Efficacy assays and methods of manufacture

Abstract

The present disclosure relates to improved cell compositions and potency assays for obtaining the improved cell compositions. Such compositions and assays may be suitable for the context of 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 the improved cell compositions.Such compositions and assays may be applicable in the context of T cell-mediated diseases such as graft-versus-host disease (GvHD). Background Art

[0002] Several cell therapy products for regenerative or immunotherapy applications have entered clinical evaluation and market authorization. However, their market access is hampered by the complexity and heterogeneity of these cell therapy products, which makes it difficult to identify relevant biological activities and, consequently, to define consistent quality of cell therapy products.

[0003] Physicochemical parameters (e.g., characterization of size, morphology, light scattering properties, tensile strength, cell number, confluence, identification of phenotypic markers, secreted substances, genotype, gene expression profile) are commonly used to identify and quantify active substances, intermediates, impurities, and contaminants. However, physicochemical parameters cannot confirm whether a product is biologically active and potent (i.e., produces the desired effect). In contrast, biological characterization considers the effect of the product on the biological system modeled in vitro or in vivo in animals and ultimately in the clinic.

[0004] There is a need to develop cell products for treating diseases (e.g., diseases requiring immunosuppression). Additionally, it is preferred to identify parameters that are critical to the efficacy of cell therapy products and to control them (e.g., through efficacy testing) so that products of consistent quality can be obtained. Summary of the Invention

[0005] The present disclosure encompasses the following unexpected findings:

[0006] i) IL-2Rα inhibition by stem cells in vitro correlates with inhibition of stem cell-directed T cell activation in vivo; and,

[0007] ii) The level of IL-2Rα inhibition by stem cells in vitro is unexpectedly effective in identifying pharmaceutical products that can improve the survival of patients with GvHD, especially those with severe GvHD.

[0008] Either or both of these findings support the use of IL-2Rα inhibition as a potency assay and further provide a reference level of inhibition that can be used as a release standard for a drug product. Thus, in a first example, the present disclosure relates to a method of selecting a cell population for treating graft-versus-host disease (GvHD), the method comprising: (i) obtaining a mesenchymal lineage precursor or stem cell (MLPSC) population; (ii) culturing the MLPSC population in culture; (iii) determining the level of IL-2RA inhibition under culture conditions; and (iv) selecting a MLPSC population that inhibits IL-2RA by at least 56% under culture conditions for use in therapy. In one example, the method comprises selecting a MLPSC population that inhibits IL-2RA by at least 60% under culture conditions for use in therapy. In one example, the method comprises selecting a MLPSC population that inhibits IL-2RA by at least 65% under culture conditions for use in therapy.

[0009] In one embodiment, the method further comprises cryopreserving the selected cells.

[0010] In another example, the present disclosure relates to a method for determining the therapeutic efficacy of a culture-expanded population of mesenchymal lineage precursor or stem cells (MLPSCs): (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 of graft-versus-host disease (GvHD), and wherein inhibition of IL-2RA by at least 56% under the culture conditions indicates therapeutic efficacy. In another example, inhibition of IL-2RA by ≥60% under the culture conditions indicates therapeutic efficacy. In another example, inhibition of IL-2RA by ≥65% under the culture conditions indicates therapeutic efficacy. For example, inhibition of IL-2RA by 65.7% under the culture conditions indicates therapeutic efficacy.

[0011] In another example, the present disclosure relates to a method for manufacturing a drug product comprising a population of MLPSCs, the method comprising: obtaining a determination result as to whether a test population of MLPSCs inhibits IL-2RA at a predetermined level under culture conditions, and processing at least a portion of the test population of MLPSCs into a drug product if the test population of MLPSCs inhibits IL-2RA at at least the predetermined level under culture conditions, thereby manufacturing the drug product; or discarding at least a portion of the test population of MLPSCs if the mesenchymal stem cell population inhibits IL-2RA at less than the predetermined level under culture conditions, wherein the predetermined level is 56%. In one example, the predetermined level is ≥ 60% under culture conditions. In one example, the predetermined level is ≥ 65% under culture conditions.

[0012] In another example, the present disclosure relates to a method of treating a subject with graft-versus-host disease (GvHD), the method comprising administering to a subject in need thereof a composition comprising a population of culture-expanded mesenchymal lineage precursor or stem cells (MLPSCs), wherein the MLPSCs suppress IL-2RA by at least 56% under culture conditions. In another example, the culture-expanded MLPSCs comprising the administered composition suppress IL-2RA by ≥60% under culture conditions. In another example, the culture-expanded MLPSCs comprising the administered composition suppress IL-2RA by ≥65% under culture conditions.

[0013] In one example, the GvHD is acute GvHD. In another example, the GvHD is chronic GvHD. In one example, the GvHD is childhood GvHD. In one example, the GvHD is refractory to steroid therapy. In one example, the GvHD is grade D GvHD.

[0014] In one example, treatment improves 100-day survival to greater than 60%, greater than 70%.

[0015] In one example, the present disclosure relates to a composition comprising a culture-expanded MLPSC population, wherein the MLPSC population is selected based on a predetermined level of IL-2RA inhibition under culture conditions, wherein the predetermined level of IL-2RA inhibition is at least 56%. In one example, the predetermined level of IL-2RA inhibition is ≥ 60%. In one example, the predetermined level of IL-2RA inhibition is ≥ 65%.

[0016] In one example, MLPSCs are mesenchymal stem cells. In one example, MSCs are one or more or all of CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, and MHC1+. In one example, MSCs are selected based on the expression of one or more or all of CD29, CD54, CD73, CD90, CD102, CD105, CD106, CD166, and MHC1.

[0017] In one example, the level of IL-2RA inhibition is ≥ 65%.

[0018] In one example, the present disclosure provides a method of manufacturing a drug product comprising a population of MLPSCs, the method comprising (i) obtaining a determination as to 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 without MLPSCs; wherein the predetermined level of IL-2RA inhibition is at least 56%.

[0019] In one example, a control population of CD3 / CD28 activated PBMCs expresses at least 12,000 pg / ml IL-2RA. In one example, a control population of CD3 / CD28 activated PBMCs expresses at least 11,000 pg / ml IL-2RA. In one example, a control population of CD3 / CD28 activated PBMCs expresses at least 13,000 pg / ml IL-2RA. In one example, a control population of CD3 / CD28 activated PBMCs expresses at least 10,000 pg / ml IL-2RA. In one example, a control population of CD3 / CD28 activated PBMCs expresses at least 12811 pg / ml IL-2RA.

[0020] In one example, CD3 / CD28 activation of PBMCs comprises stimulating PBMCs with an anti-CD3 antibody and an anti-CD28 antibody.

[0021] In one example, PBMCs were co-cultured with MLPSCs at a ratio of 5 PBMCs to 1 MLPSC.

[0022] In one example, 1×10 6 PBMCs and 2 × 10 5 MLPSCs were co-cultured.

[0023] In one example, PBMCs and MLPSCs are co-cultured for a period of about 72 hours.

[0024] 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 without MLPSCs, and a negative control population of non-activated PBMCs without MLPSCs; (ii) constructing a standard curve to determine IL-2RA levels in the positive control population, the negative control population, and the test population; and (iii) calculating the percent inhibition of IL-2RA levels in the test population of MLPSCs co-cultured with CD3 / CD28-activated PBMCs relative to the positive control population of CD3 / CD28-activated PBMCs.

[0025] In one example, IL-2RA levels are measured by enzyme-linked immunosorbent assay (ELISA).

[0026] In one example, the standard curve is generated using a four-parameter logistic (4-PL) curve fit. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : Correlation between IL-2Rα inhibition and clinical outcomes.

[0028] Figure 2: Correlation between product efficacy and survival in high-risk patients.

[0029] Figure 3: Product efficacy and changes in inflammatory biomarkers.

[0030] Figure 4 : Changes in product efficacy and activated T cells.

[0031] Figure 5: Relationship between PBMC stimulation and IL-2Rα inhibition by MSC batches.

[0032] Figure 6 : Survivors in a GvHD clinical trial received an MSC product with a higher mean percentage of IL2R inhibition.

[0033] Figure 7 : Maintenance of efficacy measured as percentage inhibition of IL2Ra in repeat testing 8 years later.

[0034] Figure 8: Accurate measurement of the percent inhibition of commercially available IL-2Rα requires adequate PBMC stimulation and induction levels of IL-2Rα expression. DETAILED DESCRIPTION

[0035] Throughout this specification, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter shall be considered to encompass both one and more of those steps, compositions of matter, groups of steps, or groups of compositions of matter (i.e., one or more).

[0036] It will be understood by those skilled in the art that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the disclosure includes all such variations and modifications. The disclosure also includes all steps, features, compositions and compounds mentioned or indicated in this specification, whether individually or collectively, and any and all combinations or any two or more of said steps or features.

[0037] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods, as described herein, are clearly within the scope of this disclosure.

[0038] Unless expressly stated otherwise, any example disclosed herein should be considered applicable mutatis mutandis to any other example.

[0039] Unless specifically defined otherwise, 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 cell culture, molecular genetics, stem cell therapy, immunology, immunohistochemistry, protein chemistry, biochemistry).

[0040] Unless otherwise indicated, the surgical techniques used in this disclosure are standard procedures well known to those skilled in the art.

[0041] Methods for obtaining and enriching populations of mesenchymal lineage stem cells or precursor cells are known in the art. For example, enriched populations of mesenchymal lineage stem cells or precursor cells can be obtained by using flow cytometry and cell sorting procedures based on the use of cell surface markers expressed on mesenchymal lineage stem cells or precursor cells.

[0042] All documents cited or referenced herein, and all documents cited or referenced in documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference in their entirety.

[0043] Selected Definitions

[0044] The method of the present disclosure is performed on a MLPSC population. Such methods can be performed on a MLPSC population representing a larger MLPSC population (e.g., a test population). In one example, the method of the present disclosure is performed on at least a portion of a test population of MLPSC. In one example, such methods include processing at least a portion of the test population of MLPSC into a drug product if the test population of MLPSC inhibits IL-2RA at least at a predetermined level under culture conditions. In one example, such methods may also include discarding at least a portion of the test population of MLPSC if the MLPSC population inhibits IL-2RA at a level lower than a predetermined level under culture conditions. In one example, the predetermined level is 56%. In one example, the predetermined level is ≥56%. In another example, the predetermined level is ≥60%.

[0045] 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-2RAlpha," and variants thereof are used interchangeably herein.

[0046] In the context of this disclosure, the term "predetermined" level refers 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 for GvHD. In another example, the predetermined level is predetermined by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In one example, the predetermined level corresponds to an improved survival rate for patients with GvHD. In one example, the predetermined level corresponds to an improved survival rate for patients with grade D GvHD.

[0047] In one example, the predetermined level is a "reference level of IL-2Rα inhibition". In one example, the reference level of inhibition is the inhibition level of an FDA-approved MLPSC population (e.g., an FDA-approved MSC population for the treatment of GvHD). In one example, the reference level of IL-2Rα provides a criterion for selecting a cell population according to the present disclosure.

[0048] In one example, the level of IL-2RA inhibition is determined under culture conditions. The term "culture conditions" is used to refer to cells grown in culture. In one example, the culture conditions refer to a population of actively dividing cells. In one example, such cells can be in an exponential growth phase.

