated with GVHD by administering mesenchymal lineage precursor or stem cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] All documents cited or referenced herein, and all documents cited or referenced within the documents cited herein, as well as manufacturer's instructions, manuals, product specifications and product brochures for any products mentioned in this specification or in any document incorporated herein by reference, are hereby incorporated by reference in their entirety.

[0002] This disclosure claims priority to Australian Provisional Application AU2017901633, entitled "Potency assay for immunosuppression," filed May 4, 2017, Australian Provisional Application AU2017901636, entitled "Method for treating Graft versus Host Disease (GVDH)," filed May 4, 2017, and Australian Provisional Patent Application AU2018900551, entitled "Potency assay for immunosuppression II," filed February 21, 2018, the entire contents of which are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to cell therapy products comprising mesenchymal progenitor or stem cells, and titration methods for these products. The present disclosure also relates to methods for treating immune or inflammatory diseases, and for treating or preventing graft-versus-host disease (GVHD) or one or more symptoms associated with GVHD, by administering mesenchymal progenitor or stem cells. [Background technology]

[0004] Several cell therapy products for regenerative or immunotherapy applications have progressed to clinical evaluation and marketing approval. However, the commercialization of these cell therapy products has been hindered by their complexity and heterogeneity, making it difficult to identify meaningful biological activity and, therefore, define the quality of a consistent cell therapy product.

[0005] Physiochemical parameters (e.g., size, morphology, light scattering properties, tensile strength, cell number, confluence characterization, identification of phenotypic markers, secreted substances, genotype, gene expression profile) are routinely used to identify and quantify active substances, intermediates, impurities, and contaminants. However, physiochemical parameters cannot confirm that a product is biologically active and efficacious (i.e., induces the desired effect). In contrast, biological characterization takes into account the effect of a product on biological mechanisms in either in vitro or in vivo models in animals and ultimately in the clinic.

[0006] Drug legislation in the United States and Europe requires that active substances whose molecular structure cannot be fully defined be evaluated for their potency before being placed on the market. It is a legal requirement to evaluate the potency of each batch of approved cell therapy products.

[0007] The potency assay should demonstrate a meaningful biological activity (or activities) of the product. The potency assay need not reflect all biological effects of the product, but should demonstrate one or more meaningful biological effects. The precision, sensitivity, specificity, and reproducibility of the analytical method used in the potency assay should be established and be suitably robust.

[0008] There is a need to develop products with improved potency for the treatment of diseases in which immunosuppression is desired. It is also desirable to identify parameters critical to the efficacy of cell therapy products and control those parameters (e.g., via potency assays) so that products of consistent quality can be manufactured. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Australian provisional application AU2017901633 [Patent Document 2] Australian provisional application AU2017901636 [Patent Document 3] Australian provisional patent application AU2018900551 Summary of the Invention [Means for solving the problem]

[0010] The applicant has developed a pre-made ex vivo expanded allogeneic mesenchymal progenitor or stem cell (MLPSC) product with improved immunosuppressive activity.

[0011] The present disclosure provides a composition comprising a mesenchymal precursor or stem cell, or progeny thereof, wherein the mesenchymal precursor or stem cell has been cryopreserved and, after thawing, inhibits the proliferation of activated T cells in a sample of PBMCs by at least about 65%.

[0012] In one embodiment, inhibition is measured by co-culturing mesenchymal progenitor or stem cells with PBMCs at a ratio of 1 mesenchymal progenitor or stem cell to 5 PBMCs or less, for example, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1 mesenchymal progenitor or stem cell to 100 PBMCs or less.

[0013] In one embodiment, co-culturing mesenchymal progenitor or stem cells with PBMCs at a ratio of 1 mesenchymal progenitor or stem cell:5 PBMCs or less inhibits T cell proliferation by at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%.

[0014] In one embodiment, inhibition of proliferation of activated T cells is measured by inhibition of IL-2R 2Rα expression in activated T cells.

[0015] In one embodiment, the mesenchymal precursor or stem cell composition expresses TNFR1 in an amount of at least 110 pg / ml. For example, the mesenchymal precursor or stem cell composition expresses TNFR1 in an amount of at least 150 pg / ml, at least 200 pg / ml, at least 250 pg / ml, at least 300 pg / ml, at least 320 pg / ml, at least 330 pg / ml, at least 340 pg / ml, or at least 350 pg / ml.

[0016] In one embodiment, the mesenchymal progenitor or stem cell has a nucleotide sequence of at least 13 pg / 10 6 For example, mesenchymal progenitor or stem cells express TNFR1 at levels of at least 15 pg / 10 6 cells, at least 20 pg / 10 6 cells, at least 25 pg / 10 6 cells, at least 30 pg / 10 6 cells, at least 35 pg / 10 6 cells, at least 40 pg / 10 6 cells, at least 45 pg / 10 6 cells, or at least 50 pg / 10 6 Amount of cells express TNFR1.

[0017] In one embodiment, mesenchymal progenitor or stem cells are isolated by immunoselection and then expanded in culture.

[0018] In one embodiment, the mesenchymal progenitor or stem cells are expanded in culture. In one embodiment, the mesenchymal progenitor or stem cells are isolated, or isolated and enriched, and expanded in culture ex vivo or in vitro prior to cryopreservation. In another example, the mesenchymal progenitor or stem cells are isolated, or isolated and enriched, cryopreserved, thawed, and subsequently expanded in culture. In yet another example, the mesenchymal progenitor or stem cells are expanded in culture before and after cryopreservation.

[0019] In one embodiment, the mesenchymal precursor or stem cells comprise at least 5% of the cell population of the composition.

[0020] In one embodiment, the composition is cryopreserved using 42.5% Profreeze™ / 50% αMEM / 7.5% DMSO.

[0021] In one embodiment, the composition is cryopreserved using Plasmalyte-A, 25% HSA and DMSO.

[0022] Although the scope of the present invention is not limited to any hypothetical theory, the inventors believe that mesenchymal progenitor or stem cells that inhibit T cell proliferation by at least about 65% are particularly useful for inhibiting immune responses, and more particularly, that such mesenchymal progenitor or stem cells are effective in treating graft-versus-host disease; solid organ transplant rejection, such as heart transplant rejection, liver transplant rejection, pancreas transplant rejection, intestinal transplant rejection, and kidney transplant rejection; and autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, type 1 diabetes mellitus, and the like. and the like.

[0023] The present inventors have also found that mesenchymal progenitor or stem cells are also useful in the treatment of inflammatory diseases, particularly T cell mediated inflammatory diseases.

[0024] The present invention also relates to a method for producing a method for manufacturing a semiconductor device, comprising the steps of: culturing a population of mesenchymal progenitor or stem cells in a medium containing recombinant trypsin; Cultivating cells in a cell factory with one or more attached air filters; Concentrating and / or washing the cells using tangential flow filtration (TFF); or Passing the harvested cells through a dual screen mesh filter to reduce visible particulates and / or cell clumps; The present invention provides a method for generating a population of mesenchymal progenitor or stem cells, comprising one or more of:

[0025] In one embodiment, the method comprises: Culturing a population of mesenchymal progenitor or stem cells in a medium containing recombinant trypsin; and concentrating and / or washing the cells using tangential flow filtration (TFF); Includes:

[0026] In one embodiment, the method comprises: culturing a population of mesenchymal progenitor or stem cells in a medium containing recombinant trypsin; Concentrating and / or washing the cells using tangential flow filtration (TFF); and Passing the harvested cells through a dual screen mesh filter to reduce visible particulates and / or cell clumps; Includes:

[0027] In one embodiment, the method comprises the steps of: culturing a population of mesenchymal progenitor or stem cells in a medium containing recombinant trypsin; Cultivating cells in a cell factory with one or more attached air filters; Concentrating and / or washing the cells using tangential flow filtration (TFF); and Passing the harvested cells through a dual screen mesh filter to reduce visible particulates and / or cell clumps; Includes:

[0028] In one embodiment, the method includes one or more or all of the steps outlined in FIG.

[0029] In one embodiment, the mesenchymal precursor or stem cells are isolated by immunoselection. For example, the mesenchymal precursor or stem cells isolated by immunoselection may be STRO-1+ mesenchymal precursor cells or their progeny.

[0030] In one embodiment, the mesenchymal precursor or stem cells are isolated by plastic adherence. For example, the mesenchymal precursor or stem cells isolated by plastic adherence may be mesenchymal stem cells or their progeny.

[0031] The present inventors have also developed a potency assay to measure the biological activity or therapeutic efficacy of cell therapy products containing mesenchymal precursor or stem cells.

[0032] Therefore, the present disclosure also provides: (i) obtaining a cell population containing mesenchymal progenitor or stem cells (cells have been cryopreserved and thawed); (ii) co-culturing the cells in culture with a cell population that includes T cells; (iii) measuring the level of inhibition of T cell IL-2Rα expression (an amount of ≥ 65% inhibition indicates biological activity or therapeutic efficacy of the mesenchymal progenitor or stem cells); For example, an amount of at least about 70% inhibition, at least about 75% inhibition, at least about 80% inhibition, at least about 85% inhibition, or at least about 90% inhibition indicates biological activity or therapeutic effect.

[0033] In one embodiment, an amount of at least about 65% inhibition indicates therapeutic efficacy of the cells in inhibiting an immune response.

[0034] In one or further embodiments, an amount of at least about 65% inhibition indicates therapeutic efficacy of the cells in preventing or treating graft-versus-host disease.

[0035] The present disclosure also provides (i) obtaining a cell population containing mesenchymal progenitor or stem cells (cells have been cryopreserved and thawed); (ii) co-culturing the cells in culture with a cell population that includes T cells; (iii) selecting cells that exhibit a level of inhibition of T cell IL-2Rα expression of ≥ 65% inhibition; The present invention provides a method for selecting potent mesenchymal precursor or stem cells, comprising:

[0036] In one embodiment, the assay or selection method described above is used to titer an enriched population of mesenchymal progenitor stem cells. For example, the mesenchymal progenitor or stem cells are enriched for mesenchymal stem cells. In another embodiment, the mesenchymal progenitor or stem cells are enriched by selection for STRO-1+ cells. In one embodiment, the mesenchymal progenitor cells are STRO-1+ cells. 明るい It is a cell.

[0037] In one or further embodiments, the assay or selection method is used to titer mesenchymal progenitor or stem cells or to select populations of mesenchymal progenitor or stem cells expanded ex vivo or in vitro. In one example, mesenchymal progenitor or stem cells are isolated, or isolated and enriched, and expanded in culture ex vivo or in vitro before cryopreservation. In another example, mesenchymal progenitor or stem cells are isolated, or isolated and enriched, cryopreserved, thawed, and subsequently expanded in culture. In yet another example, mesenchymal progenitor or stem cells are expanded in culture before and after cryopreservation.

[0038] In one embodiment, the mesenchymal precursor or stem cells are human mesenchymal precursor or stem cells.

[0039] In one embodiment, the T cells are human T cells. In another embodiment, the T cells express CD4 and CD8. In another embodiment, the T cells express CD69 and / or CD137.

[0040] In one or a further example, the population comprising T cells is a population of peripheral blood mononuclear cells (PBMCs).

[0041] In one embodiment, the assay or selection method comprises culturing mesenchymal progenitor or stem cells with T cells in a medium comprising one or more T cell stimulatory ligands. For example, the medium comprises an anti-CD3 antibody or fragment thereof and an anti-CD28 antibody or fragment thereof. In another or further embodiment, the method comprises culturing the mesenchymal progenitor or stem cells with stimulated and / or activated T cells prior to co-culturing with the mesenchymal progenitor cells.

[0042] In one embodiment, the assay or selection method involves culturing cells in DMEM supplemented with 10% FBS and 2 mM glutamine, and optionally containing one or more T cell stimulatory ligands.

[0043] In one embodiment, the method comprises co-culturing mesenchymal progenitor or stem cells and T cells at a ratio of about 1 mesenchymal progenitor or stem cell:2 T cells or less, e.g., 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1 mesenchymal progenitor or stem cell:100 T cells or less.

[0044] In one embodiment, the method comprises co-culturing the cells for 60 to 84 hours before measuring IL-2Rα expression.

[0045] In one or further embodiments, the method comprises co-culturing the cells and then lysing them to produce a cell lysate, followed by harvesting the cells.

[0046] In one or further embodiments, the method comprises measuring the amount of IL-2Rα in the cell lysate using an enzyme-linked immunosorbent assay (ELISA).

[0047] In one example, an ELISA is (i) adding sample dilutions to each well of a microplate precoated with a monoclonal antibody specific for IL-2Rα; (ii) adding the co-cultured samples to wells of a microplate precoated with a monoclonal antibody specific for IL-2Rα; (iii) incubating the microplate for a sufficient time to allow the monoclonal antibody specific for IL-2Rα to specifically bind to any IL-2Rα in the sample; (iv) washing the microplate; (v) adding an IL-2Rα conjugate to the well; (vi) incubating the microplate for a sufficient time to allow the conjugate to specifically bind to any captured IL-2Rα; (vii) washing the microplate; (viii) adding a substrate solution to the wells; (ix) incubating the microplate for a time sufficient for color development; (x) adding stop solution to the wells; (xi) reading the absorbance on a microplate reader set at 450 nm (wavelength corrected at 570 nm); (xii) measuring the concentration of IL-2Rα; Includes:

[0048] In one embodiment, the method comprises: Prepare serial dilutions of the IL-2Rα standard solution using the sample diluent to obtain final concentrations ranging from, for example, 7.8 to 500 pg / ml; adding a standard solution to the microplate before step (iii); Constructing a standard curve using four-parameter logistic curve fitting; and measuring the concentration of IL-2Rα based on a standard curve; Further includes:

[0049] In one embodiment, the method comprises: measuring TNFR1 expression in a population of mesenchymal progenitor or stem cells (an amount of ≧100 pg / ml TNFR1 indicates biological activity or therapeutic efficacy of the mesenchymal progenitor or stem cells); For example, an amount of at least about 10 pg / ml TNFR1, at least about 10 pg / ml TNFβ1, at least about 10 pg / ml TNFR1, at least about 10 pg / ml TNFR1, at least about 10 pg / ml TNFR1, at least about 10 pg / ml TNFR1, at least about 10 pg / ml TNFR1, at least about 10 pg / ml TNFR1, at least about 110 pg / ml TNFR1, at least about 150 pg / ml, at least about 200 pg / ml, at least about 250 pg / ml, at least about 300 pg / ml, at least about 320 pg / ml, at least about 330 pg / ml, at least about 340 pg / ml, or at least about 350 pg / ml indicates biological activity or a therapeutic effect.

[0050] The present disclosure also provides a method for inhibiting an immune response in a subject in need thereof, the method comprising administering to the subject a composition comprising a population of MLPSCs of the present disclosure.

[0051] The present disclosure also provides a method for preventing or treating an inflammatory disease in a subject in need thereof, the method comprising administering to the subject a composition comprising the MLPSCs of the present disclosure. The inflammatory disease may be a T cell-mediated inflammatory disease.

[0052] The present disclosure also provides a method for preventing, alleviating the progression of, or treating graft-versus-host disease in a subject, comprising administering to the subject a composition comprising the MLPSCs of the present disclosure.

[0053] The present disclosure also provides a method for preventing graft-versus-host disease in a subject, the method comprising administering to the subject hematopoietic stem cells co-cultured with MLPSCs of the present disclosure.

[0054] In one embodiment, the composition comprises 3×10 6 The subject will be administered a dose of less than 100 cells / kg body weight once weekly (qw).

[0055] The present disclosure also provides a method for preventing, attenuating the progression of, or treating graft-versus-host disease (GVHD) in a mammalian subject, comprising administering to a mammalian subject at least 3×10 MLPSCs and / or their progeny. 6 This involves administering to a subject once weekly (qw) at a dose of less than 100 cells / kg body weight.

[0056] In one embodiment, the subject receives approximately 2×10 6 In another embodiment, subjects receive MLPSCs at a dose of up to 2 x 10 cells / kg body weight qw. 6 In another embodiment, subjects are administered cells qw at a dose of 2 x 10 cells / kg body weight. 6 The maximum dose of cells / kg body weight is administered qw. For the avoidance of doubt, the terms "weekly", "once per week" or "qw" are intended to mean a period of once every seven days.

[0057] In another embodiment, the MLPSCs are administered in a single dose once a week (qw). In another embodiment, the MLPSCs are administered in divided doses per week (i.e., 7 days). For example, a subject may receive two doses (each dose of 1 x 10 6 In other embodiments, subjects may receive two or more doses per week (up to a total dose of 2 x 10 cells / kg body weight). 6 cells / kg body weight) may be administered.

[0058] In one embodiment, the graft comprises allogeneic cells, hi another embodiment, the graft comprises autologous cells.

[0059] In one embodiment, the MLPSCs administered to the subject are MPCs and / or their progeny.

[0060] In another embodiment, the MLPSCs administered to the subject are MSCs and / or their progeny.

[0061] In one embodiment, the MLPSCs and / or their progeny are delivered qw in a single dose, hi another embodiment, the MLPSCs and / or their progeny are delivered in divided doses over a period of one week.

[0062] In one embodiment, the mammalian subject is a human subject. In one embodiment, the subject is a pediatric subject. In another embodiment, the subject is an adult subject.

[0063] In another embodiment, the subject of the present disclosure is a subject with a hematological malignancy or genetic disease (e.g., cancer). In a further example, the subject has received, is receiving, or is about to receive a donor graft comprising hematopoietic cells.

[0064] The hematopoietic cell-containing graft may be selected from the group consisting of blood, peripheral blood mononuclear cells (PBMCs), blood products, or solid organs containing hematopoietic cells. In one example, the graft comprises hematopoietic stem cells (HSCs).

[0065] In one embodiment, the subject has acute GVHD. Symptoms of acute GVHD are usually graded according to standard clinical diagnostic criteria (Glucksberg H. et al. (1974) Transplantation 1974;18(4):295-304).

[0066] In one embodiment, the subject is receiving steroid treatment prior to administration of the mesenchymal precursor or stem cells. In one example, the steroid is methylprednisolone. In another example, the steroid is administered to the subject at least three (3) days prior to administration of the MLPSCs and / or their progeny.

