Potency Assays and Methods of Manufacturing

Abstract

The present disclosure relates to improved cell compositions and potency assays for obtaining same, which may be suitable for use in connection with T-cell mediated diseases such as graft-versus-host disease (GvHD).

Description

[Technical Field]

[0001] The present disclosure relates to improved cell compositions and potency assays for obtaining same. Such compositions and assays may be suitable for use in T-cell mediated diseases such as graft-versus-host disease (GvHD). [Background technology]

[0002] Although several cell therapy products for regenerative or immunotherapy applications have progressed to clinical evaluation and manufacturing approval, their market launch has been delayed due to their complexity and diversity, making it difficult to identify relevant biological activity and therefore to define consistent quality for cell therapy products.

[0003] Physiochemical parameters (e.g., size, morphology, light scattering properties, tensile strength, cell number, confluence characterization, 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 whether a product is biologically active and efficacious (i.e., whether it elicits the desired effect). Biological characterization, on the other hand, considers the product's effect on biological systems modeled either in vitro or in vivo in animals and ultimately in the clinic.

[0004] For example, there is a need to develop cell products 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 them (e.g., via potency testing) so that a consistent quality product can be obtained. Summary of the Invention

[0005] The present disclosure provides: i) IL-2Rα inhibition by stem cells in vitro correlates with stem cell suppression of T cell activation in vivo; and ii) The surprising finding that the level of IL-2Rα inhibition by stem cells in vitro is unexpectedly effective in identifying pharmaceutical agents that improve survival in GvHD patients, particularly those with severe GvHD. Either or both of these findings provide a basis for using IL-2Rα inhibition as a potency assay, and also provide a baseline level of inhibition that can be used as a release criterion for a pharmaceutical product. Thus, in a first example, the disclosure relates to a method for selecting a cell population for use in treating graft-versus-host disease (GvHD), the method comprising: (i) obtaining a population of mesenchymal progenitor or stem cells (MLPSCs); (ii) culturing the MLPSC population in a culture medium; (iii) determining the level of IL-2RA inhibition under the culture conditions; and (iv) selecting, for use in a treatment, a MLPSC population that inhibits IL-2RA by at least 56% under the culture conditions. In one example, the method comprises selecting, for use in a treatment, a MLPSC population that inhibits IL-2RA by at least 60% under the culture conditions. In one example, the method comprises selecting, for use in a treatment, a MLPSC population that inhibits IL-2RA by at least 65% under the culture conditions.

[0006] In one example, the method further comprises cryopreserving the selected cells.

[0007] In another example, the disclosure relates to a method for determining the therapeutic efficacy of a population of expanded mesenchymal progenitor or stem cells (MLPSCs), comprising (i) obtaining a population of MLPSCs, (ii) culturing the cells in a culture medium, and (iii) determining the level of IL-2RA inhibition under the culture conditions, wherein the method determines the therapeutic efficacy against graft-versus-host disease (GvHD), and IL-2RA inhibition under the culture conditions of at least 56% indicates therapeutic efficacy. In another example, IL-2RA inhibition under the culture conditions of 60% or greater indicates therapeutic efficacy. In another example, IL-2RA inhibition under the culture conditions of 65% or greater indicates therapeutic efficacy. For example, IL-2RA inhibition under the culture conditions of 65.7% indicates therapeutic efficacy.

[0008] In another example, the disclosure relates to a method for producing a pharmaceutical product comprising a population of MLPSCs, the method comprising obtaining a determination of whether a test population of MLPSCs inhibits IL-2RA under culture conditions at a predetermined level, and processing at least a portion of the test population of MLPSCs into a pharmaceutical product to produce the pharmaceutical product if the test population of MLPSCs inhibits IL-2RA under culture conditions to at least the predetermined level, or discarding at least a portion of the test population of MLPSCs if the population of mesenchymal stem cells inhibits IL-2RA under culture conditions below the predetermined level, where the predetermined level is 56%. In one example, the predetermined level is 60% or greater under culture conditions. In one example, the predetermined level is 65% or greater under culture conditions.

[0009] In another example, the disclosure relates to a method of treating a subject suffering from graft-versus-host disease (GvHD), the method comprising administering to a subject in need thereof a composition comprising an expanded population of mesenchymal progenitor or stem cells (MLPSCs), wherein the MLPSCs inhibit IL-2RA by at least 56% under culture conditions. In another example, the expanded MLPSCs contained in the administered composition inhibit IL-2RA by 60% or more under culture conditions. In another example, the expanded MLPSCs contained in the administered composition inhibit IL-2RA by 65% ​​or more under culture conditions.

[0010] In one example, the GvHD is acute GvHD. In one example, the GvHD is chronic GvHD. In one example, the GvHD is pediatric GvHD. In one example, the GvHD is refractory to steroid therapy. In one example, the GvHD is grade D GvHD.

[0011] In one example, the increase in 100-day survival rate with treatment is greater than 60%, greater than 70%.

[0012] In one example, the disclosure relates to a composition comprising a population of expanded MLPSCs, wherein the population of MLPSCs is selected based on a predetermined level of IL-2RA inhibition under culture conditions, the predetermined level of IL-2RA inhibition being at least 56%. In one example, the predetermined level of IL-2RA inhibition is 60% or greater. In one example, the predetermined level of IL-2RA inhibition is 65% or greater.

[0013] In one example, the MLPSCs are mesenchymal stem cells. In one example, the MSCs are one or more or all of CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, and MHC1+. In one example, the MSCs are selected based on the expression of one or more or all of CD29, CD54, CD73, CD90, CD102, CD105, CD106, CD166, and MHC1.

[0014] In one example, the level of IL-2RA inhibition is 65% or greater.

[0015] In one example, the disclosure provides a method for producing a pharmaceutical comprising a population of MLPSCs, the method comprising: (i) obtaining a determination of whether a test population of MLPSCs inhibits IL-2RA at a predetermined level under culture conditions, wherein step (i) comprises co-culturing the test population of MLPSCs with CD3 / CD28-activated PBMCs and determining the level of IL-2RA inhibition relative to a control population of CD3 / CD28-activated PBMCs in the absence of MLPSCs, wherein the predetermined level of IL-2RA inhibition is at least 56%.

[0016] In one example, the control population of CD3 / CD28-activated PBMCs expresses at least 12,000 pg / ml of IL-2RA. In one example, the control population of CD3 / CD28-activated PBMCs expresses at least 11,000 pg / ml of IL-2RA. In one example, the control population of CD3 / CD28-activated PBMCs expresses at least 13,000 pg / ml of IL-2RA. In one example, the control population of CD3 / CD28-activated PBMCs expresses at least 10,000 pg / ml of IL-2RA. In one example, the control population of CD3 / CD28-activated PBMCs expresses at least 12,811 pg / ml of IL-2RA.

[0017] In one example, CD3 / CD28 activation of PBMCs comprises stimulating PBMCs with anti-CD3 and anti-CD28 antibodies.

[0018] In one example, PBMCs are co-cultured with MLPSCs at a ratio of PBMC:MPLSC=5:1.

[0019] In one example, 1 x 10 6 2 x 10 PBMCs 5 are co-cultured with MLPSCs.

[0020] In one example, PBMCs and MLPSCs are co-cultured for about 72 hours.

[0021] In one example, the level of IL-2RA inhibition is determined by (i) measuring IL-2RA levels in a test population of MLPSCs co-cultured with CD3 / CD28-activated PBMCs, a positive control population of CD3 / CD28-activated PBMCs in the absence of MLPSCs, and a negative control population of unactivated PBMCs in the absence of MLPSCs; (ii) generating a standard curve to determine IL-2RA levels in the positive control population, negative control population, and test population; and (iii) calculating the percent inhibition of IL-2RA levels by the test population of MLPSCs co-cultured with CD3 / CD28-activated PBMCs compared to the positive control population, where the positive control is a CD3 / CD28-activated PBMC population.

[0022] In one example, IL-2RA levels are measured by enzyme-linked immunosorbent assay (ELISA).

[0023] In one example, the standard curve is generated using four-parameter logistic (4-PL) curve fitting. [Brief explanation of the drawings]

[0024] [Figure 1] Correlation between IL-2Rα inhibition and clinical outcomes. [Figure 2-1] Product efficacy correlates with survival in high-risk patients. [Figure 2-2] Product efficacy correlates with survival in high-risk patients. [Figure 3-1] Product efficacy and changes in inflammatory biomarkers. [Figure 3-2] Product efficacy and changes in inflammatory biomarkers. [Figure 3-3] Product efficacy and changes in inflammatory biomarkers. [Figure 3-4] Product efficacy and changes in inflammatory biomarkers. [Figure 3-5] Product efficacy and changes in inflammatory biomarkers. [Figure 4] Product efficacy and changes in activated T cells. [Figure 5-1] Relationship between PBMC stimulation and IL-2Rα inhibition by MSC lot. [Figure 5-2] Relationship between PBMC stimulation and IL-2Rα inhibition by MSC lot. [Figure 6] Survivors of GvHD clinical trials who received an MSC product with a high mean rate of IL2R inhibition. [Figure 7] Maintenance of efficacy as measured by IL2Rα inhibition rate in a repeat study after 8 years. [Figure 8-1] Accurate measurement of the rate of IL-2Rα inhibition in commercial stocks requires appropriate PBMC stimulation and induction levels of IL-2Rα expression. [Figure 8-2] Accurate measurement of the rate of IL-2Rα inhibition in commercial stocks requires appropriate PBMC stimulation and induction levels of IL-2Rα expression. DETAILED DESCRIPTION OF THE INVENTION

[0025] Throughout this specification, unless otherwise specified or the context requires otherwise, references to a single step, composition, group of steps or group of compositions should be interpreted to encompass both one and multiple (i.e., one or more) of that step, composition, group of steps or group of compositions.

[0026] Those skilled in the art will understand that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all of the steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, as well as any combination of any two or more of the steps or features.

[0027] The present disclosure is not to be limited in scope by the specific embodiments described herein for purposes of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure, as described herein.

[0028] Any example disclosed herein is deemed to apply mutatis mutandis to any other example unless otherwise specified.

[0029] 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., in the fields of cell culture, molecular genetics, stem cell therapy, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0030] Unless otherwise indicated, the surgical techniques utilized in this disclosure are standard procedures well known to those skilled in the art.

[0031] Methods for obtaining and enriching populations of mesenchymal stem cells or progenitor cells are known in the art. For example, enriched populations of mesenchymal stem cells or progenitor cells can be obtained using flow cytometry and cell sorting procedures based on the use of cell surface markers expressed on mesenchymal stem cells or progenitor cells.

[0032] All documents cited or referenced herein, and all documents cited or referenced within the documents cited herein, are hereby incorporated by reference in their entirety, along with manufacturer instructions, manuals, product specifications, and product sheets for any products mentioned herein or within any documents incorporated by reference herein.

[0033] Selected Definitions The disclosed methods are performed on a population of MLPSCs. Such methods can be performed on a population of MLPSCs, typically a large population of MLPSCs (e.g., a test population). In one example, the disclosed methods are performed on at least a portion of the test population of MLPSCs. In one example, such methods include processing at least a portion of the test population of MLPSCs as a pharmaceutical product if the test population of MLPSCs inhibits IL-2RA under culture conditions to at least a predetermined level. In one example, such methods further include discarding at least a portion of the test population of MLPSCs if the population of MLPSCs inhibits IL-2RA below a predetermined level under culture conditions. In one example, the predetermined level is 56%. In one example, the predetermined level is 56% or greater. In one example, the predetermined level is 60% or greater.

[0034] The interleukin-2 receptor alpha chain ("IL-2RA"; "IL-2Rα", also known as CD25) is a validated marker of T cell activation. In certain embodiments, the methods of the present disclosure measure the ability to inhibit T cell activation. For the avoidance of doubt, the terms "IL-2RA", "IL-2Rα", "IL-2R alpha", and variations thereof are used interchangeably herein.

