Frozen storage intermediate and potency assay thereof

Abstract

The present disclosure relates to improved cell intermediates and potency assays for obtaining the same. Such intermediates and assays may be suitable for use in providing cell compositions for treating various inflammatory disorders.

Description

Technical Field

[0001] The present disclosure relates to cell intermediates, potency assays for characterizing them, and methods of selection for culture expansion. Such intermediates, assays, and methods may be suitable for use in providing cell compositions for treating various inflammatory disorders.

Background Art

[0002] Several cell therapy products for regenerative or immunotherapeutic applications have advanced to clinical evaluation and market approval. However, the commercialization of these cell therapy products is hampered by their complexity and heterogeneity, which makes it difficult to identify appropriate biological activities and thus define consistent cell therapy product quality.

[0003] Physicochemical parameters (e.g., size, morphology, light scattering properties, tensile strength, cell number, characterization of confluence, phenotypic markers, secreted substances, genotype, identification of gene expression profiles) are routinely used for the identification and quantification of active substances, intermediates, impurities, and contaminants. However, physicochemical parameters cannot confirm whether a product is biologically active and potent (i.e., elicits the desired effect). In contrast, biological characterization takes into account the effect of a product on a biological system modeled either in vitro or in vivo, in animals and ultimately in humans.

[0004] In a potency test, it is necessary to demonstrate the appropriate biological activity(ies) of a product. A potency test does not need to reflect all of the biological functions of a product, but it is necessary to show one or more appropriate biological functions. For the analytical methods used in potency tests, accuracy, sensitivity, specificity, and reproducibility are established, and they are expected to be appropriately robust.

[0005] For example, there is a need to develop cellular products for the treatment of diseases where immunosuppression is desired. It is also preferable to identify parameters that are important to the efficacy of cell therapy products and control them (e.g., via potency testing) so that a consistent quality product can be manufactured. Summary of the Invention

[0006] Past efficacy testing has focused on characterizing the final cell product for administration. While such testing remains important, those skilled in the art should be aware that the final cell product may be incompatible with the donor cell bank. It will be understood that the cell intermediates are cultured and expanded from a cryopreserved intermediate, called a cellular intermediate. Research by the present inventors has surprisingly identified the importance of subjecting such cell intermediates to efficacy testing before culture expansion into a final cell product. For example, the present inventors have identified certain characteristics of the donor cell bank that, when fulfilled, provide a "memory" to subsequent final products derived from them. Indeed, after controlling the downstream culture expansion process, the present inventors have surprisingly discovered that certain characteristic(s) of the donor cell bank are associated with a final cell product that can improve 100-day survival rates for treating the inflammatory disease GvHD. These findings represent an important advance in the art, since only therapeutically effective intermediate cell populations are selected for large-scale expansion in 3D culture. Without these discoveries, the resulting cell product may not meet the therapeutic criteria required for administration, resulting in a waste of large-scale expansion efforts.

[0007] Accordingly, in certain embodiments, the methods of the present disclosure facilitate the selection of the most suitable cell intermediates for culturing expansion. In a first example, the present disclosure relates to a method of selecting a cell population for culturing expansion, the method comprising: (i) obtaining a population of mesenchymal progenitor lineage cells or stem cells (MLPSCs); (ii) culturing the MLPSC population in a culture medium; (iii) determining the level of IL2-RA inhibition under culture conditions; and (iv) selecting, for further expansion, an MLPSC population that inhibits IL2-RA by at least 55% under culture conditions. In another example, the method comprises selecting, for further expansion, an MLPSC population that inhibits IL2-RA by at least 56% under culture conditions. In another example, the method comprises selecting, for further expansion, an MLPSC population that inhibits IL2-RA by at least 60% under culture conditions. In another example, the method comprises selecting, for further expansion, an MLPSC population that inhibits IL2-RA by at least 70% under culture conditions. In another example, the method comprises selecting, for further expansion, an MLPSC population that inhibits IL2-RA by 55% to 75% under culture conditions. In another example, the method comprises selecting, for further expansion, an MLPSC population that inhibits IL2-RA by 60% to 75% under culture conditions. In another example, the MLPSC population selected for further expansion also expresses at least 80 pg / ml of TNF-R1 under culture conditions. In another example, the MLPSC population selected for further expansion also expresses at least 100 pg / ml or at least 105 pg / ml of TNF-R1 under culture conditions.

[0008] In one example, further expansion is performed in 2D culture. In one example, further expansion is performed in a cell factory. In another example, further expansion is performed in 3D culture. In one example, further expansion is performed in a bioreactor.