[0067] In one embodiment, the subject receives the MLPSCs and / or their progeny on the same day as administration of the graft (eg, bone marrow or PBMCs).

[0068] The MLPSCs and / or their progeny may be administered to the subject at an appropriate time, which may be during or after transplantation of the graft. For example, for prophylactic purposes, the MLPSCs and / or their progeny may be administered to the subject as early as the day of transplantation of the graft. In another example, the MLPSCs and / or their progeny may be administered to the subject before administration of the graft. In another example, the MLPSCs and / or their progeny may be administered within 7 days, 5 days, 3 days, or 2 days before administration of the graft. In another example, the MLPSCs and / or their progeny are administered to the subject the day before administration of the graft.

[0069] In another embodiment, the MLPSCs and / or their progeny are administered to a subject after the subject has been determined to be steroid-resistant. While there is no universal consensus on the definition of steroid-resistant acute GVHD, steroid-resistant acute GVHD typically refers to GVHD that worsens after 3-5 days of steroid treatment, does not improve after 5-7 days, or does not completely remit after 14 days. In another example, the MLPSCs and / or their progeny are administered to a subject at least 3 days after steroid or immunosuppressive treatment. In another example, the MLPSCs and / or their progeny are administered to a subject at least 1 month after steroid or immunosuppressive treatment. In one example, a steroid-resistant subject is one who has not responded to steroid treatment for grade B-D acute GVHD at least 3 days after steroid (e.g., methylprednisolone or equivalent). In a further example, the subject has not responded to >1 mg / kg / day of methylprednisolone or equivalent.

[0070] In another embodiment, the subject has undergone non-steroidal immunosuppressive therapy prior to administration of the MLPSCs and / or their progeny. In another example, the subject has undergone one or more non-steroidal therapies selected from the group consisting of extracorporeal photophoresis (ECP), infliximab, ruxolitinib, mycophenolate mofetil (MMF), etanacept, and basiliximab. In one example, the MLPSCs and / or their progeny are administered to a subject who is resistant to immunosuppressive therapy using non-steroidal agents.

[0071] In one example, the subject is administered MLPSCs and / or their progeny until improvement of GVHD is observed, hi another example, the subject is administered MLPSCs and / or their progeny until remission of GVHD is observed.

[0072] In one example, administration of the MLPSCs and / or their progeny prevents, alleviates, or treats an adverse event selected from one or more of the following: infusion-related reaction, hypertension, vomiting, nausea, bradycardia, and fever. In one example, administration of the MLPSCs and / or their progeny reduces the number of adverse events suffered by a subject compared to a subject not administered the MLPSCs and / or their progeny.

[0073] In one example, the subject has acute GVHD grade B, C, or D. In another example, the GVHD involves the skin, gastrointestinal tract, or liver, or a combination of any one or more of these tissues.

[0074] In one example, GVHD is the result of T cell immune response.In one example, T cells are derived from donor, and antigen is derived from recipient.For example, T cells may be present in the graft.In another embodiment, T cells are derived from recipient, and antigen is derived from donor.

[0075] In another embodiment of this method, the mesenchymal progenitor or stem cells are genetically modified to express a molecule that inhibits T cell costimulation.

[0076] In another embodiment of this method, the mesenchymal precursor or stem cells are expanded in culture prior to administration to the subject.

[0077] In one embodiment, the MLPSCs and / or their progeny are administered in the form of a pharmaceutically acceptable composition. In a further example, the pharmaceutically acceptable composition comprises a pharmaceutically acceptable carrier and / or excipient.

[0078] The MLPSCs and / or their progeny may be administered to the subject once a week for each of four (4) consecutive weeks. In another example, the MLPSCs and / or their progeny are administered to the subject once a week for each of eight (8) consecutive weeks. In another example, the subject's GVHD is assessed after four weekly infusions, and if the subject is in partial or mixed GVHD remission, the subject desirably receives four additional weekly infusions. In one example, the subject is administered up to eight doses of the MLPSCs and / or their progeny. In another example, the subject is administered a total of eight doses of the MPCs and / or their progeny. In another example, the subject is administered the MLPSCs and / or their progeny on a weekly basis until at least a partial or complete remission is achieved.

[0079] In one embodiment, at baseline (screening), day 0, subjects are assessed for GVHD status or grading.

[0080] In another embodiment, the subject is assessed for GVHD status or grading on days 14, 28, 56, and 100. In another embodiment, GVHD is assessed on some of these days, for example, at baseline (day 0), day 28, and day 100.

[0081] Subjects may be assessed as exhibiting a complete response, partial response, mixed response, worsening, or no response.

[0082] The present disclosure also provides a method for preventing, attenuating the progression of, or treating graft-versus-host disease (GVHD) in a mammalian subject, comprising administering 3×10 6 Compositions are provided that include MLPSCs and / or their progeny at a dose of less than cells / kg body weight.

[0083] In one embodiment, the composition comprises about 2×10 6 In another embodiment, the composition comprises up to 2 x 10 MLPSCs and / or their progeny at a dose of cells / kg body weight. 6 In another embodiment, the composition comprises 2 x 10 MPCs and / or their progeny at a dose of 1 x 10 cells / kg body weight. 6 Contains the maximum dose of cells / kg body weight.

[0084] In one example, the composition is a pharmaceutical composition.

[0085] The present disclosure also provides a method for preventing or treating GVHD in a mammalian subject, comprising administering to a mammal a compound comprising 3×10 6 Provided is the use of MLPSCs and / or their progeny at a dose of less than 100 cells / kg body weight. In one example, the MLPSCs and / or their progeny are meant to be administered once a week (qw) to a subject in need thereof.

[0086] In one example, the pharmaceutical agent is administered at a concentration of about 2 x 10 6 In another example, the pharmaceutical product contains up to 2 x 10 MLPSCs and / or their progeny at a dose of cells / kg body weight. 6 In another example, the medicament comprises MLPSCs and / or their progeny at a dose of 2 x 10 cells / kg body weight. 6 Contains the maximum dose of cells / kg body weight.

[0087] In one example, mesenchymal progenitor or stem cells express STRO-1 明るいIn another example, the mesenchymal progenitor or stem cells are a cell population enriched for one or more additional markers selected from TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), and / or CD146+.

[0088] In one example, the mesenchymal progenitor or stem cells are a population of mesenchymal stem cells.

[0089] In one example, the MLPSCs and / or their progeny are administered systemically. For example, the MLPSCs and / or their progeny may be administered intravenously, intraarterially, intramuscularly, or subcutaneously into the aorta, atrium, or ventricle of the heart, or into a blood vessel leading to an organ, such as the abdominal aorta, superior mesenteric artery, pancreaticoduodenal artery, or splenic artery.

[0090] In another embodiment, the method of the present disclosure further comprises administering an immunosuppressant. The immunosuppressant may be administered for a period sufficient to allow the transplanted hematopoietic cells to function. The immunosuppressant may be selected from one or more of the following, including, but not limited to, corticosteroids such as prednisone, budesonide, and prednisolone; calcineurin inhibitors such as cyclosporine and tacrolimus; mTOR inhibitors such as sirolimus and everolimus; IMDH inhibitors such as azathioprine, leflunomide, and mycophenolic acid; and biologics such as abatacept, adalimumab, etanacept, infliximab, and rituximab.

[0091] In one example, the immunosuppressant is cyclosporine, which may be administered at a dose of 5 to 40 mg / kg body weight. [Brief explanation of the drawings]

[0092] [Figure 1] Figure 1 shows the morphology of unstimulated human PBMCs, stimulated human PBMCs, and co-cultures of human MPCs (long and flat) with human PBMCs (round, circular, and slightly aggregated). [Figure 2] FIG. 1 shows the results of a T cell proliferation assay (percent inhibition of IL2R) performed on three different samples of MLPSCs generated under conventional production conditions (i.e., samples MLPSC A, MLPSC B, and MLPSC C) and three different samples of improved immune-selected MLPSCs (i.e., samples MLPSC D, MLPSC E, and MLPSC F). [Figure 3] FIG. 1 shows the results of a TNFR1 expression assay performed on three different samples of MLPSCs generated under conventional production conditions (i.e., samples MLPSC A, MLPSC B, and MLPSC C) and three different samples of improved immune-selected MLPSCs (i.e., samples MLPSC D, MLPSC E, and MLPSC F). [Figure 4] 1 shows survival rates over 100 days after infusion of MPC in responders vs. non-responders. All 9 subjects who responded at day 28 survived to day 100, while only 1 of 3 non-responders at day 28 survived to day 100 (p-value=0.0068). [Figure 5] FIG. 1 shows the steps involved in an improved process for producing culture-expanded MLPSCs. [Figure 6] FIG. 1 shows the results of a TNFR1 expression assay performed on 10 different MLPSC lot products produced under improved production conditions. [Figure 7] FIG. 1 shows the results of a T cell proliferation assay performed on 10 different MLPSC lot products produced under improved production conditions (percent inhibition of IL2R). DETAILED DESCRIPTION OF THE INVENTION

[0093] General Techniques and Definitions Throughout this specification, unless otherwise stated or otherwise required by context, references to a single step, composition of matter, group of steps, or group of compositions of matter should be interpreted as encompassing one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0094] Those skilled in the art will understand that the present disclosure described herein is capable of variations and modifications other than those specifically described herein. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, properties, compositions, and compounds referred to or indicated herein, individually or collectively, and all combinations of any two or more of the steps or properties.

[0095] The present disclosure is not limited in scope by the specific embodiments described herein, which are meant for illustrative purposes only, and functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0096] Any example disclosed herein should be construed to apply mutatis mutandis to any other example unless expressly stated otherwise.

[0097] Unless otherwise defined, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., of cell culture, molecular genetics, stem cell differentiation, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0098] Unless otherwise indicated, stem cell, cell culture, and surgical techniques utilized in this disclosure are standard procedures well known to those skilled in the art, and are described and explained throughout the literature in sources such as Perbal, 1984; Sambrook & Green, 2012; Brown, 1991; Glover & Hames, 1995 and 1996; Ausubel, 1987, including all revisions to date; Harlow & Lane, 1988; and Coligan et al., 1991, including all revisions to date.

[0099] As used in this specification and the appended claims, singular and singular forms of terms, e.g., "a," "an," and "the," optionally include plural referents unless the content clearly dictates otherwise.

[0100] The term "graft-versus-host disease" or "GVHD" refers to a complication in allogeneic hematopoietic cell transplantation in which host tissues, most frequently the skin, liver, and intestine, are damaged by donor-derived lymphocytes. This disease is described in more detail below.

[0101] As used herein, the term "subject" refers to a mammal, including humans and non-human animals. More particularly, the mammal is a human. Terms such as "subject," "patient," or "individual" are terms that may be used interchangeably in this disclosure depending on the context. In certain examples, the subject may be an adult subject or a pediatric (child) subject.

[0102] An "effective amount" refers to the minimum effective amount, at the dosage and for the period required, to achieve the desired therapeutic or preventive result. An effective amount may be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" is used to mean the amount necessary to achieve treatment of the disease or condition described above. The effective amount may vary depending on the disease or condition being treated, as well as the body weight, age, ethnic background, sex, health and / or physical condition of the mammal being treated, and other factors related to the mammal being treated. Typically, an effective amount falls within a relatively broad range (e.g., a "dosage" range) that can be determined by a physician through routine testing and experimentation. An effective amount may be administered in a single dose or in one or more repeated doses over the course of treatment.

[0103] A "therapeutically effective amount" refers to at least the minimum concentration required to achieve a measurable improvement in a particular disease (e.g., GVHD). The therapeutically effective amount herein may vary depending on factors such as the patient's condition, age, sex, and weight, as well as the ability of the cell composition to induce a desired response in an individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the composition are outweighed by the therapeutically beneficial effects. In the case of GVHD, a therapeutically effective amount may reduce the severity, inhibit or delay the progression of GVHD, and / or alleviate to some extent one or more symptoms associated with the disease.

[0104] As used herein, the terms "preventing" or "preventing" refer to preventing, delaying, and / or reducing the severity of symptoms associated with GVHD. This term is distinguished from "treating," which occurs after the onset of early symptoms of GVHD.

[0105] As used herein, the term "alleviating" means reducing the severity of a disease and / or alleviating one or more symptoms associated with a disease (e.g., GVHD). It does not mean complete inhibition or elimination of the disease.

[0106] As used herein, the term "treatment" refers to a clinical intervention aimed at altering the natural course of the treated individual or cell during the clinical pathological course. Desirable effects of treatment include slowing the rate of disease progression, alleviating or mitigating the pathology, and remission or a favorable prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with the disease are alleviated or eliminated.

[0107] As referred to herein, the term "complete remission" or "CR" is defined as the complete resolution of acute GVHD symptoms in all organs without secondary GVHD treatment.

[0108] As referred to herein, the term "partial response" or "PR" is defined as improvement in at least one stage of GVHD in all primary GVHD target organs (without deterioration of any other GVHD target organs) without complete recovery in the absence of secondary GVHD treatment.

[0109] As referred to herein, the term "no response" or "NR" is defined as the same grade of GVHD, progression of GVHD in any organ (e.g., a worsening of at least one measurable organ symptom by one stage or more), death, or the addition of secondary GVHD treatment.

[0110] As used herein, the term "worsening" means a worsening of GVHD progression in at least one organ, with or without remission in any organ.

[0111] The term "very good partial response (VGPR)" means meeting response criteria except for one or more of the following: (i) non-progressive Stage 1 rash (without residual faint erythema or hyperpigmentation), (ii) a <25% increase in total serum bilirubin variance from baseline, or (iii) minimal gastrointestinal symptoms.

[0112] As used herein, the term "mixed remission" or "MR" means improvement in the stage of at least one evaluable organ and deterioration in another organ.

[0113] The term "progression" means deterioration of at least one organ system by one stage or more without improvement in any other organ system.

[0114] As used herein, the term "adult" means a human subject 18 years of age or older.

[0115] As used herein, the term "child" means a human subject ranging in age from birth up to and including 17 years of age.

[0116] As used herein, the term "acute GVHD" refers to GVHD that usually develops within the first six months after administration of a transplant, such as a bone marrow transplant, and may develop within a few days of receiving the transplant.

[0117] As used herein, the term "chronic GVHD" refers to GVHD that usually occurs more than three months after administration of the graft. Symptoms of chronic GVHD may persist for life.

[0118] As used herein, the term "graft" refers to a biological sample selected from bone marrow, blood (e.g., whole blood or peripheral blood mononuclear cells (PBMCs)), a blood product, or a solid organ, containing hematopoietic cells.

[0119] As used herein, the term "allogeneic" refers to a graft (e.g., hematopoietic cells) donated from an individual whose genetic characteristics (particularly with respect to the major histocompatibility complex (MHC) and non-major histocompatibility factors expressed on the surface of the individual's cells) differ from those of the recipient.

[0120] As used herein, the term "autologous" refers to a graft using the subject's own cells (e.g., hematopoietic cells present in bone marrow or peripheral blood). The cells are typically harvested and stored in advance from the subject undergoing treatment (e.g., chemotherapy), and then reinfused back into the subject.

[0121] As used herein, the term "steroid-resistant" refers to GVHD that worsens after 3-5 days of steroid treatment, does not improve after 5-7 days, or does not completely remit after 14 days.

[0122] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing clear support for both meanings or either meaning.

[0123] As used herein, the term "about" means ±10%, more preferably ±5% of the specified numerical value, unless otherwise stated.

[0124] Throughout this specification the term "comprise" or variations such as "comprises" or "comprising" should be understood to mean the inclusion of the stated element, integer or step or group of elements, integers or steps, but not the exclusion of any other element, integer or step or group of elements, integers or steps.

[0125] Mesenchymal progenitor or stem cells As used herein, the term "mesenchymal progenitor or stem cell" refers to an undifferentiated pluripotent cell that has the ability to self-renew while maintaining pluripotency and to differentiate into a multitude of cell types, either mesenchymal (e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons) or non-mesodermal (e.g., hepatocytes, neurons, and epithelial cells) lineages.

[0126] The term "mesenchymal progenitor or stem cell" includes both parent cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells (MPCs), multipotent stromal cells, mesenchymal stem cells, perivascular mesenchymal precursor cells, and their undifferentiated progeny.

[0127] Mesenchymal progenitor or stem cells may be autologous, allogeneic, xenogeneic, syngeneic, or isogeneic. Autologous cells are isolated from the same individual into whom they will be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or syngeneic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0128] Mesenchymal progenitor or stem cells reside primarily within bone marrow, but have also been found to reside within a wide variety of host tissues, including, for example, umbilical cord blood and cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp.

[0129] Mesenchymal progenitor or stem cells can be isolated from host tissue and enriched by immunoselection.For example, bone marrow aspirate from a subject can be further treated with antibodies against STRO-1 or TNAP to allow for the selection of mesenchymal progenitor or stem cells.In one example, mesenchymal progenitor or stem cells can be enriched by using STRO-1 antibodies as described in Simmons & Torok-Storb, 1991.

[0130] STRO-1+ cells are present in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph nodes, thymus, bone, ligaments, tendons, skeletal muscle, dermis, and periosteum, and can differentiate into germline cells such as mesoderm and / or endoderm and / or ectoderm. STRO-1+ cells can therefore differentiate into numerous cell types, including, but not limited to, adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue, and fibrous connective tissue. The specific lineage commitment and differentiation pathways these cells follow depend on various influences, such as mechanical and / or endogenous bioactive factors (e.g., growth factors, cytokines, etc.), and / or the local microenvironmental conditions established by the host tissue.

[0131] As used herein, the term "enriched" refers to a cell population that has an increased proportion of one particular cell type or a plurality of particular cell types when compared to an untreated cell population (e.g., cells in their natural environment). In one example, a population enriched for STRO-1+ cells comprises at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% STRO-1+ cells. In this regard, the term "cell population enriched for STRO-1+ cells" is understood to provide clear support for the term "cell population comprising X% STRO-1+ cells" (where X% is a percentage as recited herein). In some examples, STRO-1+ cells are capable of forming clonogenic colonies; for example, CFU-F (fibroblasts) or a subset thereof (e.g., 50%, 60%, 70%, 70%, 90%, or 95%) may possess this activity.