[0035] The term "predetermined" level is used in the context of the present disclosure to refer to a therapeutically effective amount of IL-2Rα inhibition. In one example, the predetermined level is a clinically proven effective predetermined level. In one example, the level is clinically proven to be effective against GvHD. In another example, the predetermined level is predetermined by a regulatory agency, such as the US Food and Drug Administration (FDA). In one example, the predetermined level corresponds to an increased survival rate of GvHD patients. In one example, the predetermined level corresponds to an increased survival rate of GvHD patients with grade D GvHD.

[0036] In one example, the predetermined level is a "baseline level of IL-2Rα inhibition." In one example, the baseline level of inhibition is the level inhibited by an FDA-approved MLPSC population (e.g., an FDA-approved MSC population for treating GvHD). In one example, the baseline level of IL-2Rα serves as a basis for selecting a cell population according to the present disclosure.

[0037] In one example, the level of IL-2RA inhibition is determined under culture conditions. The term "culture conditions" refers to cells growing in culture. In one example, culture conditions refer to a cell population actively dividing. Such cells may, in one example, be in exponential growth phase.

[0038] In one example, the culture conditions include co-culturing a population of MLPSCs disclosed herein with a second cell population, such as a population comprising peripheral blood mononuclear cells (PBMCs). In one example, the co-culturing includes culturing a population of MLPSCs disclosed herein with an activated population of PBMCs. For example, the PBMCs can be activated with anti-CD3 and anti-CD28 antibodies prior to co-culturing with the population of MLPSCs disclosed herein.

[0039] In one example, the "culture conditions" include co-culturing MLPSCs and activated PBMCs at a ratio of MLPSC:activated PBMC of approximately 1:2 or less. For example, the MLPSC:activated PBMC ratio is 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 1:100 or less. In this example, the level of IL-2RA inhibition is determined after approximately 30-84 hours of cell culture under the culture conditions.

[0040] In one example, the "culture conditions" include co-culturing MLPSCs and T cells at a ratio of MLPSC:T cells of approximately 1:2 or less. For example, the MLPSC:T cell ratio may be 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 1:100 or less. In this example, the level of IL-2RA inhibition is determined after approximately 30-84 hours of cell culture under the culture conditions.

[0041] In one example, the level of a particular marker can be determined by taking a sample of cell culture medium and measuring the level of the marker in the sample. In another example, the level of a particular marker can be determined by taking a sample of cells and measuring the level of the marker in a cell lysate. Those skilled in the art will understand that secreted markers can be measured by sampling the culture medium, while markers expressed on the cell surface can be measured by evaluating a sample of cell lysate. In one example, a sample is taken when the cells are in exponential growth phase. In one example, a sample is taken after at least 2-3 days of culture. In another example, a sample is taken after approximately 30-84 hours of co-culture. In one example, a sample is taken from a co-culture of MLPSCs and activated PBMCs. In this example, the cell sample can be lysed and the level of the marker determined.

[0042] In one example, the level of IL-2RA can be determined using various methods, such as enzyme-linked immunosorbent assay (ELISA)-based methods. Suitable ELISA kits are known in the art and include, for example, the Quantikine Human CD25 / IL-2Rα ELISA. In one example, the ELISA is (i) adding sample dilutions to each well of a microplate pre-coated with a monoclonal antibody specific for IL-2RA; (ii) adding the co-culture samples to wells of a microplate pre-coated with a monoclonal antibody specific for IL-2RA; (iii) incubating the microplate for a period of time sufficient for the monoclonal antibody specific for IL-2RA to specifically bind to IL-2RA in the sample; (iv) washing the microplate; (v) adding an IL-2RA conjugate to the well; (vi) incubating the microplate for a time sufficient to allow the conjugate to specifically bind to the captured IL-2RA; (vii) washing the microplate; (viii) adding a substrate solution to the wells; (ix) incubating the microplate for a sufficient time to allow color to develop; (x) adding stop solution to the wells; (xi) reading the optical density using a microplate reader with a measurement wavelength set at 450 nm and a compensation wavelength set at 570 nm; (xii) determining the level of IL-2RA.

[0043] In another example, the level of IL-2RA is determined using fluorescence-activated cell sorting (FACS) with an appropriate antibody, such as anti-CD25. Additional antibodies may be used if it is necessary to distinguish between types of CD25+ cells.

[0044] In one example, the level of IL-2RA in the co-culture is compared with the level of IL-2RA in the cultured activated PBMC population.Then, the level of IL-2RA is compared to obtain the level of IL-2RA inhibition in the co-culture.For example, the level of IL-2RA can be inhibited by at least 60% in the co-culture (i.e., MLPSC:activated PBMC) compared with the monoculture (activated PBMC alone).Those skilled in the art will understand other suitable methods for determining the level of IL-2RA under culture conditions.

[0045] In one example, the level of IL-2RA is determined using a standard curve. A "standard curve" refers to a common method for determining the concentration of a substance in an unknown sample by comparing the unknown sample with a set of standard samples of known concentrations. In one example, the standard curve is generated using four-parameter logistic (4-PL) curve fitting. As those skilled in the art will appreciate, four-parameter logistic (4-PL) curves are a regression model commonly used in the analysis of bioassays such as ELISA. These curves are sigmoidal, or "s" shaped. This type of curve is particularly useful for characterizing bioassays, as bioassays are often linear only within a specific concentration range.

[0046] Expanding cells from a cryopreserved intermediate means thawing cryogenically frozen cells and culturing them in vitro under conditions suitable for cell growth.

[0047] In one example, the "level" or "amount" of a particular marker, such as IL-2RA, is determined before the cells are cryopreserved. In another example, the level or amount of a particular marker is determined after the first cryopreservation of the cells. In another example, the level is determined after a second cryopreservation of the cells. For example, cells may be expanded to obtain an intermediate, cryopreserved, thawed before being replated in culture, and further expanded before the level of the particular marker can be determined under culture conditions.

[0048] The term "intermediate" is used in the context of the present disclosure to refer to an intermediate population of MLPSCs that has been freshly isolated and subjected to early-stage expansion (e.g., approximately two passages). For example, generation of an intermediate population of MLPSCs can include isolation of nucleated bone marrow cells (NBMCs) from bone marrow aspirate (BMA), cell culture, and two passages to expand the relevant cells. In one example, the cells are then harvested and prepared for analysis according to methods described herein. In one example, the harvested cells are cryopreserved. Thus, generation of the intermediate population is the first step in the process of manufacturing a composition (i.e., a pharmaceutical product) to be administered to a subject. While the above referenced example refers to isolation of NBMCs from BMA, one skilled in the art will understand that relevant cells can be obtained from other suitable source(s) (e.g., adipose tissue) as discussed herein and subjected to the necessary expansion to form the intermediate and pharmaceutical products.

[0049] In certain examples disclosed herein, the intermediates of the present disclosure are expanded to produce populations of MLPSCs for administration. In one example, such populations may be referred to as pharmaceutical compositions or drug products (DPs). For example, expansion of the intermediates includes cell culture and further passages (e.g., three additional passages) for expansion.

[0050] "Isolated" or "purified" means that a cell has been separated from at least some components of its natural environment. The term also includes gross physical separation of the cell from its natural environment (e.g., removal from a donor). The term "isolated" includes alteration of the cell's direct relationship to neighboring cells, e.g., by dissociation. The term "isolated" does not refer to cells in a tissue section. When used to refer to a population of cells, the term "isolated" includes a population of cells resulting from propagation of isolated cells of the present disclosure.

[0051] The terms "passage," "passaging," or "subculture" are used in the context of this disclosure to refer to known cell culture techniques used to keep cells alive and propagate under culture conditions for extended periods of time so that cell numbers can be continually increased. The degree of subculture a cell line has undergone is often expressed as "passage number," which generally refers to the number of times the cells have been subcultured. In one example, a single passage involves removing non-adherent cells and leaving adherent mesenchymal progenitor or stem cells. Such mesenchymal progenitor or stem cells can then be dissociated from the substrate or flask (e.g., with a protease such as trypsin or collagenase), medium can be added, and optionally washed (e.g., by centrifugation), after which the mesenchymal progenitor or stem cells can be replated or reseeded into one or more culture vessels with a larger total surface area. The mesenchymal progenitor or stem cells can then continue to expand in culture. In another example, a method for removing non-adherent cells includes a non-enzymatic treatment step (e.g., with EDTA). In one example, mesenchymal progenitor or stem cells are passaged at or near confluence (e.g., about 75% to about 95% confluence). In one example, mesenchymal progenitor or stem cells are seeded at a concentration of about 10%, about 15%, or about 20% cells per ml of culture medium.

[0052] The terms "medium" or "media" as used in the context of this disclosure include components of the environment surrounding cells in culture. A medium is intended to contribute to and / or provide the appropriate conditions for growing cells. A medium can be a solid, liquid, gas, or a mixture of phases or materials. A medium can include liquid growth media as well as liquid media that do not support cell growth. Exemplary gaseous media include the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed.

[0053] As used herein, the terms "treating," "treat," or "treatment" include alleviating or eliminating at least one symptom of GvHD by administering a population of mesenchymal stem cells or progenitor cells and / or their progeny and / or soluble factors derived therefrom. In one example, the treatment comprises administering a population of expanded mesenchymal stem cells or progenitor cells disclosed herein. In one example, the therapeutic response is determined relative to a baseline. In one example, the therapeutic response is determined relative to a control patient population.

[0054] In one example, the method of the present disclosure inhibits disease progression or disease complications in a subject. "Inhibiting" disease progression or disease complications in a subject means preventing or alleviating disease progression and / or disease complications in a subject. Thus, in one example, the method of the present disclosure inhibits the progression of disease severity. For example, such a method can inhibit the progression of GvHD severity (i.e., inhibit the stepwise progression).

[0055] As used herein, the term "prevent" or "preventing" includes arresting or inhibiting the onset of at least one symptom of an inflammatory disease by administering a population of mesenchymal stem or progenitor cells and / or their progeny and / or soluble factors derived therefrom.

[0056] The term "subject" as used herein refers to a human subject. For example, the subject may be an adult. In another example, the subject is a pediatric subject. Terms such as "subject," "patient," or "individual" are terms that can be used interchangeably in this disclosure, depending on the context. In one example, the subject is suffering from GvHD.

[0057] As used herein, the term "non-genetically modified" refers to cells that have not been modified by transfection with a nucleic acid. For the avoidance of doubt, in the context of the present disclosure, mesenchymal progenitor or stem cells transfected with a nucleic acid encoding Ang1 are considered to be genetically modified.

[0058] The term "clinically proven" (used independently or modifying the term "effective") means that efficacy has been demonstrated by a clinical trial, and that the clinical trial meets the approval standards of the US Food and Drug Administration, EMEA, or a corresponding national regulatory agency. For example, the clinical study may be a randomized, double-blind study of appropriate size used to clinically prove the efficacy of the composition. In one example, a clinically proven effective amount is an amount shown by a clinical trial to meet a specified endpoint. In one example, the endpoint is prevention of death. In other words, the endpoint is an increase in survival rate. For example, the 100-day survival rate may be increased when a cell population that meets the release criteria of the present disclosure is administered.

[0059] Thus, the terms "clinically proven efficacy" and "clinically proven effect" can be used in the context of the present disclosure to refer to a dosage, dosing regimen, treatment, or method disclosed herein. Efficacy can be measured based on a change in the course of a disease in response to administration of a composition disclosed herein. For example, a composition of the present disclosure is administered to a subject in an amount and for a time sufficient to induce an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of the inflammatory disease. To determine whether the amount and time of treatment are sufficient, various indicators reflecting the severity of the disease can be evaluated. Such indicators include, for example, clinically recognized indicators of disease severity or symptoms. In one example, the degree of improvement is determined by a physician, who can make this determination based on signs, symptoms, or other test results. In one example, a clinically proven effective dose improves patient survival. In another example, a clinically proven effective dose reduces a subject's risk of death. In another example, a clinically proven effective dose increases 100-day survival. In one example, the disclosed method involves administering a clinically proven effective amount of a composition disclosed herein.