[0049] In one example, the culture conditions encompass co-culturing of an MLPSC population disclosed herein with a second cell population, such as a population comprising peripheral blood mononuclear cells (PBMCs). In one example, co-culturing includes culturing an MLPSC population disclosed herein and an activated PBMC population. For example, PBMCs can be activated using anti-CD3 and anti-CD28 antibodies prior to co-culturing with an MLSPC population disclosed herein.

[0050] In one example, "culture conditions" include co-culturing MLPSCs and activated PBMCs at a ratio of about 1 MLPSC to 2 activated PBMCs or less. For example, MLPSCs and activated PBMCs are co-cultured at a ratio of 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 MLPSC to 100 activated PBMCs or less. In this example, the level of IL-2RA inhibition is determined after culturing the cells under the culture conditions for about 30 to 84 hours.

[0051] In one example, "culture conditions" include co-culturing MLPSCs and T cells at a ratio of about 1 MLPSC:2 T cells or less. For example, MLPSCs and T cells are co-cultured at a ratio of 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 MLPSC:100 T cells or less. In this example, the level of IL-2RA inhibition is determined after culturing the cells under the culture conditions for about 30 to 84 hours.

[0052] In one example, the level of a specific marker can be determined by obtaining a cell culture medium sample and measuring the level of the marker in the sample. In another example, the level of a specific marker can be determined by obtaining a cell sample and measuring the level of the marker in a cell lysate. It will be understood by those skilled in the art 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 the cell lysate. In one example, the sample is obtained when the cell is in the exponential growth phase. In one example, the sample is obtained after culturing for at least two or three days. In another example, the sample is collected after about 30 to 84 hours of co-cultivation. In one example, the sample is collected from the co-cultivation of MLPSC and activated PBMC. In this example, the cell sample can be lysed and the level of the marker can be determined.

[0053] In one example, various methods, such as enzyme-linked immunosorbent assay (ELISA)-based methods, can be used to determine the level of IL-2RA. Suitable ELISA kits are known in the art and include, for example, the Quantikine human CD25 / IL-2Rα ELISA. In one example, the ELISA comprises:

[0054] (i) adding sample diluent to each well of a microplate pre-coated with a monoclonal antibody specific for IL-2RA;

[0055] (ii) adding the co-cultured samples to the wells of a microplate pre-coated with a monoclonal antibody specific for IL-2RA;

[0056] (iii) incubating the microplate for a time sufficient to allow the monoclonal antibody specific for IL-2RA to specifically bind to any IL-2RA in the sample;

[0057] (iv) washing the microplate;

[0058] (v) adding IL-2RA conjugate to the well;

[0059] (vi) incubating the microplate for a time sufficient to allow the conjugate to specifically bind to any captured IL-2RA;

[0060] (vii) washing the microplate;

[0061] (viii) adding substrate solution to the wells;

[0062] (ix) incubating the microplate for a sufficient time to allow color development;

[0063] (x) Add stop solution to the wells;

[0064] (xi) reading the optical density on a microplate reader set to 450 nm and wavelength calibrated at 570 nm;

[0065] (xii) Determining the level of IL-2RA.

[0066] In another example, fluorescence activated cell sorting (FACS) is used to determine IL-2RA levels using appropriate antibodies, such as anti-CD25. Additional antibodies can also be used if differentiation of CD25+ cell types is desired.

[0067] In one example, the IL-2RA levels in the co-culture are compared to the IL-2RA levels in the culture population of activated PBMCs. The IL-2RA levels are then compared to provide the level of IL-2RA inhibition in the co-culture. For example, the IL-2RA levels in the co-culture (i.e., MLPSC: activated PBMCs) can be inhibited by at least 60% relative to the monoculture (activated PBMCs alone). Other suitable methods for determining IL-2RA levels under culture conditions will be appreciated by those skilled in the art.

[0068] In one example, the level of IL-2RA is determined using a standard curve. A "standard curve" refers to a general method for determining the concentration of a substance in an unknown sample by comparing it to a set of standard samples of known concentrations. In one example, a standard curve is constructed using a four-parameter logistic (4-PL) curve fit. As will be understood by those skilled in the art, four-parameter logistic (4PL) curves are regression models commonly used to analyze bioassays, such as ELISAs. They follow a sigmoidal or "s" shaped curve. This type of curve is particularly useful for characterizing bioassays, as bioassays are typically linear only within a specific concentration range.

[0069] Culturing and expanding cells from a cryopreserved intermediate means thawing the cells that have been subjected to cryopreservation and culturing them in vitro under conditions suitable for the growth of the cells.

[0070] 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 cells are cryopreserved for the first time. In another example, the level is determined after the cells are cryopreserved for the second time. For example, the cells can be cultured and expanded to provide an intermediate, cryopreserved, thawed, then re-seeded in culture, and further cultured and expanded, and the level of the particular marker can then be determined under culture conditions.

[0071] The term "intermediate" used in the context of this disclosure refers to an intermediate population of MLPSC that is freshly isolated and amplified in early culture (e.g., about 2 passages). For example, the generation of an intermediate population of MLPSC can include isolating nucleated bone marrow cells (NBMC), cell culture, and passage twice from bone marrow aspirate (BMA) to amplify the relevant cells. In one example, the cells are then harvested and prepared for analysis according to the methods described herein. In one example, the harvested cells are cryopreserved. Therefore, the generation of an intermediate population is the first stage in the process of producing a composition (i.e., a drug product) for administration to a subject. Although the examples cited above relate to the separation of NBMC from BMA, it will be understood by those skilled in the art that relevant cells can be obtained from other suitable sources (e.g., adipose tissue) discussed herein, and the relevant cells are subjected to necessary culture amplification to obtain intermediates and drug products.

[0072] In certain examples disclosed herein, the intermediates of the present disclosure are cultured and expanded to provide MLPSC populations for administration. In one example, such populations can be referred to as pharmaceutical compositions or pharmaceutical products (DPs). For example, the culture expansion of the intermediates involves cell culture and more passages (e.g., more than three passages) for culture expansion.

[0073] "Isolated" or "purified" means a cell that has been separated from at least some components of its natural environment. The term includes the total physical separation of a cell from its natural environment (e.g., removal from a donor). The term "isolated" includes altering the relationship of a cell to its directly associated neighboring cells, such as by dissociation. The term "isolated" does not refer to cells in a tissue section. When used to refer to a cell population, the term "isolated" includes a cell population produced by proliferation of an isolated cell of the present disclosure.

[0074] The term "passage (passaging)" or "passage culture" is used in the context of the present disclosure to refer to the known cell culture techniques for keeping cells alive and growing for a long time under culture conditions so that cell number is constantly increased. The degree of passage culture cell lines is usually expressed as "passage number" and is usually used to refer to the quantity of the number of times a cell has been passaged and cultured. In one example, a passage includes removing non-adhesive cells and leaving adherent mesenchymal lineage precursors or stem cells. Such mesenchymal lineage precursors or stem cells can then be dissociated from matrix or flask (for example, by using protease, such as trypsin or collagenase), medium can be added, optional washing (for example, by centrifugation), and then mesenchymal lineage precursors or stem cells can be re-plated or re-seeded into one or more culture vessels with a total surface area larger. Then, mesenchymal lineage precursors or stem cells can continue to expand in culture. In another example, the method for removing non-adhesive cells includes the step of non-enzymatic treatment (for example, with EDTA). In one example, mesenchymal lineage precursors or stem cells are passaged at or near confluence (e.g., about 75% to about 95% confluence). In one example, mesenchymal lineage precursors or stem cells are seeded at a concentration of about 10%, about 15%, or about 20% cells / ml of culture medium.

[0075] The terms "culture medium" or "medium" as used in the context of this disclosure include components of the environment surrounding cells in culture. It is contemplated that the culture medium facilitates and / or provides conditions suitable for cell growth. The culture medium can be solid, liquid, gaseous, or a mixture of phases and materials. The culture medium can include liquid growth media as well as liquid media that do not sustain cell growth. Exemplary gaseous media include the gaseous phase to which cells grown on a culture dish or other solid or semisolid support are exposed.

[0076] As used herein, the term "treating," "treat," or "treatment" includes administering a population of mesenchymal stem cells or precursor cells and / or their progeny and / or soluble factors therefrom to alleviate or eliminate at least one symptom of GvHD. In one example, treatment includes administering a population of culture-expanded mesenchymal stem cells or precursor 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.

[0077] In one example, the methods of the present disclosure inhibit disease progression or disease complications in a subject. "Inhibiting" disease progression or disease complications in a subject means preventing or reducing disease progression and / or disease complications in the subject. Thus, in one example, the methods of the present disclosure inhibit progression of disease severity. For example, such methods can inhibit progression of GvHD severity (i.e., inhibit progression of grade).

[0078] As used herein, the terms "prevent" or "preventing" include administering a population of mesenchymal lineage stem or precursor cells and / or progeny thereof and / or soluble factors derived therefrom to prevent or inhibit the development of at least one symptom of an inflammatory disease.

[0079] As used herein, the term "subject" refers to a human subject. For example, the subject can 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 the context of this disclosure. In one example, the subject has GvHD.

[0080] 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 lineage precursors or stem cells transfected with a nucleic acid encoding Ang1 will be considered genetically modified.

[0081] The term "clinically proven" (used independently or as a modification of the term "effective") means that efficacy has been demonstrated through clinical trials, wherein the clinical trials have met the approval criteria of the U.S. Food and Drug Administration, EMEA or corresponding national regulatory agencies. For example, the clinical study can be a randomized, double-blind study of sufficient scale to clinically demonstrate the effect of the composition. In one example, a clinically proven effective amount is an amount that the clinical trial shows meets a specified endpoint. In one example, the endpoint is protection from death. In other words, the endpoint increases survival rate. For example, when a cell population that meets the release criteria according to the present disclosure is administered, 100-day survival rate can be increased.

[0082] Thus, the terms "clinically demonstrated efficacy" and "clinically proven effective" can be used in the context of the present disclosure to refer to a dosage, dosage regimen, treatment or method disclosed herein. Efficacy can be measured based on changes in the disease process in response to the administration of a composition disclosed herein. For example, a composition of the present disclosure is administered to a subject in an amount and duration sufficient to cause an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of the inflammatory disease. Various indicators reflecting the severity of the disease can be assessed to determine whether the amount and duration of treatment are sufficient. 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 amount increases patient survival. In another example, a clinically proven effective amount reduces the risk of death in a subject. In another example, a clinically proven effective amount increases 100-day survival. In one example, the method of the present disclosure administers a clinically proven effective amount of a composition disclosed herein.

[0083] "Therapeutic efficacy" in the context of the present disclosure refers to MLPSCs and compositions disclosed herein that can treat, inhibit, and / or prevent disease. For example, therapeutically effective MLPSCs and compositions disclosed herein can treat, inhibit, and / or prevent inflammatory diseases (such as GvHD). In one example, therapeutically effective MLPSCs and compositions disclosed herein increase the 100-day survival rate of subjects with GvHD.

[0084] The term "and / or," such as "X and / or Y," should be understood to mean "X and Y" or "X or Y," and should be used to provide clear support for both meanings or either meaning.

[0085] Unless stated to the contrary, the term "about" as used herein means + / - 10%, more preferably + / - 5% of the specified value.

[0086] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0087] As used herein, the singular forms "a," "an," and "the" include singular and plural references unless the context dictates otherwise.