[0132] In one example, a cell population is enriched in a selectable manner from a cell sample containing STRO-1+ cells. In this context, the term "selectable manner" is understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for the selection of STRO-1+ cells. The marker may be, but need not be, STRO-1. For example, as described and / or exemplified herein, cells (e.g., MPCs) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 may also be enriched for STRO-1 (STRO-1 明るい (which may be TNAP+). Thus, a statement that a cell is STRO-1+ does not mean that the cell is selected due to STRO-1 expression. In one example, the cells are selected on the basis of at least STRO-3 expression, e.g., the cells are STRO-3+ (TNAP+).

[0133] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells can be selected, isolated or enriched from a wide variety of sources. That is, in some instances, these terms provide support for selection from any tissue containing STRO-1+ cells, vascularized tissue, tissue containing pericytes (e.g., STRO-1+ pericytes), or any one or more of the tissues listed herein.

[0134] In one example, the mesenchymal progenitor or stem cells of the present disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+.

[0135] By "individually" it is meant that the present disclosure encompasses the listed markers or groups of markers separately; notwithstanding that individual markers or groups of markers may not be separately listed herein, the appended claims may define such markers or groups of markers separately and divisible from one another.

[0136] By "collectively," it is meant that the present disclosure encompasses any number or combination of the listed markers or groups of markers, and that notwithstanding that such number or combination of markers or groups of markers is not expressly recited herein, the appended claims may define such combinations or subcombinations separately and divisibly from any other combinations of markers or groups of markers.

[0137] Cells referred to as "positive" for a given marker may express that marker at either low (lo, dim, or dull), medium (intermediate), or high (bright, bri) levels, depending on the degree to which the marker is present on the cell surface; these terms relate to the intensity of the fluorescence or other marker used in the cell separation process or flow cytometric analysis of the cells. The distinction between low (lo, dim, or dull), medium (intermediate), and high (bright, bri) is understood in relation to the marker used in the particular cell population being separated or analyzed. Cells referred to as "negative" for a given marker do not necessarily mean that the marker is completely absent from the cell. This term means that the marker is expressed by the cell at a relatively very low level and that the marker, when detectably labeled, produces a very low signal, or that the marker is undetectable above background levels, e.g., levels detected using an isotype control antibody.

[0138] As used herein, the term "bright" or "bri" means that the marker on the cell surface produces a relatively high signal when detectably labeled. Without being limited by theory, it is proposed that "bright" cells express more of the target marker protein (e.g., the antigen recognized by the STRO-1 antibody) than do other cells in the sample. For example, STRO-1 bri Cells were labeled with FITC-conjugated STRO-1 antibody and analyzed by fluorescence-activated cell sorting (FACS) analysis to identify non-bright cells (STRO-1 lo / 薄暗い / 鈍い / 中 / 中間 ) produce a higher fluorescent signal compared to STRO-1. In one example, mesenchymal progenitor or stem cells are isolated from bone marrow and enriched by selection for STRO-1+ cells. In this example, the "bright" cells comprise at least about 0.1% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. In other examples, the "bright" cells comprise at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. In one example, STRO-1 明るい The cells exhibit a 2 log increase in STRO-1 surface expression compared to "background" (i.e., cells that are STRO-1-). lo / 薄暗い / 鈍い and / or STRO-1 中 / 中間 Cells exhibit less than 2 logarithmic magnitude of STRO-1 surface expression (usually about 1 log, or below "background").

[0139] In one example, STRO-1+ cells are STRO-1 明るい In one example, STRO-1 明るい STRO-1 cells lo / 薄暗い / 鈍い or STRO-1 中 / 中間 are preferentially enriched relative to cells.

[0140] In one example, STRO-1 明るいThe cells may further be one or more of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), and / or CD146+. For example, the cells may be selected for and / or shown to express one or more of the above markers. In this regard, cells shown to express a marker need not be specifically tested; rather, previously enriched or isolated cells may be tested before use, and it can be reasonably assumed that the isolated or enriched cells will express the same markers.

[0141] In one example, STRO-1 明るい The cells are perivascular mesenchymal progenitor cells as defined in WO2004 / 85630 (characterized by the presence of the perivascular marker 3G5).

[0142] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, the term encompasses the liver isoform (LAP), bone isoform (BAP), and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP refers to a molecule capable of binding to the STRO-3 antibody produced by the hybridoma cell line deposited with the American Type Culture Collection (ATCC) on December 19, 2005 under the terms of the Budapest Treaty under Accession Number PTA-7282.

[0143] Furthermore, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0144] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germ cell lines. Non-limiting examples of lineages to which cells can be committed include bone progenitor cells; hepatocyte progenitor cells (which have the potential to differentiate into bile duct epithelial cells and hepatocytes); neural restricted cells (which can give rise to glial cell precursors that progress to oligodendrocytes and astrocytes); neural cell precursors that progress to neurons; and precursors of cardiac muscle and cardiomyocytes, a glucose-responsive, insulin-secreting pancreatic beta cell line. Other lineages include, but are not limited to, progenitor cells of odontoblasts, dentin-producing cells, and chondrocytes, as well as the following: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal duct epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendon cells, ligament cells, chondrocytes, adipocytes, fibroblasts, bone marrow stromal cells, cardiac muscle cells, smooth muscle cells, skeletal muscle cells, pericytes, vascular cells, epithelial cells, glial cells, nerve cells, astrocytes, and oligodendrocytes.

[0145] In one example, the mesenchymal progenitor or stem cell is a mesenchymal stem cell (MSC). The MSC may be a homogenous composition or a mixed cell population enriched for MSCs. A homogenous MSC composition may be obtained by culturing adherent bone marrow or periosteal cells, or MSCs may be identified by specific cell surface markers identified with unique monoclonal antibodies. Methods for obtaining MSC-enriched cell populations using plastic adherence are described, for example, in U.S. Patent No. 5,486,359. MSCs prepared by conventional plastic adherence isolation methods rely on the nonspecific plastic adhesion properties of CFU-Fs. Other sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium.

[0146] The mesenchymal precursor or stem cells may be cryopreserved prior to administration to a subject.

[0147] In a preferred embodiment of the invention, the mesenchymal precursor or stem cells are obtained from a master cell bank derived from mesenchymal precursor or stem cells enriched from the bone marrow of healthy volunteers. The use of mesenchymal precursor or stem cells from such a source is particularly advantageous for subjects who do not have suitable relatives who can serve as mesenchymal precursor or stem cell donors, or who require immediate treatment, and who are at high risk of relapse into disease-related debilitation or death during the period required to generate mesenchymal precursor or stem cells.

[0148] The present inventors demonstrate that the mesenchymal progenitor cells of the present disclosure are unexpectedly effective in terms of their ability to inhibit T cell proliferation after cryopreservation and thawing. In contrast, previous publications have taught that cryopreserved mesenchymal stem cells exhibit impaired immunosuppressive properties after thawing (Francois et al., 2012; Chinnadurai et al., 2016).

[0149] The culture medium may be used to expand the isolated or enriched mesenchymal progenitor or stem cells ex vivo or in vitro. As will be appreciated by those skilled in the art, the isolated or enriched mesenchymal progenitor or stem cells may be cryopreserved, thawed, and subsequently or further expanded ex vivo or in vitro in culture medium.

[0150] The cultured mesenchymal progenitor or stem cells are phenotypically distinct from in vivo cells, for example, in one embodiment, the cultured mesenchymal progenitor or stem cells express one or more of the following markers: CD44, NG2, DC146, and CD140b.

[0151] Cultured mesenchymal progenitor or stem cells are biologically distinct from in vivo cells, exhibiting a higher proliferation rate compared to most non-cycling (quiescent) in vivo cells.

[0152] In one example, the mesenchymal progenitor or stem cell enriched cell population is grown at approximately 6000-7000 viable cells / cm in serum-supplemented medium, e.g., Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine. 2 and allowed to adhere to the culture vessel overnight at 37° C. under 20% O. In one embodiment, the cells are at a density of about 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6810, 6820, 6830, 6840, 6850, 6860, 6870, 6880, 6890, 6890, 6900, 6910, 6920, 6930, 6940, 6970, 6980, 6990, or 7000 viable cells / cm. 2 , preferably about 6850-6860 viable cells / cm 2 Following this, the medium is changed and the cells are cultured at 37°C under 5% O for a total of 68-72 hours before co-culture with T cells, and the amount of IL-2Rα expressed by the T cells is measured.

[0153] Ang1 and VEGF levels In one example, the mesenchymal progenitor or stem cells are at least 0.1 μg / 10 6 However, in other instances, the MLPSCs express Ang1 in amounts of at least 0.2 μg / 10 6 cells, 0.3μg / 10 6 cells, 0.4μg / 10 6 cells, 0.5μg / 10 6 cells, 0.6μg / 10 6 cells, 0.7μg / 10 6 cells, 0.8μg / 10 6 cells, 0.9μg / 10 6 cells, 1μg / 10 6 cells, 1.1μg / 10 6 cells, 1.2μg / 10 6 cells, 1.3μg / 10 6 cells, 1.4μg / 10 6 cells, 1.5μg / 10 6 The amount of cells expressing Ang1.

[0154] In another example, the MLPSCs are about 0.05 μg / 10 6 However, in other instances, mesenchymal precursor cells express VEGF in amounts less than about 0.05 μg / 10 6 cells, 0.04μg / 10 6 cells, 0.03μg / 10 6 cells, 0.02μg / 10 6 cells, 0.01μg / 10 6 cells, 0.009μg / 10 6 cells, 0.008μg / 10 6 cells, 0.007μg / 10 6 cells, 0.006μg / 10 6 cells, 0.005μg / 10 6 cells, 0.004μg / 10 6 cells, 0.003μg / 10 6 cells, 0.002μg / 10 6 cells, 0.001μg / 10 6 They express VEGF in subcellular amounts.

[0155] The amount of cellular Ang1 and / or VEGF expressed in a composition or culture of MLPSCs can be measured using methods well known to those skilled in the art. Such methods include, but are not limited to, quantitative assays such as quantitative ELISA assays. In this example, cell lysates from a culture of mesenchymal progenitor cells are added to wells of an ELISA plate. The wells may be coated with a primary antibody (either monoclonal or polyclonal) against Ang1 or VEGF. The wells are then washed and then contacted with a secondary antibody (either monoclonal or polyclonal) against the primary antibody. The secondary antibody is conjugated to a suitable enzyme, such as horseradish peroxidase. The wells may then be incubated and then washed after the incubation period. The wells are then contacted with a suitable substrate for the enzyme conjugated to the secondary antibody, such as one or more chromogens. Usable chromogens include, but are not limited to, hydrogen peroxide and tetramethylbenzidine. After the addition of the substrate(s), the wells are incubated for a suitable period of time. Upon completion of incubation, a "stop" solution is added to the well to stop the reaction with the enzyme's substrate(s). The optical density (OD) of the sample is then measured. To determine the amount of Ang1 or VEGF expressed by the culture medium of the stem cells being tested, the optical density of the sample is correlated with the optical density of a sample containing a known amount of Ang1 or VEGF.

[0156] In another example, the MLPSCs express Ang1:VEGF at a ratio of at least about 2: 1. However, in other examples, the mesenchymal precursor cells express Ang1:VEGF at a ratio of at least about 10:1, 15:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 50:1.

[0157] Methods for measuring the Ang1:VEGF expression ratio are readily apparent to those skilled in the art. For example, the expression levels of Ang1 and VEGF can be quantified via quantitative ELISA as described above. After quantifying the levels of Ang1 and VEGF, a ratio based on the quantified levels of Ang1 and VEGF can be expressed as the Ang1:VEGF ratio (Ang1 level / VEGF level).

[0158] In one example, the MLPSCs of the present disclosure have not been genetically modified to express Ang1 and / or VEGF at the levels or ratios exemplified above. Cells that have not been genetically modified to express Ang1 and / or VEGF have not been modified by transfection with a nucleic acid that expresses or encodes Ang1 and / or VEGF. For the avoidance of doubt, in the context of the present disclosure, mesenchymal precursor cells transfected with a nucleic acid encoding Ang1 and / or VEGF are considered genetically modified. In the context of the present disclosure, cells that have not been genetically modified to express Ang1 and / or VEGF naturally express Ang1 and / or VEGF to some extent without transfection with a nucleic acid encoding Ang1 and / or VEGF.

[0159] T cells The titration method of the present disclosure requires co-culturing mesenchymal progenitor or stem cells with T cells. In one embodiment, the mesenchymal progenitor or stem cells are co-cultured with the T cells in a medium containing at least one T cell stimulatory ligand. In one or further embodiments, the T cells are activated. The T cells may be first stimulated or activated prior to co-culture with the mesenchymal progenitor or stem cells.

[0160] As used herein, the term "T cells" refers to thymus-derived cells that are involved in a variety of cell-mediated immune responses.

[0161] As used herein, the term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can be mediated by altered expression of specific molecules, such as downregulation of TGF-β, and / or reorganization of cytoskeletal structure.

[0162] As used herein, the term "activated" refers to a state of T cells that have been stimulated sufficiently to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers specifically to T cells that have undergone cell division.

[0163] Recently activated T cells typically express a series of activation markers at different time points following activation. Activation markers include receptors, such as chemokine and cytokine receptors, adhesion molecules, costimulatory molecules, and MHC class II proteins. Flow cytometry can be used to assess various types of surface or intracellular markers that indicate the activation state of T cells. Two of the most commonly used immediate-early activation markers for assessing the activation state of human peripheral blood mononuclear cells (PBMCs) T cells are CD69 and CD40L.

[0164] CD69 (AIM, Leu23, MLR3) is a signaling membrane glycoprotein involved in inducing T cell proliferation. Although CD69 is typically expressed at very low levels (<5–10%) on resting CD4+ or CD8+ T cells in PBMCs, it is rapidly upregulated on CD4+ or CD8+ T cells within 1 hour of TCR stimulation or other T cell activators (e.g., phorbol esters) via a protein kinase C (PKC)-dependent pathway, making it one of the earliest markers of activation. CD69 expression typically peaks between 16–24 hours and then declines, becoming barely detectable by 72 hours after the stimulus is removed.

[0165] CD40L (CD154) is a member of the TNF receptor superfamily that functions as a costimulatory molecule by binding to CD40, which is constitutively expressed on antigen-presenting cells (APCs). Ligation of CD40L to CD40 activates multiple downstream pathways, including MAPK (JNK, p38, ERK1 / 2), NF-κB, and STAT3 transcription factors. CD40L expression is rapidly upregulated within 1–2 h after TCR stimulation via the transcription factors NFAT and AP-1. CD40L expression peaks around 6 h after stimulation and declines around 16–24 h. However, CD40L expression is biphasic, and addition of anti-CD28 or IL-2 along with TCR stimulation typically results in sustained expression for several days.

[0166] As used herein, the term "specifically binds" refers to a ligand, e.g., an antibody, that recognizes and binds to a relevant binding partner present in a sample (e.g., a stimulatory and / or costimulatory molecule present on a T cell), but does not substantially recognize or bind to other molecules in the sample.

[0167] As used herein, the term "stimulatory ligand" refers to a ligand that can specifically bind to a cognate binding partner (herein referred to as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell. Stimulatory ligands are well known in the art and include, among others, peptide-bound MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies. Stimulatory ligands may be used in soluble form, expressed on or bound to the surface of a cell, or immobilized on a surface.

[0168] As used herein, the term "superagonist antibody" refers to an antibody that can specifically bind to a molecule on a T cell and mediate a primary activation signal event in the T cell without interacting with the TCR / CD3 complex or CD2 on the T cell. Exemplary superagonist antibodies include, but are not limited to, superagonist anti-CD28 antibodies and superagonist anti-CD2 antibodies. Unless referred to as a "superagonist," an anti-CD2 antibody or anti-CD28 antibody, etc., is a costimulatory ligand as defined elsewhere herein and provides a costimulatory signal rather than a primary activation signal.

[0169] As used herein, the term "stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand.

[0170] As used herein, the term "costimulatory signal" refers to a signal that cooperates with a primary signal, such as TCR / CD3 ligation, to mediate a T cell response, such as, but not limited to, activation, proliferation, differentiation into effector cells, cytotoxicity, or induction of cytokine secretion.

[0171] As used herein, the term "costimulatory ligand" includes a molecule on an antigen-presenting cell (APC) (e.g., a dendritic cell, a B cell, etc.) or an artificial APC (aAPC) that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing signals that mediate a T cell response, including, but not limited to, activation, proliferation, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, in addition to the primary signal provided, for example, by binding of the TCR / CD3 complex to a peptide-bound MHC molecule. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands also include antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, ligands that specifically bind to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83, among others. These and other ligands are well known in the art and have been well characterized, for example, as described in Schwartz et al., 2001; Schwartz et al., 2002; and Zhang et al., 2004. Those skilled in the art will understand that variants or mutants of known ligands can be used, and methods for preparing such variants or mutants are well known in the art.

[0172] As used herein, the term "aAPC" includes, but is not limited to, cell-based aAPCs, bead-based APCs, microparticulate aAPCs, and nanoparticle aAPCs. Materials that have been used include glass, poly(glycolic acid), poly(lactic-co-glycolic acid), iron oxide, liposomes, lipid bilayers, Sepharose, and polystyrene. aAPCs contain a stimulatory ligand, for example, a stimulatory ligand that specifically binds to the TCR / CD3 complex, thereby transducing a primary signal. aAPCs may further contain at least one costimulatory ligand that specifically binds to at least one costimulatory molecule present on T cells.

[0173] For purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to a stimulatory molecule on a T cell via an antigen-binding domain contained within an Fv. This term includes four-chain antibodies (e.g., two light (L) chains and two heavy (H) chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, deimmunized antibodies, synthetic humanized antibodies, half antibodies, bispecific antibodies).

[0174] As used herein, "variable region" refers to a portion of the light and / or heavy chain of an antibody as defined herein that is capable of specifically binding to an antigen and includes the amino acid sequences of the complementarity-determining regions (CDRs), i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, a variable region includes three CDRs as well as three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4). H V means the variable region of the heavy chain. L means the variable region of the light chain.