[0060] "Therapeutic effect" is used in the context of the present disclosure to refer to the MLPSCs and compositions disclosed herein that can treat, inhibit, and / or prevent a disease. For example, the therapeutically effective MLPSCs and compositions disclosed herein can treat, inhibit, and / or prevent an inflammatory disease such as GvHD. In one example, the therapeutically effective MLPSCs and compositions disclosed herein increase the 100-day survival rate of a subject suffering from GvHD.

[0061] 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.

[0062] As used herein, the term "about" refers to + / - 10%, more preferably + / - 5% of the specified value, unless otherwise specified.

[0063] It will be understood that throughout this specification words such as "comprise" or variations such as "comprises" or "comprising" refer to the inclusion of a particular element, component or step, or group of elements, components or steps, but do not imply the exclusion of any other element, component or step, or group of elements, components or steps.

[0064] As used herein, the singular forms "a," "an," and "the" include singular and plural references unless the context indicates otherwise.

[0065] Graft-versus-host disease (GvHD) In one example, the disclosed method relates to providing a cell population effective for treating graft-versus-host disease (GvHD). "Graft-versus-host disease (GvHD)" is an immunological disorder that is a major factor limiting the success and efficacy of allogeneic bone marrow or stem cell transplantation. GvHD can occur in acute (aGvHD) or chronic (cGvHD) forms. Acute GvHD usually appears within 100 days of bone marrow or stem cell transplantation. Chronic GvHD generally appears later than aGvHD (more than 100 days after transplantation) and has some characteristics of an autoimmune disease. It can also develop de novo after resolution of aGvHD or as an extension of aGvHD. Chronic GvHD can cause multiple, often debilitating symptoms, including widespread skin rash, painful mouth ulcers, shortness of breath, and limb and joint pain. In one example, cGvHD patients have impaired CD5+ B cell reconstitution. In one example, cGvHD is refractory to steroid therapy. In one example, the cGvHD is refractory to biological therapy. In one example, the cGvHD is refractory to steroid therapy and biological therapy.

[0066] The severity of GvHD is graded according to the pattern of organ dysfunction and clinical performance status. Multi-organ dysfunction can include skin rash, liver dysfunction, and / or gastrointestinal (GI) dysfunction. Examples of skin rash, liver dysfunction, and GI dysfunction are shown in Tables 1 and 2. In one example, a subject suffers from GvHD accompanied by multi-organ dysfunction. In one example, a subject treated according to the present disclosure suffers from severe GvHD. In one example, severe GvHD is graded according to the Glucksberg scale (Glucksberg et al., 1974; Thomas et al., 1975) (Table 1). For example, a subject may suffer from Grade II GvHD or Grade III / IV GvHD according to the Glucksberg scale. In one example, a subject suffers from Grade II GvHD. In another example, a subject suffers from Grade III / IV GvHD.

[0067] In another example, severe GvHD is graded according to the IBMTR Severity Index (Table 2) (Rowlings et al., 1997). In one example, the subject has Grade B, Grade C, or Grade D GvHD according to the IBMTR severity scale. In one example, the subject has Grade D GvHD.

[0068] In another example, the subject has Minnesota high-risk GvHD. Minnesota high-risk acute GvHD is defined as GvHD of either cutaneous stage 4; lower gastrointestinal (GI) stage 3-4 or hepatic stage 3-4; or cutaneous stage 3+ and lower GI stage 2-4 or hepatic stage 2-4 (MacMillan et al., 2015). In each of these examples, the subject may also have a high MAP score. For example, the subject may have a MAP score of 0.29 or greater. [Table 1] [Table 2]

[0069] In one example, a subject with severe GvHD does not respond to treatment with a primary therapy. In one example, the subject deteriorates within three days of primary therapy. In this example, a subject deteriorates if the severity of their GvHD increases. For example, the subject's GvHD severity increases according to the MAP. In another example, the subject's GvHD severity increases according to the Glucksberg scale. In another example, the subject's GvHD severity increases according to the IMBTR scale. In another example, the subject's GvHD severity increases according to organ dysfunction.

[0070] In another example, the subject does not respond within 7 days of the first-line therapy. For example, the subject is refractory to the first-line therapy. In one example, the first-line therapy is systemic steroids. In one example, the subject has severe GvHD and is refractory to steroids. In one example, the steroid is a corticosteroid. In another example, the steroid is a glucocorticoid. In another example, the steroid is prednisone. In another example, the subject has severe GvHD and is refractory to steroids and a second-line therapy. For example, the second-line therapy can include extracorporeal photopheresis, etanercept, infliximab, ruxolitinib, antithymocyte globulin, mycophenolic acid, alemtuzumab, basiliximab, or tocilizumab.

[0071] Treatment response The methods of the present disclosure relate to the treatment of GvHD. As used herein, the terms "treating," "treat," "treatment," and "alleviating progression" include alleviating or eliminating at least one symptom of GvHD by administering a population of mesenchymal stem cells or progenitor cells and / or their progeny and / or soluble factors derived therefrom and / or extracellular vesicles derived therefrom.

[0072] The term "response" as used herein refers to a response to therapy. In one example, a subject is considered to have responded if there is an improvement in at least one organ, no progression in other organs, and no need for additional therapy. In another example, a subject is considered to have not responded if the GvHD condition remains unchanged or progresses, or if additional secondary therapy is subsequently required. In this example, the subject that has not responded is a non-responder.

[0073] In one example, the treatment induces a partial response. In one example, the partial response is induced at least 28 days after treatment is initiated. In one example, the partial response is induced 28 days after treatment is initiated. In one example, the partial response is induced at least 30 days after treatment is initiated. In one example, the partial response is induced at least 2 months after treatment is initiated. In another example, the partial response is induced at least 3 months after treatment is initiated. In another example, the partial response is induced within 3 months. In another example, the partial response is induced 28-56 days after treatment is initiated. In another example, the partial response is induced 100 days after treatment is initiated. In another example, the partial response is induced 160 days after treatment is initiated. In another example, the partial response is induced 180 days after treatment is initiated.

[0074] In another example, a partial response is induced after two doses. In another example, a partial response is induced after two doses, once weekly. In another example, a partial response is induced after two doses, once weekly every two weeks. In another example, a partial response is induced after three or more doses. In one example, in the context of GvHD, a partial response is - A decrease of at least 1 point in the Skin %BSA score, - A decrease of at least 1 point in the oral score, - A decrease of at least 1 point in ocular score, - A decrease of at least 1 point in the skin feature score, - A decrease of at least 1 point in the gastrointestinal score, - A decrease of at least 1 point in liver score, - A reduction of at least 1 point in the pulmonary symptom score, - A decrease of at least 1 point in pulmonary FEV1 score, - A decrease of at least 1 point in the joint and fascia score, - A decrease of at least 1 point in the genital score.

[0075] In one example, a partial response is characterized by a decrease of at least one point in the skin %BSA score. In another example, a partial response is characterized by a decrease of at least one point in the oral score. In another example, a partial response is characterized by a decrease of at least one point in the ocular score. In these examples, scores can be taken using the NIH Consensus Criteria 2014 for GvHD.

[0076] In another example, a partial response is: - A decrease of at least 1 point in the Skin %BSA score, - A decrease of at least 1 point in the oral score, - A decrease of at least 1 point in the ocular score.

[0077] There are various classification systems for characterizing GvHD (Lee, S., (2017) Blood., 129(1):30-37). In one example, the NIH Consensus Criteria 2014 can be used to score the outcomes disclosed herein (Jagasia et al., (2015) Biol Blood Marrow Transplant., 21:389-401). The following table shows the components of the NIH Consensus Criteria 2014. [Table 3-1] [Table 3-2]

[0078] In one example, a partial response is a reduction of 1 or more points from the NIH Consensus Criteria 2014 organ-specific score in the table above. Thus, in one example, treatment reduces the skin %BSA score by 1 or more points. In another example, treatment reduces the oral score by 1 or more points. In another example, treatment reduces the eye score by 1 or more points. In another example, treatment reduces the skin characteristic score by 1 or more points. In another example, treatment reduces the gastrointestinal score by 1 or more points. In another example, treatment reduces the liver score by 1 or more points. In another example, treatment reduces the pulmonary symptom score by 1 or more points. In another example, treatment reduces the pulmonary FEV1 score by 1 or more points. In another example, treatment reduces the joint and fascia score by 1 or more points. In another example, treatment reduces the genital score by 1 or more points.

[0079] In one example, the treatment induces a complete response after treatment is initiated. In one example, a complete response is complete resolution of GvHD symptoms in all organs. In one example, a complete response is induced 28 days after treatment is initiated. In one example, a complete response is induced at least 28 days after treatment is initiated. In one example, a complete response is induced at least 30 days after treatment is initiated. In one example, a complete response is induced at least 2 months after treatment is initiated. In another example, a complete response is induced at least 3 months after treatment is initiated. In another example, a complete response is induced 28-56 days after treatment is initiated. In another example, a complete response is induced 100 days after treatment is initiated. In another example, a complete response is induced 160 days after treatment is initiated. In another example, a complete response is induced 180 days after treatment is initiated.

[0080] In another example, a complete response is induced after two doses. In another example, a complete response is induced after two doses, once weekly. In another example, a complete response is induced after two doses, once weekly every two weeks. In another example, a complete response is induced after three or more doses.

[0081] In another example, again with respect to GvHD, treatment increases the subject's chance of survival. For example, treatment increases the chance that the subject will survive for at least 20 to 200 days after initiation of treatment. In one example, treatment increases the chance that the subject will survive for at least 180 days after initiation of treatment. In another example, treatment increases the chance that the subject will survive for at least 100 days. In one example, the increased chance is determined compared to a subject not treated with a composition of the present disclosure. In one example, treatment increases 100-day survival by more than 60%. In another example, treatment increases 100-day survival by more than 60%. In another example, treatment increases 100-day survival by more than 65%. In another example, treatment increases 100-day survival by more than 68%. In another example, treatment increases 100-day survival by more than 70%. In another example, treatment increases 100-day survival by more than 71%. In another example, treatment increases 100-day survival by more than 72%. In another example, treatment increases 100-day survival by 60% to 75%. In another example, treatment increases 100-day survival by 65% ​​to 75%. In another example, treatment increases 100-day survival by 70% to 75%. In one example, 100-day survival is measured across a population of patients, and the average 100-day survival observed in that population serves as a measure of the 100-day survival for each patient in the population. In one example, this measure is compared to a subject (or population of subjects) not treated with a composition of the present disclosure.

[0082] Mesenchymal progenitor or stem cells As used herein, the term "mesenchymal progenitor or stem cell (MLPSC)" refers to an undifferentiated, multipotent cell that has the ability to self-renew while maintaining multipotency and to differentiate into many cell types of mesenchymal origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, tendons, or non-mesodermal origin, such as hepatocytes, neurons, and epithelial cells. For the avoidance of doubt, "mesenchymal progenitor cells" refers to cells that can differentiate into mesenchymal cells, such as bone, cartilage, muscle, and adipocytes, as well as fibrous connective tissue.

[0083] The term "mesenchymal progenitor or stem cells" includes both parent cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal precursor cells, and their undifferentiated progeny.

[0084] Mesenchymal progenitor or stem cells can be autologous, allogeneic, xenogeneic, syngenic, or isogenic. Autologous cells are isolated from the same individual into which 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. Syngenic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0085] In one example, the mesenchymal progenitor or stem cells are allogeneic. In one example, the allogeneic mesenchymal progenitor or stem cells are expanded and cryopreserved.

[0086] Mesenchymal progenitor or stem cells reside primarily in bone marrow, but have also been shown to reside in a variety of host tissues, including umbilical cord blood and umbilical cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp. They are also found in the skin, spleen, pancreas, brain, kidney, liver, heart, retina, hair follicles, intestine, lung, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis, and periosteum, and can differentiate into germline cells such as mesoderm and / or endoderm and / or ectoderm. Thus, mesenchymal progenitor or stem cells can differentiate into numerous cell types, including, but not limited to, adipose, osseous, cartilaginous, elastic, muscular, and fibrous connective tissue. The specific lineage commitment and differentiation pathways these cells follow depend on a variety of influences, including mechanical and / or endogenous bioactive factors, such as growth factors and cytokines, and / or local microenvironmental conditions established by the host tissue.