[0088] Graft-versus-host disease (GvHD)

[0089] In one example, the method of the present disclosure relates to providing a cell colony that can effectively treat graft-versus-host disease (GvHD). "Graft-versus-host disease (GvHD)" is an immune disorder that is a major factor limiting the success and availability of allogeneic bone marrow or stem cell transplants. GvHD occurs in acute (aGvHD) or chronic (cGvHD) forms. Acute GvHD usually manifests within 100 days after bone marrow or stem cell transplantation. Chronic GvHD usually manifests later than aGvHD (>100 days after transplantation) and has some characteristics of autoimmune diseases. It can develop from scratch, after aGvHD subsides, or as an extension of aGvHD. Chronic GvHD can cause a variety of symptoms that are often debilitating, including large-area rashes, painful oral ulcers, shortness of breath, and pain in the limbs and joints. For example, CD5+B cell reconstruction is impaired in patients with cGvHD. In one example, cGvHD is refractory to steroid therapy. In one example, cGvHD is refractory to biological therapy. In one example, cGvHD is refractory to steroid therapy and biologic therapy.

[0090] The severity of GvHD can be graded according to the organ involvement pattern and clinical performance status. Multi-organ involvement includes rash, liver involvement and / or gastrointestinal (GI) involvement. Table 1 and Table 2 provide examples of rash, liver involvement and gastrointestinal involvement. In one example, the subject suffers from GvHD with multi-organ involvement. In one example, the 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, according to the Glucksberg scale, the subject can suffer from grade II GvHD or grade III / IV GvHD. In one example, the subject suffers from grade II GvHD. In another example, the subject suffers from grade III / IV GvHD.

[0091] 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, C, or D GvHD according to the IBMTR severity scale. In one example, the subject has grade D GvHD.

[0092] In another example, the subject has Minnesota high-risk GvHD. Minnesota high-risk acute GvHD is defined as skin stage 4; lower gastrointestinal (GI) stage 3-4 or liver stage 3-4; or skin stage 3+ and lower GI stage 2-4 or liver stage 2-4 GvHD (MacMillan et al., 2015). In each of these examples, the subject can also have a high MAP score. For example, a subject can have a MAP score of ≥0.29:

[0093] Table 1: Glucksberg acute GvHD clinical stages and grades (Rowlings et al., 1997).

[0094]

[0095]

[0096] Table 2: IBMTR Severity Index criteria for acute GvHD (Rowlings et al., 1997).

[0097]

[0098] *Index assigned based on maximum value of individual organ systems

[0099] In one example, a subject with severe GvHD is unresponsive to treatment with primary therapy. In one example, the subject worsens within 3 days of primary therapy. In this example, worsening is indicated if the severity of the subject's GvHD increases. For example, the severity of the subject's GvHD has increased according to the MAP. In another example, the severity of the subject's GvHD has increased according to the Glucksberg scale. In another example, the severity of the subject's GvHD has increased according to the IMBTR scale. In another example, the severity of the subject's GvHD has increased according to organ involvement.

[0100] In another example, the subject has no response within 7 days of the primary therapy. For example, the subject is refractory to the primary therapy. In one example, the primary therapy is systemic steroids. In one example, the subject suffers from 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 suffers from severe GvHD and is refractory to steroids and second-line therapy. For example, second-line therapy can include extracorporeal photopheresis, etanercept, infliximab, ruxolitinib, antithymocyte globulin, mycophenolic acid, alemtuzumab, basiliximab or tocilizumab.

[0101] Treatment response

[0102] The methods disclosed herein relate to the treatment of GvHD. As used herein, "treating," "treat," "treatment," and "slowing progression" include administering a population of mesenchymal stem or precursor cells and / or their progeny and / or soluble factors derived therefrom and / or extracellular vesicles derived therefrom, thereby reducing or eliminating at least one symptom of GvHD.

[0103] As used herein, the term "response" refers to a response to therapy. In one example, a subject is considered to have responded if at least one organ improves without progression in any other organ, and if no additional therapy is required. In another example, a subject is considered to have not responded if they have stable or progressive GvHD or if secondary therapy is subsequently required. In this example, a subject who does not respond is a non-responder.

[0104] In one example, treatment induces a partial response. In one example, the partial response is induced at least 28 days after the start of treatment. In one example, the partial response is induced 28 days after the start of treatment. In one example, the partial response is induced at least 30 days after the start of treatment. In one example, the partial response is induced at least 2 months after the start of treatment. In another example, the partial response is induced at least 3 months after the start of treatment. In another example, the partial response is induced within 3 months. In another example, the partial response is induced 28 to 56 days after the start of treatment. In another example, the partial response is induced 100 days after the start of treatment. In another example, the partial response is induced 160 days after the start of treatment. In another example, the partial response is induced 180 days after the start of treatment.

[0105] In another example, a partial response is elicited after two doses. In another example, a partial response is elicited after two doses administered once weekly. In another example, a partial response is elicited after two doses administered once weekly for two weeks. In another example, a partial response is elicited after three or more doses. In one example, in the context of GvHD, a partial response is characterized by one, more, or all of the following:

[0106] - a decrease in skin %BSA score of at least one point;

[0107] - Oral score decreased by at least one point;

[0108] - Eye score decreased by at least one point;

[0109] - a decrease in skin feature score of at least one point;

[0110] - A decrease in the GI score of at least one point;

[0111] - liver score decreased by at least one point;

[0112] - Pulmonary symptom score decreases by at least one point;

[0113] - A decrease in lung FEV1 score of at least one point;

[0114] - Joint and fascia scores decreased by at least one point;

[0115] - A decrease in the reproductive tract score of at least one point.

[0116] In one example, a partial response is characterized by a decrease in the skin % BSA score of at least one point. In another example, a partial response is characterized by a decrease in the oral cavity score of at least one point. In another example, a partial response is characterized by a decrease in the eye score of at least one point. In these examples, the score can be obtained using the NIH Consensus Criteria 2014 for GvHD.

[0117] In another example, the partial response is characterized by one or more or all of the following:

[0118] - a decrease in skin %BSA score of at least one point;

[0119] - Oral score decreased by at least one point;

[0120] - Eye score decreased by at least one point.

[0121] There are multiple 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 results disclosed herein (Jagasia et al., (2015) Biol Blood Marrow Transplant., 21:389-401). The components of the NIH Consensus Criteria 2014 are shown in the following table:

[0122] Table 3: Organ Scoring of GvHD

[0123]

[0124]

[0125] In one example, a partial response is a decrease of ≥1 point in the organ-specific NIH Consensus Criteria 2014 score in the table above. Thus, in one example, treatment causes a decrease of ≥1 point in the skin % BSA score. In another example, treatment causes a decrease of ≥1 point in the oral cavity score. In another example, treatment causes a decrease of ≥1 point in the eye score. In another example, treatment causes a decrease of ≥1 point in the skin feature score. In another example, treatment causes a decrease of ≥1 point in the gastrointestinal tract score. In another example, treatment causes a decrease of ≥1 point in the liver score. In another example, treatment causes a decrease of ≥1 point in the pulmonary symptom score. In another example, treatment causes a decrease of ≥1 point in the lung FEV1 score. In another example, treatment causes a decrease of ≥1 point in the joint and fascia score. In another example, treatment causes a decrease of ≥1 point in the reproductive tract score.

[0126] In one example, treatment induces a complete response after the start of treatment. In one example, a complete response is complete resolution of GvHD symptoms in all organs. In one example, the complete response is induced 28 days after the start of treatment. In one example, the complete response is induced at least 28 days after the start of treatment. In one example, the complete response is induced at least 30 days after the start of treatment. In one example, the complete response is induced at least 2 months after the start of treatment. In another example, the complete response is induced at least 3 months after the start of treatment. In another example, the complete response is induced 28 to 56 days after the start of treatment. In another example, the complete response is induced 100 days after the start of treatment. In another example, the complete response is induced 160 days after the start of treatment. In another example, the complete response is induced 180 days after the start of treatment.

[0127] In another example, a complete response is elicited after two doses. In another example, a complete response is elicited after two doses administered once weekly. In another example, a complete response is elicited after two doses administered once weekly for two weeks. In another example, a complete response is elicited after three or more doses.

[0128] In another example, also in the context of GvHD, treatment increases the subject's probability of survival. For example, treatment increases the probability that the subject will survive at least 20 to 200 days after the start of treatment. In one example, treatment increases the probability that the subject will survive at least 180 days after the start of treatment. In another example, treatment increases the probability that the subject will survive at least 100 days. In one example, the increase in probability is determined relative to a subject not treated with the composition of the present disclosure. In one example, treatment increases the 100-day survival rate by greater than 60%. In another example, treatment increases the 100-day survival rate by greater than 60%. In another example, treatment increases the 100-day survival rate by greater than 65%. In another example, treatment increases the 100-day survival rate by greater than 68%. In another example, treatment increases the 100-day survival rate by greater than 70%. In another example, treatment increases the 100-day survival rate by greater than 71%. In another example, treatment increases the 100-day survival rate by greater than 72%. In another example, treatment increases the 100-day survival rate by 60% to 75%. In another example, treatment improves the 100-day survival rate by 65% ​​to 75%. In another example, treatment improves the 100-day survival rate by 70% to 75%. In one example, the 100-day survival rate of a patient population is determined, and the average 100-day survival rate observed in the population provides a measure of the 100-day survival rate of each patient in the population. In one example, the measure is compared with a subject (or subject population) not treated with the composition of the present invention.

[0129] Mesenchymal lineage precursors or stem cells

[0130] As used herein, the term "mesenchymal lineage precursor or stem cell (MLPSC)" refers to an undifferentiated multipotent cell that has the ability to self-renew while maintaining multipotency and the ability to differentiate into a number of cell types of mesenchymal origin (e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons) or non-mesoderm origin (e.g., hepatocytes, neural cells, and epithelial cells). For the avoidance of doubt, "mesenchymal lineage precursor cell" refers to a cell that can differentiate into mesenchymal cells (such as bone, cartilage, muscle, and adipocytes) as well as fibrous connective tissue.

[0131] The term "mesenchymal lineage precursor or stem cell" includes both parental cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSC), perivascular mesenchymal precursor cells and their undifferentiated progeny.

[0132] Mesenchymal lineage precursors or stem cells can be autologous, allogeneic, xenogeneic, syngeneic, or isogenic. Autologous cells are isolated from the same individual into which they will be re-implanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0133] In one example, the mesenchymal lineage precursors or stem cells are allogeneic. In one example, the allogeneic mesenchymal lineage precursors or stem cells are culture expanded and cryopreserved.

[0134] Mesenchymal lineage precursors or stem cells are mainly located in the bone marrow, but have also been shown to be present in a variety of host tissues, including, for example, umbilical cord blood and umbilical cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp. They are also present in skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestines, lungs, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis, and periosteum; and can be differentiated into reproductive system such as mesoderm and / or endoderm and / or ectoderm. Therefore, mesenchymal lineage precursors or stem cells can be differentiated into a large number of cell types, including but not limited to adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue, and fibrous connective tissue. The specific lineage-stereotype and differentiation pathways that these cells enter depend on the various effects of mechanical influences and / or endogenous bioactive factors (such as growth factors, cytokines, and / or the local microenvironmental conditions established by host tissues).