[0009] In one example, the method further comprises culturing and expanding the selected MLPSC population to provide a pharmaceutical composition. In one example, culturing and expanding the selected MLPSC population comprises at least 3 to 5 passages.

[0010] In one example, the culture expansion of a selected MLPSC population provides an MLPSC population characterized by greater than 60% inhibition of IL2-RA under culture conditions. In another example, the culture expansion of a selected MLPSC population provides an MLPSC population characterized by greater than 70% inhibition of IL2-RA under culture conditions. In another example, the culture expansion of a selected MLPSC population provides an MLPSC population characterized by greater than 75% inhibition of IL2-RA under culture conditions. In another example, the culture expansion of a selected MLPSC population provides an MLPSC population characterized by 60 - 80% inhibition of IL2-RA under culture conditions.

[0011] Furthermore, due to the inventors' discovery, it has been revealed that administration of a cell composition cultured from a cell intermediate selected according to the method of the present disclosure surprisingly increased the 100-day survival rate of patients with inflammatory diseases such as GvHD. In the context of GvHD, the increase in 100-day survival rate was particularly prominent in patients with severe disease, grade D GvHD. Thus, in another embodiment, the present disclosure encompasses a method for determining the therapeutic efficacy of a population of mesenchymal progenitor lineage cells or stem cells (MLPSCs), the method comprising: (i) obtaining a population of MLPSCs; (ii) culturing the cells in a culture medium; and (iii) determining the level of IL2-RA inhibition under culture conditions, wherein at least 55% inhibition of IL2-RA under culture conditions indicates therapeutic efficacy. In one example, greater than 56% inhibition of IL2-RA under culture conditions indicates therapeutic efficacy. In one example, greater than 60% inhibition of IL2-RA under culture conditions indicates therapeutic efficacy. In one example, greater than 70% inhibition of IL2-RA under culture conditions indicates therapeutic efficacy. In another example, greater than 75% inhibition of IL2-RA under culture conditions indicates therapeutic efficacy. In another example, 55% - 75% inhibition of IL2-RA under culture conditions indicates therapeutic efficacy. In another example, greater than 75% inhibition of IL2-RA under culture conditions indicates therapeutic efficacy. In another example, 60% - 75% inhibition of IL2-RA under culture conditions indicates therapeutic efficacy.

[0012] In another example, the method of determining therapeutic efficacy further comprises determining the level of TNF-R1 expression under culture conditions. In one example, an expression of at least 100 pg / ml of TNF-R1 under culture conditions indicates therapeutic efficacy. In one example, an expression of at least 105 pg / ml of TNF-R1 under culture conditions indicates therapeutic efficacy. In certain examples, it will be appreciated that after determining therapeutic efficacy, an intermediate population of MLPSCs can be cultured and expanded to provide a pharmaceutical composition. In such examples, the pharmaceutical composition can comprise a population of MLPSCs characterized by greater than 70% IL2-RA inhibition under culture conditions. In another example, the pharmaceutical composition can comprise a population of MLPSCs characterized by greater than 75% IL2-RA inhibition under culture conditions. In another example, the pharmaceutical composition can comprise a population of MLPSCs characterized by 60% to 75% IL2-RA inhibition under culture conditions.

[0013] The discoveries of the present inventors also support new treatments for inflammatory diseases. Thus, in one embodiment, the present disclosure includes a method of treating a subject suffering from an inflammatory disease, the method comprising administering to a subject in need thereof a composition comprising a cultured and expanded population of mesenchymal progenitor or stem cells (MLPSCs), wherein the MLPSCs are cultured and expanded from a population of MLPSCs that inhibit IL2-RA by at least 55% under culture conditions. In one example, the cultured and expanded MLPSCs included in the administered composition inhibit IL2-RA by at least 56% under culture conditions. In one example, the cultured and expanded MLPSCs included in the administered composition inhibit IL2-RA by at least 60% under culture conditions. In one example, the cultured and expanded MLPSCs included in the administered composition inhibit IL2-RA by at least 70% under culture conditions. In another example, the cultured and expanded MLPSCs included in the administered composition inhibit IL2-RA by at least 75% under culture conditions. In another example, the cultured and expanded MLPSCs included in the administered composition inhibit IL2-RA by 55% to 75% under culture conditions. In another example, the cultured and expanded MLPSCs included in the administered composition inhibit IL2-RA by 55% to 75% under culture conditions. In another example, the cultured and expanded MLPSCs included in the administered composition inhibit IL2-RA by 60% to 75% under culture conditions. In another example, the MLPSCs are cultured and expanded from a population of MLPSCs that also express at least 80 pg / ml of TNF-R1 under culture conditions. In another example, the MLPSCs are cultured and expanded from a population of MLPSCs that also express at least 105 pg / ml of TNF-R1 under culture conditions.