[0087] As used herein, the terms "enriched," "enriched," or variations thereof are used to refer to a population of cells in which the proportion of one particular cell type or the proportion of multiple particular cell types is increased compared to a population of untreated cells (e.g., cells in their native environment). In one example, a population enriched for mesenchymal progenitor or stem cells contains at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% mesenchymal progenitor or stem cells. Note that the term "a population of cells enriched for mesenchymal progenitor or stem cells" is interpreted as expressly supporting the term "a population of cells comprising X% mesenchymal progenitor or stem cells (where X% is a percentage described herein)." Mesenchymal progenitor or stem cells, in some instances, can form clonogenic colonies, e.g., CFU-F (fibroblasts) or a subset thereof (e.g., 50% or 60% or 70% or 70% or 90% or 95%) can have this activity.

[0088] In one example of the present disclosure, the mesenchymal progenitor or stem cells are mesenchymal stem cells (MSCs). The MSCs may be a homogenous composition or a mixed cell population enriched for MSCs. A homogenous MSC composition can be obtained by culturing adherent bone marrow or periosteal cells, and MSCs can be identified by specific cell surface markers identified by unique monoclonal antibodies. Methods for obtaining MSC-enriched cell populations are described, for example, in U.S. Patent No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, the MSCs are allogeneic. In one example, the MSCs are cryopreserved. In one example, the MSCs are expanded and cryopreserved.

[0089] In another example, the mesenchymal progenitor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs.

[0090] Isolated or enriched mesenchymal progenitor or stem cells can be expanded in vitro by culture. Isolated or enriched mesenchymal progenitor or stem cells can be cryopreserved, thawed, and then expanded in vitro by culture.

[0091] In one example, isolated or enriched mesenchymal progenitor or stem cells are cultured in a culture medium (serum-free or serum-supplemented medium), such as alpha minimal essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, at a density of 50,000 viable cells / cm. 2 The cells are seeded at 100°C and allowed to adhere to the culture vessel overnight at 37°C and 20% O. The culture medium is then replaced and / or changed as needed, and the cells are cultured for an additional 68-72 hours at 37°C and 5% O.

[0092] As will be appreciated by those skilled in the art, cultured mesenchymal progenitor or stem cells are phenotypically distinct from in vivo cells. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal progenitor or stem cells are also biologically distinct from in vivo cells, having a higher proliferation rate than most non-cycling (quiescent) cells in vivo.

[0093] In one example, a population of cells is enriched in a selectable form from a cell preparation containing STRO-1+ cells. The term "selectable form" 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., mesenchymal progenitor cells) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (which may be STRO-1 bright). Thus, the statement that cells are STRO-1+ does not mean that the cells are selected solely by STRO-1 expression. In one example, cells are selected based on at least STRO-3 expression, e.g., they are STRO-3+ (TNAP+). For example, MPCs can be isolated from bone mononuclear cells using anti-STRO-3 antibodies.

[0094] 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 also support selection from any tissue containing STRO-1+ cells (e.g., mesenchymal progenitor cells), or vascular tissue, or tissue containing pericytes (e.g., STRO-1+ pericytes), or one or more of the tissues listed herein.

[0095] In one example, the cells used in 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+, or any combination thereof.

[0096] By "individually" it is meant that the present disclosure encompasses the listed markers or groups of markers separately, and that even if individual markers or groups of markers cannot be separately recited herein, the appended claims may define such markers or groups of markers separately and divisibly from one another.

[0097] "Collectively" means that the disclosure encompasses any number or combination of the listed markers or markers, and that notwithstanding that such number or combination of markers or markers may not be specifically recited herein, the appended claims may define such combination or subcombination separately and divisibly from any other combination of markers or markers.

[0098] The term "TNAP" as used herein is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, this term encompasses liver isoform (LAP), bone isoform (BAP) and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP as used herein refers to a molecule capable of binding to STRO-3 antibody produced by the hybridoma cell line deposited with ATCC on December 19, 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0099] Additionally, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0100] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two distinct germ cell lineages. Non-limiting examples of lineages to which STRO-1+ cells can commit include bone progenitors, hepatocyte precursors that are multipotent to bile duct epithelial cells and hepatocytes, neural-restricted cells that can produce glial cell precursors that develop into oligodendrocytes and astrocytes, neuronal precursors that develop into neurons, cardiac muscle and cardiomyocyte precursors, and glucose-responsive insulin-secreting pancreatic beta cell lines. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular precursors, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes, and oligodendrocyte precursors.

[0101] In one example, mesenchymal progenitor or stem cells are obtained from a single donor or multiple donors, and the donor samples, or mesenchymal progenitor or stem cells, are then pooled and expanded in culture.

[0102] The mesenchymal precursor or stem cells encompassed by the present disclosure can also be cryopreserved before administration to a subject, hi one example, the mesenchymal precursor or stem cells are expanded and cryopreserved before administration to a subject.

[0103] In one example, the present disclosure encompasses mesenchymal progenitor or stem cells, their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal progenitor or stem cells, and extracellular vesicles isolated therefrom. For example, mesenchymal progenitor or stem cells of the present disclosure can be added to a cell culture medium and expanded under conditions and for a period of time suitable for secreting extracellular vesicles. The secreted extracellular vesicles can then be collected from the culture medium and used for therapy.

[0104] As used herein, the term "extracellular vesicles" refers to lipid particles ranging in size from about 30 nm to 10 microns that are naturally released from cells, but typically they are less than 200 nm in size. These are released from the releasing cells (e.g., mesenchymal stem cells, STRO-1). + The sample may contain proteins, nucleic acids, lipids, metabolites, or organelles from a cell.

[0105] As used herein, the term "exosome" refers to a type of extracellular vesicle, generally ranging in size from about 30 nm to about 150 nm, that originates from the endosomal compartment of mammalian cells, from which it is transported to the plasma membrane and released. They may contain nucleic acids (e.g., RNA, microRNA), proteins, lipids, and metabolites, and function in intercellular communication by delivering cargo that is secreted from one cell and taken up by another.

[0106] In one example, the composition of the present disclosure includes cells that induce new blood vessel formation in a target tissue. In one example, the target tissue is the heart. In another example, the cells secrete factors that protect at-risk or damaged myocardium. In one example, the at-risk or damaged myocardium is exposed to insufficient blood flow due to an ischemic event. In one example, the cells secrete factors that reduce apoptosis of cardiomyocytes.

[0107] Cell expansion culture In one example, mesenchymal progenitor or stem cells are expanded. "Expanded" mesenchymal progenitor or stem cells are distinguished from freshly isolated cells in that they are cultured and passaged (i.e., subcultured) in cell culture medium. In one example, freshly isolated cells are expanded for approximately one or two passages to form an intermediate population. In one example, freshly isolated cells are expanded for two passages to form an intermediate population. In another example, freshly isolated cells are expanded for approximately one to three passages to form an intermediate population.

[0108] Thus, in one example, the relevant cells are isolated and expanded through two passages to form an intermediate MLPSC population. This intermediate MLPSC population is then expanded to form a DP. For example, the intermediate cell population is expanded three additional passages (i.e., a total of five passages) to form a DP.

[0109] In one example, mesenchymal progenitor cells or stem cells are expanded for approximately 4 to 10 passages. In one example, mesenchymal progenitor cells or stem cells are expanded for at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 passages. For example, mesenchymal progenitor cells or stem cells can be expanded for at least 5 passages. In one example, mesenchymal progenitor cells or stem cells can be expanded for at least 5 to 10 passages. In one example, mesenchymal progenitor cells or stem cells can be expanded for at least 5 to 8 passages. In one example, mesenchymal progenitor cells or stem cells can be expanded for at least 5 to 7 passages. In one example, mesenchymal progenitor cells or stem cells can be expanded for more than 7 passages. In these examples, MLPSCs can be expanded before cryopreservation and further expanded to form a cryopreserved intermediate MLPSC population.

[0110] In one example, the DP compositions of the present disclosure are made from an intermediate population of cryopreserved MLPSCs, or stated differently, by culturing cells from a cryopreserved intermediate.

[0111] In one example, a composition of the present disclosure includes mesenchymal progenitor cells or stem cells expanded from a cryopreserved intermediate. In one example, the cells expanded from a cryopreserved intermediate are expanded for at least three, at least five, at least six, at least seven, at least eight, at least nine, or at least ten passages. For example, the mesenchymal progenitor cells or stem cells can be expanded for at least three passages. In one example, the mesenchymal progenitor cells or stem cells can be expanded for at least three to ten passages. In one example, the mesenchymal progenitor cells or stem cells can be expanded for at least three to eight passages. In one example, the mesenchymal progenitor cells or stem cells can be expanded for at least three to seven passages.

[0112] In one example, IL-2RA inhibition by mesenchymal progenitor or stem cells expanded from the cryopreserved intermediates disclosed herein is assessed under culture conditions (e.g., co-culture with T cells). In one example, these MLPSCs exhibit greater than 56% IL-2RA inhibition under culture conditions. In another example, these MLPSCs exhibit greater than 60% IL-2RA inhibition under culture conditions.

[0113] In one example, the expanded mesenchymal progenitor or stem cells can be expanded in an animal protein-free medium (e.g., a xeno-free medium). In one example, the mesenchymal progenitor or stem cells can be expanded in a medium that does not contain fetal bovine serum.

[0114] In one embodiment, mesenchymal progenitor or stem cells can be obtained from a single donor, or from multiple donors, and the donor samples, or mesenchymal progenitor or stem cells, are then pooled and optionally expanded. In one example, the expansion process includes: i. increasing the number of viable cells by passage expansion to provide a preparation of at least about 1 billion viable cells, the passage expansion comprising establishing a primary culture of isolated mesenchymal progenitor or stem cells, followed by establishing an initial non-primary (P1) culture of the isolated mesenchymal progenitor or stem cells from the previous culture; ii. Expanding the P1 culture of isolated mesenchymal progenitor or stem cells by passage expansion into a second, non-primary (P2) culture of mesenchymal progenitor or stem cells; and iii. preparing and cryopreserving an in-process intermediate mesenchymal progenitor or stem cell preparation obtained from the P2 culture of mesenchymal progenitor or stem cells; iv. Optionally, thawing a cryopreserved in-process intermediate mesenchymal progenitor or stem cell preparation and expanding the in-process intermediate mesenchymal progenitor or stem cell preparation by passage expansion.

[0115] In the context of the present disclosure, a specific assay may be performed between steps iii and iv. For example, IL-2RA inhibition may be determined under culture conditions after step iii. In one example, step iv is performed only when a desired level of IL-2RA inhibition is observed under culture conditions. In this example, a cell population is selected for expansion based on IL-2RA inhibition under culture conditions.

[0116] In one example, the expanded mesenchymal progenitor or stem cell preparation comprises: i. less than about 0.75% CD45+ cells; ii. at least about 95% CD105+ cells; iii. have an antigenic profile and activity profile comprising at least about 95% CD166+ cells.

[0117] In one example, the expanded mesenchymal progenitor or stem cell population is expanded from an intermediate MLPSC population and is capable of inhibiting IL-2RA expression by CD3 / CD28-activated PBMCs by at least 56% compared to a control. In another example, the expanded mesenchymal progenitor or stem cell population is expanded from an intermediate MLPSC population and is capable of inhibiting IL-2RA expression by CD3 / CD28-activated PBMCs by at least 60% compared to a control.

[0118] The process of isolating and ex vivo expanding mesenchymal progenitor or stem cells can be carried out using any device and cell handling method known in the art. Various embodiments of the expansion cultures disclosed herein use steps that require cell handling, such as seeding, feeding, dissociating adherent cultures, or washing. Any cell handling step can potentially injure the cells. While mesenchymal progenitor or stem cells can generally tolerate some damage during preparation, it is preferable to handle the cells using handling procedures and / or devices that adequately perform a given step(s) while minimizing damage to the cells.