[0135] The term "enriched", "enrichment" or variations thereof are used herein to describe such cell colonies, in which the ratio of a particular cell type or the ratio of many particular cell types increases when compared to an untreated cell colony (e.g., cells in their native environment). In one example, the colony enriched for mesenchymal lineage precursors or stem cells comprises at least about 0.1%, or 0.5%, or 1%, or 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 50%, or 75% mesenchymal lineage precursors or stem cells. In this regard, the term "cell colony enriched for mesenchymal lineage precursors or stem cells" will be considered to provide clear support for the term "cell colony comprising X% mesenchymal lineage precursors or stem cells", where X% is a percentage as enumerated herein. In some examples, mesenchymal lineage precursors or stem cells can form clonogenic colonies, such as CFU-F (fibroblasts) or a subset thereof (e.g., 50%, or 60%, or 70%, or 70%, or 90%, or 95%) can have such activity.

[0136] In one example of the present disclosure, the mesenchymal lineage precursor or stem cell is a mesenchymal stem cell (MSC). MSC can be a homogeneous composition or a mixed cell population enriched in MSC. Homogeneous MSC compositions can be obtained by culturing adhered bone marrow or periosteal cells, and MSC can be identified by specific cell surface markers identified with unique monoclonal antibodies. Methods for obtaining cell populations enriched in MSC are described in, for example, U.S. Patent No. 5,486,359. Alternative sources of MSC include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, MSC is allogenic. In one example, MSC is cryopreserved. In one example, MSC is cultured, expanded, and cryopreserved.

[0137] In another example, the mesenchymal lineage precursor or stem cell is a CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSC.

[0138] The isolated or enriched mesenchymal lineage precursors or stem cells can be expanded in vitro by culture.The isolated or enriched mesenchymal lineage precursors or stem cells can be cryopreserved, thawed and subsequently expanded in vitro by culture.

[0139] In one example, isolated or enriched mesenchymal lineage precursors or stem cells are plated at 50,000 viable cells / cm 2Cells are plated in culture medium (serum-free or serum-supplemented), for example, alpha minimum essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, and allowed to adhere to the culture vessels overnight at 37° C., 20% O 2 . The culture medium is then replaced and / or changed as needed, and the cells are cultured at 37° C., 5% O 2 for an additional 68 to 72 hours.

[0140] As will be understood by those skilled in the art, cultured mesenchymal lineage precursors or stem cells are phenotypically different from cells in vivo. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal lineage precursors or stem cells are also biologically different from cells in vivo, having a higher proliferation rate than most non-circulating (quiescent) cells in vivo.

[0141] In one example, a cell population is enriched from a cell preparation comprising STRO-1+ cells in a selectable form. In this regard, the term "selectable form" is understood to mean that the cells express markers (e.g., cell surface markers) that allow the selection of STRO-1+ cells. The marker can be STRO-1, but not necessarily. For example, as described and / or exemplified herein, cells (e.g., mesenchymal precursor 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 (and may be STRO-1bright). Therefore, the indication that a cell is STRO-1+ does not mean that the cell is selected only by STRO-1 expression. In one example, cells are selected based on at least STRO-3 expression, for example, they are STRO-3+ (TNAP+). For example, MPCs can be isolated from bone mononuclear cells using anti-STRO-3 antibodies.

[0142] 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 from, isolated from, or enriched from a variety of sources. That is, in some instances, these terms provide support for selection from any tissue or vascularized tissue containing STRO-1+ cells (e.g., mesenchymal precursor cells) or tissue containing pericytes (e.g., STRO-1+ pericytes) or any one or more of the tissues listed herein.

[0143] In one example, the cells used in the present disclosure express one or more markers, alone 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.

[0144] "Individually" means that the disclosure individually encompasses the recited marker or marker groups, and although individual markers or marker groups may not be individually listed herein, the appended claims may define such markers or marker groups individually and separately from each other.

[0145] "Collectively" means that the present disclosure encompasses any number or combination of the listed markers or marker groups, and although such number or combination of markers or marker groups may not be specifically listed herein, the appended claims may define such combinations or subcombinations individually and separately from any other combination of markers or marker groups.

[0146] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue nonspecific alkaline phosphatase. For example, the term encompasses the liver isoform (LAP), the bone isoform (BAP), and the kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP, as used herein, refers to a molecule that can bind to the STRO-3 antibody produced by the hybridoma cell line deposited with the ATCC on December 19, 2005, under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0147] Furthermore, in one example, STRO-1+ cells were able to generate clonogenic CFU-F.

[0148] In one example, a significant proportion of STRO-1+ cells are capable of differentiating into at least two different germ lineages. Non-limiting examples of lineages to which STRO-1+ cells can be committed include bone progenitor cells; hepatocyte progenitor cells, which are multipotent for bile duct epithelial cells and hepatocytes; neural-restricted cells, which can give rise to glial cell precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; myocardium and precursors of cardiomyocytes; and pancreatic beta cell lineages that secrete glucose-responsive insulin. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as precursors of 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 progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiomyocytes, smooth muscle cells, skeletal muscle cells, pericytes, vascular cells, epithelial cells, glial cells, neurons, astrocytes, and oligodendrocytes.

[0149] In one example, mesenchymal lineage precursors or stem cells are obtained from a single donor or multiple donors, wherein the donor samples or mesenchymal lineage precursors or stem cells are subsequently pooled and then expanded in culture.

[0150] Mesenchymal lineage precursors or stem cells encompassed by the present disclosure can also be cryopreserved prior to administration to a subject. In one example, the mesenchymal lineage precursors or stem cells are culture-expanded and cryopreserved prior to administration to a subject.

[0151] In one example, the present disclosure encompasses mesenchymal lineage precursors or stem cells and their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal lineage precursors or stem cells and extracellular vesicles isolated therefrom. For example, the mesenchymal lineage precursors or stem cells of the present disclosure can be cultured and expanded for a period of time under conditions suitable for secretion of extracellular vesicles into cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

[0152] As used herein, the term "extracellular vesicle" refers to lipid particles that are naturally released from cells and range in size from about 30 nm to as large as 10 microns, but the size of the lipid particles is generally less than 200 nm. The lipid particles may contain extracellular vesicles from releasing cells (e.g., mesenchymal stem cells; STRO-1 + proteins, nucleic acids, lipids, metabolites or organelles of cells).

[0153] As used herein, the term "exosome" refers to a type of extracellular vesicle that is typically sized in the range of about 30 nm to about 150 nm and originates from the endosomal compartment of mammalian cells, from which it is transported to the cell membrane and released. The extracellular vesicle may contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolites, and plays a role in intercellular communication by being secreted from one cell and taken up by other cells to deliver their cargo.

[0154] In one example, the compositions of the present disclosure comprise cells that induce neovascularization 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 has experienced a lack of blood flow caused by an ischemic event. In one example, the cells secrete factors that reduce cardiomyocyte apoptosis.

[0155] Cell culture and expansion

[0156] In one example, mesenchymal lineage precursors or stem cells are cultured and expanded. The difference between " culture-expanded " mesenchymal lineage precursors or stem cells and freshly separated cells is that they have been cultured and passed down (i.e., passage culture) in cell culture medium. In one example, freshly separated cell culture expansion is approximately 1 or 2 passages, to provide an intermediate colony. In one example, freshly separated cell culture expansion is 2 passages, to provide an intermediate colony. In another example, freshly separated cell culture expansion is approximately 1 to 3 passages, to provide an intermediate colony.

[0157] Thus, in one example, the relevant cells are isolated and cultured and expanded for 2 passages to provide an intermediate MLPSC population. The intermediate MLPSC population is then cultured and expanded to provide DP. For example, the intermediate cell population can be cultured for another three passages (i.e., a total of 5 passages) to provide DP.

[0158] In one example, the mesenchymal lineage precursors or stem cells are cultured and expanded for about 4–10 passages. In one example, the mesenchymal lineage precursors or stem cells are cultured and expanded for at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, or at least 10 passages. For example, the mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5 passages. In one example, the mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-10 passages. In one example, the mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-8 passages. In one example, the mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-7 passages. In one example, the mesenchymal lineage precursors or stem cells can be cultured and expanded for more than 7 passages. In these examples, the MLPSCs can be cultured and expanded prior to cryopreservation to provide a cryopreserved intermediate MLPSC population, which can then be further cultured and expanded.

[0159] In one example, the DP compositions of the present disclosure are produced by culturing cells from a cryopreserved intermediate MLPSC population, or in other words, a cryopreserved intermediate.

[0160] In one example, the compositions of the present disclosure include mesenchymal lineage precursors or stem cells cultured and amplified from an intermediate stored at low temperatures. In one example, cells cultured and amplified from an intermediate stored at low temperatures are cultured and amplified for at least 3 generations, at least 5 generations, at least 6 generations, at least 7 generations, at least 8 generations, at least 9 generations, at least 10 generations. For example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 3 generations. In one example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 3-10 generations. In one example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 3-8 generations. In one example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 3-7 generations.

[0161] In one example, the effect of IL-2RA inhibition by mesenchymal lineage precursors or stem cells expanded from cryopreserved intermediates disclosed herein is assessed under culture conditions (e.g., co-cultured with T cells). In one example, these MLPSCs inhibit IL-2RA by greater than 56% under culture conditions. In another example, these MLPSCs inhibit IL-2RA by greater than 60% under culture conditions.

[0162] In one example, the culture-expanded mesenchymal lineage precursors or stem cells can be cultured and expanded in a medium without animal protein (e.g., a medium without exogenous components). In one example, the mesenchymal lineage precursors or stem cells can be cultured and expanded in a medium without fetal bovine serum.

[0163] In one embodiment, mesenchymal lineage precursors or stem cells can be obtained from a single donor or multiple donors, wherein the donor samples or mesenchymal lineage precursors or stem cells are then pooled and then cultured and expanded as needed. In one example, the culture expansion process includes:

[0164] i. expanding said number of viable cells by passage expansion to provide a preparation of at least about 1 billion viable cells, wherein the passage expansion comprises establishing a primary culture of the isolated mesenchymal lineage precursors or stem cells, and then serially establishing a first non-primary (P1) culture of the isolated mesenchymal lineage precursors or stem cells from the previous culture;

[0165] ii. expanding the isolated P1 culture of mesenchymal lineage precursors or stem cells into a second non-primary (P2) culture of mesenchymal lineage precursors or stem cells by passage expansion; and,

[0166] iii. preparing and cryopreserving an in-process intermediate mesenchymal lineage precursor or stem cell preparation obtained from a P2 culture of mesenchymal lineage precursor or stem cells; and, optionally

[0167] iv. Thawing the cryopreserved in-process intermediate mesenchymal lineage precursor or stem cell preparation and expanding the in-process intermediate mesenchymal lineage precursor or stem cell preparation by passage expansion.

[0168] In the context of the present disclosure, certain assays can be performed between steps iii and iv. For example, IL-2RA inhibition can be determined under culture conditions after step iii. In one example, step iv is only performed if the desired level of IL-2RA inhibition is observed under culture conditions. In this example, a cell population is selected for culture expansion based on IL-2RA inhibition under culture conditions.

[0169] In one example, the expanded mesenchymal lineage precursor or stem cell preparation has an antigenic profile and an activity profile comprising:

[0170] i. less than about 0.75% CD45+ cells;

[0171] ii. at least about 95% CD105+ cells;

[0172] iii. at least about 95% CD166+ cells.