[0014] In another embodiment, the present disclosure also relates to new cryopreserved cell intermediates. Such intermediates are generally passaged only 2 - 5 times after isolation and before cryopreservation. Thus, in one example, the present disclosure relates to a cryopreserved cell intermediate comprising a population of cultured and expanded mesenchymal progenitor cells or stem cells (MLPSC), where the population of MLPSC is passaged 2 - 5 times in culture, and the cultured and expanded MLPSC: a) inhibits IL2 - RA by at least 55% under culture conditions, and / or b) expresses at least 80 pg / ml of TNF - R1 under culture conditions. In one example, the cell intermediate: a) inhibits IL2 - RA by at least 55% under culture conditions, and / or b) expresses at least 100 pg / ml of TNF - R1 under culture conditions. In one example, the cell intermediate: a) inhibits IL2 - RA by at least 60% under culture conditions, and / or b) expresses at least 80 pg / ml of TNF - R1 under culture conditions. In one example, the cell intermediate: a) inhibits IL2 - RA by at least 60% under culture conditions, and / or b) expresses at least 100 pg / ml or at least 105 pg / ml of TNF - R1 under culture conditions. In one example, the cell intermediate: a) inhibits IL2 - RA by at least 70% or at least 75% under culture conditions, and / or b) expresses at least 100 pg / ml or at least 105 pg / ml of TNF - R1 under culture conditions. In another example, the cell intermediate: a) inhibits IL2 - RA by 55% - 75% under culture conditions, and / or b) expresses 80 pg / m - 105 pg / m of TNF - R1 under culture conditions.

[0015] In the above reference example, IL2 - RA inhibition was measured by co - culturing MLPCS and activated PBMC.

[0016] In one example, the cell intermediates of the present disclosure are cultured and grown in 2D culture. For example, 2D culture may include cell culture in a multi-layer cell factory. In one example, the cell intermediates of the present disclosure are cultured and grown in about 5% CO2, for example, 5% + / - 2% CO2. In one example, cell culture is performed using passive CO2 priming. For example, the cell intermediates of the present disclosure are cultured and grown using passive priming in about 5% CO2, for example, 5% + / - 2% CO2. To avoid misunderstanding, cell culture according to such an example does not include active CO2 priming.

[0017] In one example, the inflammatory disease treated according to the present disclosure is graft-versus-host disease (GvHD). In one example, GvHD is classified as grade D. In one example, GvHD is classified as chronic GvHD. In one example, the subject treated according to the present disclosure is refractory to steroids, immunosuppressive agents, and / or biologic therapies. In one example, the subject is refractory to a single biologic agent. In one example, the subject is refractory to multiple biologic agents.

[0018] In one example, treatment according to the present disclosure increases the 100-day survival rate to more than 60%. In another example, treatment increases the 100-day survival rate to more than 70%. In another example, treatment according to the present disclosure increases the 100-day survival rate by 60 - 80%.

[0019] In one example, the subject treated according to the present disclosure receives at least two doses of the composition disclosed herein.

[0020] In another example, the present disclosure relates to a method of manufacturing a formulation comprising a population of mesenchymal progenitor or stem cells (MLPSCs). In one example, the method comprises obtaining a determination as to whether a test population of MLPSCs has a predetermined level of IL2-RA inhibition under culture conditions, and culturing and expanding at least a portion of the test population of MLPSCs to a formulation if the test population of MLPSCs has at least the predetermined level of IL2-RA inhibition under culture conditions, thereby manufacturing the formulation, or discarding at least a portion of the test population of MLPSCs if the population of MLPSCs has less than the predetermined level of IL2-RA inhibition under culture conditions. In one example, the predetermined level of IL2-RA inhibition is at least 55% under culture conditions. In one example, the predetermined level of IL2-RA inhibition is at least 60% under culture conditions. In one example, the predetermined level of IL2-RA inhibition is at least 70% under culture conditions. In another example, the method comprises obtaining a determination as to whether a test population of MLPSCs has a predetermined level of TNF-R1 under culture conditions, and culturing and expanding at least a portion of the test population of MLPSCs to a formulation if the test population of MLPSCs has at least the predetermined level of IL2-RA inhibition and the predetermined level of TNF-R1 under culture conditions, thereby manufacturing the formulation, or discarding at least a portion of the test population of MLPSCs if the population of MLPSCs has less than the predetermined level of IL2-RA inhibition and less than the predetermined level of TNF-R1 under culture conditions. In one example, the predetermined level of TNF-R1 is at least 80 pg / ml. In another example, the predetermined level of TNF-R1 is at least 100 pg / ml. In another example, the predetermined level of TNF-R1 is at least 105 pg / ml.