[0175] As used herein, the term "complementarity determining region" (also known as CDR, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues in the variable region of an antibody, the presence of which is primarily responsible for specific antigen binding. Each variable region domain (V H or V L) usually have three CDR regions, identified as CDR1, CDR2 and CDR3.

[0176] "Framework region" (FR) refers to the region of variable domain residues other than the CDR residues.

[0177] As used herein, the term "Fv" refers to a V, whether composed of multiple polypeptides or a single polypeptide. L and V H The term "antigen-binding domain" should be taken to mean any protein that binds to form a complex having an antigen-binding domain capable of specifically binding to an antigen. H and V L The V may be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the present disclosure (as well as any protein of the present disclosure) may have multiple antigen-binding domains that may or may not bind to the same antigen. This term should be understood to encompass fragments derived directly from antibodies, as well as proteins corresponding to such fragments produced using recombinant techniques. In some instances, the V H is the heavy chain constant domain (C H ) 1 and / or V L is the light chain constant domain (C L Exemplary Fv-containing polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabodies, triabodies, tetrabodies or higher order complexes, or constant regions or domains thereof, e.g., C H 2 or C H Any of the above linked to three domains, such as a minibody.

[0178] A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digestion of whole antibodies with the enzyme papain to generate fragments consisting of an intact light chain and part of the heavy chain, or can be produced using recombinant techniques.

[0179] An antibody "Fab' fragment" is produced by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a V H Fab' fragments can be obtained by generating a molecule consisting of a portion of a heavy chain containing the Fab' fragment and a single constant domain. Two Fab' fragments are obtained per antibody produced in this manner. Fab' fragments can also be produced using recombinant techniques.

[0180] An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments linked together by two disulfide bonds, and can be obtained by treating whole antibody molecules with the enzyme pepsin (without subsequent reduction).

[0181] "Fab2" fragments may be prepared using, for example, leucine zipper or C H It is a recombinant fragment containing two Fab fragments linked together using a three-domain technique.

[0182] A "single-chain Fv" or "scFv" is a recombinant molecule comprising the variable region fragment of an antibody (Fv) in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0183] T cell stimulation In one embodiment of the present disclosure, T cells may be stimulated with a single factor, hi another embodiment, T cells are stimulated with two factors, one factor inducing a primary signal and the second factor being a costimulatory signal.

[0184] Ligands useful for stimulating a single signal or for stimulating a primary signal and accessory molecules for stimulating a second signal may be used in soluble form, expressed on or bound to the surface of a cell, or immobilized on a surface.

[0185] The surface may be any surface to which a factor / ligand can be attached or associated and which is biocompatible, i.e., substantially non-toxic to the target cells to be stimulated. Biocompatible surfaces may be biodegradable or non-biodegradable. Surfaces may be natural or synthetic, and synthetic surfaces may be polymeric.

[0186] The agent may be bound, linked, or integrated with the surface using various methods well known and available in the art. The agent may be a natural ligand, a protein ligand, or a synthetic ligand. Binding may be covalent or non-covalent, electrostatic, or hydrophobic, and may be performed using various binding methods, including, for example, chemical, mechanical, enzymatic, electrostatic, or other methods by which the ligand can stimulate the cell. For example, an antibody against the ligand may be first bound to the surface, or avidin or streptavidin may be bound to the surface for binding to the biotinylated ligand. An antibody against the ligand may be bound to the surface via an anti-idiotypic antibody. Another example is the use of protein A or protein G or other nonspecific antibody-binding molecules bound to the surface for antibody binding. Alternatively, the ligand may be bound to the surface by chemical or other methods, such as cross-linking to the surface using commercially available cross-linking reagents (Pierce, Rockford, IL).

[0187] The amount of a particular ligand bound to a surface can be readily measured using flow cytometric analysis if the surface is that of a bead, or using enzyme-linked immunosorbent assays (ELISA) if the surface is, for example, a tissue culture dish, mesh, fiber, or bag.

[0188] When surface-linked, the factors may be linked to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one factor may be linked to a surface and the other factor in solution. In one embodiment, the factor providing the costimulatory signal is bound to the cell surface, and the factor providing the primary activation signal is in solution or linked to a surface. In a preferred embodiment, the two factors are immobilized on beads, either on the same bead, i.e., in "cis," or on separate beads, i.e., in "trans."

[0189] In one embodiment, the molecule that provides the primary activation signal is a CD3 ligand, and the costimulatory molecule is a CD28 ligand. In a preferred embodiment, the CD3 ligand is an anti-CD3 antibody or fragment thereof, and the CD28 ligand is an anti-CD28 antibody or fragment thereof. In one embodiment, the anti-CD3 antibody or fragment thereof and the anti-CD28 antibody or fragment thereof are used in soluble form. In an alternative embodiment, one or both of the anti-CD3 antibody or fragment thereof and the anti-CD28 antibody or fragment thereof are immobilized on a surface. In one embodiment, both the anti-CD3 antibody or fragment thereof and the anti-CD28 antibody or fragment thereof are immobilized on the same surface, e.g., a bead.

[0190] In one embodiment, the ratio of CD3 antibody to CD28 antibody bound to the beads ranges from 100:1 to 1:100 (all integer values ​​in between). In one aspect of the invention, more anti-CD28 antibody is bound to the particles compared to anti-CD3 antibody, i.e., the CD3:CD28 ratio is less than 1. In specific embodiments of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads is greater than 2:1. In one specific embodiment, a 1:100 CD3:CD28 ratio of antibody bound to the beads is used. In another embodiment, a 1:75 CD3:CD28 ratio of antibody bound to the beads is used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to the beads is used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody bound to the beads is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio of antibody bound to the beads is used. In another embodiment, a 1:3 CD3:CD28 ratio of antibody bound to the beads is used. In yet another embodiment, a 3:1 CD3:CD28 ratio of antibodies bound to beads is used.

[0191] Those skilled in the art will understand that the factor(s) used to stimulate T cells are provided in an amount sufficient to mediate a T cell response, such as, but not limited to, activation, proliferation, differentiation into effector cells, induction of cytotoxicity or cytokine secretion. Preferably, the factor(s) used to stimulate T cells are provided in an amount sufficient to mediate T cell proliferation.

[0192] Source of T cells Prior to stimulation / activation, a source of T cells is obtained from a subject. The term "subject" is intended to include any organism (e.g., a mammal) capable of inducing an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Certain embodiments of the present invention may utilize many T cell lines available in the art. In certain embodiments of the present invention, T cells can be obtained from a unit of blood drawn from a subject using many techniques well known to those skilled in the art, such as Ficoll separation. In one preferred embodiment, cells from an individual's circulating blood are obtained by apheresis or leukopheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, and other nucleated white blood cells, as well as red blood cells and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction before placing the cells in an appropriate buffer or medium for subsequent processing. In one embodiment, cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the wash solution may lack calcium, magnesium, or many, if not all, divalent cations. Those skilled in the art will readily appreciate that the washing step can be performed using methods well known to those skilled in the art, such as a semi-automated "flow-through" centrifuge (e.g., the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, cells may be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffers. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in medium.

[0193] Enrichment of T cell populations by negative selection can be achieved by combining antibodies directed against surface markers specific to the negatively selected cells. One method is cell separation and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, monoclonal antibody cocktails for enriching T cells (CD3+) by negative selection typically include antibodies specific for B cells (CD19), monocytes (CD14), NK cells (CD56), etc. The antibodies are typically immobilized on a surface (e.g., a particle such as a bead).

[0194] In isolating a desired cell population by positive or negative selection, the concentration of cells relative to beads can be varied. In certain embodiments, to ensure maximum contact between the cells and beads, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the cell concentration). For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, a concentration of 125 or 150 million cells / ml may be used. Using higher concentrations can result in increased cell yield, cell activation, and cell proliferation. Furthermore, using a high cell concentration allows for more efficient capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells.

[0195] If desired or necessary, monocyte populations (i.e., CD14) can be isolated from blood products prior to co-culture with mesenchymal progenitor or stem cells or ex vivo expansion using a variety of methods, including anti-CD14 coated beads or columns. +The removal of T cells may be facilitated by utilizing the phagocytic activity of these cells, or by using convective centrifugal elutriation. Thus, in one embodiment, the present invention utilizes paramagnetic particles of a size sufficient for engulfment by phagocytic monocytes. In certain embodiments, the paramagnetic particles are commercially available beads, such as those manufactured by Dynal AS under the trade name Dynabeads™. In this context, exemplary Dynabeads™ are M-280, M-450, and M-500. In one aspect, other nonspecific cells are removed by coating the paramagnetic particles with "irrelevant" proteins (e.g., serum proteins or antibodies). Irrelevant proteins and antibodies include proteins and antibodies or fragments thereof that do not specifically target the T cells being expanded. In certain embodiments, irrelevant beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin.

[0196] T cell proliferation The stimulated or activated T cells may be further expanded in cell culture using methods generally known in the art.

[0197] In one embodiment, the medium used to expand T cells comprises a factor capable of stimulating CD3 and a factor capable of stimulating CD28 on T cells.

[0198] Co-culture of stimulated or activated T cells with mesenchymal progenitor or stem cells The assay of the present invention measures inhibition of T cell IL-2Rα expression after co-culturing T cells with mesenchymal precursor or stem cells. Inhibition of IL-2Rα expression is associated with an inhibitory effect on T cell proliferation. Without being limited by theory, one skilled in the art will understand that mesenchymal precursor or stem cells may inhibit or suppress T cell stimulation and / or activation, thereby suppressing T cell proliferation, or that mesenchymal precursor or stem cells may act to suppress the proliferation of activated T cells.

[0199] In one embodiment, T cells are co-cultured with mesenchymal progenitor or stem cells in medium containing at least one T cell stimulatory factor (preferably at a concentration capable of stimulating and / or activating the T cells). In another embodiment, the T cells are first stimulated and / or activated prior to co-culture with the mesenchymal progenitor or stem cells.

[0200] In one embodiment, the mesenchymal progenitor or stem cells are co-cultured with PBMCs.

[0201] In one embodiment, mesenchymal progenitor or stem cells are co-cultured with PBMCs in a medium containing an agent capable of stimulating CD3 and CD28 on T cells, e.g., an antibody against CD3 and an antibody against CD28, e.g., mouse anti-human CD3 and mouse anti-human CD28. In one embodiment, the antibody against CD3 and / or the antibody against CD28 are added to the medium in soluble form, each at a concentration of about 2 μg / ml.

[0202] In one embodiment, PBMCs are co-cultured with mesenchymal progenitor or stem cells at a ratio of 5 PBMCs:1 mesenchymal progenitor or stem cells. For example, 1 x 10 6 PBMCs were expanded from enriched populations of mesenchymal progenitor or stem cells (2 x 10 5 In a further embodiment, the cells are co-cultured in a final volume of 1 ml.

[0203] In one embodiment, isolated or enriched mesenchymal progenitor or stem cells are first expanded ex vivo or in vitro in culture medium and then co-cultured with PBMCs.

[0204] In an alternative embodiment, the isolated, enriched or cultured mesenchymal progenitor or stem cells are co-cultured with an enriched and / or expanded population of T cells.

[0205] Preferably, the mesenchymal precursor or stem cells are co-cultured with T cells in medium containing one or more T cell stimulatory factors / ligands. Those skilled in the art will appreciate that T cells may be first stimulated and / or activated and then co-cultured with mesenchymal precursor or stem cells in the presence or absence of at least one T cell stimulatory factor.

[0206] Measurement of IL-2Rα levels This disclosure contemplates assays of any format, including Western blots, enzyme-linked immunosorbent assays (ELISAs), fluorescent immunoassays (FLISAs), competitive assays, radioimmunoassays, lateral flow immunoassays, flow-through immunoassays, electrochemiluminescence assays, nephelometry-based assays, turbidity-based assays, fluorescence-activated cell sorting (FACS)-based assays for detecting TGFβ1 in media used to culture mesenchymal or progenitor cells, and surface plasmon resonance (SPR or Biacore).

[0207] After co-culture of mesenchymal progenitor or stem cells and T cells, cells can be collected and lysed using methods well known in the art.The cell lysate can then be assayed for the presence of IL-2Rα, for example, using ELISA or FLISA.Alternatively, the level of IL-2Rα expression can be measured by assaying intact cells, for example, using flow cytometry.

[0208] One suitable assay format is, for example, ELISA or FLISA.

[0209] In one format, such assays involve immobilizing an IL-2Rα-binding protein on a solid matrix, such as a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g., a glass slide). The test sample is then directly contacted with the IL-2Rα-binding protein, resulting in binding or capture of IL-2Rα in the sample. After washing to remove any unbound protein in the sample, a protein that binds to IL-2Rα at a different epitope is directly contacted with the captured IL-2Rα. This detector protein is usually labeled with a detectable reporter molecule, such as an enzyme (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), or β-galactosidase) in the case of ELISA, or a fluorophore in the case of FLISA. Alternatively, a secondary labeled protein that binds to the detector protein may be used. After washing to remove any unbound protein, the detectable reporter molecule is detected by adding a substrate, such as hydrogen peroxide, TMB or toluidine, or 5-bromo-4-chloro-3-indole-β-D-galactopyranoside (x-gal) in the case of ELISA. Of course, the immobilized (capture) and detector proteins can also be used in reverse.

[0210] The level of IL-2Rα in the sample is then determined using a standard curve generated with known amounts of markers or by comparison with a control sample.

[0211] In one embodiment, inhibition of IL-2Rα expression is measured by comparing the level of IL-2Rα expression in a cell population comprising T cells with the level of IL-2Rα in the cell population after co-culture of the cell population comprising T cells with a cell population comprising mesenchymal precursor or stem cells, and the difference is expressed as a "percent inhibition."

[0212] The above assays are easily adapted to use chemiluminescence or electrochemiluminescence as the detection standard.

[0213] Those skilled in the art will recognize that other detection methods based on immunosorbent assays are useful in practicing the present disclosure, such as immunosorbent assays based on the above description that use detectable radiolabels, detectable gold labels (e.g., colloidal gold), detectable liposomes encapsulating, for example, NAD+, or acridinium immunoassays.

[0214] In some examples of the present disclosure, levels of IL-2Rα are measured using a surface plasmon resonance detector (e.g., BIAcore™, GE Healthcare, Piscataway, NJ), a flow-through device (e.g., as described in U.S. Patent 7,205,159), a micro- or nano-immunoassay device (e.g., as described in U.S. Patent 7,271,007), a lateral flow device (e.g., as described in U.S. Publication 20040228761 or U.S. Publication 20040265926), a fluorescence polarization immunoassay (FPIA, e.g., as described in U.S. Patent 4,593,089 or U.S. Patent 4,751,190), or an immunoturbidimetric assay (e.g., as described in U.S. Patent 5,571,728 or U.S. Patent 6,248,597).

[0215] Measurement of TNFR1 levels The titration method of the present disclosure may also include a step of measuring the expression of TNFR1 by mesenchymal progenitor or stem cells. TNFR1 may be soluble TNFR1 (sTNFR1). This step may be performed after co-culture of mesenchymal progenitor or stem cells with T cells. Alternatively, TNFR1 expression may be measured in a cell lysate of isolated, enriched, or expanded mesenchymal progenitor or stem cells prior to co-culture with T cells. In one embodiment, TNFR1 expression is measured in a cell lysate of cryopreserved enriched and / or expanded mesenchymal progenitor or stem cells.

[0216] Those skilled in the art will appreciate that the methods described above for detecting IL-2Rα expression can also be used to detect TNFR1 expression. In a preferred embodiment, cell lysates are assayed using ELISA or FLISA.

[0217] In one format, such an assay involves immobilizing a TNFR1-binding protein on a solid matrix.Then, a test sample is directly contacted with the TNFR1-binding protein, resulting in the binding or capture of TNFR1 in the sample.After washing to remove all unbound proteins in the sample, a protein that binds to TNFR1 at a different epitope is directly contacted with the captured TNFR1.This detection protein is usually labeled as described above.Alternatively, a secondary labeled protein that binds to the detection protein may be used.After washing to remove all unbound proteins, in the case of the above-mentioned ELISA, a substrate is added to detect the detectable reporter molecule.Then, the level of TNFR1 in the sample is measured using a standard curve prepared using a known amount of marker or by comparing with a control sample.

[0218] In one embodiment, TNFR1 expression is measured in cell lysates of cryopreserved enriched and / or expanded mesenchymal progenitor or stem cells, where at least 100 pg / mL TNFR1 indicates biological activity or therapeutic effect.

[0219] Compositions and Administration A pharmaceutically acceptable carrier may be used to prepare a composition comprising mesenchymal precursor or stem cells. As used herein, the term "pharmaceutically acceptable carrier" refers to a composition of matter that facilitates the storage, administration, and / or maintains the biological activity of mesenchymal precursor or stem cells.

[0220] In one example, the carrier does not cause significant local or systemic adverse events in recipients. Pharmaceutically acceptable carriers can be solid or liquid. Useful examples of pharmaceutically acceptable carriers include, but are not limited to, diluents, solvents, surfactants, excipients, suspending agents, buffers, lubricants, adjuvants, vehicles, emulsifiers, absorbing agents, dispersion media, coating agents, stabilizers, protective colloids, adhesives, thickeners, thixotropic agents, penetrating agents, sequestering agents, scaffolds, isotonicity agents, and absorption retardants, which do not affect the viability and activity of mesenchymal progenitor or stem cells. The selection of suitable carriers is within the skill of those skilled in the art.

[0221] The compositions of the present disclosure may be conveniently provided in unit dosage form and may be prepared by any method known in the art. As used herein, the term "unit dosage form" refers to a physically discrete unit suitable for a single dose for a subject to be treated, each unit containing a predetermined amount of active compound calculated to cooperate with a pharmaceutical carrier to produce the desired therapeutic or preventive effect. The dose of mesenchymal precursor or stem cells may vary depending on factors such as the condition, age, sex, and weight of the subject to be treated.

[0222] The term "subject" refers to an animal, preferably a mammal, including non-primates (e.g., cows, pigs, horses, cats, dogs, rats, or mice) and primates (e.g., monkeys or humans). In a preferred embodiment, the subject is a human.