[0173] In one example, the expanded population of mesenchymal lineage precursors or stem cells is expanded from an intermediate MLPSC population and is capable of inhibiting IL-2RA expression by CD3 / CD28-activated PBMCs by at least 56% relative to a control. In another example, the expanded population of mesenchymal lineage precursors or stem cells is expanded from an intermediate MLPSC population and is capable of inhibiting IL-2RA expression by CD3 / CD28-activated PBMCs by at least 60% relative to a control.

[0174] Any equipment and cell processing methods known in the art can be used to carry out the process of separation and ex vivo expansion of mesenchymal lineage precursors or stem cells. Various culture expansion embodiments disclosed herein adopt steps that require manipulation of cells, such as steps of inoculation, feeding, dissociation of adherent cultures or washing. Any step of manipulating cells is likely to damage the cells. Although mesenchymal lineage precursors or stem cells can generally tolerate a certain amount of damage during preparation, it is preferred to manipulate cells by fully performing the operating procedures and / or equipment for one or more given steps while minimizing damage to the cells.

[0175] In one example, mesenchymal lineage precursors or stem cells are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculating wash bag, a rotating membrane filter having an inlet port and an outlet port, a filtrate bag, a mixing zone, a final product bag for the washed cells, and appropriate tubing, e.g., as described in U.S. Pat. No. 6,251,295, which is hereby incorporated by reference.

[0176] In one example, a mesenchymal lineage precursor or stem cell composition cultured according to the present disclosure is 95% homogeneous with respect to being CD105 positive and CD166 positive and CD45 negative. In one example, this homogeneity persists through ex vivo expansion (ie, through multiple population doublings).

[0177] In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded in 2D culture. For example, the mesenchymal lineage precursors or stem cells of the present disclosure can be cultured and expanded in a cell factory. In certain examples, 3D culture of the intermediates disclosed herein can be performed using, for example, a bioreactor. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are initially cultured and expanded in 2D culture and then further expanded in 3D culture. In one example, the intermediate cell population of the present disclosure has not yet been cultured and expanded in 3D culture. In one example, IL-2RA inhibition is evaluated after culture expansion in 3D culture.

[0178] In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded from an intermediate population. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded from an intermediate population in 2D culture prior to seeding in 3D culture.

[0179] In the context of intermediate populations and therapeutic compositions expanded therefrom, in one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded in 2D culture for at least 3 days and then seeded in another culture system (such as a cell factory or 3D culture in a bioreactor). In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded in 2D culture for at least 4 days and then seeded in another culture system. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded in 2D culture for 3 to 5 days and then seeded in another culture system. In these examples, 2D culture can be carried out 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 5 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 carried out in various types of bioreactors (such as stirred tanks, wave bags, and vertical wheels).

[0180] In one example, CO2 is provided during the culture expansion of MLPSC. In one example, MLPSC is cultured and expanded in less than 9% CO2. In one example, MLPSC is cultured and expanded in less than 8% CO2. In one example, MLPSC is cultured and expanded in 5% CO2. For example, MLPSC can be cultured and expanded in 5% + / - 2% CO2. In one example, MLPSC is cultured and expanded under passive pretreatment of CO2. For example, the cell factory can be passively pretreated with 5% CO2.

[0181] Pretreatment of the cell factory maintains CO2 tension between the cell factory and the incubator and stabilizes the pH level of the growth medium. Active pretreatment involves actively passing CO2 gas through a bacterial ventilation filter into each culture dish (e.g., a cell factory) for a specified period of time (e.g., about 10 minutes). However, active pretreatment may introduce contamination into the culture because it requires an open port to provide gas. Passive pretreatment involves placing the closed culture system in an incubator with an appropriate CO2 concentration and then inoculating the cells (e.g., about 12 to 72 hours).

[0182] In one example, the cells of the present disclosure are STRO-3+ prior to expansion in culture to provide an intermediate cell population.

[0183] Cell culture medium

[0184] The mesenchymal lineage precursors or stem cells disclosed herein can be cultured and expanded in a variety of suitable growth media.

[0185] The term "medium" or "media" as used in the context of the present disclosure includes components of the environment surrounding cells. The medium helps and / or provides conditions suitable for allowing cell growth. The medium can be solid, liquid, gaseous, or a mixture of phases and materials. The medium can include liquid growth media as well as liquid media that do not maintain cell growth. Medium also includes gel-like media such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous medium includes the gas phase to which cells grown on a culture dish or other solid or semisolid support are exposed.

[0186] Cell culture media used for cell culture and expansion contain all essential amino acids and 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).

[0187] Those skilled in the art will appreciate that, in order to obtain optimal results, the basal medium must be appropriate for the cell line of interest. For example, if it is found that the glucose (or other energy source) in the basal medium is depleted and therefore restricts growth, it may be necessary to increase the glucose (or other energy source) level in the basal medium, or to add glucose (or other energy source) during the culture process. In one example, the dissolved oxygen (DO) level may also be controlled.

[0188] In one embodiment, the cell culture medium contains additives of human origin. For example, human serum and human platelet cell lysate can be added to the cell culture medium.

[0189] In one embodiment, the cell culture medium contains only additives of human origin. Thus, in one embodiment, the cell culture medium is free of exogenous components. For the avoidance of doubt, in these embodiments, the culture medium does not contain animal proteins. In one embodiment, the cell culture medium used in the methods of the present disclosure does not contain animal components.

[0190] In one example, the culture medium comprises serum. For example, fetal bovine serum and / or newborn calf serum. In other examples, the culture medium is a culture medium that does not contain fetal bovine serum and contains growth factors that promote the proliferation of mesenchymal lineage precursors or stem cells. In one embodiment, the culture medium is a serum-free stem cell culture medium. In one example, the cell culture medium comprises:

[0191] basal culture medium;

[0192] Platelet-derived growth factor (PDGF);

[0193] Fibroblast growth factor 2 (FGF2).

[0194] In one example, the culture medium comprises platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2), wherein the level of FGF2 is less than about 6 ng / ml. For example, the level of FGF2 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, less than about 1 ng / ml. In one example, the level of FGF2 is about 1 ng / ml.

[0195] In one example, the PDGF is PDGF-BB. In one example, the level of PDGF-BB is between about 1 ng / ml and 150 ng / ml. In another example, the level of PDGF-BB is between about 7.5 ng / ml and 120 ng / ml. In another example, the level of PDGF-BB is between about 15 ng / ml and 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.

[0196] 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 stimulates cell proliferation by binding to its receptor EGFR. In one example, the method of the present disclosure includes culturing a stem cell colony in a cell culture medium free of fetal bovine serum that further comprises EGF. In one example, the level of EGF is between about 0.1 ng / ml and 7 ng / ml. For example, the level of EGF can be at least about 5 ng / ml.

[0197] In another example, the level of EGF is about 1 ng / ml to 8 ng / ml. In another example, the level of EGF is between about 3 ng / ml and 6 ng / ml. In another example, the level of EGF is about 5 ng / ml. In another example, the level of EGF is about 5 ng / ml or less.

[0198] In the above examples, a basal culture medium such as αMEM or StemSpan TM In one example, the culture medium comprises AlphaMEM or StemSpan supplemented with 10 ng / ml PDGF-BB, 5 ng / ml EGF, and 1 ng / ml FGF. TM .

[0199] In other examples, additional factors can be added to the cell culture medium. For example, 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-1β (IL-1β) and matrix-derived factor 1α (SDF-1α) can be supplemented into the cell culture medium. In another embodiment, cells can also be cultured in the presence of at least one cytokine in an amount sufficient to support cell growth. In another embodiment, cells can be cultured in the presence of heparin or its derivatives. In one example, the heparin derivative is sulfate. Various forms of heparin sulfate are known in the art and include heparin sulfate 2 (HS2). HS2 can be derived from various sources, including, for example, the liver of male and / or female mammals. Therefore, exemplary heparin sulfates include male liver heparin sulfate (MML HS) and female liver heparin sulfate (FML HS).

[0200] In another example, the cell culture medium of the present disclosure maintains stem cells in an undifferentiated state. When stem cells have not yet committed to a specific differentiation lineage, they are considered to be undifferentiated. As discussed above, stem cells exhibit morphological characteristics that distinguish them from differentiated cells. In addition, undifferentiated stem cells express genes that can be used as markers for detecting differentiation states. Polypeptide products can also be used as markers for detecting differentiation states. Therefore, those skilled in the art can easily determine whether the methods of the present disclosure maintain stem cells in an undifferentiated state using conventional morphological analysis, genetic analysis, and / or proteomic analysis.

[0201] Co-culture of mesenchymal lineage precursors or stem cells with stimulated or activated T cells

[0202] Interleukin-2 receptor alpha chain ("IL-2RA"; "IL-2Rα" is also known as CD25) is a validated marker of T cell activation. In certain embodiments, the methods of the present disclosure measure inhibition of T cell activation. In one example, T cell activation is determined based on the level of T cell IL-2RA expression following co-culture of activated PBMCs with mesenchymal lineage precursors or stem cells. Inhibition of IL-2RA expression correlates with inhibition of T cell activation.

[0203] In one example, activated PBMCs are co-cultured with mesenchymal lineage precursors or stem cells in a culture medium comprising at least one T cell stimulator, the concentration of which is preferably sufficient to stimulate and / or activate T cells. In another embodiment, T cells are first stimulated and / or activated prior to co-culturing with mesenchymal lineage precursors or stem cells.

[0204] In one example, mesenchymal lineage precursors or stem cells are co-cultured with PBMCs in a culture medium containing an agent that can stimulate CD3 and an agent that can stimulate CD28 on T cells, such as an antibody to CD3 and an antibody to CD28, for example, mouse anti-human CD3 and mouse anti-human CD28. In one example, the antibody to CD3 and / or the antibody to CD28 are added to the culture medium in a soluble form at a concentration of about 2 μg / ml for each antibody.

[0205] In one embodiment, PBMCs are co-cultured with mesenchymal lineage precursors or stem cells at a ratio of 5 PBMCs to 1 mesenchymal lineage precursors or stem cells. 6 PBMCs and 2 × 10 5 In one example, cells can be co-cultured in a final volume of 1 ml.

[0206] In one example, MLPSCs were co-cultured with PBMCs stimulated with anti-CD3 / CD28 antibodies, and IL-2Rα expression of the activated PBMCs was measured by enzyme-linked immunosorbent assay (ELISA).

[0207] In one example, the level of IL-2Rα is determined relative to a control, such as the IL-2Rα expression level of activated PBMCs without MLPSCs.

[0208] Determining treatment efficacy

[0209] In one embodiment, the present disclosure relates to methods of determining the efficacy of a treatment of a population of MLPSCs (eg, DP).

[0210] In one example, a method for determining the efficacy of a treatment of a culture-expanded mesenchymal lineage precursor or stem cell (MLPSC) population comprises: (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 efficacy of a treatment for graft-versus-host disease (GvHD), and wherein inhibition of IL-2RA by at least 56% under the culture conditions indicates therapeutic efficacy. In another example, inhibition of IL-2RA by ≥60% indicates therapeutic efficacy. In another example, inhibition of IL-2RA between 56% and 60% indicates therapeutic efficacy.