[0021] In one example, culturing and expanding at least a portion of the test population of MLPSCs to a formulation provides a population of MLPSCs characterized by greater than 70% IL2-RA inhibition under culture conditions. In another example, culturing and expanding at least a portion of the test population of MLPSCs to a formulation provides a population of MLPSCs characterized by greater than 75% IL2-RA inhibition under culture conditions.

[0022] In one example, the MLPSC is a mesenchymal stem cell. In another example, the MLPSC is a mesenchymal progenitor cell.

[0023] In one example, the compositions disclosed herein are administered intravenously.

[0024] In one example, the method of the present disclosure involves administering 1×10 7 ~2×10 8 cells. For example, multiple doses of 1×10 7 ~2×10 8 cells may be administered at regular intervals, such as on days 0, 30, 60, and 90. In one example, the method of the present disclosure involves administering about 1×10 8 cells per dose.

[0025] In one example, a subject being treated according to the present disclosure receives a second dose within 7 days after the first dose is administered. In one example, the second dose is administered 4 days after the first dose. In one example, the dose contains 2×10 6 cells / kg body weight.

[0026] In another example, the compositions of the present disclosure further comprise Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA). In one example, the composition further comprises a Plasma-Lyte A (70%), DMSO (10%), HSA (25%) solution, and the HSA solution contains 5% HSA and 15% buffer.

[0027] In one example, the compositions of the present disclosure contain more than 6.68×10 6 viable cells / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0028]

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Mode for Carrying Out the Invention

[0029] Throughout this specification, unless otherwise specifically defined or required by the context, references to a single step, the composition of a problem, a group of steps or a group of compositions of a problem shall be construed to include one and a plurality of (i.e., one or more) of those steps, the composition of a problem, a group of steps or a group of compositions of a problem.

[0030] Those skilled in the art will understand that the disclosure described in this specification is capable of variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all of the steps, features, compositions and compounds individually or collectively mentioned or shown in this specification, as well as any combination or any two or more of the above steps or features.

[0031] This disclosure is not limited in scope by the specific embodiments described herein for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure as described herein.

[0032] Any example disclosed in this specification should be construed as applicable mutatis mutandis to any other example, unless otherwise specified.

[0033] Unless otherwise specifically defined, all technical and scientific terms used in this specification shall be construed 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, and biochemistry).

[0034] Unless otherwise indicated, the surgical techniques utilized in this disclosure are standard procedures well known to those of ordinary skill in the art.

[0035] Methods for obtaining and enriching populations of mesenchymal stem cells or progenitor cells are known in the art. For example, enriched populations of mesenchymal stem cells or progenitor cells can be obtained by using flow cytometry and cell sorting procedures based on the use of cell surface markers expressed on mesenchymal stem cells or progenitor cells.

[0036] All documents cited or referenced in this specification, and all documents cited or referenced within the documents cited in this specification, are hereby incorporated by reference in their entirety into this specification, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any product mentioned in this specification or within any document incorporated by reference herein.

[0037] Selected Definitions The term "intermediate" is used in the context of the present disclosure to refer to an intermediate population of MLPSCs that have been newly isolated and subjected to initial stage culture expansion (e.g., about 2 passages). For example, the generation of an intermediate population of MLPSCs may include the isolation of nucleated bone marrow cells (NBMCs) from bone marrow aspirate (BMA), cell culture, and 2 passages for growing the relevant cells. In one example, the cells are then collected and formulated for analysis according to the methods described herein. In one example, the collected cells are cryopreserved. Thus, the generation of the intermediate disclosed herein is the first step in the process of making a composition for administration to a subject. The above reference example refers to the isolation of NBMCs from BMA, but those skilled in the art will understand that relevant cells can be obtained from other suitable source(s) (e.g., adipose tissue) as discussed herein and subjected to the same initial stage culture expansion to obtain the intermediate included in the present disclosure.

[0038] In certain examples disclosed herein, the intermediate of the present disclosure is cultured and expanded to provide a population of MLPSCs for administration. In one example, such a population may be referred to as a pharmaceutical composition or dosage form (DP). For example, the culture expansion of the intermediate includes cell culture and more passages (e.g., an additional 3 passages) for culture expansion. The culture expansion of the intermediate will be described further below.

[0039] The cryopreserved intermediate according to the present disclosure may also be referred to as a donor cell bank (DCB).