[0119] In one example, mesenchymal progenitor or stem cells are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculating wash bag, a spinning membrane filter with inlet and outlet ports, a filtrate bag, a mixing zone, a final product bag for the washed cells, and appropriate tubing, for example, as described in U.S. Pat. No. 6,251,295, which is incorporated herein by reference.

[0120] In one example, mesenchymal progenitor or stem cell compositions cultured according to the present disclosure are 95% homogeneous for being CD105 positive, CD166 positive, and CD45 negative. In one example, this homogeneity persists through ex vivo expansion, i.e., multiple population doublings.

[0121] In one example, mesenchymal progenitor cells or stem cells of the present disclosure are expanded in 2D culture. For example, mesenchymal progenitor cells or stem cells of the present disclosure can be expanded in a cell factory. In certain examples, this can be followed by 3D culture of the intermediates disclosed herein, for example, using a bioreactor. In one example, mesenchymal progenitor cells or stem cells of the present disclosure are first expanded in 2D culture and then further expanded in 3D culture. In one example, the intermediate cell population of the present disclosure has not been expanded in 3D culture. In one example, IL-2RA inhibition is evaluated before expansion in 3D culture.

[0122] In one example, the mesenchymal progenitor or stem cells of the present disclosure are expanded from an intermediate population. In one example, the mesenchymal progenitor or stem cells of the present disclosure are expanded from an intermediate in 2D culture before seeding in 3D culture.

[0123] With respect to both the intermediate population and the therapeutic composition expanded from the intermediate population, in one example, the mesenchymal progenitor or stem cells of the present disclosure are expanded in 2D culture for at least three days before seeding in a further culture system, such as a cell factory or 3D culture in a bioreactor. In one example, the mesenchymal progenitor or stem cells of the present disclosure are expanded in 2D culture for at least four days before seeding in a further culture system. In one example, the mesenchymal progenitor or stem cells of the present disclosure are expanded in 2D culture for three to five days before seeding in a further culture system. In these examples, the 2D culture can be performed in a cell factory. Various cell factory products are commercially available (e.g., Thermofisher, Sigma, Corning). In one example, the cell factory has at least five layers. In one example, the cell factory has at least 10 layers. In one example, the cell factory has at least 20 layers. 3D culture can be performed in various types of bioreactors, such as stirred tanks, wave bags, and vertical wheels.

[0124] In one example, CO2 is supplied during expansion of MLPSCs. In one example, MLPSCs are expanded in less than 9% CO2. In one example, MLPSCs are expanded in less than 8% CO2. In one example, MLPSCs are expanded in 5% CO2. For example, MLPSCs are expanded in 5% + / - 2% CO2. In one example, MLPSCs are expanded with passive priming of CO2. For example, a cell factory can be passively primed with 5% CO2.

[0125] Priming cell factories maintains CO2 tension between the cell factory and the incubator and stabilizes the pH level of the growth medium. Active priming involves actively pumping CO2 gas into each culture vessel (e.g., cell factory) through a bacterial vent filter for a set period of time (e.g., approximately 10 minutes). However, active priming requires an open port to deliver gas, potentially introducing contaminants into the culture. Passive priming involves placing the sealed culture system in an incubator with the appropriate CO2 concentration prior to cell seeding (e.g., approximately 12–72 hours).

[0126] In one example, the cells of the present disclosure are STRO-3+, which are later expanded in culture to provide an intermediate somatic cell population.

[0127] Cell culture medium The mesenchymal progenitor or stem cells disclosed herein can be expanded in a variety of suitable growth media.

[0128] The terms "medium" or "media" as used in the context of this disclosure include the components of the environment surrounding cells. Media contribute to and / or provide conditions suitable for cell growth. Media can be solid, liquid, gaseous, or a mixture of phases or materials. Media can include liquid growth media and liquid media that do not support cell growth. Media also include gel-like media such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous media include the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed.

[0129] The cell culture medium used for expansion contains all essential amino acids, but may also contain non-essential amino acids. Generally, amino acids are classified into essential amino acids (Thr, Met, Val, Leu, Ile, Phe, Trp, Lys, His) and non-essential amino acids (Gly, Ala, Ser, Cys, Gln, Asn, Asp, Tyr, Arg, Pro).

[0130] Those skilled in the art will understand that for optimal results, the basal medium must be appropriate for the cell line of interest. For example, if growth is found to be limited due to energy source depletion, it may be necessary to increase the level of glucose (or other energy source) in the basal medium or add glucose (or other energy source) during culture. In one example, dissolved oxygen (DO) levels may also be controlled.

[0131] In one example, the cell culture medium contains human-derived additives, for example, the cell culture medium can be supplemented with human serum and human platelet cell lysate.

[0132] In one example, the cell culture medium contains only human-derived additives. Thus, in one example, the cell culture medium is xeno-free. For the avoidance of doubt, in these examples, the culture medium does not contain animal proteins. In one example, the cell culture medium used in the methods of the present disclosure does not contain animal components.

[0133] In one example, the culture medium contains serum, such as fetal bovine serum and / or newborn bovine serum. In another example, the culture medium does not contain fetal bovine serum and contains growth factors that promote the proliferation of mesenchymal progenitor cells or stem cells. In one embodiment, the culture medium is a serum-free stem cell culture medium. In one example, the cell culture medium is basal medium, Platelet-derived growth factor (PDGF), Contains fibroblast growth factor 2 (FGF2).

[0134] In one example, the culture medium contains platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2), and the FGF2 level is less than about 6 ng / ml. For example, the FGF2 level can be less than about 5 ng / ml, less than about 4 ng / ml, less than about 3 ng / ml, less than about 2 ng / ml, or less than about 1 ng / ml. In one example, the FGF2 level is about 1 ng / ml.

[0135] In one example, the PDGF is PDGF-BB. In one example, the level of PDGF-BB is about 1 ng / ml to 150 ng / ml. In another example, the level of PDGF-BB is about 7.5 ng / ml to 120 ng / ml. In another example, the level of PDGF-BB is about 15 ng / ml to 60 ng / ml. In another example, the level of PDGF-BB is about 10 ng / ml. In another example, the level of PDGF-BB is at least about 10 ng / ml or less.

[0136] In other examples, additional factors can be added to the cell culture medium. In one example, the culture medium further comprises EGF. EGF is a growth factor that promotes cell proliferation by binding to its receptor, EGFR. In one example, the method of the present disclosure includes culturing a population of stem cells in a cell culture medium that does not contain fetal bovine serum and further comprises EGF. In one example, the level of EGF is about 0.1 to 7 ng / ml. For example, the level of EGF can be at least about 5 ng / ml.

[0137] In another example, the EGF level is about 1 ng / ml to 8 ng / ml. In another example, the EGF level is about 3 ng / ml to 6 ng / ml. In another example, the EGF level is about 5 ng / ml. In another example, the EGF level is about 5 ng / ml or less.

[0138] In the above example, a basal medium such as Alpha MEM or StemSpan™ can be supplemented with the reference amounts of growth factors. In one example, the culture medium comprises Alpha MEM or StemSpan™ supplemented with 10 ng / ml PDGF-BB, 5 ng / ml EGF, and 1 ng / ml FGF.

[0139] In other examples, additional factors can be added to the cell culture medium. For example, the cell culture medium can be supplemented with one or more stimulatory factors selected from the group consisting of epidermal growth factor (EGF), lα, 25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor α (TNF-α), interleukin-lβ (IL-lβ), and stromal-derived factor lα (SDF-lα). In another embodiment, the cells can be cultured in the presence of at least one cytokine in an amount sufficient to support cell growth. In another embodiment, the cells can be cultured in the presence of heparin or a derivative thereof. In one example, the heparin derivative is sulfate. Various forms of heparin sulfate are known in the art, including heparin sulfate 2 (HS2). HS2 can be derived from various sources, including, for example, male and / or female mammalian liver. Thus, exemplary heparin sulfates include male liver heparin sulfate (MML HS) and female liver heparin sulfate (FML HS).

[0140] In another example, the cell culture medium of the present disclosure maintains stem cells in an undifferentiated state. Stem cells are considered undifferentiated when they are not committed to a specific lineage. As described above, stem cells exhibit morphological characteristics that distinguish them from differentiated cells. Furthermore, undifferentiated stem cells express genes that can be used as markers for detecting a differentiated state. Polypeptide products can also be used as markers for detecting a differentiated state. Therefore, those skilled in the art can easily determine whether the method of the present disclosure maintains stem cells in an undifferentiated state using routine morphological, genetic, and / or proteomic analysis.

[0141] Co-culture of mesenchymal progenitor or stem cells with stimulated or activated T cells Interleukin-2 receptor alpha chain ("IL-2RA"; "IL-2Rα", also known as CD25) is a validated marker of T cell activation. In certain embodiments, the disclosed methods measure inhibition of T cell activation. In one example, T cell activation is determined based on the expression level of IL-2RA on T cells after co-culture of activated PBMCs with mesenchymal progenitor cells or stem cells. Inhibition of IL-2RA expression is associated with a suppressive effect on T cell activation.

[0142] In one example, activated PBMCs are co-cultured with mesenchymal progenitor or stem cells in a culture medium containing at least one T cell stimulatory agent, 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.

[0143] In one example, mesenchymal progenitor or stem cells are co-cultured with PBMCs in a culture medium containing an agent capable of stimulating CD3 and CD28 on T cells, such as an antibody against CD3 and an antibody against CD28, e.g., mouse anti-human CD3 and mouse anti-human CD28. In one example, the antibody against CD3 and / or the antibody against CD28 are added to the culture medium in soluble form at a concentration of about 2 μg / ml each.

[0144] In one embodiment, PBMCs are co-cultured with mesenchymal progenitor or stem cells at a PBMC:mesenchymal progenitor or stem cell ratio of 5:1. For example, 1 x 10 6 2 x 10 PBMCs 5 Co-culturing with MLPSCs is also contemplated. In one example, the cells may be co-cultured in a final volume of 1 ml.

[0145] In one example, MLPSCs are co-cultured with anti-CD3 / CD28 antibody-stimulated PBMCs, and IL-2Rα expression by activated PBMCs is measured by enzyme-linked immunosorbent assay (ELISA).

[0146] In one example, the level of IL-2Rα is determined relative to a control, such as the level of IL-2Rα expression by activated PBMCs in the absence of MLPSCs.

[0147] Assessment of treatment effectiveness In one embodiment, the present disclosure relates to a method for determining the therapeutic efficacy of a population of MLPSCs (eg, DP).

[0148] In one example, a method for determining the therapeutic efficacy of a population of expanded mesenchymal progenitor or stem cells (MLPSCs) includes (i) obtaining a population of MLPSCs, (ii) culturing the cells in a medium, and (iii) determining the level of IL-2RA inhibition under the culture conditions, wherein the method determines the therapeutic efficacy against graft-versus-host disease (GvHD), and IL-2RA inhibition under the culture conditions of at least 56% indicates therapeutic efficacy. In another example, IL-2RA inhibition of 60% or greater indicates therapeutic efficacy. In another example, IL-2RA inhibition of 56%-60% indicates therapeutic efficacy.

[0149] In one example, a therapeutic population of mesenchymal progenitor or stem cells can inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by at least 56% compared to a control. In another example, a therapeutic population of mesenchymal progenitor or stem cells can inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by at least 60% compared to a control. In another example, a therapeutic population of mesenchymal progenitor or stem cells can inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by 56-70% compared to a control.

[0150] In one example, the resulting population of MLPSCs is representative of a larger population of MLPSCs. For example, the resulting population of MLPSCs can be representative of a cryopreserved population of MLPSCs that is administered in a series, such as a series of doses. For example, the doses are from the same population, and one or more doses are obtained according to the methods disclosed herein and evaluated for therapeutic effect. In this example, the resulting dose(s) are representative of the remaining doses from the same MLPSC population. In one example, the resulting population of MLPSCs is representative of a 3D-cultured population of MLPSCs.