[0211] In one example, the therapeutically effective mesenchymal lineage precursor or stem cell population is capable of inhibiting IL-2RA expression by CD3 / CD28 activated PBMCs by at least 56% relative to a control. In another example, the therapeutically effective mesenchymal lineage precursor or stem cell population is capable of inhibiting IL-2RA expression by CD3 / CD28 activated PBMCs by at least 60% relative to a control. In another example, the therapeutically effective mesenchymal lineage precursor or stem cell population is capable of inhibiting IL-2RA expression by CD3 / CD28 activated PBMCs by 56% to 70% relative to a control.

[0212] In one example, the MLPSC population obtained represents a larger MLPSC population. For example, the MLPSC population obtained can represent a series of cryopreserved MLPSC populations, such as a series of doses. For example, the dose can be derived from the same population, and one or more doses are obtained according to the methods disclosed herein and the therapeutic efficacy of these doses is evaluated. In this example, the dose obtained represents the remaining doses derived from the same MLPSC population. In one example, the MLPSC population obtained represents an MLPSC population in 3D culture.

[0213] Performing the methods disclosed herein for determining therapeutic efficacy can satisfy release criteria for therapeutically effective cell compositions as DPs. In one example, such culture-expanded compositions are characterized by a threshold level of IL-2RA inhibition. In one example, the level of IL-2RA inhibition under culture conditions is between 56% and 60%. In another example, the level of IL-2RA inhibition under culture conditions is ≥ 60%.

[0214] Methods of manufacturing pharmaceutical products

[0215] In one example, the present disclosure relates to a method for manufacturing a drug product comprising a population of MLPSCs, the method comprising: obtaining a determination result as to whether a test population of MLPSCs inhibits IL-2RA at a predetermined level under culture conditions, and processing at least a portion of the test population of MLPSCs into a drug product if the test population of MLPSCs inhibits IL-2RA at at least the predetermined level under culture conditions, thereby manufacturing the drug product; or discarding at least a portion of the test population of MLPSCs if the mesenchymal stem cell population inhibits IL-2RA at less than the predetermined level under culture conditions, wherein the predetermined level is 56%. In one example, the predetermined level is ≥ 60% under culture conditions. In one example, the predetermined level is ≥ 65% under culture conditions.

[0216] In one example, the test population is obtained from a MLPSC population in 3D culture. For example, MLPSCs can be cultured in a bioreactor. In one example, the test population is obtained from a cryopreserved MLPSC population. In one example, the test population represents a larger MLPSC population, such as a plurality of cryopreserved MLPSC populations. In one example, a plurality of cryopreserved MLPSC populations have been cultured and expanded from the same intermediate MLPSC population.

[0217] In one embodiment, the drug product comprises a composition disclosed herein. In one embodiment, the drug product comprises 2×10 6 MLPSCs.

[0218] In one example, obtaining a determination as to whether a test population of MLPSCs inhibits IL-2RA at a predetermined level under culture conditions comprises co-culturing a representative number of MLPSCs according to a method disclosed herein.

[0219] In one example, the present disclosure provides a method for manufacturing an MSC drug product, the method comprising: a first step of providing (e.g., culture expansion (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 contracted third party or a non-contracted (e.g., independent) third party)) a test MSC population (e.g., a sample of the test MSC population); a second step of acquiring (e.g., detecting, measuring, receiving, or obtaining) at least one value (e.g., 1, 2, 3, and / or 4) for an MSC parameter listed in Table A for the test MSC population; and a third step of processing at least a portion of the test MSC population (e.g., processing a portion of a manufacturing batch, culture, or run, an entire manufacturing batch, culture, or run, or a plurality of manufacturing batches, cultures, or runs) into an MSC drug product (e.g., in a form or packaging for administration as described later herein; optionally cryopreserved) if the at least one value for the test MSC population meets a reference standard for the parameter shown in Table A, thereby manufacturing the MSC drug product. In one example, the value comprises parameter number 1. In another example, the value comprises parameter number 2. In another example, the value includes parameter number 3. In another example, the value includes parameter number 4. In another example, the value includes parameter numbers 1 and 2. In another example, the value includes parameter numbers 1 and 3. In another example, the value includes parameter numbers 1, 2, and 4.

[0220] In one example, such a method comprises a second step comprising obtaining values ​​for any combination of two or more MSC parameters listed in Table A, and a third step of such a method comprises processing at least a portion of the test MSC population into an MSC drug product if the values ​​for any combination of two or more MSC parameters meet the corresponding reference standards for the parameters shown in Table A.

[0221] Table A

[0222]

[0223] Selecting cells for treating graft-versus-host disease (GvHD)

[0224] In one embodiment, the present disclosure encompasses selecting cells for treating GvHD. In one example, a population of MLPSCs is assessed for compliance with necessary criteria, and when the criteria are met, the population is selected for treating GvHD.

[0225] In one example, if the MLPSC population suppresses IL-2RA by at least 56% to 60% under culture conditions, the MLPSC population is selected for treating GvHD. In one example, if the MLPSC population suppresses IL-2RA by ≥60% under culture conditions, the MLPSC population is selected for treating GvHD. In one example, if the MLPSC population suppresses IL-2RA by ≥65% under culture conditions, the MLPSC population is selected for treating GvHD.

[0226] The selection process is not particularly limited, as long as it is capable of selecting cell populations characterized by relevant criteria (such as % IL-2RA inhibition). In one example, a series of intermediate MLPSC populations are evaluated for IL-2RA inhibition under culture conditions, and populations that inhibit IL-2RA inhibition by at least 60% under culture conditions are selected for further expansion. In another example, a series of intermediate MLPSC populations are evaluated for IL-2RA inhibition under culture conditions, and populations that inhibit IL-2RA inhibition by at least 65% under culture conditions are selected for further expansion.

[0227] In one example, the selected mesenchymal lineage precursor or stem cell population is capable of inhibiting IL-2RA expression by CD3 / CD28 activated PBMCs by at least 56% relative to a control. In another example, the selected mesenchymal lineage precursor or stem cell population is capable of inhibiting IL-2RA expression by CD3 / CD28 activated PBMCs by at least 60% relative to a control. In another example, the selected mesenchymal lineage precursor or stem cell population is capable of inhibiting IL-2RA expression by CD3 / CD28 activated PBMCs by 56% to 70% relative to a control.

[0228] In one example, CD3 / CD28 activated PBMCs express at least 10,00 pg / mL IL-2RA. In one example, CD3 / CD28 activated PBMCs express at least 11,00 pg / mL IL-2RA. In one example, CD3 / CD28 activated PBMCs express at least 12,00 pg / mL IL-2RA. In one example, CD3 / CD28 activated PBMCs express at least 13,000 pg / mL IL-2RA. In one example, CD3 / CD28 activated PBMCs express at least 12,811 pg / mL IL-2RA.

[0229] In one example, the MLPSC population obtained represents a larger MLPSC population. For example, the MLPSC population obtained can represent a series of cryopreserved MLPSC populations, such as a series of doses. For example, the dose can be derived from the same population, and one or more doses are obtained according to the methods disclosed herein and the therapeutic efficacy of these doses is evaluated. In this example, the dose representative obtained is derived from the remaining doses of the same MLPSC population. In one example, the MLPSC population obtained represents an MLPSC population in 3D culture. In these examples, cells from alternative populations of MLPSC can be selected based on the evaluation of a representative test population of MLPSC.

[0230] In one example, selected cell populations are cryopreserved for later administration.

[0231] In another example, the present disclosure relates to a composition comprising a selected MLPSC population. In one example, the composition comprises a culture-expanded MLPSC population, wherein the MLPSC population is selected based on a predetermined level of IL-2RA inhibition under culture conditions, wherein the predetermined level of IL-2RA inhibition is at least 56%. In another example, the predetermined level of IL-2RA inhibition is ≥ 60%. In another example, MLPSC are also selected based on expression of one or more or all of CD29, CD54, CD73, CD90, CD102, CD105, CD106, CD166, MHC1.

[0232] In one example, a population of MLPSCs comprising a composition of the present disclosure is selected according to one or more parameters described in Table A.

[0233] Determine the amount of IL-2RA levels

[0234] The present disclosure contemplates any form of assay for determining IL-2RA levels, including, for example, Western blot, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), competition assay, radioimmunoassay, lateral flow immunoassay, flow-through immunoassay, electrochemiluminescence assay, turbidimetric-based assay, nephelometry-based assay, fluorescence-activated cell sorting (FACS)-based assay.

[0235] After co-culture of MLPSCs and T cells, the cells can be harvested and lysed using methods well known in the art. The cell lysate can then be assayed for the presence of IL-2RA, for example, by ELISA or FLISA. Alternatively, the level of IL-2RA expression can be determined by, for example, flow cytometry of intact cells.

[0236] In one example, inhibition of IL-2RA expression is measured by comparing the level of IL-2RA expression in a cell population comprising T cells to the level of IL-2RA in a cell population following co-culture of a cell population comprising T cells and a cell population comprising mesenchymal lineage precursors or stem cells, and expressing the difference as "percent inhibition."

[0237] The assay described above can be easily modified to use chemiluminescence or electrochemiluminescence as the basis for detection.

[0238] It will be understood by those skilled in the art that other detection methods based on immunosorbent assays can also be used to perform the present disclosure. For example, based on the immunosorbent method described above, detection can be performed using a radiolabel, or using a gold label (e.g., colloidal gold), or using liposomes (e.g., encapsulating NAD+), or using an acridine-linked immunosorbent assay.

[0239] In some examples of the present disclosure, the level of IL-2RA is measured using a surface plasmon resonance detector (e.g., BIAcore TM , GE Healthcare, Piscataway, NJ), flow-through devices (e.g., as described in U.S. Pat. No. 7,205,159), micro- or nanoimmunoassay devices (e.g., as described in U.S. Pat. No. 7,271,007), lateral flow devices (e.g., as described in U.S. Publication No. 20040228761 or U.S. Publication No. 20040265926), fluorescence polarization immunoassay (FPIA, e.g., as described in U.S. Pat. No. 4,593,089 or U.S. Pat. No. 4,751,190), or immunoturbidimetric assays (e.g., as described in U.S. Pat. No. 5,571,728 or U.S. Pat. No. 6,248,597).

[0240] Composition

[0241] The present disclosure encompasses compositions comprising a population of MLPSCs selected according to the methods disclosed herein. In an example, the compositions of the present disclosure comprise a therapeutically effective amount of MLPSCs, wherein the therapeutic efficacy is determined according to the methods disclosed herein. In one example, the composition is a pharmaceutical product manufactured according to the present disclosure to have a predetermined level of IL-2Rα inhibition.

[0242] In certain embodiments, the compositions of the present disclosure comprise a pharmaceutically acceptable carrier and / or excipient.

[0243] The terms "carrier" and "excipient" refer to compositions of matter that are routinely 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 edition, Mac Publishing Company (1980)). Carriers can also reduce any undesirable side effects of the active compound. Suitable carriers are, for example, stable, e.g., unable to react with other ingredients in the carrier. In one example, the carrier does not produce significant local or systemic side effects in the recipient at the dosage and concentration used for treatment.

[0244] Suitable carriers for the present disclosure include those conventionally used, for example, water, saline, aqueous dextrose, lactose, Ringer's solution, buffered solutions, hyaluronic acid and glycols are exemplary liquid carriers, particularly (when isotonic) for solutions. Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, etc.

[0245] In another example, the carrier is a medium composition, such as a medium in which the cells are grown or suspended. For example, such a medium composition does not cause any side effects in a subject to which it is administered.

[0246] 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.