[0040] The terms "level" and "amount" are used to define the amount of a particular substance in a cell preparation. For example, the level of a particular substance can be defined using a specific concentration, weight, percentage (e.g., v / v%), or ratio. In one example, the level is expressed with respect to how much a particular marker is expressed in the cells disclosed herein under culture conditions. In one example, the expression represents cell surface expression. In another example, the level is expressed with respect to whether a particular marker is released from the cells described herein into their culture medium under culture conditions.

[0041] In one example, the level is expressed in pg / ml. In another example, the level is expressed in pg per 10 cells 6 The level of pg / ml may be converted to pg per 10 cells as needed. For example, in the context of TNF-R1, in one example, 80 pg / ml of TNF-R1 corresponds to approximately 9.4 pg of TNF-R1 per 10 cells 6 In one example, in the context of TNF-R1, in one example, 100 pg / ml of TNF-R1 corresponds to approximately 11.5 pg of TNF-R1 per 10 cells 6 In one example, in the context of TNF-R1, in one example, 105 pg / ml of TNF-R1 corresponds to approximately 12.5 pg of TNF-R1 per 10 cells 6 In one example, 105 pg / ml of TNF-R1 corresponds to approximately 12.5 pg of TNF-R1 per 10 cells 6 In another example, 150 pg / ml of TNF-R1 corresponds to approximately 17.5 pg of TNF-R1 per 10 cells 6 In another example, 200 pg / ml of TNF-R1 corresponds to approximately 23.5 pg of TNF-R1 per 10 cells, etc. 6

[0042] In one example, the level of a particular marker is determined under culture conditions. The term "culture conditions" is used to refer to cells that are growing in culture. In one example, the culture conditions refer to a population of cells that are actively dividing. Such cells may, in one example, be in the exponential growth phase. In one example, the cells may be in the stationary phase

[0043] In one example, the culture conditions include co-culture of the MLPSC population disclosed herein and a second cell population such as a population containing peripheral blood mononuclear cells (PBMC). In one example, the co-culture includes culturing the MLPSC population and a population of activated PBMCs disclosed herein. For example, PBMCs may be activated using anti-CD3 and anti-CD28 antibodies prior to co-culture with the MLSPC population disclosed herein

[0044] ​In one example, the "culture conditions" include co-culturing MLPSC and T cells with a ratio of MLPSC:T cells of about 1:2 or less. For example, the MLPSC:T cell ratio is 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or less. In this example, the level of IL2-RA inhibition is determined at about 30 - 84 hours after cell culture under the culture conditions.

[0045] In one example, the level of a specific marker can be determined by collecting a sample of the cell culture medium and measuring the level of the marker in the sample. In another example, the level of a specific marker can be determined by collecting a sample of the cells and measuring the level of the marker in the cell lysate. Those skilled in the art will understand that secreted markers can be measured by sampling the culture medium, while markers expressed on the cell surface can be measured by evaluating a sample of the cell lysate. In one example, the sample is collected when the cells are in the exponential growth phase. In one example, the sample is collected at least 2 or 3 days after the culture. In another example, the sample is collected about 30 - 84 hours after co-culture. In one example, the sample is collected from the co-culture of MLPSC and activated PBMC. In this example, the cell sample can be lysed and the level of the marker can be determined. For example, the level of IL2-RA can be determined using various methods such as an enzyme-linked immunosorbent assay (ELISA)-based method. In one example, ELISA involves (i) adding a sample diluent to each well of a microplate pre-coated with a monoclonal antibody specific for IL2-RA, (ii) adding a co-culture sample to the wells of a microplate pre-coated with a monoclonal antibody specific for IL2-RA, (iii) incubating the microplate for a time sufficient to allow the monoclonal antibody specific for IL2-RA to specifically bind to any IL2-RA in the sample, (iv) washing the microplate, (v) Adding the IL2-RA complex to the wells, and (vi) Incubating the microplate for a time sufficient to allow the complex to specifically bind to any captured IL2-RA, and (vii) Washing the microplate, and (viii) Adding the substrate solution to the wells, and (ix) Incubating the microplate for a time sufficient for color development, and (x) Adding the stop solution to the wells, and (xi) Reading the optical density with a microplate reader set at 450 nm with wavelength correction at 570 nm, and (xii) Determining the concentration of IL2-RA.

[0046] In another example, the level of IL2-RA is determined using fluorescence-activated cell sorting (FACS) with a suitable antibody such as anti-CD25. Additional antibodies may also be used if it is necessary to distinguish CD25+ cell types.