[0151] By carrying out the method for assessing therapeutic efficacy disclosed herein, a therapeutically effective cell composition can meet the release criteria for release as a DP. In one example, such an expanded composition is characterized by a threshold level of IL-2RA inhibition. In one example, the level of IL-2RA inhibition is 56% to 60% under culture conditions. In another example, the level of IL-2RA inhibition is 60% or more under culture conditions.

[0152] Pharmaceutical manufacturing methods In one example, the disclosure relates to a method for producing a pharmaceutical product comprising a population of MLPSCs, the method comprising obtaining a determination of whether a test population of MLPSCs inhibits IL-2RA under culture conditions at a predetermined level, and processing at least a portion of the test population of MLPSCs into a pharmaceutical product to produce the pharmaceutical product if the test population of MLPSCs inhibits IL-2RA under culture conditions to at least the predetermined level, or discarding at least a portion of the test population of MLPSCs if the population of mesenchymal stem cells inhibits IL-2RA under culture conditions below the predetermined level, where the predetermined level is 56%. In one example, the predetermined level is 60% or greater under culture conditions. In one example, the predetermined level is 65% or greater under culture conditions.

[0153] In one example, the test population is obtained from a 3D-cultured MLPSC population. For example, the MLPSCs can be in a bioreactor culture. In one example, the test population is obtained from a population of cryopreserved MLPSCs. In one example, the test population represents a larger MLPSC population, such as a plurality of cryopreserved MLPSC populations. In one example, the plurality of cryopreserved MLPSC populations are expanded from the same MLPSC intermediate population.

[0154] In one example, the pharmaceutical comprises a composition disclosed herein. In one example, the pharmaceutical comprises 2×10 6 Includes MLPSCs.

[0155] In one example, obtaining a determination of whether a test population of MLPSCs inhibits IL-2RA at a predetermined level under culture conditions includes co-culturing a representative amount of MLPSCs according to the methods disclosed herein.

[0156] In one example, the present disclosure provides a method for producing an MSC medicine, the method comprising a first step of supplying (e.g., expanding (e.g., small-scale or large-scale cell culture) or manufacturing) or obtaining (e.g., receiving and / or purchasing from a third party (including a contractually related third party or a non-contractually related (e.g., independent) third party) a test population of MSCs (e.g., a sample of the test population of MSCs); a second step of obtaining (e.g., detecting, measuring, receiving, or acquiring) a value (e.g., 1, 2, 3, and / or 4) of an MSC parameter set forth in Table A for the test population of MSCs; and, if the value of the at least one of the test population of MSCs meets the reference standard set forth in Table A for the parameter, determining that the MSC medicine (e.g., administering as described below in this specification) is a therapeutically effective amount of the MSC medicine. and a third step of processing at least a portion of the test population of MSCs (e.g., processing a portion of a manufacturing lot, culture, or production run, an entire manufacturing lot, culture, or production run, or multiple manufacturing lots, cultures, or production runs) in its intended form or packaging, optionally cryopreserved, to produce the MSC medicine. In one example, the value(s) comprises parameter number 1. In another example, the value(s) comprises parameter number 2. In another example, the value(s) comprises parameter number 3. In another example, the value(s) comprises parameter number 4. In another example, the value(s) comprises parameter numbers 1 and 2. In another example, the value(s) comprises parameter numbers 1 and 3. In another example, the value(s) comprises parameter numbers 1, 2, and 4.

[0157] In one example, the method includes a second step that includes obtaining values ​​for any combination of two or more MSC parameters listed in Table A, and a third step of the method includes processing at least a portion of the test population of MSCs as an MSC pharmaceutical product if the values ​​for any combination of two or more MSC parameters meet the corresponding reference standards listed in Table A for the parameters. [Table 4]

[0158] Selection of cells for use in treating graft-versus-host disease (GvHD) In one embodiment, the present disclosure encompasses selecting cells for use in treating GvHD. In one example, a population of MLPSCs is evaluated for required criteria, and if the criteria are met, the population is selected for use in treating GvHD.

[0159] In one example, an MLPSC population is selected for use in treating GvHD if it inhibits IL-2RA by at least 56-60% under culture conditions. In one example, an MLPSC population is selected for use in treating GvHD if it inhibits IL-2RA by 60% or more under culture conditions. In one example, an MLPSC population is selected for use in treating GvHD if it inhibits IL-2RA by 65% ​​or more under culture conditions.

[0160] The selection process is not particularly limited, as long as it allows the selection of cell populations characterized by relevant criteria, such as IL-2RA inhibition rate. In one example, a series of intermediate MLPSC populations are evaluated for IL-2RA inhibition under culture conditions, and a population that inhibits IL-2RA inhibition by at least 60% under culture conditions is selected for further expansion. In one example, a series of intermediate MLPSC populations are evaluated for IL-2RA inhibition under culture conditions, and a population that inhibits IL-2RA inhibition by at least 65% under culture conditions is selected for further expansion.

[0161] In one example, the selected mesenchymal progenitor cell or stem cell preparation can inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by at least 56% compared to a control. In another example, the selected mesenchymal progenitor cell or stem cell preparation can inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by at least 60% compared to a control. In another example, the selected mesenchymal progenitor cell or stem cell preparation can inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by 56%-70% compared to a control.

[0162] In one example, the CD3 / CD28-activated PBMCs express at least 10,00 pg / mL of IL-2RA. In one example, the CD3 / CD28-activated PBMCs express at least 11,00 pg / mL of IL-2RA. In one example, the CD3 / CD28-activated PBMCs express at least 12,00 pg / mL of IL-2RA. In one example, the CD3 / CD28-activated PBMCs express at least 13,000 pg / mL of IL-2RA. In one example, the CD3 / CD28-activated PBMCs express at least 12,811 pg / mL of IL-2RA.

[0163] In one example, the resulting population of MLPSCs is representative of a larger population of MLPSCs. For example, the resulting population of MLPSCs can be representative of a cryopreserved population of MLPSCs that is administered in a series, such as a series of doses. For example, the doses are from the same population, and one or more doses are obtained according to the methods disclosed herein and evaluated for therapeutic effect. In this example, the resulting dose(s) are representative of remaining doses from the same MLPSC population. In one example, the resulting population of MLPSCs is representative of a 3D-cultured MLPSC population. In these examples, cells from another MLPSC population can be selected based on evaluation of a representative test population of MLPSCs.

[0164] In one example, the selected cell population is cryopreserved for later administration.

[0165] In another example, the disclosure relates to a composition comprising a selected population(s) of MLPSCs. In one example, the composition comprises an expanded population of MLPSCs, wherein the population of MLPSCs is selected based on a predetermined level of IL-2RA inhibition under culture conditions, the predetermined level of IL-2RA inhibition being at least 56%. In another example, the predetermined level of IL-2RA inhibition is 60% or greater. In another example, the MLPSCs are selected based on expression of one or more or all of CD29, CD54, CD73, CD90, CD102, CD105, CD106, CD166, and MHC1.

[0166] In one example, the MLPSC population comprising the composition of the present disclosure is selected based on one or more of the parameters set forth in Table A.

[0167] Determination of IL-2RA levels The present disclosure contemplates any form of assay for determining levels of IL-2RA, including, for example, Western blot, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), competitive assay, radioimmunoassay, lateral flow immunoassay, flow-through immunoassay, electrochemiluminescence assay, nephelometry-based assay, turbidimetric titration-based assay, fluorescence-activated cell sorting (FACS)-based assay.

[0168] After co-culture of MLPSCs and T cells, the cells can be harvested and lysed using methods well known in the art. Cell lysates can then be analyzed for the presence of IL-2RA, for example, by ELISA or FLISA. Alternatively, the level of IL-2RA expression can be determined by analyzing intact cells, for example, by flow cytometry.

[0169] In one example, inhibition of IL-2RA expression is measured by comparing the level of IL-2RA expression in a population of cells comprising T cells to the level of IL-2RA in a population of cells after co-culture of the population of cells comprising T cells with a population of cells comprising mesenchymal progenitor or stem cells, the difference being expressed as "percentage inhibition."

[0170] The assay described above is easily modified to use chemiluminescence or electrochemiluminescence as the basis of detection.

[0171] Those skilled in the art will appreciate 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 radioactive labels for detection, or gold labels (e.g., colloidal gold) for detection, or liposomes encapsulating, for example, NAD+ for detection, or acridinium-linked immunosorbent assays.

[0172] In some examples of the present disclosure, the level of IL-2RA is determined using a surface plasmon resonance detector (e.g., BIAcore™, GE Healthcare, Piscataway, NJ), a flow-through device (e.g., that described in U.S. Pat. No. 7,205,159), a micro- or nano-immunoassay device (e.g., that described in U.S. Pat. No. 7,271,007), a lateral flow device (e.g., that described in U.S. Pat. No. 20040228761 or U.S. Pat. No. 20040265926), a fluorescence polarization immunoassay (FPIA, e.g., that described in U.S. Pat. No. 4,593,089 or U.S. Pat. No. 4,751,190), or an immunoturbidimetric assay (e.g., that described in U.S. Pat. No. 5,571,728 or U.S. Pat. No. 6,248,597).

[0173] composition The present disclosure encompasses compositions comprising a population of MLPSCs selected according to the methods disclosed herein. For example, the compositions of the present disclosure comprise a therapeutically effective amount of MLPSCs, and the therapeutic effect is determined according to the methods disclosed herein. In one example, the composition is a pharmaceutical product manufactured according to the present disclosure, thereby inhibiting IL-2Rα at a predetermined level.

[0174] In certain examples, the compositions of the present disclosure include a pharmaceutically acceptable carrier and / or excipient.

[0175] The terms "carrier" and "excipient" refer to compositions conventionally used in the art to facilitate the storage, administration, and / or biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980)). A carrier may also reduce undesirable side effects of an active compound. A suitable carrier is, for example, stable and, for example, incapable of reacting with other components in the carrier. In one example, a carrier does not cause significant local or systemic side effects in a recipient at dosages and concentrations used for therapy.

[0176] Suitable carriers for the present disclosure include those conventionally used, for example, water, saline, aqueous dextrose, lactose, Ringer's solution, buffers, hyaluronan, and glycols are exemplary liquid carriers, particularly for solutions (when isotonic). Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

[0177] In another example, the carrier is a media composition, e.g., the cells are grown or suspended in. For example, such a media composition does not induce any adverse effects in the subject to which it is administered.

[0178] Exemplary carriers and excipients do not adversely affect the viability of the cells and / or the ability of the cells to reduce, prevent or delay metabolic syndrome and / or obesity.

[0179] In one example, the carrier or excipient provides buffering activity to maintain the cells and / or soluble factors at an appropriate pH, thereby exerting biological activity; for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it minimally interacts with the cells and factors, allowing for rapid release of the cells and factors; in such cases, the compositions of the present disclosure can be formulated as a liquid for direct application to the bloodstream, or to tissues, or areas surrounding or adjacent to the tissues, for example, by injection.

[0180] In one example, the composition of the present disclosure comprises 1.5 million to 3 million cells / kg, hi one example, the composition of the present disclosure comprises 2 million cells / kg.

[0181] In one example, the composition contains 5.00 x 10 6 In another example, the composition contains more than 5.50 x 10 viable cells / mL. 6 In another example, the composition contains more than 6.00 x 10 viable cells / mL. 6 In another example, the composition contains more than 6.50 x 10 viable cells / mL. 6 In another example, the composition contains more than 6.68 x 10 viable cells / mL. 6 Contains more than 100 viable cells / mL.

[0182] The compositions of the present disclosure can be cryopreserved. Cryopreservation of mesenchymal progenitor or stem cells can be performed using slow cooling or "rapid" freezing protocols known in the art. Preferably, the cryopreservation method maintains the phenotype, cell surface markers, and proliferation rate of the cryopreserved cells to a similar extent as unfrozen cells.

[0183] The cryopreservation composition may contain a cryopreservation solution, the pH of which is typically 6.5 to 8, preferably 7.4.