[0223] An exemplary dose is at least about 1 x 10 6 For example, the dose may be about 1.0 x 10 cells. 6 ~Approx. 1×10 10 cells, e.g., about 1.1 x 10 6 ~Approx. 1×10 9 Cells, e.g., about 1.2 x 10 6 ~Approx. 1×10 8 cells, e.g., about 1.3 x 10 6 ~Approx. 1×10 7 cells, e.g., about 1.4 x 10 6 ~Approx. 9×10 6cells, e.g., about 1.5 x 10 6 ~Approx. 8×10 6 cells, e.g., about 1.6 x 10 6 ~Approx. 7×10 6 cells, e.g., about 1.7 x 10 6 ~Approx. 6×10 6 Cells, e.g., about 1.8 x 10 6 ~Approx. 5×10 6 cells, e.g., about 1.9 x 10 6 ~Approx. 4×10 6 Cells, e.g., about 2 x 10 6 ~Approx. 3×10 6 It may contain cells.

[0224] In one example, the dose is about 5 x 10 5 ~2×10 7 Cells, e.g., about 6 x 10 6 Cells ~ approx. 1.8×10 7 The dose may be, for example, about 6 x 10 cells. 6 cells or approximately 1.8 x 10 7 It may also be a cell.

[0225] Mesenchymal progenitor or stem cells comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the cell population of the composition.

[0226] The compositions of the present disclosure may be cryopreserved. Cryopreservation of mesenchymal progenitor or stem cells can be performed using slow-cooling or "rapid" freezing protocols well known in the art. Preferably, the cryopreservation method maintains the phenotype, cell surface markers, and proliferation rate of the cryopreserved cells similar to those of unfrozen cells.

[0227] The cryopreserved composition may contain a cryopreservation solution, the pH of which is usually 6.5 to 8, preferably 7.4.

[0228] The cryopreservation solution may contain a sterile, non-pyrogenic, isotonic solution, such as PlasmaLyte A™. 100 mL of PlasmaLyte A™ contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H 11 Contains 368 mg of sodium acetate trihydrate, USP (C2H3NaO2·3H2O); 37 mg of potassium chloride, USP (KCl); and 30 mg of magnesium chloride, USP (MgCl2·6H2O). Contains no antimicrobial agents. pH is adjusted with sodium hydroxide. pH is 7.4 (6.5-8.0).

[0229] The cryopreservation medium may include Profreeze™. The cryopreservation medium may additionally or alternatively include a culture medium.

[0230] To facilitate freezing, cryoprotectants, such as dimethyl sulfoxide (DMSO), are typically added to cryopreservation solutions. Ideally, cryoprotectants should be nontoxic, nonantigenic, and chemically inert to cells and patients, confer high post-thaw survival rates, and allow for transplantation without irrigation. However, DMSO, the most commonly used cryoprotectant, exhibits some degree of cytotoxicity. Hydroxyethyl starch (HES) may be used as an alternative or in combination with DMSO to reduce the cytotoxicity of cryopreservation solutions.

[0231] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulking agents. In one example, the cryopreservation solution includes about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further include one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0232] In one embodiment, cells are suspended in 42.5% Profreeze™ / 50% αMEM / 7.5% DMSO and allowed to cool in a temperature-controlled freezer.

[0233] The cryopreserved composition may be thawed and administered directly to a subject, or may be added to another solution containing, for example, HA. Alternatively, the cryopreserved composition may be thawed and the mesenchymal progenitor or stem cells resuspended in another carrier before administration.

[0234] The compositions of the present disclosure may be administered via a route suitable for the particular condition to be treated.For example, the compositions of the present disclosure may be administered systemically, i.e., parenterally, intravenously, or by injection.The compositions of the present disclosure may be targeted to a specific tissue or organ.

[0235] Dosage regimens may be adjusted to provide the optimal therapeutic effect, for example, by administering a single bolus or by gradually administering several divided doses, or the dose may be reduced or increased proportionately according to the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage.

[0236] In some embodiments, it may not be necessary or desirable to immunosuppress a patient before initiating treatment with a cell composition. Indeed, transplantation of allogeneic STRO-1+ cells in sheep has been well tolerated without immunosuppression. However, in other instances, it may be desirable or appropriate to administer pharmacological immunosuppression to a patient before initiating cell therapy. This can be achieved by using systemic or local immunosuppressants, or by delivering the cells using an encapsulation device. The cells may be encapsulated within a capsule that is permeable to the nutrients and oxygen required by the cells and the cell therapy factors, but impermeable to immune humoral factors and the cells. Preferably, the encapsulation material is hypoallergenic, easily and stably positioned in the target tissue, and provides additional protection to the transplant construct. These and other methods for reducing or eliminating immune responses to transplanted cells are well known in the art. Alternatively, the cells may be genetically modified to reduce their immunogenicity.

[0237] It is understood that the mesenchymal progenitor or stem cells may be administered with other beneficial drugs or biomolecules (growth factors, trophic factors). When administered with other factors, the mesenchymal progenitor or stem cells may be administered in a single pharmaceutical composition or in separate pharmaceutical compositions, simultaneously or sequentially with the other factors (either before or after the other factors are administered). Bioactive factors that may be co-administered include anti-apoptotic factors (e.g., EPO, EPO mimetibody, TPO, IGF-I and IGF-II, HGF, caspase inhibitors), anti-inflammatory agents (e.g., p38 MAPK inhibitors, TGF-β inhibitors, statins, IL-6 and IL-1 inhibitors, Pemirolast™, Tranilast™, Remicade™, Sirolimus™, and nonsteroidal anti-inflammatory drugs (NSAIDs), such as Tepoxalin™, Tolmetin™, and Suprofen™; immunosuppressants / immunomodulators (e.g., calcineurin inhibitors, such as cyclosporine and tacrolimus); mTOR inhibitors (e.g., Sirolimus™, Everolimus™); antiproliferative agents (e.g., azathioprine, mycophenolate mofetil); corticosteroids (e.g., prednisolone, hydrocortisone); antibodies, such as monoclonal anti-IL-2Rα receptor antibodies (e.g., basiliximab, dapagliflozin ... clizumab), polyclonal anti-T cell antibodies (e.g., antithymocyte globulin (ATG); antilymphocyte globulin (ALG); monoclonal anti-T cell antibody OKT3); antithrombotic drugs (e.g., heparin, heparin derivatives, urokinase, PPack (dextrophenylalanine proline arginine chloromethyl ketone), antithrombin compounds, platelet receptor antagonists, antithrombin antibodies, antiplatelet receptor antibodies, aspirin, dipyridamole, protamine, hirudin, prostaglandin inhibitors, and antiplatelet drugs); and antioxidants (e.g., probucol, vitamin A, ascorbic acid, tocopherol, coenzyme Q-10, glutathione, L-cysteine, N-acetylcysteine), as well as local anesthetics.

[0238] Graft-versus-host disease and its staging Acute and chronic graft-versus-host disease (GVHD) are multisystem disorders that are complications of allogeneic hematopoietic cell transplantation (usually in the form of bone marrow or peripheral blood stem cell harvest). GVHD develops when transplanted immune cells (the graft) from a different donor recognize the transplant recipient (the host) as foreign, thereby initiating a disease-causing immune response in the transplant recipient. Clinical symptoms of acute GVHD include a classic maculopapular rash, persistent nausea and / or vomiting, abdominal cramps with diarrhea, and elevated serum bilirubin levels. In contrast, patients with chronic GVHD typically exhibit skin infiltrates resembling lichen planus or scleroderma, dry oral mucosa with gastrointestinal ulceration and sclerosis, and elevated serum bilirubin levels.

[0239] GVHD is commonly classified as acute or chronic based on the time of onset, using a 100-day cutoff. However, this general classification has been challenged by the recognition that symptoms of acute and chronic GVHD can appear outside of these designated time periods. This observation has led to the increasing use of clinical findings, rather than a set time period, to distinguish between acute and chronic GVHD. The widely accepted National Institutes of Health (NIH) consensus criteria for diagnosing GVHD classify GVHD symptoms as "diagnostic" or "specific" for chronic GVHD, or as common to both acute and chronic GVHD (Filipovich AH et al. (2005) Biol Blood Marrow Transplant 11:945).

[0240] Patients with GVHD are subdivided based on the timing of onset and presenting characteristics: Classical acute GVHD - Cases that occur within 100 days of hematopoietic cell transplantation (HCT) and show features of acute GVHD without diagnostic and specific features of chronic GVHD. Persistent, recurrent, or late-onset acute GVHD - cases occurring >100 days after HCT and showing features of acute GVHD, without diagnostic and specific features of chronic GVHD. Classic chronic GVHD - may occur at any time after HCT, has diagnostic and specific features of chronic GVHD, and lacks features of acute GVHD. Overlap syndrome - a condition that can occur any time after HCT and exhibits features of both chronic and acute GVHD. This is sometimes colloquially referred to as an "acute exacerbation" of "chronic" GVHD.

[0241] The pathophysiology of acute GVHD has been well described and extensively reviewed (Ferrara JL et al. (2006) Semin Hematol 43(1):3-10). Briefly, events leading to the development of clinically apparent GVHD begin during conditioning, when transplant recipients receive high-dose chemotherapy and / or radiation therapy. Tissue damage caused by high-dose therapy leads to the activation of host antigen-presenting cells (APCs), upregulation of major histocompatibility antigens on the APC surface, and presentation of host antigens. T lymphocytes from the donor infused with the stem cell graft respond to antigenic differences in this environment with clonal proliferation, tissue migration, and direct cell-to-cell cytotoxicity. High levels of inflammatory cytokines (especially tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and interleukin-2 (IL-2)) and abundant host antigens trigger an inflammatory cascade that can result in severe tissue damage, organ dysfunction, and death.

[0242] Clinically significant acute GVHD develops in patients undergoing allogeneic hematopoietic cell transplantation (HCT) despite intensive immunosuppressive prophylaxis. The exact incidence of acute GVHD after allogeneic HCT is unknown. Incidence rates in patients undergoing allogeneic HCT from genotypically HLA-identical siblings have been reported to range from 9 to 50 percent (Lee SE et al. (2013) Bone Marrow Transplant 48:587).

[0243] Acute GVHD is also common in matched unrelated donors and haploidentical related donors.

[0244] Numerous studies have identified the following risk factors for developing acute graft-versus-host disease (GVHD) (Hahn T et al. (2008) J Clin Oncol 26:5728): · HLA mismatch degree (HLA-mismatched donor or unrelated donor); · Donor-recipient gender mismatch (female donor vs. male recipient); · Intensity of transplant conditioning; acute GVHD prophylaxis used; and · Source of graft (peripheral blood or bone marrow in larger quantities than cord blood)

[0245] Less established risk factors include increasing host age, donor and host cytomegalovirus (CMV) status, donor Epstein-Barr virus (EBV) seropositivity (Styczynski J et al. (2016) J Clin Oncol 34:2212), peripheral blood stem cell transplantation versus bone marrow transplantation, the presence of a sterile environment (including enteric sterilization), and certain HLA haplotypes. However, risk factors for acute GVHD differ from the underlying disease, and different risk models for each condition are required (Hahn T et al. (2008) J Clin Oncol 26:5728).

[0246] Graft-versus-host disease (GVHD) is a common complication of allogeneic hematopoietic cell transplantation (HCT), typically manifesting early after transplantation. Early signs and symptoms of acute GVHD most commonly appear around the time of leukocyte engraftment. While the original definition of acute GVHD required symptom onset before 100 days post-transplant, current National Institutes of Health (NIH) consensus criteria use clinical findings rather than a defined time frame to distinguish acute from chronic GVHD. Thus, patients who exhibit typical features of acute GVHD before day 100 are considered to have "classic acute GVHD," while patients who exhibit the same features after day 100 (usually once immunosuppression has worn off) are classified as having "late-onset acute GVHD" (Vigorito AC et al. (2009) Blood 114:702). Some clinicians also use the terms "early-onset acute GVHD" or "hyperacute GVHD" to describe symptoms of acute GVHD that appear within 14 days of transplantation (Sullivan KM et al. (1986) Blood 67:1172).

[0247] organ invasion The skin, gastrointestinal tract, and liver are the primary target organs in patients with acute GVHD. The first (and most common) clinical symptom of acute GVHD in most patients is a maculopapular rash, which usually appears at or around the time of leukocyte engraftment. The rash first appears on the nape of the neck, ears, shoulders, palms of the hands, and soles of the feet. The rash may resemble sunburn and may be itchy or painful. Histological examination of the skin reveals changes in the dermal and epidermal layers (Sale GE et al. (1977) Am J Pathol 89:621). Characteristic findings include lymphocytes released by exocytosis, abnormally keratinized epidermal keratinocytes, follicular infiltrates, satellite lymphocytes adjacent to or surrounding abnormally keratinized epidermal keratinocytes, and lymphocytic infiltrates around dermal vessels (Darmstadt GL et al. (1992) J Invest Dermatol 99:397). The stage of skin infiltration is combined with information on the stage of gastrointestinal and hepatic infiltration to determine the overall severity grade of acute GVHD.

[0248] Acute GVHD often involves both the upper and lower gastrointestinal tract. Gastrointestinal involvement usually manifests with diarrhea and abdominal pain, but nausea, vomiting, and anorexia may also occur. Pathological evaluation of tissue obtained by upper endoscopy, rectal biopsy, or colonoscopy provides a definitive diagnosis. Diagnosis of gastrointestinal involvement requires pathological evaluation of tissue. Once diagnosed, the degree of gastrointestinal involvement is graded based on the severity of diarrhea: Stage 1—500–1000 mL / day diarrhea; Stage 2—1000–1500 mL / day diarrhea; Stage 3—1500–2000 mL / day diarrhea; and Stage 4—>2000 mL / day diarrhea or pain or bowel obstruction.

[0249] Involvement of the lower gastrointestinal tract by acute GVHD is often severe and is characterized by diarrhea (with or without bloody stool) and abdominal cramps. Diagnosis is confirmed by pathological evaluation of tissue obtained by rectal biopsy or colonoscopy. Patients with acute GVHD may develop severe diarrhea (occasionally exceeding 10 liters per day). Stool may initially be watery but often becomes bloody. Diarrhea is secretory and characteristically persistent and unremitting, despite fasting. Diarrhea may be accompanied by crampy abdominal pain, which can also be difficult to manage. Severe intestinal obstruction may develop in association with acute GVHD or due to increased opioid use required to control physical discomfort. Rectal biopsy is generally useful in making the diagnosis of acute GVHD affecting the gastrointestinal tract. Histological examination reveals crypt cell necrosis due to the accumulation of degenerative material within the dead crypts.

[0250] Involvement of the upper gastrointestinal tract by acute GVHD often manifests as anorexia, dyspepsia, food intolerance, nausea, and vomiting (Weisdorf DJ et al. (1990) Blood 76:624). Patients may also present with gingivitis and mucositis, although these findings are more commonly due to the effects of transplant conditioning. Positive biopsies from upper endoscopy of the esophagus and stomach confirmed the diagnosis. Differential diagnoses include herpes simplex virus or candida esophagitis, gastritis, peptic ulcer disease, and gastrointestinal toxicity from chemotherapy and / or radiation.

[0251] Liver infiltration is usually seen in patients with signs of acute skin and / or gastrointestinal GVHD (Ratanatharathorn V et al. (1998) Blood 92:2303). Patients rarely have moderate to severe liver GVHD without evidence of other organ involvement. Although liver infiltration can be suggested by abnormal liver function tests in the setting of skin or gastrointestinal GVHD, a liver biopsy is required to confirm hepatic GVHD. Liver infiltration is revealed by abnormal liver function tests, with elevated serum levels of conjugated bilirubin and alkaline phosphatase being the earliest and most common findings. While serum cholesterol is usually elevated, coagulation disorders and hyperammonemia, although very rare, may develop in severe cases. Patients may also present with painful hepatomegaly, dark urine, pale stools, fluid retention, and itching. Fever, anorexia, and nausea are common nonspecific symptoms. Although the occurrence of a characteristic rash provides suggestive clinical evidence, a biopsy is the most definitive method for diagnosing liver GVHD. However, this biopsy may not be feasible immediately after HCT due to the potential for acute bleeding due to severe thrombocytopenia.

[0252] Diagnostic biomarkers for acute GVHD The use of serum biomarkers to diagnose acute GVHD is an area of ​​active research. Biomarkers or panels of biomarkers are usually used in combination with each other or with other findings. An ideal biomarker would not only predict the onset of clinical acute GVHD but also guide its management. Many candidate biomarkers exist, but none are ready for clinical application.

[0253] One candidate biomarker is tumor suppressor 2 (ST2), a member of the interleukin-1 receptor family.

[0254] Proteomic analysis of plasma and urinary polypeptide patterns has demonstrated promise for early diagnosis of acute GVHD (Srinivasan R et al. (2006) Exp Hematol 34:796). For example, it has been proposed that a panel of markers including interleukin-2 receptor-α, tumor necrosis factor receptor-1, interleukin-8, and hepatocyte growth factor can confirm the diagnosis of acute GVHD at the onset of clinical symptoms and provide prognostic information independent of GVHD severity (Paczesny S et al. (2009) Blood 113:273). The use of Reg3 has also proven useful in the diagnosis of acute gastrointestinal GVHD (Ferrara JL et al. (2011) Blood 118:6702). In addition, plasma levels of CD30 have been found to be elevated in patients with acute GVHD (Chen YB et al. (2012) Blood 120:691).

[0255] Analysis of plasma microRNA signatures may provide noninvasive biomarkers for acute GVHD. In one study, evaluating a panel of six microRNAs was able to distinguish HCT recipients with acute GVHD from those without and predict the severity of acute GVHD (Xiao B et al. (2013) Blood 122:3365). When four markers were incorporated into a panel predicting acute GVHD, the levels of miRNA biomarkers were clearly associated with the severity of acute GVHD. More importantly, elevated levels of these miRNAs can be detected before the onset of acute GVHD. These data remain to be confirmed in a larger patient cohort.