[0047] In one example, the level of IL2-RA in co-culture is compared to the level of IL2-RA in a cultured population of activated PBMCs. Subsequently, each level of IL2-RA is compared to obtain the level of IL2-RA inhibition in co-culture. For example, the level of IL2-RA can be inhibited by at least 60% in co-culture (i.e., MLPSC: activated PBMC) compared to single culture (activated PBMC alone). Those skilled in the art will understand other suitable methods for determining the level of IL2-RA under culture conditions.

[0048] Culturing and growing cells from cryopreserved intermediates means thawing cryopreserved cells and culturing them in vitro under conditions suitable for cell growth.

[0049] In one example, the “level” or “amount” of a particular marker, such as IL2-RA or TNF-R1, is determined before the cells are cryopreserved. For example, the level may be determined after the first 2 to 5 passages of the cells. In another example, the level or amount of a particular marker is determined after the first cryopreservation of the cells. In another example, the level is determined after the second cryopreservation of the cells. For example, the cells can be cultured and expanded to obtain an intermediate, cryopreserved, thawed before being re-seeded into culture so that the level of a particular marker can be determined under culture conditions, and then cryopreserved again.

[0050] “Isolated” or “purified” means a cell that is separated from at least some of the components of its natural environment. This term includes the complete physical separation of the cell from its natural environment (e.g., removal from a donor). The term “isolated” includes, for example, altering the direct relationship of the cell with adjacent cells by dissociation. The term “isolated” does not refer to cells present in a tissue section. When used to refer to a population of cells, the term “isolated” includes a population of cells resulting from the growth of the isolated cells of the present disclosure.

[0051] The terms "passage", "passaging", or "subculture" are used in the context of this disclosure to refer to known cell culture techniques used to maintain the survival and growth of cells over an extended period of time under culture conditions such that the number of cells continuously increases. The extent of subculture that a cell line has undergone is often expressed as the "passage number", which is generally used to refer to the number of times the cells have been subcultured. In one example, one passage includes removing non-adherent cells and leaving adherent mesenchymal progenitor cells or stem cells. Next, such mesenchymal progenitor cells or stem cells can be detached from the substrate or flask (e.g., by using a protease such as trypsin or collagenase), medium can be added, an optional wash can be performed (e.g., by centrifugation), and then the mesenchymal progenitor cells or stem cells can be replated or reseeded into one or more culture vessels that generally have a larger surface area. Next, the mesenchymal progenitor cells or stem cells can continue to grow in culture. In another example, the method of removing non-adherent cells includes a step of non-enzymatic treatment (e.g., using EDTA). In one example, mesenchymal progenitor cells or stem cells are passaged at confluence or near confluence (e.g., about 75% to about 95% confluence). In one example, mesenchymal progenitor cells or stem cells are seeded at a concentration of about 10%, about 15%, or about 20% cells / ml of the culture medium.

[0052] The term "medium" or "media" as used in the context of this disclosure includes the components of the environment surrounding the cells during culture. The medium is assumed to contribute to and / or provide conditions suitable for growing the cells. The medium can be solid, liquid, gaseous, or a mixture of these phases and materials. The medium can include a liquid growth medium as well as a liquid medium that does not maintain cell growth. An exemplary gaseous medium includes the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed.

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

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

[0055] As used herein, the terms "preventing," or "prevention" include administering a population of mesenchymal stem cells or progenitor cells and / or their progeny and / or soluble factors derived therefrom, thereby stopping or inhibiting the onset of at least one symptom of an inflammatory disease.

[0056] As used herein, the term "subject" refers to a human subject. For example, the subject can be an adult. Terms such as "subject," "patient," or "individual" are terms that can be used interchangeably in the context of the present disclosure.

[0057] As used herein, the term "not genetically engineered" refers to cells that have not been recombined by transfection with a nucleic acid. To avoid misunderstanding, in the context of the present disclosure, mesenchymal progenitor cells or stem cells transfected with a nucleic acid encoding Ang1 are considered to be genetically engineered.

[0058] The term "total dose" is used in the context of the present disclosure to refer to the total number of cells received by a subject being treated in accordance with the present disclosure. In one example, the total dose consists of a single administration of cells. In another example, the total dose consists of two administrations of cells. In another example, the total dose consists of three administrations of cells. In another example, the total dose consists of four or more administrations of cells. For example, the total dose consists of two to four administrations of cells.

[0059] The term "clinically proven" (used either independently or to modify the term "effective") means that efficacy has been demonstrated by clinical trials, where the clinical trials meet the approval criteria of the U.S. Food and Drug Administration, the EMEA or the corresponding national regulatory agency. For example, the clinical study can be a properly sized and randomized double-blind study used to clinically prove the effect of a composition. In one example, a clinically proven effective amount is the amount shown by clinical trials to meet a specified endpoint. In one example, the endpoint is the prevention of death. In other words, the endpoint is to increase the survival rate. For example, the 100-day survival rate can increase when a therapeutic agent according to the present disclosure is administered.