[0184] Cryopreservation solutions can include, for example, sterile, non-pyrogenic, isotonic solutions such as PlasmaLyte A™. 100 mL of PlasmaLyte A™ contains 526 mg of sodium chloride (NaCl), USP, sodium gluconate (C6H 11 Contains 502 mg of USP sodium acetate trihydrate (C2H3NaO2·3H2O), 368 mg of USP sodium acetate trihydrate (C2H3NaO2·3H2O), 37 mg of USP potassium chloride (KCl), and 30 mg of USP magnesium chloride (MgCl2·6H2O). Contains no antimicrobial agents. pH is adjusted with sodium hydroxide. pH is 7.4 (6.5-8.0).

[0185] The cryopreservation solution may include Profreeze™. The cryopreservation solution may additionally or alternatively include culture medium, such as αMEM.

[0186] To facilitate freezing, cryoprotectants, such as dimethyl sulfoxide (DMSO), are typically added to cryopreservation solutions. Ideally, cryoprotectants should be non-toxic to cells and patients, non-antigenic, and chemically inert, provide high post-thaw survival rates, and allow for irrigation-free transplantation. However, DMSO, the most commonly used cryoprotectant, exhibits some cytotoxicity. Hydroxyethyl starch (HES) can be used as an alternative or in combination with DMSO to reduce the cytotoxicity of cryopreservation solutions.

[0187] 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.

[0188] In one embodiment, cells are suspended in 42.5% Profreeze™ / 50% αMEM / 7.5% DMSO and cooled in a controlled rate freezer.

[0189] The cryopreserved composition may be thawed and administered directly to a subject, or may be added to another solution containing, for example, HA, or the cryopreserved composition may be thawed and the mesenchymal progenitor cells or stem cells resuspended in an alternative carrier prior to administration.

[0190] In one example, a cell composition of the present disclosure can include Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA). For example, a composition of the present disclosure can include a Plasma-Lyte A (70%), DMSO (10%), and HSA (25%) solution, where the HSA solution includes 5% HSA and 15% buffer.

[0191] It will be apparent to those skilled in the art that many modifications and / or variations may be made to the above-described embodiments without departing from the broad and general scope of the present disclosure, and the present embodiments are therefore considered in all respects to be illustrative and not restrictive.

[0192] The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. [Example]

[0193] Example 1: Clinical Trial A mesenchymal stem cell (MSC) drug (DP) was administered as salvage therapy to 241 children with steroid-refractory acute graft-versus-host disease (aGvHD) who had failed one or more additional therapies. Children with grade D disease accounted for approximately 50% of patients recommended MSCs as salvage therapy, as this stage of disease is the most refractory to all treatments and has the highest mortality rate. Study subjects had failed not only steroid therapy but also multiple salvage interventions. This group of subjects provided insight into the efficacy of DP in a patient population previously unresponsive to other conventional therapies. The 28-day overall response rate (OR) and 100-day overall survival (OS) were high across all grades of aGVHD, particularly in subjects with grade D disease. These data highlight the efficacy of DP in a highly refractory patient population.

[0194] Example 2: IL-2Rα Inhibition The inhibitory effect on activated T cells is thought to reflect an important mechanism of action through which MSC DP has a therapeutic effect in aGVHD. An IL-2Rα potency assay was developed to functionally measure the ability of MSC DP to inhibit IL-2Rα expression by activated T cells in vitro.

[0195] We evaluated day 100 OS and IL-2Rα inhibition rates for each MSC DP administered to 241 children with steroid-refractory acute graft-versus-host disease (aGvHD). Next, we performed a sensitivity analysis to assess whether a minimum IL-2Rα inhibition threshold could be identified to explain the higher survival rates conferred by specific DPs. In the sensitivity analysis, we evaluated survival rates for children who received DPs with mean IL-2Rα inhibition rates above or below thresholds of 66% to 50%. The results are shown in Table 4. [Table 5-1] [Table 5-2]

[0196] Table 4 shows that the 100-day survival outcomes for children receiving DP lots with mean IL-2Rα inhibition rates exceeding any threshold between 54% and 66% ranged from 67% to 69%. In contrast, children receiving DP lots with mean IL-2Rα inhibition rates below 60% experienced a linear and gradual decline in 100-day survival rates. Therefore, we have confirmed that a threshold of IL-2Rα inhibition of 60% or greater will lead to the commercialization of DP lots that confer a significant survival benefit in this disease. This represents a significant advance in the manufacturing of DPs, particularly for DPs used in the treatment of T-cell-mediated diseases such as GvHD.

[0197] Example 3: Additional Analysis As shown in Figure 1, there was a positive correlation between IL-2Rα inhibition rate and 180-day survival (85% with 180-day OS above the median vs. 54% with 180-day OS below the median, p=0.01). This survival benefit was preceded by a longer 28-day OR among responders receiving products with higher IL-2Rα inhibition rates. The relationship between higher survival and mean IL-2Rα inhibition rates (above vs. below the median) was most pronounced in patients with the most severe disease and highest risk of death. a.Minnesota high-risk (OS at 180 days was 89% vs. 50%, p=0.01) b. MAGIC Algorithm Probability (MAP) ≥ 0.29 (OS at 180 days was 100% vs. 17%, p = 0.003) c. IBMTR grade D disease (OS at 180 days was 91% vs. 50%, p=0.03)

[0198] As shown in Figure 2, in high-risk MAP patients (MAP ≥ 0.29), there was a significant correlation between the mean IL-2Rα inhibition rate of each product, which increased progressively, and the increase in survival days up to 180 days (Pearson correlation coefficient = 0.81, p = 0.0013) (left panel). The lack of correlation between product efficacy and survival days in low-risk patients likely reflects that the efficacy of all drugs was sufficient to keep low-risk patients alive (right panel).

[0199] As shown in Figure 3, in all children with high MAP scores (≥0.29), there was a linear negative correlation between incremental IL-2Rα inhibition by MSC lots over 28 days and the reduction in various inflammatory biomarkers.

[0200] As shown in Figure 4, in patients treated with a single lot of DP, efficacy correlated significantly with the decrease in the percentage of in vivo activated T cells from baseline to day 28, as measured by T cells exhibiting the CD3+CD4+CD25+HLA-DR+ phenotype.

[0201] summary

[0202] There was a significant relationship between the rate of in vitro IL-2Rα inhibition and the reduction in circulating levels of activated T cells from screening to day 28 (p=0.009).

[0203] Increased product efficacy correlated with a gradual decrease in inflammatory biomarkers in vivo and improved survival.

[0204] This was most pronounced in children with the most severe disease, the highest baseline levels of inflammatory biomarkers, and the highest risk of death.

[0205] The most severely ill (Grade D) patients receiving medications with mean IL-2Rα inhibition rates above the median had a significantly higher survival rate at 180 days compared with patients receiving products with mean IL-2Rα inhibition rates below the median (91% vs. 50%, p=0.03).

[0206] Even the 50% survival rate achieved in patients with grade D SR-aGVHD who received products with mean IL-2Rα inhibition below the median is significantly higher than that seen with other treatment options over the past 20 years, where 180-day survival rates have remained low at 12%–23% even with the best available treatment.

[0207] Thus, the IL-2Rα inhibition assay is a direct measure of a drug's ability to inhibit T cell activation in vitro and correlates with the drug's biological activity in vivo, as demonstrated by improved survival outcomes and reduced circulating levels of inflammatory biomarkers and activated T cells.

[0208] Example 4: Measurement of IL-2Rα inhibition Ce-MSCs (either intermediate populations or pharmaceuticals) are co-cultured with unstimulated or stimulated (CD3; CD28 antibodies) peripheral blood mononuclear cells (PBMCs) as described below, typically at a stem cell:PBMC ratio of approximately 1:5. [Table 6]

[0209] Plates were incubated at 37°C ± 2°C and 5 ± 2% CO for 60–84 hours, after which cells were harvested, lysed, and IL-2RA expression by activated PBMCs was assessed using a commercially available ELISA kit according to the manufacturer's instructions (R&D Systems).

[0210] Inhibition of IL-2RA expression on PBMCs by MSC lots (IL-2Rα inhibition assay) is a quantitative bioactivity assay that directly measures the inhibitory effect of a cell population (e.g., final product). Measurement of bioactivity allows for evaluation of the inhibitory effect of the population on activated T cells in vitro.

[0211] Example 5: Relationship between PBMC stimulation and IL-2Rα inhibition The IL-2RA inhibition assay involves culturing MSCs under standard culture conditions for 72 hours, followed by co-culture with PBMCs for an additional 72 hours. IL-2RA inhibition is measured on day 6 of culture. IL-2Rα inhibition is a key quality characteristic that measures the effect of MSCs on alloreactive T cell activation, proliferation, and inflammatory cytokine production, key mechanisms by which the immune system regulates disease severity in SR-aGVHD.

[0212] When measuring IL-2RA inhibition, the degree of PBMC stimulation and subsequent proinflammatory cytokine release is a critical factor in the ability of an MSC lot to release immunomodulatory factors necessary for IL-2RA inhibitory activity, such as IDO-1, PGE2, and PD-L1. To assess the minimum level of PBMC stimulation with anti-CD3 and anti-CD28 antibodies required to obtain reliable measurements of lot potency, regression analyses were performed to evaluate the relationship between IL-2RA produced by stimulated PBMCs and the degree of suppressive activity elicited by the MSC lot.

[0213] As shown in Figure 5, a correlation was observed between the amount of IL-2RA produced by stimulated PBMCs and the percent IL-2RA inhibition across all 40 lots tested (p=0.012). This association remained significant at thresholds of 10,000, 11,000, and 12,000 pg / ml of IL-2RA expression in stimulated PBMCs but was no longer evident when the amount of IL-2RA produced by stimulated PBMCs exceeded 13,000 pg / ml. The 30 lots tested with PBMCs producing >13,000 pg / ml of IL-2RA achieved a mean 83% inhibition (range 71-91%), compared with 77% inhibition (range 63-86%) for the 10 lots tested with PBMCs producing <13,000 pg / ml of IL-2RA (p=0.005).

[0214] These results indicate that IL-2RA production of 12,000–13,000 pg / ml reflects the minimum threshold of PBMC stimulation required to ensure sufficient activation of an MSC lot for maximal IL-2RA inhibitory activity. Although the mean IL-2RA inhibitory activity of lots tested with PBMCs producing less than 13,000 IL-2RA was significantly lower than that of PBMCs producing more than 13,000 pg / ml IL-2RA, these data indicate that the potency of MSC lots tested with insufficiently stimulated PBMCs is uncertain and that the actual potency values ​​of these lots may be underestimated.

[0215] Example 6: Survivors of a GvHD clinical trial administered MSCs with high mean IL2R inhibition rates Next, we used inverse cumulative distribution curves to evaluate the relationship between product potency and clinical outcomes for patients (n=34) who received lots for which potency values ​​were measured using fully stimulated PBMCs (n=30 lots, IL-2RA >12811 pg / ml) (Figure 6).

[0216] As can be seen in Figure 6, the inverse cumulative distribution curve showed a significant relationship (p=0.03) between the weighted mean IL-2RA inhibition rate for the administered lots and survival at day 100. Because the mean IL-2RA inhibition across these 30 lots was 83.1% + 5.8%, we used 65.7% as the minimum threshold for IL-2RA inhibition required for lot release by the IL-2RA inhibition assay using fully stimulated PBMCs (IL-2RA >12811 pg / ml).

[0217] A clinical lot tested in 2015 in an IL-2RA inhibition assay using fully stimulated PBMCs (IL-2RA >12811 pg / ml) was retested in 2023 to assess whether potency remained when cryopreserved. As can be seen in Figure 7, IL-2Ra inhibition was measured for the same clinical lot at two time points: first in 2015 and then again in 2023, demonstrating high stability of the clinical lot for at least 8 years when cryopreserved.