[0256] Staging of GVHD Several systems for grading acute GVHD have been developed. The two most common are the Glucksberg grade (I-IV) (Glucksberg H et al. (1974) Transplantation 18(4):295) and the International Bone Marrow Transplant Registry (IBMTR) grading system (A-D) (Rowlings PA et al., (1997) Br J Hematol 97(4):855). The severity of acute GVHD is determined by assessing the degree of involvement of the skin, liver, and gastrointestinal tract. The stage of individual organ involvement, with or without Glucksberg (IBMTR), is combined with the patient's performance status to create an overall grade that has prognostic significance. Grade I (A) GVHD is described as mild disease, grade II (B) GVHD as moderate disease, grade III (C) GVHD as severe disease, and grade IV (D) GVHD as life-threatening disease (Przepiorka D, Weisdorf D, Martin P, Klingemann HG, Beatty P, Hows J, Thomas ED (1995) Bone Marrow Transplant. 1995;15(6):825; Cahn JY et al. (2005) Blood 106(4):1495).

[0257] The IBMTR grading system grades the severity of acute GVHD as follows: Grade A - Stage 1 skin involvement (maculopapular rash covering <25 percent of the body) only, without hepatic or gastrointestinal involvement Grade B - Stage 2 skin involvement, Stage 1-2 gastrointestinal involvement or liver involvement Grade C - Stage 3 infiltration in any organ system (generalized erythroderma, bilirubin 6.1-15.0 mg / dL, diarrhea 1500-2000 mL / day) Grade D - Stage 4 infiltration in any organ system (generalized erythroderma with blistering, bilirubin >15 mg / dL, diarrhea >2000 mL / day or pain or bowel obstruction) Define

[0258] Grading is important in assessing the effectiveness of prophylaxis or treatment, its impact on survival, and the graft-versus-leukemia effect. Patients with moderate to severe GVHD have a significantly higher mortality rate than those with mild disease. As an example, the estimated 5-year survival rates for patients with grade III (C) and grade IV (D) acute GVHD are 25% and 5%, respectively (Przepiorka D et al. (1995) Bone Marrow Transplant 15:825). However, caution must be exercised when applying these survival estimates to the current patient population, where post-HCT care has changed. Current preventive treatments may alter overall outcomes and disease manifestations. Typically, initial grading for each organ is calculated within a 10-day window (-5 to +5 days) of initiation of steroid treatment. Thereafter, real-time staging and grading are determined weekly by the attending physician based on laboratory and clinical information and histological confirmation, if possible.

[0259] Recent studies (see, e.g., MacMillan ML et al (2010) Blood 115:5412-5417) have proposed that 28-day responses, including PR and Cr, be incorporated as early targets to predict later, more definitive outcomes in patients with acute GVHD.

[0260] Hematopoietic stem cell transplant (HSCT) A "hematopoietic stem cell transplant (HSCT)" refers to a transplant containing multipotent hematopoietic stem cells, which can be derived, for example, from bone marrow or peripheral blood. The transplant may also contain some non-stem cells, such as APCs, including DCs and / or lymphocytes.

[0261] "Hematopoietic stem cells" are capable of self-renewal and differentiation to give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, dendritic cells), erythroid (red blood cells), megakaryocytic (platelets), and lymphoid (T cells, B cells, NK cells) lineages. During differentiation, hematopoietic stem cells first lose their self-renewal capacity and then progressively lose lineage potential as they commit to becoming mature effector cells. Typically, Lin-, CD34+, CD38-, CD90+, CD45RA- human cells are hematopoietic stem cells. In one example, expression of CD34 is used to identify hematopoietic stem cells in peripheral blood isolated from a human donor.

[0262] HSCT can be used to treat diseases and conditions requiring stem cell transplants. For example, stem cells can be used to treat disorders or dysfunction of normal blood cell production and maturation, hematopoietic malignancies, autoimmune diseases, liver diseases, or immunodeficiencies (e.g., due to radiation exposure, chemotherapy, or infection with pathogens).

[0263] The stem cells may be expanded or differentiated ex vivo prior to administration to a subject.

[0264] Allogeneic hematopoietic stem cell transplants may be used to treat one or more of the following conditions: acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, myelodysplastic syndromes, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, aplastic anemia, pure red cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, hemophagocytic lymphohistiocytosis (HLH), inborn errors of metabolism (e.g., mucopolysaccharidoses, Gaucher disease, metachromatic leukodystrophy, and adrenoleukodystrophy).

[0265] Preventing or treating GVHD The mesenchymal precursor or stem cells and / or progeny thereof may be used in a method for preventing or treating a subject with GVHD, comprising administering to the subject the cells in an amount effective to prevent or treat. In one embodiment, the subject has received one or more allogeneic hematopoietic stem cell transplants that result in GVHD.

[0266] Symptoms of GVHD include skin hardening, limited oral intake, dry eyes, gastrointestinal (GI) tract symptoms such as dysphagia, loss of appetite, nausea, vomiting, abdominal pain, or diarrhea, liver symptoms manifested by elevated bilirubin, elevated alkaline phosphatase, and elevated alanine aminotransferase (ALT) / aspartate aminotransferase (AST) ratios, shortness of breath, and / or stiffness in the arms or legs.

[0267] A subject may exhibit multiple symptoms depending on the tissues affected by graft-versus-host disease. Some subjects may exhibit 4-5 symptoms, while others may exhibit 1-2 symptoms. Mesenchymal progenitor or stem cells and / or their progeny may be used to treat one, more, or all of the symptoms associated with GVHD in a subject.

[0268] The effective amount per injection of mesenchymal progenitor or stem cells and / or their progeny for treating GVHD or symptoms of GVHD in mammals, particularly humans, is 1 x 104 cells / kg (weight)~1×10 8 cells / kg (body weight), 1×10 4 cells / kg (weight)~1×10 8 cells / kg (body weight), 2×10 4 cells / kg (weight)~1×10 8 cells / kg (body weight), 2.5×10 4 cells / kg (weight)~1×10 8 cells / kg (body weight), 2×10 4 cells / kg (weight)~1×10 7 cells / kg (body weight), 2.5×10 4 cells / kg (weight)~I×10 7 cells / kg (body weight), 2×10 4 cells / kg (weight)~3×10 6 cells / kg (body weight), 2.5×10 4 cells / kg (weight)~3×10 6 cells / kg (body weight), 2×10 4 cells / kg (weight)~2×10 6 cells / kg (body weight), 2.5×10 4 cells / kg (weight)~2×10 6 cells / kg (body weight), 2×10 4 cells / kg (weight)~1×10 6 cells / kg (body weight), 2.5×10 4 cells / kg (weight)~1×10 6 cells / kg (body weight), 2×10 4 cells / kg (weight)~1×10 5 cells / kg body weight, or 2.5 x 10 4 cells / kg (weight)~1×10 5 It may be cells / kg body weight.

[0269] The mesenchymal precursor or stem cells and / or their progeny may be surgically implanted, injected, delivered (e.g., using a catheter or syringe), or otherwise administered directly or indirectly to a subject's site in need of repair or augmentation, e.g., an organ, or into the blood system.

[0270] In one embodiment, mesenchymal precursor or stem cells and / or their progeny are delivered to the subject's bloodstream.For example, mesenchymal precursor or stem cells and / or their progeny are delivered parenterally.Exemplary routes of parenteral administration include, but are not limited to, intravenous, intramuscular, subcutaneous, intraarterial, intraperitoneal, intraventricular, intracerebroventricular, and intradural.

[0271] In one embodiment, the mesenchymal precursor or stem cells and / or their progeny are injected into the delivery site, for example, using a syringe or via a catheter or central line.

[0272] The choice of administration regimen for a therapeutic formulation depends on several factors, including the rate of metabolism of the component in serum or tissue, the level of symptoms, and the immunogenicity of the component. Preferably, the administration regimen maximizes the amount of therapeutic compound delivered to the patient while accommodating an acceptable level of side effects.

[0273] In one example, the mesenchymal precursor or stem cells and / or their progeny are delivered in a single bolus dose. Alternatively, the mesenchymal precursor or stem cells and / or their progeny are administered by continuous infusion.

[0274] Although not limited to any particular method of administration, parenteral administration is preferred. Administration can be systemic or local, but systemic administration is preferred, with intravenous injection being most preferred.

[0275] The mesenchymal precursor or stem cells and / or their progeny, or compositions comprising them, can be used in combination with other active agent(s). For example, the mesenchymal precursor or stem cells of the present disclosure may be combined with corticosteroids, nonsteroidal anti-inflammatory compounds, or other agents effective in treating inflammation. Combinations of mesenchymal precursor or stem cells with these other agents may be administered simultaneously or sequentially; that is, the mesenchymal precursor or stem cells, or compositions comprising them, may be administered before or after treatment with one or more other active agents.

[0276] In one embodiment, the mesenchymal precursor or stem cells and / or their progeny are administered prior to, simultaneously with, or following administration of the hematopoietic stem cells.

[0277] In another embodiment, the hematopoietic stem cells are co-cultured with mesenchymal precursor or stem cells and / or their progeny prior to administration to the subject.

[0278] The attending physician may determine the appropriate sequence for administering the mesenchymal precursor or stem cells, or compositions comprising them, in combination with other active agents.

[0279] Treatment of inflammatory diseases The present disclosure also provides a method for treating an autoimmune disease in a subject. The method comprises administering to the subject a mesenchymal precursor or stem cell, or progeny thereof, described herein, in an amount effective to treat the autoimmune disease in the subject. Autoimmune diseases that can be treated according to the present disclosure include, but are not limited to, multiple sclerosis, type 1 diabetes, rheumatoid arthritis, uveitis, celiac disease, lupus, autoimmune thyroid disease, inflammatory bowel disease, autoimmune lymphoproliferative disorder (ALPS), demyelinating diseases, autoimmune encephalomyelitis, autoimmune gastritis (AIG), and autoimmune glomerular disease.

[0280] genetically modified cells In one embodiment, mesenchymal precursor or stem cells are genetically modified, eg, to express and / or secrete a protein of interest, eg, a protein that provides a therapeutic and / or prophylactic effect.

[0281] The method for genetically modifying cells is clear to those skilled in the art.For example, the nucleic acid to be expressed in cells is functionally linked to a promoter for inducing expression in cells.For example, the nucleic acid is functionally linked to various intracellular promoters of interest, such as viral promoters, for example, CMV promoters (for example, CMV-IE promoters) or SV-40 promoters.Other suitable promoters are well known in the art.

[0282] It is preferred to provide the nucleic acid in the form of an expression construct. As used herein, the term "expression construct" refers to a nucleic acid that functions to confer expression on an operably linked nucleic acid in a cell (e.g., a reporter gene and / or a counterselectable reporter gene). Within the context of the present disclosure, it is understood that an expression construct can include or be a plasmid, bacteriophage, phagemid, cosmid, viral subgenomic or genomic fragment, or other nucleic acid capable of maintaining and / or replicating heterologous DNA in an expressible form.

[0283] Methods for constructing suitable expression constructs for carrying out the present invention will be clear to those skilled in the art and are described, for example, in Ausubel FM, 1987 (including all current revisions); or Sambrook & Green, 2012. For example, each of the components of the expression construct may be amplified from a suitable template nucleic acid, for example, using PCR, and subsequently cloned into a suitable expression construct, such as a plasmid or phagemid.

[0284] Suitable vectors for such expression constructs are well known in the art and / or described herein. For example, suitable expression vectors for the methods of the present invention for use in mammalian cells include, for example, vectors from the pcDNA vector series (Invitrogen), pCI vector series (Promega), pCMV vector series (Clontech), pM vector (Clontech), pSI vector (Promega), VP16 vector (Clontech), or vectors from the pcDNA vector series (Invitrogen).

[0285] Those skilled in the art will be aware of alternative vectors and sources of such vectors, such as, for example, Invitrogen Corporation, Clontech or Promega.

[0286] Methods for introducing isolated nucleic acid molecules or gene constructs containing them into cells for expression are well known to those skilled in the art. The techniques used for any given organism depend on well-known successful techniques. Methods for introducing recombinant DNA into cells include, inter alia, microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection using, for example, Lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and microparticle bombardment using, for example, DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0287] Alternatively, the expression construct of the present invention is a viral vector. Suitable viral vectors are well known in the art and commercially available. Conventional viral-based systems for delivering nucleic acids and integrating them into the host cell genome include, for example, retroviral vectors, lentiviral vectors, or adeno-associated viral vectors. Alternatively, adenoviral vectors are useful for introducing episomally maintained nucleic acids into host cells. Viral vectors are an efficient and versatile method for introducing genes into target cells and tissues. In addition, high transduction efficiency has been observed in many different cell types and target tissues.

[0288] For example, retroviral vectors typically contain cis-acting long terminal repeats (LTRs) capable of packaging up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for vector replication and packaging, and are used to integrate expression constructs into target cells for long-term expression. Widely used retroviral vectors include vectors based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SrV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., International Publication WO 1994 / 026877; Buchschacher & Panganiban, 1992; Johann et al., 1992; Sommerfelt & Weiss, 1990; Wilson et al., 1989; Miller et al., 1991; Lynch, et al., 1991; Miller & Rosman, 1989; Miller, 1990; Scarpa et al., 1991; Burnset et al., 1993).

[0289] Various adeno-associated virus (AAV) vector systems have also been developed for nucleic acid delivery. AAV vectors can be easily constructed using techniques well known in the art (see, for example, U.S. Patents 5,173,414 and 5,139,941; International Publications WO92 / 01070 and WO93 / 03769; Lebkowski et al., 1988; Vincent et al., 1990; Carter, 1992; Muzyczka, 1992; Kotin, 1994; Shelling & Smith, 1994; Zhou et al., 1994).

[0290] Other viral vectors useful for delivering the expression constructs of the invention include, for example, viral vectors derived from poxviruses, such as vaccinia virus and avipox virus, or alphaviruses, or conjugated viral vectors (e.g., as described in Fisher-Hoch et al., 1989).

[0291] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0292] hematopoietic cells In some instances, the recipient subject receives a donor graft containing hematopoietic cells. In further instances, the donor graft is bone marrow or peripheral blood mononuclear cells (PBMCs) collected from blood. Such grafts include hematopoietic cells. Terms such as graft, bone marrow graft, and PBMC graft are used to refer to grafts containing hematopoietic cells. Hematopoietic cell transplantation (HCT) is an important and potentially curative treatment option for a wide variety of malignant and non-malignant diseases. The multipotent hematopoietic stem cells required for this procedure are typically obtained from the bone marrow or peripheral blood of related or unrelated donors. Umbilical cord blood (blood remaining in the umbilical cord and placenta after the birth of an infant) has emerged as an established alternative source of hematopoietic stem cells in allogeneic HCT.

[0293] As referred to herein, the term "hematopoietic cells" is a general term meaning progenitor / hematopoietic stem cells derived from any source (e.g., bone marrow, peripheral blood, umbilical cord blood). In other cases, the source of such cells is specifically described (e.g., autologous peripheral blood progenitor cell transplantation).

[0294] In some cases, PBMCs are obtained by apheresis of a donor. In this case, the donor may be an unrelated donor, in which case the graft is called an allogeneic PBMC graft. In other cases, PBMCs are obtained from the recipient before receiving treatment (e.g., chemotherapy treatment). In this case, the graft is called an autologous PBMC graft.

[0295] In some cases, PBPCs are obtained after the donor (or recipient) receives a series of granulocyte colony stem cell factor (G-CSF) injections. Without being bound by theory, the subject typically receives a daily subcutaneous injection of G-CSF, and the subject's white blood cell count is monitored every few days to monitor stem cell mobilization into the peripheral blood. In some cases, the subject receives G-CSF injections every day for at least 7 consecutive days. In some cases, the subject receives G-CSF injections every day for at least 10 consecutive days. In some cases, G-CSF may be combined with another factor (e.g., plerixafor injection or stem cell factor (SCF)).

[0296] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. [Example]

[0297] Example 1 Materials and Methods Mesenchymal progenitor or stem cells (MLPSCs) prepared using the plastic adhesion method MLPSCs were generated fresh from bone marrow as described in US 5,837,539. Approximately 80–100 ml of bone marrow was aspirated into a sterile heparin-containing syringe and sent to the MDACC Cell Therapy Laboratory for MSC generation. Bone marrow mononuclear cells were isolated using Ficoll-Hypaque and placed into two T175 flasks containing 50 ml per flask of MLPSC growth medium containing alpha-modified MEM (αMEM) supplemented with gentamicin, glutamine (2 mM), and 20% (vol / vol) fetal bovine serum (FBS) (Hyclone).

[0298] The cells were cultured at 37°C under 5% CO for 2–3 days, at which point nonadherent cells were removed and the remaining adherent cells were continuously cultured (7–10 days) until the cells reached 70% or greater confluence, at which point the cells were trypsinized and transferred into six T175 flasks containing growth medium (50 ml of medium per flask).

[0299] Immunoselection of mesenchymal progenitor or stem cells (MLPSCs) Bone marrow (BM) was collected from healthy, normal adult volunteers (20-35 years of age). Briefly, 40 ml of BM was aspirated from the posterior iliac crest into a tube containing lithium heparin anticoagulant.

[0300] Bone marrow mononuclear cells (BMMNC) were prepared by density gradient separation using Lymphoprep™ (Nycomed Pharma, Oslo, Norway) as previously described by Zannettino et al., 1998. After centrifugation at 400 × g for 30 min at 4°C, the buffy coat was removed using a transfer pipette and washed three times with "HHF," which consisted of Hank's Balanced Salt Solution (HBSS; Life Technologies, Gaithersburg, MD) containing 5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

[0301] Subsequently, STRO-3+ (or TNAP+) cells were isolated by magnetic-activated cell sorting as previously described by Gronthos & Simmons, 1995; and Gronthos, 2003. Briefly, approximately 1-3 × 10 cells were sorted using a blocking buffer consisting of 10% (vol / vol) normal rabbit serum in HHF. 8BMMNCs are incubated on ice for 20 minutes. Cells are incubated with 200 μl of a 10 μg / ml solution of STRO-3 mAb in blocking buffer for 1 hour on ice. Subsequently, cells are washed twice with HHF by centrifugation at 400 × g. A 1 / 50 dilution of goat anti-mouse γ-biotin (Southern Biotechnology Associates, Birmingham, UK) in HHF buffer is added, and cells are incubated on ice for 1 hour. BMMNCs are incubated in MACS buffer (1% BSA, 5 mM EDTA, and 0.01% sodium azide supplemented with CaCl ) as described above. 2+ and Mg 2+ The cells are washed twice with PBS (without HCl) and resuspended in a final volume of 0.9 ml of MACS buffer.