[0060] Accordingly, the terms "clinically proven efficacy" and "clinically proven effect" may be used in the context of the present disclosure to refer to the dosages, dosing regimens, treatments, or methods disclosed herein. Efficacy can be measured based on changes in the course of a disease in response to administration of the compositions disclosed herein. For example, the compositions of the present disclosure are administered to a subject in an amount and for a time sufficient to induce improvement, preferably sustained improvement, in at least one metric reflecting the severity of an inflammatory disease. The various metrics reflecting the severity of the disease are evaluable to determine whether the amount and time of treatment are sufficient. Such metrics include, for example, clinically recognized metrics 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 improves the survival rate of a patient. In another example, a clinically proven effective amount reduces the risk of death of a subject. In another example, a clinically proven effective amount increases the 100-day survival rate. In one example, the methods of the present disclosure administer a clinically proven effective amount of the compositions disclosed herein.

[0061] "Therapeutic efficacy" is used in the context of the present disclosure to refer to the MLPSCs and compositions disclosed herein that can treat, inhibit, and / or prevent a disease. For example, the therapeutically effective MLPSCs and compositions disclosed herein can treat, inhibit, and / or prevent an inflammatory disease such as GvHD. In one example, the therapeutically effective MLPSCs and compositions disclosed herein increase the 100-day survival rate of a subject suffering from GvHD.

[0062] The term "and / or", e.g., the term "X and / or Y", is understood to mean either "X and Y" or "X or Y" and should be construed as providing clear support for both meanings or either meaning.

[0063] As used herein, the term "about" refers to + / - 10% of the specified value, more preferably + / - 5%, unless otherwise specified.

[0064] Throughout this specification, words such as "comprise", "comprises" or "comprising" are to be understood to mean that they include a particular element, component or step, or group of elements, components or steps, but are not meant to exclude any other element, component or step, or group of elements, components or steps.

[0065] As used herein, the singular forms "a", "an", and "the" include both singular and plural references unless the context indicates otherwise.

[0066] Inflammatory disease As used herein, the term "inflammatory disease" is to be construed to include, but is not limited to, diseases including graft-versus-host disease (GvHD), pruritus, dermatitis, psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, myasthenia gravis, type I or type II diabetes, diabetic nephropathy, asthma, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular disorders, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, seborrheic dermatitis, Sjogren's syndrome, keratoconjunctivitis, uveitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory diseases of joints, skin, or muscle, acute or chronic idiopathic inflammatory arthritis, myositis, demyelinating diseases, chronic obstructive pulmonary disease, interstitial lung disease, interstitial nephritis, and chronic active hepatitis.

[0067] In one example, an inflammatory disease is mediated by activation of T cells. Examples of such diseases include GvHD, rheumatoid arthritis, and diabetes. In one example, an inflammatory disease mediated by activation of T cells is refractory to at least one biological therapy.

[0068] In one example, the inflammatory disease is hyperinflammation. The term "hyperinflammation" is used in the context of the present disclosure to refer to a severe and ongoing inflammatory process in the body. For example, hyperinflammation may refer to a severe and ongoing inflammatory process in the airways and / or lungs, kidneys, or liver. Thus, hyperinflammation can affect multiple organs in the body and their vasculature. In one example, hyperinflammation is associated with a cytokine storm or cytokine release syndrome (CRS). In one example, a cytokine storm or CRS is accompanied by a massive release of inflammatory cytokines such as IL-6.

[0069] In one example, hyperinflammation results in secondary (or acquired) hemophagocytic lymphohistiocytosis (sHLH). Thus, in one example, the methods of the present disclosure include the treatment of hemophagocytic lymphohistiocytosis (HLH).

[0070] In one example, hyperinflammation is caused by a viral infection. For example, hyperinflammation may be caused by rhinovirus, influenza virus, respiratory syncytial virus (RSV), or coronavirus. In one example, hyperinflammation may be caused by a coronavirus. For example, the coronavirus can be severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or COVID-19. In one example, hyperinflammation is caused by Epstein-Barr virus (EBV) or herpes simplex virus (HSV).

[0071] In one example, the inflammatory disease is a multisystem inflammatory syndrome (MIS).