[0218] Example 7: Sufficient PBMC stimulation and IL-2Ra expression induction levels required for accurate measurement of the inhibition rate of IL-2Rα inhibition The slope of degradation of IL-2Rα inhibition values ​​was 1.2% per year, indicating that DP lots of MSCs remain highly stable over time with respect to IL-2Rα inhibition. Comparison of the regression line to the actual assay values ​​demonstrates the precision of the sensitive, real-world IL-2Rα inhibition assay, as assessed across 10 lots using fully stimulated PBMCs at both time points. The ability of linear modeling with a common slope to predict actual test values ​​was used to calculate the assay standard deviation based on the linear regression residuals. This calculation indicates that the standard deviation of the IL-2Rα inhibition assay is less than 7%, indicating that the assay is sufficiently precise for use in measuring critical quality attributes.

[0219] To confirm the relationship between sufficient PBMC stimulation and MSC lot potency, as measured by IL-2Rα production, observed in the clinical lots, we performed a regression analysis based on these same parameters for 61 lots. As can be seen in Figure 8 (left box), for these additional 61 MSC lots, there was also a significant linear correlation between PBMC IL-2Rα production following anti-CD3 / CD28 stimulation and the degree of IL-2Rα inhibition (p<0.0001). This is consistent with IL2R production by activated PBMCs serving as a biomarker for the secretion of pro-inflammatory cytokines, such as IFN-gamma, TNF-alpha, and IL-1, which are involved in the induction of anti-inflammatory factors secreted by Ryoncil lots, including IDO-1, PGE2, and PD-L1.

[0220] As with previous clinical lots, the correlation between PBMC IL-2RA production and IL-2RA inhibition by cocultured MSC lots was observed only up to 12,000–13,000 pg / ml of IL-2RA production by stimulated PBMCs, indicating that maximal MSC suppression potency was induced above these PBMC stimulation levels. Of the lots tested with PBMCs stimulated to produce >13,000 pg / ml of IL-2RA, 95% showed >60% inhibition of IL-2RA, whereas only 70% of the lots tested with PBMCs suboptimally stimulated to produce less IL2R alpha achieved this level of inhibition (Figure 8, right box).

[0221] These results confirm that optimal PBMC stimulation and inflammatory cytokine secretion are required to accurately measure the potency of MSC lots with respect to IL-2RA inhibition, and that this can be achieved through quality control and strict pass / fail criteria of anti-CD3 / CD28 antibody-stimulated PBMCs.

[0222] Example 8: Pass / Fail Criteria for IL-2RA Inhibition Assay Considering the above data, the value of 65.7% represents the minimum threshold of IL-2RA inhibition met by 95% of all lots used in clinical trials, as measured by the IL-2RA inhibition assay when PBMCs were stimulated to IL-2RA levels of 12811 pg / ml or greater.

[0223] Example 9: IL2Ra Inhibition Assay Protocol and Validation Procedures Cell surface expression of interleukin-2 receptor alpha (IL-2Rα / IL-2RA) is an early marker of T cell activation. IL-2 / IL-2Rα primarily functions to promote the proliferation of CD4+ (helper) and CD8+ (cytotoxic) T cells. Inhibition of IL-2Rα expression suppresses T cell proliferation and further amplifies the immune response. An IL-2Rα inhibition assay was performed using ceMSCs as follows.

[0224] MSC(2.00×10 5cells / well) were treated with mouse anti-human CD3 / CD28 stimulated PBMCs (1.00 × 10 6 Co-culture with 1000 cells / well and incubate at 37°C ± 2°C, 5% ± 2% CO2 for 72 ± 2 hours.

[0225] Unstimulated (without CD3 / CD28) and stimulated (with CD3 / CD28 but without ceMSC) PBMCs are included as negative and positive controls, respectively.

[0226] At the end of the incubation period, cells are harvested and lysates are prepared, which are then aliquoted into sterile tubes and stored at -60°C or colder for up to 29 days before use in the IL-2Rα ELISA.

[0227] IL-2Rα levels are measured in lysates of unstimulated and stimulated PBMCs alone and in co-cultures using a Quantikine human CD25 / IL-2Rα ELISA kit at three serial dilutions.

[0228] IL-2Rα levels in controls and samples were determined using a standard curve constructed using four-parameter logistic (4-PL) curve fitting. Final ceMSC efficacy was assessed by the percentage of ceMSC-mediated inhibition of IL-2Rα levels in CD3 / CD28-stimulated PBMCs relative to control CD3 / CD28-stimulated PBMCs cultured without ceMSCs.

[0229] verification Due to the inherent variability in potency testing of cellular material against secondary reagent cell lines, additional measures must be considered. To address this inherent assay variability, we utilized a normalization factor to correct for donor-to-donor variability inherent in the assay's PBMC reagent.

[0230] In investigating the variability of the IL-2Rα inhibition assay, the combined effect of PBMC donor on the inhibitory mechanism and the variability of PBMC response to anti-CD3 / anti-CD28 stimulation were shown to be important parameters.

[0231] Therefore, appropriate controls would include screening PBMC lots to confirm reproducibility of PBMCs stimulating IL-2Rα above 12,811 pg / mL, and verifying pass / fail determination of whether the tested PBMCs meet the standard range of the ceMSC reference cell line on an average of at least four vials tested (i.e., four runs).

[0232] The ceMSC reference cell line can be generated from the same three lots of ceMSCs (generated from three bone marrow donors) used in the validation of the IL-2Rα inhibition assay, and the tolerance range is calculated based on the values ​​obtained in the validation. The ceMSC reference cell line is then used to qualify new PBMC lots for use in the IL-2Rα inhibition assay. The ceMSC reference cell line is qualified to ensure accurate generation of normalization factors for each PBMC lot and continued suitability of assay performance over time.

[0233] Those skilled in the art will appreciate that many modifications and / or variations may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described, and the present embodiments are therefore considered in all respects to be illustrative and not restrictive.

[0234] This application claims priority to US 63 / 444,198, filed February 8, 2023, and US 63 / 445,588, filed February 14, 2023, the disclosures of which are incorporated herein by reference.

[0235] All publications discussed above are incorporated herein in their entirety.

[0236] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and is not to be construed as an admission that any or all of such matters form part of the prior art or were common general knowledge in the art relevant to the present invention existing before the priority date of each claim in this application.

Claims

1. 1. A method for selecting a cell population for use in treating graft-versus-host disease, the method comprising: (i) obtaining a population of mesenchymal progenitor or stem cells (MLPSCs); (ii) culturing the MLPSC population in a culture medium; (iii) determining a level of IL-2RA inhibition under the culture conditions; and (iv) selecting an MLPSC population that inhibits IL-2RA by at least 56% under the culture conditions for use in the treatment.

2. 10. The method of claim 1, comprising selecting a population of MLPSCs that inhibits IL-2RA by at least 60% under culture conditions for use in therapy.

3. 3. The method of claim 1 or claim 2, further comprising cryopreserving the selected cells.

4. 1. A method for determining the therapeutic effect of a population of expanded mesenchymal progenitor or stem cells (MLPSCs), comprising: (i) obtaining a population of MLPSCs; (ii) culturing the cells in a culture medium; and (iii) determining a level of IL-2RA inhibition under the culture conditions, wherein the method also comprises determining the therapeutic effect on graft-versus-host disease (GvHD), wherein IL-2RA inhibition under the culture conditions is at least 56%, indicating a therapeutic effect.

5. The method of claim 4, wherein IL-2RA inhibition of 60% or more under culture conditions indicates a therapeutic effect.

6. 1. A method for producing a pharmaceutical product comprising a population of MLPSCs, the method comprising: obtaining a determination of whether a test population of MLPSCs inhibits IL-2RA under culture conditions to a predetermined level; and if the test population of MLPSCs inhibits IL-2RA under culture conditions to at least the predetermined level, processing at least a portion of the test population of MLPSCs as a pharmaceutical product to produce the pharmaceutical product; or discarding at least a portion of the test population of MLPSCs if the population of mesenchymal stem cells inhibits IL-2RA under culture conditions to less than the predetermined level, wherein the predetermined level is 56%.

7. The method of claim 6 , wherein the predetermined level is 60% or more under culture conditions.

8. 1. A method for treating a subject suffering from graft-versus-host disease (GvHD), comprising administering to a subject in need thereof a composition comprising an expanded population of mesenchymal progenitor or stem cells (MLPSCs), wherein the MLPSCs inhibit IL-2RA by at least 56% under culture conditions.

9. The method of claim 8, wherein the expanded MLPSCs contained in the composition administered inhibit IL-2RA by 60% or more under culture conditions.

10. The method according to any one of claims 1 to 5, 8 and 9, wherein the GvHD is acute GvHD.

11. The method according to any one of claims 1 to 5, 8 and 9, wherein the GvHD is chronic GvHD.

12. The method according to any one of claims 1 to 5 and 8 to 11, wherein the GvHD is pediatric GvHD.

13. The method according to any one of claims 1 to 5 and 8 to 12, wherein the GvHD is refractory to steroid therapy.

14. The method according to any one of claims 1 to 5 and 8 to 13, wherein the GvHD is grade D GvHD.

15. The method of any one of claims 8 to 14, wherein the increase in 100-day survival rate with treatment is greater than 60%, greater than 70%.

16. The method of any one of claims 1 to 15, wherein the MLPSCs are mesenchymal stem cells.

17. A composition comprising a population of expanded MLPSCs, wherein the population of MLPSCs is selected based on a predetermined level of IL-2RA inhibition under culture conditions, and wherein the predetermined level of IL-2RA inhibition is at least 56%.

18. 18. The composition of claim 17, wherein the predetermined level of IL-2RA inhibition is 60% or greater.

19. The composition of claim 17 or 18, wherein the MLPSCs are mesenchymal stem cells.

20. 20. The composition of claim 19, wherein the mesenchymal stem cells are also selected based on expression of one or more or all of CD29, CD54, CD73, CD90, CD102, CD105, CD106, CD166, MHC1.

21. The method of any one of claims 1 to 16 or the composition of any one of claims 17 to 20, wherein the level of IL-2RA inhibition is 65% or greater.

22. A method for producing a pharmaceutical product comprising a population of MLPSCs, (i) obtaining a determination of whether a test population of MLPSCs inhibits IL-2RA at a predetermined level under culture conditions, wherein step (i) comprises co-culturing the test population of MLPSCs with CD3 / CD28-activated PBMCs and determining the level of IL-2RA inhibition relative to a control population of CD3 / CD28-activated PBMCs in the absence of MLPSCs, wherein the predetermined level of IL-2RA inhibition is at least 56%.

23. 23. The method of claim 22, wherein the control population of CD3 / CD28-activated PBMCs expresses at least 12,000 pg / ml of IL-2RA.

24. The method of claim 22 or 23, wherein activating PBMCs with CD3 / CD28 comprises stimulating PBMCs with an anti-CD3 antibody and an anti-CD28 antibody.

25. The method of any one of claims 22 to 24, wherein the PBMCs are co-cultured with MLPSCs at a ratio of PBMC:MPLSC=5:

1.

26. 1 x 10 ６ PBMCs are 2 x 10 ５ The method of claim 25, wherein the cells are co-cultured with MLPSCs.

27. The method of any one of claims 22 to 26, wherein the PBMCs and MLPSCs are co-cultured for about 72 hours.

28. The level of IL-2RA inhibition is (i) measuring IL-2RA levels in a test population of MLPSCs co-cultured with CD3 / CD28-activated PBMCs, a positive control population of CD3 / CD28-activated PBMCs in the absence of MLPSCs, and a negative control population of non-activated PBMCs in the absence of MLPSCs; (ii) generating standard curves for determining IL-2RA levels in the positive control population, negative control population, and test population; and (iii) determining the rate of inhibition of IL-2RA levels by a test population of MLPSCs co-cultured with CD3 / CD28-activated PBMCs compared to the positive control CD3 / CD28-activated PBMC population.

29. 29. The method of claim 28, wherein the IL-2RA level is measured by enzyme-linked immunosorbent assay (ELISA).

30. 30. The method of claim 29 or 29, wherein the standard curve is generated using four-parameter logistic (4-PL) curve fitting.

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