[0302] 100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) were added to the cell suspension and incubated on ice for 15 minutes. The cell suspension was washed twice and resuspended in 0.5 ml of MACS buffer. Subsequently, the cell suspension was loaded onto a mini MACS column (MS Columns, Miltenyi Biotec) and washed three times with 0.5 ml of MACS buffer to recover cells that did not bind to STRO-3 mAb (deposited with the American Type Culture Collection (ATCC) on December 19, 2005, under accession number PTA-7282 (see International Publication WO 2006 / 108229)). After adding an additional 1 ml of MACS buffer, the column was removed from the magnet and TNAP+ cells were isolated using positive pressure. An aliquot of cells from each fraction was stained with streptavidin-FITC and purity was assessed by flow cytometry.

[0303] Measurement of IL-2Rα expression Preparation of culture medium DMEM containing 10% fetal bovine serum and 2 mM GlutaMax-I was prepared, filtered through a 0.2 μm filter, and labeled with a 1-month expiration date at 2–8°C. Before use, prewarm the medium for a minimum of 30 min in a 37°C water bath.

[0304] Preparation of 0.5% trypsin / EDTA EDTA containing 0.5% EDTA was prepared, filtered through a 0.2 μm filter, labeled with a 12-month expiration date or the expiration date of the reagent (whichever came first), and stored in aliquots at −20°C.

[0305] Thawing MLPSC Sample vials for the MLPSC batch were thawed. A maximum of two samples were thawed per batch. Sample vials were removed from the vapor-phase LN2 freezer and transferred to dry ice if thawing occurred within ≤15 minutes, or to wet ice if thawing occurred immediately (≤1 minute). Sample vials were placed in a 37°C ± 2°C water bath for ≤5 minutes to thaw, then removed from the water bath, sprayed or wiped with 70% isopropyl alcohol, and transferred to a safety cabinet. Using a 10 ml syringe fitted with a 16 g or larger needle, cells were transferred dropwise into a 50 ml centrifuge tube containing 15 ml of pre-warmed medium and mixed thoroughly. Subsequently, cells were passed through a 40 μm cell strainer and collected into a new 50 ml tube. Next, cells were centrifuged at 250 × g for 8 minutes at room temperature or 2–8°C. The supernatant was discarded and 8 ml of medium (target concentration 3–5 × 10) was added. 6 Cells were resuspended in 0.2 ml of PBS (0.01% of cells / ml). Cells were gently triturated to ensure a uniform suspension. A trypan blue cell count was performed on a 0.2 ml sample. The average viability was ≥ 70%. The above procedure was repeated when testing duplicate samples.

[0306] Preliminary culture of MLPSCs At least three, preferably four, T175 flasks at 6,857 cells / cm 2Cells were seeded with 1000 cells / ml of 10000 cells. The flasks were gently shaken to evenly distribute the cells, followed by overnight incubation at 37°C ± 2°C, 5 ± 2% CO2 for up to 24 hours. Cells were removed from the incubator and examined under a microscope to ensure ≥30% confluence, and cell morphology was observed for visible signs of contamination. The medium was aseptically aspirated from each flask and replaced with 30 mL of pre-warmed medium. Flasks were incubated at 37°C ± 2°C, 5 ± 2% CO2 for a total of 60–84 hours from the start of incubation on day 1.

[0307] Preparation of co-culture medium Co-culture medium was prepared by adding equal volumes of medium and DMEM. Before use, the co-culture medium was pre-warmed in a 37°C water bath for a minimum of 30 minutes.

[0308] Thawing PBMCs Thaw vials of PBMCs as required. The number of vials to thaw depends on the total number of cells per vial (based on manufacturer's claims). >2 x 10 cells per vial. 7 For viable cells, only one vial was thawed. The vial(s) were removed from the vapor-phase LN2 freezer and placed on dry ice if thawing occurred within ≤15 minutes, or on wet ice if thawing occurred immediately (≤1 minute). The vials were placed in a 37°C ± 2°C water bath for 2-3 minutes to thaw, then removed from the water bath, sprayed or wiped with 70% isopropyl alcohol, and transferred to a safety cabinet. Using a 10 ml syringe fitted with a 16 g or larger needle, the cells were transferred dropwise into a 50 ml centrifuge tube containing 20 ml of prewarmed medium and mixed thoroughly. The vial was rinsed with medium, and any remaining cells were added to the tube. The cells were then passed through a 40 μm cell strainer and collected in a new 50 ml tube. The cells were then centrifuged at 350 × g for 5 minutes at room temperature or 2-8°C. The supernatant was discarded, and 2.5 × 10 6 Cells were resuspended in co-culture medium at 2 × 10 cells / ml. Trypan blue cell counts were performed using 0.2 ml samples. The average viability was ≥ 70%. 68 ml of PBMCs (1.6 x 10 cells / ml) 7 PBMCs were cultured on ice while MLPSCs were prepared. After cell counting, the total viable cells were <1.6 × 10 7 If so, thaw another vial of PBMCs and repeat the above procedure.

[0309] Preparation of MLPSCs The mesenchymal cells were removed from the incubator and examined under a microscope to observe cell adhesion and cell morphology (long, flat fibroblasts). The medium was aspirated from the flasks and the cells were washed with 9 ml of pre-warmed DPBS. Subsequently, the DPBS was aspirated and 4 ml of pre-warmed 0.05% trypsin / EDTA was added per flask. The flasks were shaken to cover and incubated at 37°C for 3-6 minutes. The sides of the flasks were gently tapped during incubation. The cells were inspected under a microscope to ensure complete detachment. If not, the flasks were gently tapped again. 7 ml of pre-warmed medium was added to each flask to transfer the cells. The cells were pooled from the flasks into a 50 ml conical tube. An additional 1 ml of pre-warmed medium was added to the tube to rinse the flasks and remove any remaining cells. The cells were then centrifuged at 350 x g for 5 minutes at room temperature or 2-8°C. The supernatant was aspirated and the cells were resuspended in 2 ml of co-culture medium per flask. Trypan blue cell counts were performed using 0.2 ml samples. The average viability was ≥ 70%. 4 × 10 5 1.5 ml of MLC (6 x 10 cells / ml) 5 Total MPCs) were prepared and set aside while PBMCs were stimulated.

[0310] Stimulation of PBMCs Five ml of the PBMC suspension was transferred to a 15 ml conical tube and 20 μl of 1 mg / ml CD3 antibody stock and 20 μl of 1 mg / ml CD28 antibody stock were added to give final concentrations of 4 μg / ml anti-CD3 antibody and 4 μg / ml anti-CD28 antibody (stimulated PBMCs). The remaining PBMCs were not stimulated. The 24-well plate layout was as shown below. [Table 1]

[0311] 500 μl of unstimulated PBMCs (negative control) was added to wells B2, B3, C2, and C3 of a 24-well plate. 500 μl of stimulated PBMCs (positive control) was added to wells B4 and C4. 500 μl of stimulated PBMCs was added to wells A5, B5, C5, and D5. When only one MLPSC sample was tested, 500 μl of stimulated PBMCs was added to wells A5 and B5 only. 500 μl of medium-grown MLPSCs (sample 1) was added to co-culture wells A5 and B5. 500 μl of medium-grown MLPSCs (sample 2) was added to co-culture wells C5 and D5. 500 μl of co-culture medium was added to wells B2, B3, B4, C2, C3, and C4. To limit evaporation loss, all empty wells were filled with 1000 μl of co-culture medium. These conditions represented a ratio of approximately 1 MLC:5 PMBC.

[0312] The plates were cultured at 37°C ± 2°C under 5 ± 2% CO2 for 60 to 84 hours, and the morphology was observed (see Figure 1).

[0313] ELISA IL-2Rα expression was measured using a commercially available ELISA kit (R&D Systems) according to the manufacturer's instructions. ELISA was performed according to the manufacturer's protocol. This assay employs a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) technique. The assay employs microplates containing wells precoated with a monoclonal antibody specific for IL-2Rα. IL-2R present in standard samples, quality control samples, or coculture samples was captured with the immobilized IL-2Rα antibody. After washing away all unbound material, an enzyme-linked polyclonal antibody specific for IL-2Rα was added to the wells. After a washing step to remove all unbound enzyme-linked antibody, a substrate solution was added to the wells, allowing color development proportional to the amount of bound IL-2Rα. Color development was then stopped, and the color intensity was measured using an ELISA reader. Details of the ELISA are described below.

[0314] Using a 1000 μl pipette, cells in each well were collected and transferred to their corresponding microcentrifuge tubes. The wells were rinsed with 200 μl of prewarmed DPBS, and any remaining cells were added to the tubes. The culture plate was observed under a microscope to confirm complete cell removal. The cells were centrifuged at maximum rpm for 90 seconds at room temperature or 2–8°C. The supernatant was aspirated, and the cell pellet was placed on ice while the lysis buffer was prepared. The lysis buffer was prepared by adding one complete Mini-Tablet to 10 ml of CellLytic-M Cell Lysis Extraction Reagent. The lysis buffer was vortexed to mix and stored on ice. 250 μl of lysis buffer was added to each tube. Each pellet was resuspended using a 1000 μl pipette and then vortexed. The cells were incubated on ice for 15 minutes. The replicate lysates were then pooled and mixed. The lysates were centrifuged at maximum rpm for 10 minutes at room temperature or 2-8°C, and the lysates were transferred to new tubes, taking care to avoid any pelleted material. Lysates were stored at ≤-60°C for up to 29 days, or the ELISA was performed the same day. If the ELISA was performed the same day, the lysates were first frozen on dry ice or in a ≤-60°C freezer for a minimum of 15 minutes.

[0315] All samples and reagents were allowed to warm to ambient temperature and left for at least 30 minutes before use. 20 ml of concentrated wash buffer was diluted into 480 ml of NANO water (to obtain a 1:25 dilution) and mixed thoroughly. The solutions were labeled with a one-month expiration date or the kit expiration date (whichever came first) and stored at 2–8°C. Three rows of wells on a microplate were assigned to standards, controls, and samples. Extra rows were omitted from the microplate. Lysis buffer was prepared by adding two complete Mini-Tablets to 20 ml of CellLytic-M Reagent. The lysis buffer was mixed by vortexing and stored on ice. 1 ml of lysis buffer was added to the two IL-2Rα standards to prepare 5000 pg / ml standards. The standards were incubated at room temperature for a minimum of 30 minutes with gentle agitation. After incubation, the volumes of each standard were pooled, and serial dilutions and control dilutions were prepared according to Tables 1 and 2 below. [Table 2] [Table 3]

[0316] Unstimulated samples were prepared according to Table 3 below. [Table 4]

[0317] Co-culture samples were prepared according to Table 4 below. [Table 5]

[0318] 100 μl of Assay Diluent RD1-1 was added to all wells of a microplate precoated with anti-IL-2Rα monoclonal antibody. 50 μl of standard, control, or sample was added to the appropriate wells. Polyclonal anti-IL-2Rα HRP conjugate was added to each well, the plate was covered, and the plate was incubated at room temperature for 3 hours ± 20 minutes with gentle agitation on an orbital shaker. The plate was then washed four times with 300 μl of wash buffer per well. After the final wash, residual liquid was removed by blotting onto absorbent paper. Within 15 minutes of use, a substrate solution was prepared by mixing equal parts of Reagent A and Reagent B. 200 μl of substrate solution was added to each well, the plate was covered, and the plate was incubated at room temperature in the dark for 20 ± 5 minutes. 50 μl of stop solution was added to each well, and the absorbance (OD) of each sample was read within 5 to 30 minutes on a microplate reader set to 450 nm (wavelength correction at 570 nm). A standard curve was constructed using four-parameter logistic curve fitting. The concentration of IL-2Rα in each sample was derived from the standard curve and corrected for dilution to obtain the final result.

[0319] Measurement of TNFR1 expression Preparation of culture medium DMEM containing 10% fetal bovine serum and 1 mM GlutaMax-I was prepared, filtered through a 0.2 μm filter, and labeled with a 1-month expiration date at 2–8°C. Before use, prewarm the medium for a minimum of 30 min in a 37°C water bath.

[0320] Preparation of 0.5% trypsin / EDTA EDTA containing 0.5% EDTA was prepared, filtered through a 0.2 μm filter, labeled with a 12-month expiration date or the expiration date of the reagent (whichever came first), and stored in aliquots at −20°C.

[0321] Thawing of mesenchymal cells Sample vials for the MLPSC batch were thawed. A maximum of two samples were thawed per batch. Sample vials were removed from the vapor-phase LN2 freezer and transferred to dry ice if thawing occurred within ≤15 minutes, or to wet ice if thawing occurred immediately (≤1 minute). The sample vials were placed in a 37°C ± 2°C water bath for ≤5 minutes to thaw, then removed from the water bath, sprayed or wiped with 70% isopropyl alcohol, and transferred to a safety cabinet. Using a 10 ml syringe fitted with a 16 g or larger needle, 2 ml of cells were transferred dropwise into a 50 ml centrifuge tube containing 8 ml of pre-warmed medium and mixed thoroughly. The cells were then passed through a 40 μm cell strainer and collected in a new 50 ml tube. The cells were then centrifuged at 250 × g for 5 minutes at room temperature or 2–8°C. The supernatant was discarded, and 5 ml of medium (target concentration 3–5 × 10) was added. 6 Cells were resuspended in 0.2 ml of PBS (0.01% of cells / mL). Cells were gently triturated to ensure a uniform suspension. A trypan blue cell count was performed on a 0.2 ml sample. The average viability was ≥ 70%. The above procedure was repeated when testing duplicate samples.

[0322] ELISA TNFRI expression was measured using a commercially available ELISA kit, Quantikine (R&D Systems), according to the manufacturer's instructions. The ELISA was performed according to the manufacturer's protocol. This assay provides measurements of both soluble and cell-bound TNFRI (Qjwang et al., 1997). This assay employs quantitative sandwich enzyme immunoassay technology. The assay employs microplates containing wells precoated with a monoclonal antibody specific for TNFRI. TNFRI present in standard samples, quality control samples, or MPC cell lysate samples was captured with the immobilized TNFRI antibody. After washing away all unbound material, an enzyme-linked polyclonal antibody specific for TNFRI was added to the wells. After a washing step to remove all unbound enzyme-linked antibody, a substrate solution was added to the wells, allowing color development proportional to the amount of bound TNFRI. Color development was then stopped, and the color intensity was measured using an ELISA reader. Details of the ELISA are described below.

[0323] Cells were centrifuged at 250 x g for 5 minutes at room temperature or 2-8°C. The supernatant was aspirated, and the cell pellet was placed on ice while the lysis buffer was prepared. Lysis buffer was prepared by adding two complete Mini-Tablets to 20 ml of CellLytic-M cell lysis extraction reagent. The lysis buffer was mixed by vortexing and stored on ice. Lysis buffer was added. Each pellet was resuspended using a 1000 μl pipette and then vortexed. The cells were incubated on ice for 10-15 minutes, vortexing every 5 minutes. Duplicate lysates were then pooled, mixed, and transferred to one or more 2 ml microcentrifuge tubes. The lysates were centrifuged at maximum rpm for 10 minutes at 2-8°C, and the lysates were transferred to a new tube, taking care to avoid all pelleted material. Lysates were stored at ≤-60°C for up to 29 days, or ELISA was performed the same day. If the ELISA was performed on the same day, the lysate was first frozen on dry ice or in a <-60°C freezer for a minimum of 1 hour.

[0324] All samples and reagents were allowed to warm to ambient temperature and left for at least 30 minutes before use. 20 ml of concentrated wash buffer was diluted into 480 ml of NANO Water (to obtain a 1:25 dilution) and mixed thoroughly. The solutions were labeled with a one-month expiration date or the kit expiration date (whichever came first) and stored at 2–8°C. Three rows of wells on the microplate were assigned to standards, controls, and samples. Extra rows were omitted from the microplate. Lysis buffer was prepared by adding two complete Mini-Tablets to 20 ml of CellLytic-M Reagent. The lysis buffer was mixed by vortexing and stored on ice. 0.5 ml of lysis buffer was added to the sTNFR1 standard solution to prepare a 5000 pg / ml standard. The standard solution was incubated at room temperature with gentle agitation for a minimum of 15 minutes. After incubation, serial dilutions and control dilutions were prepared according to Tables 5 and 6 below. [Table 6] [Table 7]

[0325] Samples were prepared according to Table 7 below. [Table 8]

[0326] 50 μl of Assay Diluent HD1-7 was added to all wells of a microplate precoated with anti-TNFR1 monoclonal antibody. 200 μl of standard, control, or sample was added to the appropriate wells. The plate was covered and incubated for 2 hours ± 10 minutes at room temperature with gentle agitation on an orbital shaker. The plate was then washed three times with 300 μl of wash buffer per well. After the final wash, residual liquid was removed by blotting onto absorbent paper. Polyclonal anti-TNFR1 HRP conjugate was added to each well, the plate was covered, and the plate was incubated for 2 hours ± 10 minutes at room temperature with gentle agitation on an orbital shaker. The plate was then washed three times with 300 μl of wash buffer per well. After the final wash, residual liquid was removed by blotting onto absorbent paper. Within 15 minutes of use, a substrate solution was prepared by mixing equal parts of Reagent A and Reagent B. 200 μl of substrate solution was added to each well, and the plate was covered and incubated in the dark at room temperature for 20 ± 10 minutes. 50 μl of stop solution was added to each well, and the optical density (OD) of each sample was read within 5–30 minutes on a microplate reader set at 450 nm (wavelength corrected at 570 nm). A standard curve was constructed using four-parameter logistic curve fitting. The concentration of TNFR1 in each sample was derived from the standard curve and corrected for dilutions to obtain the final results.

[0327] statistical analysis The objective assessment of acute GVHD (aGVHD) response to treatment with MSCs or MPCs was determined by the overall response rate at day 28. To indicate changes in aGVHD organ stage, response data from baseline to day 28 in each organ were classified as complete remission, partial remission, worsening, or stable disease.