[0247] In one example, the carrier or excipient provides buffering activity to maintain cells and / or soluble factors at a suitable pH to exert biological activity, for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it has minimal interaction with cells and factors and allows for rapid release of cells and factors, in which case the compositions of the present disclosure can be produced as a liquid for application, for example, by injection, directly to the bloodstream or to tissue or to an area surrounding or adjacent to a tissue.

[0248] In one example, the composition of the present disclosure comprises 1.5 to 3 million cells / kg. In one example, the composition of the present disclosure comprises 2 million cells / kg.

[0249] In one embodiment, the composition comprises greater than 5.00×10 6 In another example, the composition comprises greater than 5.50×10 6 In another example, the composition comprises greater than 6.00×10 6 In another example, the composition comprises greater than 6.50×10 6 In another example, the composition comprises greater than 6.68×10 6 viable cells / mL.

[0250] The compositions of the present disclosure can be cryopreserved. Slow freezing methods or rapid freezing protocols known in the art can be used to cryopreserve mesenchymal lineage precursors or stem cells. Preferably, the cryopreservation method maintains similar phenotypes, cell surface markers, and growth rates of cryopreserved cells compared to unfrozen cells.

[0251] The cryopreserved composition may comprise a cryopreservation solution. The pH of the cryopreservation solution is generally 6.5 to 8, preferably 7.4.

[0252] The cryopreservation solution may comprise a sterile, pyrogen-free isotonic solution such as, for example, PlasmaLyte A TM 100mLPlasmaLyte A TM Contains 526 mg sodium chloride, USP (NaCl); 502 mg sodium gluconate (C6H 11 NaO7); 368 mg sodium acetate trihydrate, USP (C2H3NaO2·3H2O); 37 mg potassium chloride, USP (KCl); and 30 mg magnesium chloride, USP (MgCl2·6H2O). It does not contain an antimicrobial agent. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).

[0253] Cryopreservation solutions may contain Profreeze TM The cryopreservation solution may additionally or alternatively comprise a culture medium, such as αMEM.

[0254] In order to promote freezing, cryoprotectants such as dimethyl sulfoxide (DMSO) are usually added to the cryopreservation solution. Ideally, cryoprotectants should be nontoxic to cells and patients, non-antigenic, chemically inert, provide high survival rates after thawing and allow transplantation without the need for washing. However, the most commonly used cryoprotectant DMSO shows some cytotoxicity. Hydroxyethyl starch (HES) can be used as an alternative or in combination with DMSO to reduce the cytotoxicity of the cryopreservation solution.

[0255] The cryopreservation solution may comprise one or more of DMSO, hydroxyethyl starch, human serum fractions, and other protein fillers. In one example, the cryopreserved solution comprises about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further comprise one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0256] In one embodiment, cells are suspended in 42.5% Profreeze TM / 50% αMEM / 7.5% DMSO and cooled in a controlled-rate refrigerator.

[0257] The cryopreserved composition can be thawed and administered directly to a subject or added to another solution, e.g., a solution comprising HA. Alternatively, the cryopreserved composition can be thawed and the mesenchymal lineage precursors or stem cells resuspended in an alternative carrier prior to administration.

[0258] In one example, the cell composition of the present disclosure may include Plasma-Lyte A, dimethyl sulfoxide (DMSO) and human serum albumin (HSA). For example, the composition of the present disclosure may include Plasma-Lyte A (70%), DMSO (10%), HSA (25%) solution, or an HSA solution comprising 5% HSA and 15% buffer.

[0259] It will be appreciated by those skilled in the art that many changes and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure. Therefore, the embodiments of the present invention are to be considered in all respects as illustrative and not restrictive.

[0260] The following specific examples should be interpreted as merely illustrative, and not limiting in any way the remainder of the disclosure. 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.

[0261] Example

[0262] Example 1: Clinical trial

[0263] 241 children with steroid-refractory acute graft-versus-host disease (aGvHD) who had failed one or more additional therapies received a mesenchymal stem cell (MSC) drug product (DP) as salvage therapy. Children with grade D disease comprised approximately 50% of the patients who received MSCs as salvage therapy, as this stage of disease is the most difficult to treat and has the highest mortality rate of all treatment modalities. Subjects in this study had failed steroid therapy as well as multiple salvage treatment interventions. This group of subjects provided an assessment of the effectiveness of DP in a patient population that had previously not responded to other conventional therapies. Overall response rates (OR) at day 28 and overall survival (OS) at day 100 were higher for all grades of aGVHD, particularly for subjects with grade D disease. These data highlight the effectiveness of DP in a highly refractory patient population.

[0264] Example 2: IL-2Rα inhibition

[0265] The inhibitory effect on activated T cells is thought to reflect a key mechanism of action by which MSC DPs exert their therapeutic effects in aGVHD. An IL-2Rα potency assay has been developed to provide a functional measure of the ability of MSC DPs to inhibit IL-2Rα expression by activated T cells in vitro.

[0266] For each MSC DP administered to 241 children with steroid-refractory acute graft-versus-host disease (aGvHD), day 100 OS and IL-2Rα inhibition percentage were evaluated. A sensitivity analysis was then performed to evaluate whether a threshold for minimum IL-2Rα inhibition percentage was determined to explain the higher survival rates conferred by certain DPs. The sensitivity analysis evaluated the survival of children who received DPs with an average IL-2Rα inhibition percentage greater than or less than a threshold of 66% to 50%. The results are shown in Table 4.

[0267] Table 4: Day 100 Overall Survival Analysis by Mean IL-2Rα Inhibition Percentage Threshold by Drug Product Received

[0268]

[0269]

[0270] Table 4 shows that children who received DP batches with a mean IL-2Rα inhibition percentage greater than any threshold between 54% and 66% had day 100 survival outcomes ranging from 67% to 69%. In contrast, children who received DP batches with a mean IL-2Rα inhibition percentage less than 60% had a linear decrease in day 100 survival. Thus, the inventors have determined that an IL-2Rα inhibition threshold of ≥60% will release DP batches, thereby providing a significant survival benefit for this disease. Therefore, the inventors' findings represent a significant advance in DP manufacturing, particularly in the context of DP for the treatment of T cell-mediated diseases such as GvHD.

[0271] Example 3: Further analysis

[0272] like Figure 1 As shown, percent IL-2Rα inhibition was positively correlated with day 180 survival (85% for day 180 OS > median vs 54% for day 180 OS ≤ median, p = 0.01). This survival benefit was achieved by a longer duration of OR at day 28 in responders who received products with higher percent IL-2Rα inhibition. The relationship between better survival and mean percent IL-2Rα inhibition > median, relative to mean percent IL-2Rα inhibition ≤ median, was most pronounced in patients with the most severe disease and the highest risk of death:

[0273] a. Minnesota high risk (OS at day 180 89% vs. 50%, p = 0.01);

[0274] b. MAGIC algorithm probability (MAP) ≥ 0.29 (OS at day 180 100% vs. 17%, p = 0.003);

[0275] c. IBMTR grade D disease (Day 180 OS 91% vs. 50%, p=0.03).

[0276] As shown in Figure 2, in patients with high-risk MAP (MAP ≥ 0.29), there was a significant correlation between increasing mean product inhibition percentage of IL-2Rα and increased survival days within 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 may reflect that all drug products have sufficient efficacy to achieve survival in low-risk patients (right panel).

[0277] As shown in Figure 3, in all children with high MAP scores (≥0.29), there was a linear inverse correlation between the gradual increase in IL-2Rα inhibition by MSC batch and the reduction in various inflammatory biomarkers over 28 days.

[0278] like Figure 4 As shown, efficacy was significantly associated with a decrease in the proportion of in vivo activated T cells between baseline and day 28 for patients treated with a single batch of DP, as measured by T cells expressing the CD3+CD4+CD25+HLA-DR+ phenotype.

[0279] In summary:

[0280] The percentage inhibition of IL-2Rα in vitro was significantly correlated with the reduction in circulating levels of activated T cells from screening to day 28 (p=0.009).

[0281] Increased product efficacy was associated with a gradual reduction in inflammatory biomarkers and better survival.

[0282] The beneficial effects on survival were greatest for children with the most severe disease, the highest baseline levels of inflammatory biomarkers, and the highest risk of death.

[0283] For patients with the most severe disease (grade D) who received drug products with mean IL-2Rα inhibition > median, day 180 survival was significantly higher than for patients who received products with mean IL-2Rα inhibition ≤ median (91% vs. 50%, p = 0.03).

[0284] Even patients with grade D SR-aGVHD who received products with a mean IL-2Rα inhibition percentage ≤ the median achieved a 50% survival rate, which is significantly higher than the survival seen with other treatment options, which have remained low at 12% to 23% at day 180 even with optimal treatment over the past two decades.

[0285] Therefore, the IL-2Rα inhibition assay is a direct measure of the ability of a drug product to inhibit T cell activation in vitro and correlates with the drug product's bioactivity in vivo, as shown by improved survival outcomes and reductions in circulating levels of inflammatory biomarkers and activated T cells.

[0286] Example 4: Measurement of IL-2Rα inhibition

[0287] Ce-MSCs (intermediate population or drug product) were co-cultured with unstimulated or stimulated (CD3; CD28 antibodies) peripheral blood mononuclear cells (PBMCs) as indicated below. These conditions represent a ratio of approximately 1 stem cell: 5 PBMCs:

[0288]

[0289] The plates were incubated at 37°C + / - 2°C, 5% + / - 2% CO2 for 60 to 84 hours, after which the cells were harvested, lysed, and activated PBMCs assessed for IL-2RA expression using a commercially available ELISA kit according to the manufacturer's instructions (R&D systems).

[0290] The inhibition of IL-2RA expression by MSC batches on PBMC (IL-2Rα inhibition assay) is a quantitative bioactivity assay that provides a direct measure of the inhibitory effect of a cell population (e.g., the final product). This bioactivity measurement allows the evaluation of the inhibitory effect of the population on activated T cells in vitro.

[0291] Example 5: Relationship between PBMC stimulation and IL-2Rα inhibition

[0292] 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 attribute that measures the effect of MSCs on key immune system mechanisms mediating disease severity in SR-aGVHD, namely alloreactive T cell activation, proliferation, and inflammatory cytokine production.

[0293] When measuring IL-2RA inhibition, the degree of PBMC stimulation and subsequent inflammatory cytokine release is a key component of the ability to induce the MSC batch to release immunomodulatory factors (such as IDO-1, PGE2, and PD-L1) necessary for IL-2RA inhibitory activity. To assess the minimum level of PBMC stimulation with anti-CD3 and anti-CD28 antibodies necessary to obtain reliable measurements of batch potency, regression analysis was performed to assess the relationship between IL-2RA production by stimulated PBMCs and the degree of inhibitory activity induced by the MSC batch.

[0294] As shown in Figure 5, a correlation was observed between the amount of IL-2RA produced by stimulated PBMCs and the percentage of IL-2RA inhibition in all 40 batches tested (p = 0.012). This correlation remained significant at post-stimulation PBMC IL-2RA expression thresholds of 10,000 pg / ml, 11,000 pg / ml, and 12,000 pg / ml, but was no longer significant when the amount of IL-2RA produced by stimulated PBMCs exceeded 13,000 pg / ml. The average IL-2RA inhibition for the 30 batches tested with PBMCs producing >13,000 pg / ml IL-2RA was 83% (range 71% to 91%), while the inhibition for the 10 batches tested with PBMCs producing <13,000 pg / ml IL-2RA was 77% (range 63% to 86%) (p = 0.005).