[0328] To assess the impact of response on overall survival, two Kaplan-Meier survival analyses were performed up to day 100. A Kaplan-Meier curve was generated for patients who achieved an overall response (complete or partial response) at day 28, and a separate Kaplan-Meier curve was generated for non-responders at day 28. The null hypothesis of no difference in overall survival between the two groups was tested using the log-rank test using commercially available software. The test was performed at a significance level of p<0.05.

[0329] Categorical variables were summarized as frequencies and percentages. Continuous variables were summarized using descriptive statistics (number, mean, standard deviation, median, and range). All confidence intervals had a 95% confidence level.

[0330] Example 2: Immunoselected mesenchymal progenitor or stem cells with improved immunosuppressive properties The immunosuppressive potential of immunoselected mesenchymal progenitor or stem cells (MLPSCs) was evaluated by comparing them with those obtained by a conventional production process (such as that described in U.S. Pat. No. 9,828,586). Figure 2 shows a comparison of the results of a T cell proliferation assay (IL2R inhibition rate) performed on three different samples of MLPSCs generated under conventional production conditions (i.e., samples MLPSC A, MLPSC B, and MLPSC C) with the results of a T cell proliferation assay (IL2R inhibition rate) performed on three different samples of improved immunoselected MLPSCs (i.e., samples MLPSC D, MLPSC E, and MLPSC F). These results demonstrate that the assay distinguishes between class 1 and class 2 pluripotent lineage cells based on their ability to inhibit T cell proliferation. Notably, class 2 cells inhibit T cell proliferation substantially more effectively than class 1 cells.

[0331] In contrast, the results of the TNFR1 expression assay shown in Figure 3 demonstrate no significant differences between the same two classes of mesenchymal progenitor or stem cells. TNFR1 expression has previously been described as a marker for identifying cells that inhibit T cell proliferation (see US20140248244).

[0332] The results provided herein in Figures 2 and 3 demonstrate that it is possible to generate improved mesenchymal progenitor or stem cell populations with enhanced ability to inhibit T cell proliferation. These improved mesenchymal progenitor or stem cell populations are subsets of cells that express high levels of TNFR1. In other words, TNFR1 expression can be separated from the T cell inhibitory properties of a particular mesenchymal progenitor or stem cell population. The fact that it is possible to generate mesenchymal progenitor or stem cell populations that exhibit enhanced ability to inhibit T cell proliferation, even after cryopreservation and thawing, is particularly surprising in light of common knowledge that cryopreserved mesenchymal stem cells exhibit impaired immunosuppressive properties after thawing (Francois et al., 2012; Chinnadurai et al., 2016).

[0333] Example 3: IL-2R-low inhibited mesenchymal progenitor or stem cells (MLPSCs) infused to treat steroid-resistant pediatric subjects This study is designed to treat pediatric patients who are not responding to steroid therapy for acute graft-versus-host disease (GVHD). Failure to steroid therapy for acute GVHD is defined as any grade B-D (IBMTR grading) acute GVHD without improvement after at least 3 days of methylprednisolone (≥ 1 mg / kg / day) or equivalent.

[0334] 2 × 10 MLPSCs were cultured ex vivo with low IL-2R inhibition, twice a week for each of four consecutive weeks. 6 Patients were treated with a dose of hMSCs / kg (actual body weight), with infusions spaced at least 3 days apart.

[0335] Study design: A total of 241 pediatric patients undergoing HSCT were enrolled and treated at 50 centers in North America and Europe between 2007 and 2014. Age: 2 months to 17 years Acute GvHD grade B-D (CIBMTR) Failed steroid treatment and multiple other medications aGVHD that has not improved after at least 3 days of methylprednisolone (at least 1 mg / kg / day or equivalent)

[0336] result: The overall response rate (CR+PR) at 28 days in 241 children under EAP was 65% (95% CI: 58.9%, 70.9%). Survival at 100 days was consistent with overall response and was significantly improved in children who responded at 28 days (82% vs. 39%, p<0.0001).

[0337] Example 4: IL-2R-inhibited high mesenchymal progenitor or stem cells (MLPSC) (low dose) infused to treat steroid-resistant pediatric subjects Test Purpose The primary objectives were to gather safety information for repeat doses of highly IL-2R-inhibiting MLPSCs administered intravenously to subjects with grade B-D aGVHD unresponsive to steroid treatment after allogeneic HSCT, and to evaluate the efficacy of repeat doses of MPCs administered intravenously to subjects with grade B-D aGVHD unresponsive to steroid treatment after allogeneic HSCT.

[0338] A secondary objective of this study was to determine the correlation between the effect on MPCs at 28 days and survival at 100 days.

[0339] Treatment planning 2 × 10 MPCs administered intravenously (IV) once a week for each of four consecutive weeks. 6 MPC / kg (weight at screening) dose * Pediatric subjects were treated with

[0340] If eligible, subjects will receive four additional MPC infusions after the initial four doses, with a dose of 1 x 10 6 MPC / kg once weekly. Eligibility for further treatment was determined by the subject's acute GVHD (aGVHD) response assessment (partial or combined) performed on Day 28.

[0341] Subjects received a maximum of eight infusions.

[0342] Evaluation of acute GVDH Conducted at screening, days 14, 28, 56, and 100 / end of study.

[0343] Target demographics Twelve (12) subjects were treated at seven transplant centers, with six of the subjects treated at the Fed Hutchinson Cancer Research Center and each of the remaining subjects treated at a different transplant center (as shown in Table 10).

[0344] All subjects were under 18 years of age, ranging from 3 to 17 years. The mean age was 10.3 years, and the median age was 10.5 years.

[0345] Subjects eligible for participation in the trial had to have not responded to steroid therapy for grade B to D acute GVHD (aGVHD) after allogeneic hematopoietic stem cell therapy (HSCT). Non-response to steroid therapy for acute GVHD was defined as any grade B to D acute GVHD that did not improve after at least three (3) days of methylprednisolone (>1 mg / kg / day) or equivalent.

[0346] All subjects had visceral disease involving the lower gastrointestinal tract (GI) and / or liver: 9 subjects had lower GI only (8 grade D, 1 grade B), 1 subject had lower GI grade C and liver grade D, 1 subject had upper GI grade B and lower GI grade C, and 1 subject had liver grade C.

[0347] At baseline, 9 of 12 subjects (9 / 12, or 75%) had grade D graft-versus-host disease (GVHD), 2 of 12 subjects (2 / 12, or 17%) had grade C GVHD, and 1 subject (1 / 12, or 18%) had grade B GVHD.

[0348] Subjects were treated with an average of 4.5 GVHD treatments prior to MPC treatment, including the following nonsteroidal treatments: extracorporeal photopheresis (ECP, 7 subjects), infliximab (5 subjects), ruxolitinib (3 subjects), mycophenolate (MMF, 3 subjects), etanacept (1 subject), or basiliximab (1 subject).

[0349] Based on the subject's weight at screening, 2 x 10 6 Subjects were treated intravenously (IV) at a dose of cells / kg. Cells were delivered to subjects once weekly (qw) for each of four consecutive weeks.

[0350] Eligible subjects will receive four additional cell infusions after the initial four doses, with a dose of 1 x 10 6 Patients received a reduced dose of 100 cells / kg once weekly. Eligibility for additional treatment was determined by the subject's acute GVHD (aGVHD) response assessment (partial or combined) on Day 28. Subjects received a maximum of eight infusions.

[0351] GVHD assessments were performed at screening, days 14, 28, 56, and 100 (end of study).

[0352] Response to treatment and survival rate up to 100 days Treatment with MLPSCs with high IL-2R inhibition (2 × 10 6 The effect on MPC / kg body weight (administered weekly) and subject survival is summarized in Table 9 below. After receiving cell infusion, GVHD assessments were performed at screening (day 0), 14, 28, 56, and 100 days. Body weight for calculating cell doses was based on the subject's weight at screening.

[0353] Ten of 12 subjects (10 / 12) (83%) survived to day 100, of whom 7 received 8 infusions and 3 received 4 infusions. All 12 subjects had lower GI and / or hepatic GVHD, 9 / 12 subjects (75%) had grade D GVHD at baseline, and only 1 subject (8%) had grade B GVHD at baseline. The mean number of prior therapies that led to acute GVHD was 4.25. [Table 9]

[0354] Figure 4 graphically depicts survival to day 100 for responders versus non-responders, with the probability of survival plotted on the y-axis as the number of days since first study treatment, with baseline day 0. All nine responders at day 28 survived to day 100, compared with one of three non-responders at day 28 who survived to day 28 (i.e., 75% vs. 8%).

[0355] The results show that the overall response rate (i.e., complete and partial response rates) at day 28 was 75%, which is higher than the average overall response rate observed after MSC infusion. Furthermore, the response rate observed at day 28 was a strong predictor of overall survival at day 100 (see Table 9).

[0356] Notably, no specific safety issues were observed after infusion of MLPSCs with high IL-2R inhibition.

[0357] The response to treatment with MLPSCs with high IL-2R inhibition and survival of individual subjects is shown in Table 10 provided below. [Table 10-1] [Table 10-2]

[0358] No safety issues were observed in treated patients, and a pilot study in GI tract disease demonstrated an overall response rate of 75% (Table 10). Of those subjects who responded at 28 days after infusion of highly IL-2R-inhibiting MLPSCs, 100% survived to day 100. This indicates that the response at day 28 is a sign of a long-lasting beneficial effect and is consistent with survival at day 100.

[0359] Therefore, the twice-weekly dose of 2 × 10 required when administering MLPSCs with low IL-2R inhibition. 6 Results indicate that comparable, if not higher efficacy rates could be achieved in subjects receiving high IL-2R inhibitory MLPSCs (in this case MPCs) at a once-weekly dose of half the cells / kg body weight dose (see Comparative Example 3 above).

[0360] Example 5 Improved production process of MSCs Given the excellent results obtained by treating GvHD with MLPSCs that exhibit high IL-2R inhibition (as described in Example 4), studies were conducted on a process for generating high-titer MLPSCs from stem cells isolated using plastic adherence methods.

[0361] A conventional production process for producing MLPSCs from cells isolated using plastic adhesion is described in US 9,828,586. A number of modifications were introduced into this conventional production process, as described below. Surprisingly, this improved production process (described below) resulted in the production of MLPSCs with improved immunosuppressive properties.

[0362] Equipment changes One equipment change concerns the method used to wash, transfer, and concentrate cells. The previous process used a Cytomate Cell Processor (Baxter) at several different steps in these workflows. This single device is a benchtop instrument with disposable instruments uniquely designed for washing, concentrating, and transferring white blood cells. In the previous process, the Cytomate was used to seed cells into culture vessels (cell factories), and upon harvest, the Cytomate was used to wash the cells and concentrate them by reducing their volume. In the new process, the Cytomate replaces the procedure for the cell seeding step (syringe tree) and the procedure for the cell washing and concentration steps (tangential flow filtration (TFF)).

[0363] Other changes in process and testing methods The following aspects of the production and assay of ceMSC constructs were also performed using the improved production process. To reduce the risk of aseptic processing, a cell factory was used with an air filter installed (pre-assembled) before sterilization. To minimize the risk of animal-derived adventitious agents, such as parvovirus and circovirus, the use of porcine trypsin was eliminated and replaced with recombinant trypsin (produced using yeast), which required changes to the cells and solutions used in the process, as well as changes to the trypsin incubation time. ·Introduced the use of blood filters to reduce / minimize the possibility of cell clumps and visible particulate matter. Introduced the use of cryovials with a 4.3 mL fill volume as the final container instead of cryobags with a 15 mL fill volume. The same concentration of cells / mL as in the previous process was maintained, but instead of one cryobag, an equivalent number of cells was distributed across four vials.

[0364] After thawing, all final product assays were performed on samples of cryopreserved cells in the final product. In the conventional process, some assays were performed on cell aliquots stored in tubes (not representative of the final container). Figure 5 shows all steps from thawing the donor cell bank (DCB) to cryopreservation and assay of the ce-MSC product.

[0365] Example 6 - Assay of MLPSC constructs obtained by the improved production process The final product lot of MLPSCs produced according to the improved production process described in Example 5 was tested for the sales standards listed in Table 8 below. [Table 11]

[0366] Table 8 shows consistently high levels of TNFR1 expression across all of the product lots tested. As shown in Figure 6, each product lot exhibited TNFR1 expression levels >275 pg / ml.

[0367] Table 8 also shows unexpectedly high levels of inhibition of IL-2Rα expression by the 10 product lots tested. As shown in Figure 7, each product lot exhibited levels of inhibition of 80% or greater.

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Claims

1. A composition comprising mesenchymal progenitor or stem cells, wherein the mesenchymal progenitor or stem cells have been cryopreserved and, after thawing, inhibits the proliferation of activated T cells in a sample of PBMCs by at least about 65%.

2. 2. The composition of claim 1, wherein said inhibition of proliferation of activated T cells is measured by inhibition of IL-2R 2Rα expression in said activated T cells.

3. 3. The composition of claim 1 or claim 2, wherein the mesenchymal progenitor or stem cells inhibit T cell proliferation by at least about 65% when co-cultured with PMBCs at a ratio of no more than 1 mesenchymal progenitor or stem cell:5 PMBCs.

4. 3. The composition of claim 1 or claim 2, wherein the mesenchymal progenitor or stem cells inhibit T cell proliferation by at least about 65% when co-cultured with PMBCs at a ratio of 1 mesenchymal progenitor or stem cell:10 PMBCs or less.

5. 3. The composition of claim 1 or claim 2, wherein the mesenchymal progenitor or stem cells inhibit T cell proliferation by at least about 65% when co-cultured with PMBCs at a ratio of 1 mesenchymal progenitor stem cell:50 PMBCs or less.

6. 3. The composition of claim 1 or claim 2, wherein the mesenchymal stem cells inhibit T cell proliferation by at least about 65% when co-cultured with PBMCs at a ratio of 1 mesenchymal progenitor or stem cell:100 PBMCs or less.

7. 7. The composition of any one of claims 1 to 6, wherein the composition inhibits T cell proliferation by at least about 70% when mesenchymal progenitor or stem cells are co-cultured with PMBCs at a ratio of no more than 1 mesenchymal progenitor or stem cell:5 PMBCs.

8. 8. The composition of any one of claims 1 to 7, wherein the composition inhibits T cell proliferation by at least about 80% when co-cultured with mesenchymal progenitor or stem cells with PMBCs at a ratio of no more than 1 mesenchymal progenitor or stem cell:5 PMBCs.

9. 9. The composition of any one of claims 1 to 8, wherein the mesenchymal progenitor or stem cells express TNFR1 in an amount of at least 270 pg / ml.

10. 9. The composition of any one of claims 1 to 8, wherein the mesenchymal progenitor or stem cells express TNFR1 in an amount of at least 300 pg / ml.

11. 9. The composition of any one of claims 1 to 8, wherein the mesenchymal progenitor or stem cells express TNFR1 in an amount of at least 320 pg / ml.

12. 12. The composition of any one of claims 1 to 11, wherein the mesenchymal progenitor or stem cells are isolated by immunoselection.

13. 13. The composition of any one of claims 1 to 12, wherein the mesenchymal progenitor or stem cells are mesenchymal stem cells grown in culture.

14. 14. A method for treating an inflammatory disease in a subject in need thereof, the method comprising administering to the subject a composition comprising mesenchymal precursor or stem cells according to any one of claims 1 to 13.

15. 15. The method of claim 14, wherein the inflammatory disease is a T cell-mediated inflammatory disease.

16. 14. A method for preventing, alleviating the progression of, or treating graft-versus-host disease (GVHD) in a subject, the method comprising administering to the subject a composition comprising mesenchymal precursor or stem cells described in any one of claims 1 to 13.

17. The composition is 3×10 ６ 17. The method of any one of claims 14 to 16, wherein the subject is administered once a week (qw) at a dose of less than 100 cells / kg body weight.

18. The composition contains about 2×10 ６ 18. The method of any one of claims 14 to 17, wherein the subject is administered a dose of 1000 cells / kg body weight qw.

19. The composition is 2×10 ６ 19. The method of any one of claims 14 to 18, wherein the maximum dose of cells / kg body weight is administered qw to the subject.

20. 20. The method of any one of claims 14 to 19, wherein the composition is administered in a single dose or in divided dose(s).

21. 1. A method for preventing, attenuating the progression of, or treating graft-versus-host disease (GVHD) in a mammalian subject, comprising administering to a mammalian subject 3×10 6 mesenchymal progenitor or stem cells (MLPSCs) and / or their progeny. ６ administering to the subject once weekly (qw) at a dose of less than MPC / kg body weight.

22. The subject receives approximately 2 x 10 MLPSCs and / or their progeny. ６ 22. The method of claim 21, wherein the antibody is administered qw at a dose of cells / kg body weight.

23. The subject receives 2×10 MLPSCs and / or their progeny. ６ 23. The method of claim 21 or claim 22, wherein the maximum dose of cells / kg body weight is administered qw.

24. 24. The method of any one of claims 14 to 23, wherein the mammal is a pediatric human subject.

25. 25. The method of any one of claims 14 to 24, wherein the subject has a hematological malignancy or genetic disorder.

26. 26. The method of any one of claims 14 to 25, wherein the subject has received, is receiving, or is about to receive a donor graft comprising hematopoietic cells.

27. 27. The method of claim 26, wherein the graft comprises hematopoietic stem cells (HSCs).

28. 28. The method of claim 26 or claim 27, wherein the graft comprises allogeneic hematopoietic cells.

29. 29. The method of any one of claims 14 to 28, wherein the MLPSCs are mesenchymal progenitor cells (MPCs).

30. 29. The method of any one of claims 14 to 28, wherein the MLPSCs and / or their progeny are administered early on the day of transplantation of the graft.

31. 29. The method of any one of claims 14 to 28, wherein the MLPSCs and / or their progeny are administered after the subject has been determined to be steroid-resistant.

32. 31. The method of any one of claims 14 to 30, wherein the subject has acute GVHD.

33. 32. The method of any one of claims 14 to 31, wherein the cells are administered in the form of a pharmaceutically acceptable composition.

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