[0295] These results suggest that 12,000 pg / ml to 13,000 pg / ml of IL-2RA production reflects the minimum threshold of PBMC stimulation necessary to ensure that the MSC batch is sufficiently activated to induce maximal IL-2RA inhibitory activity. Although the average IL-2RA inhibitory activity of batches tested with PBMCs producing <13,000 pg / ml IL-2RA was significantly lower than the average IL-2RA inhibitory activity of batches tested with PBMCs producing >13,000 pg / ml IL-2RA, these data suggest that the potency of MSC batches tested with insufficiently stimulated PBMCs is unreliable and may underestimate the true potency values ​​of these batches.

[0296] Example 6: Survivors in a GvHD clinical trial received MSCs with a higher mean IL2R inhibition percentage

[0297] The inverse cumulative distribution curve was then used to assess the relationship between product potency and clinical outcomes in patients (n=34) who received batches with potency values ​​measured using adequately stimulated PBMCs (n=30 batches, >12811 pg / ml IL-2RA). Figure 6 ).

[0298] from Figure 6 As can be seen from the inverse cumulative distribution curve, there is a significant relationship between the weighted average of the IL-2RA inhibition percentage of the received batches and the survival rate at day 100 (p = 0.03). Since the average IL-2RA inhibition of these 30 batches was 83.1%, the survival rate at day 100 was significantly higher than that of the control group (P = 0.03). + 5.8%, 65.7% was the minimum IL-2RA inhibition threshold required for batch release using the IL-2RA inhibition assay performed with fully stimulated PBMCs (≥12811 pg / ml IL-2RA).

[0299] Clinical batches were tested using the IL-2RA inhibition assay in 2015, with fully stimulated PBMCs (≥12811 pg / ml IL-2RA) retested in 2023 to evaluate whether cryopreservation can maintain potency. Figure 7 As shown, IL-2Rα inhibition measured at two time points using the same clinical batch (first tested in 2015 and then again in 2023) demonstrated high stability of the clinical batch when stored frozen for at least 8 years.

[0300] Example 7: Accurate measurement of percent inhibition of IL-2Rα inhibition requires adequate PBMC stimulation and induction levels of IL-2Rα expression

[0301] The slope of the decline in IL-2Rα inhibition values ​​was 1.2% per year, indicating that the MSC DP batches remained highly stable over time with respect to IL-2Rα inhibition. Comparing the regression line to the actual assay values, as assessed across 10 batches using fully stimulated PBMCs at two time points, provided a highly sensitive, true test of the IL-2Rα inhibition assay precision. The ability of linear stability modeling to predict actual assay values ​​using a common slope was used to calculate the assay standard deviation from the residuals in the linear regression. This calculation indicated that the standard deviation of the IL-2Ra inhibition assay was less than 7%, demonstrating adequate precision for measuring critical quality attributes.

[0302] To confirm the relationship between adequate PBMC stimulation (measured as IL-2Rα production) and MSC batch potency observed in the clinical batches, 61 batches were regressed based on these same parameters. As can be seen in Figure 8 (left box), a significant linear correlation also existed between PBMC production of IL-2RA and the percentage degree of IL-2R alpha inhibition after anti-CD3 / CD28 stimulation in these 61 additional MSC batches (p<0.0001). This is consistent with IL2R produced by activated PBMCs being a biomarker of inflammatory cytokine secretion (such as IFNγ, TNFα, and IL-1, which are responsible for the induction of anti-inflammatory factors secreted by Ryoncil batches, including IDO-1, PGE2, and PD-L1).

[0303] As with earlier clinical batches, a correlation between PBMC production of IL-2RA and IL-2RA suppression by co-cultured MSC batches was only observed when the stimulated PBMCs produced a maximum of 12,000 pg / ml to 13,000 pg / ml IL-2RA, suggesting that above these levels of PBMC stimulation, induction of MSC suppressive potency reached a maximum. While 95% of batches tested with stimulated PBMCs producing >13,000 pg / ml IL-2RA showed >60% IL-2RA suppression, only 70% of batches tested with suboptimally stimulated PBMCs producing lower amounts of IL2Ralpha achieved this level of suppression, as shown in Figure 8 (right box).

[0304] These results demonstrate that accurate measurement of the potency of MSC batches to inhibit IL-2RA requires optimal PBMC stimulation and inflammatory cytokine secretion, which can be achieved through quality control and strict acceptance criteria for PBMC stimulation with anti-CD3 / CD28 antibodies.

[0305] Example 8: Acceptance Criteria for IL-2RA Inhibition Assay

[0306] Given the above data, a value of 65.7% represents a minimum threshold of IL-2RA inhibition met by 95% of all batches used in the clinical trial when PBMCs were stimulated to achieve IL-2RA levels ≥ 12811 pg / ml as measured by the IL-2RA inhibition assay.

[0307] Example 9: IL2Ra Inhibition Assay Protocol and Validation Procedure

[0308] Expression of interleukin-2 receptor alpha (IL-2Rα / IL-2RA) on the cell surface is an early marker of T cell activation. The primary function of IL-2 / IL-2Rα is to promote the proliferation of CD4+ (helper) and CD8+ (cytotoxic) T cells. Inhibition of IL-2Rα expression leads to suppressed T cell proliferation and further expansion of the immune response. ceMSCs were used in the IL-2Rα inhibition assay according to the following steps:

[0309] MSCs (2.00×10 5 cells / well) and PBMCs stimulated with mouse anti-human CD3 / CD28 (1.00×10 6 The cells were co-cultured at 37°C ± 2°C and 5% ± 2% CO2 for 72 ± 2 hours.

[0310] Unstimulated PBMCs (without CD3 / CD28) and stimulated PBMCs (with CD3 / CD28 but without ceMSCs) were included as negative and positive controls, respectively.

[0311] At the end of the incubation period, cells were harvested for lysate preparation. The cell lysate was then aliquoted into sterile tubes and stored at ≤-60°C for up to 29 days until use in the IL-2Rα ELISA.

[0312] IL-2Rα levels were measured in triplicate at one dilution in lysates of unstimulated and stimulated PBMCs alone and in co-cultures using the Quantikine Human CD25 / IL-2Rα ELISA Kit.

[0313] A standard curve constructed using four-parameter logistic (4-PL) curve fitting was used to determine IL-2Rα levels in controls and samples. CeMSC efficacy was evaluated by the percentage inhibition of IL-2Rα production by ceMSCs against CD3 / CD28-stimulated PBMCs relative to control CD3 / CD28-stimulated PBMCs cultured without ceMSCs.

[0314] verify

[0315] The inherent variability introduced when performing potency testing of cell material against secondary reagent cell lines required additional measurements to be considered. To account for this inherent assay variability, it was decided to utilize a normalization factor to correct for the inherent donor-to-donor variability in the assayed PBMC reagents.

[0316] Studies investigating the variability of the IL-2Rα inhibition assay revealed that the combined effect of PBMC donor in the mechanism of inhibition and the variability of PBMC response to anti-CD3 / anti-CD28 stimulation are important parameters.

[0317] Therefore, appropriate controls would include screening of PBMC batches to confirm the reproducibility of PBMC stimulation of IL-2Rα greater than 12,811 pg / mL, and verification that the test PBMCs meet the acceptability of the ceMSC reference cell line standard range in the average of at least 4 vials (i.e., 4 runs) tested.

[0318] The ceMSC reference cell line can be generated from the same three batches of ceMSCs (generated from three bone marrow donors) used in the validation of the IL-2Rα inhibition assay, and the acceptance range will be calculated based on the values ​​generated in the validation. The ceMSC reference cell line will then be utilized to qualify new PBMC batches for use in the IL-2Rα inhibition assay. The ceMSC reference cell line is qualified and utilized to ensure that the normalization factor is accurately generated for each PBMC batch and that the assay performance remains consistent over time.

[0319] It will be appreciated by those skilled in the art that many changes and / or modifications may be made to the present invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Therefore, the embodiments of the present invention are considered to be illustrative and not restrictive in all aspects.

[0320] This application claims priority to US63 / 444,198 filed on February 8, 2023 and US63 / 445,588 filed on February 14, 2023, the disclosures of which are incorporated herein by reference.

[0321] All publications discussed above are incorporated herein in their entirety.

[0322] Any discussion of documents, acts, materials, devices, articles and the like has been included in this specification solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Claims

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

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

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

4. A method for determining therapeutic efficacy of a culture-expanded population of mesenchymal lineage precursor or stem cells (MLPSCs): (i) obtaining a population of MLPSCs; (ii) culturing the cells in culture; and (iii) determining the level of IL-2RA inhibition under the culture conditions, wherein the method determines efficacy of a treatment for graft-versus-host disease (GvHD), and wherein inhibition of IL-2RA by at least 56% under the culture conditions indicates therapeutic efficacy.

5. The method of claim 4, wherein inhibition of IL-2RA by ≥60% under culture conditions indicates therapeutic efficacy.

6. A method of manufacturing a pharmaceutical product comprising a population of MLPSCs, comprising: Obtaining a determination as to whether the test population of MLPSCs inhibits IL-2RA at a predetermined level under the culture conditions, and processing at least a portion of the test population of MLPSCs into a drug product if the test population of MLPSCs inhibits IL-2RA at at least the predetermined level under the culture conditions, thereby manufacturing the drug product; or discarding at least a portion of the test population of MLPSCs if the mesenchymal stem cell population inhibits IL-2RA at less than the predetermined level under the culture conditions, wherein the predetermined level is 56%. The method according to claim 6 , wherein the predetermined level is ≥ 60% under culture conditions.

8. A method of treating a subject having graft-versus-host disease (GvHD), the method comprising administering to a subject in need thereof a composition comprising a population of culture-expanded mesenchymal lineage precursor 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 culture-expanded MLPSCs comprising the administered composition suppress IL-2RA by ≥60% under culture conditions.

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

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

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

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

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

15. The method of any one of claims 8 to 14, wherein treatment improves 100-day survival to 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 culture-expanded population of MLPSCs, wherein the population of MLPSCs is selected based on a predetermined level of IL-2RA inhibition under culture conditions, wherein the predetermined level of IL-2RA inhibition is at least 56%.

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

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

20. The composition of claim 19, wherein the mesenchymal stem cells are also selected based on the 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%.

22. A method of manufacturing a pharmaceutical product comprising a population of MLPSCs, the method comprising (i) obtaining a determination as to 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 without MLPSCs; wherein the predetermined level of IL-2RA inhibition is at least 56%.

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

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

25. The method according to any one of claims 22 to 24, wherein the PBMCs are co-cultured with MLPSCs at a ratio of 5 PBMCs:1 MLPSC.

26. The method according to claim 25, wherein 1×10 6 PBMCs and 2 × 10 5 MLPSCs were co-cultured.

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

28. The method of any one of claims 22 to 27, wherein 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 without MLPSCs, and a negative control population of non-activated PBMCs without MLPSCs; (ii) constructing a standard curve to determine the IL-2RA levels in the positive control population, the negative control population, and the test population; and (iii) Calculate the percentage of inhibition of IL-2RA levels of the positive control population by the test population of MLPSCs co-cultured with CD3 / CD28 activated PBMCs relative to the positive control population of CD3 / CD28 activated PBMCs.

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

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

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