[0072] In one example, the inflammatory disease is ARDS. In one example, ARDS is mild ARDS. In another example, ARDS is moderate ARDS. In another example, ARDS is severe ARDS. In another example, ARDS is moderate or severe ARDS. In another example, ARDS is moderate, severe, or very severe ARDS.

[0073] In one example, the inflammatory disease is graft-versus-host disease (GvHD). "Graft-versus-host disease (GvHD)" is an immunological disorder that limits the success and effectiveness of allogeneic bone marrow or stem cell transplantation. GvHD occurs in acute (aGvHD) or chronic (cGvHD) forms. Acute GvHD usually appears within 100 days of bone marrow or stem cell transplantation. Chronic GvHD generally appears later than aGvHD (more than 100 days after transplantation) and has some characteristics of autoimmune diseases. It may develop newly after recovery from aGvHD or as an extension of aGvHD. Chronic GvHD can cause multiple, often debilitating symptoms, including extensive skin rashes, painful oral ulcers, shortness of breath, and limb and joint pain. In one example, cGvHD patients have a reconstitution disorder of CD5+ B cells. 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 both steroid therapy and biological therapy.

[0074] The severity of GvHD is graded according to the pattern of organ damage and clinical performance status. Examples of multi-organ damage include skin rash, liver damage, and / or gastrointestinal (GI) damage. Examples of skin rash, liver damage, and GI damage are presented in Tables 1 and 2. In one example, the subject has GvHD with multi-organ damage. In one example, the subject being treated according to the present disclosure has 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, the subject may have GvHD of grade II or grade III / IV according to the Glucksberg scale. In one example, the subject has GvHD of grade II. In another example, the subject has GvHD of grade III / IV.

[0075] 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 GvHD of grade B, grade C, or grade D according to the IBMTR severity scale. In one example, the subject has grade D GvHD.

[0076] In another example, the subject has Minnesota high-risk GvHD. Minnesota high-risk acute GvHD is defined as GvHD with skin stage 4; lower gastrointestinal (GI) stage 3 - 4 or liver stage 3 - 4; or skin stage 3+ and lower GI 2 - 4 or liver stage 2 - 4 (MacMillan et al., 2015). In each of these examples, the subject may also have a high MAP score. For example, the subject may have a MAP score of 0.29 or higher.

Table 1

Table 2

[0077] In one example, a subject with severe GvHD does not respond to treatment with primary therapy. In one example, the subject deteriorates within 3 days of primary therapy. In this example, the subject is deteriorating if the severity of their GvHD increases. For example, the severity of the subject's GvHD is increasing according to MAP. In another example, the severity of the subject's GvHD is increasing according to the Glucksberg scale. In another example, the severity of the subject's GvHD is increasing according to the IMBTR scale. In another example, the severity of the subject's GvHD is increasing according to organ damage.

[0078] In another example, the subject has not responded within 7 days of primary therapy. For example, the subject is refractory to primary therapy. In one example, the primary therapy is a systemic steroid drug. In one example, the subject has severe GvHD and is refractory to steroids. In one example, the steroid is a corticosteroid. In another example, the steroid is a glucocorticoid. In another example, the steroid is prednisone. In another example, the subject has severe GvHD and is refractory to steroids and second-line therapy. For example, second-line therapies can include extracorporeal photopheresis, etanercept, infliximab, ruxolitinib, antithymocyte globulin, mycophenolic acid, alemtuzumab, basiliximab, or tocilizumab.

[0079] Treatment response The methods of the present disclosure relate to the treatment of inflammatory diseases such as GvHD. As used herein, the terms "treating," "treat," "treatment," "reducing progression" include administering a population of mesenchymal stem cells or progenitor cells and / or their progeny and / or soluble factors derived therefrom and / or extracellular vesicles derived therefrom, thereby reducing or eliminating at least one symptom of the inflammatory disease.

[0080] As used herein, the term "response" means 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 no additional therapy is required. In another example, a subject is considered not to have responded if their GvHD is stable or progressive or if additional second-line therapy is subsequently required. In this example, a non-responding subject is a non-responder.

[0081] In one example, the treatment induces a partial remission. In one example, the partial remission is induced at least 28 days after treatment is initiated. In one example, the partial remission is induced 28 days after treatment is initiated. In one example, the partial remission is induced at least 30 days after treatment is initiated. In one example, the partial remission is induced at least 2 months after treatment is initiated. In another example, the partial remission is induced at least 3 months after treatment is initiated. In another example, the partial remission is induced within 3 months. In another example, the partial remission is induced 28 to 56 days after treatment is initiated. In another example, the partial remission is induced 100 days after treatment is initiated. In another example, the partial remission is induced 160 days after treatment is initiated. In another example, the partial remission is induced 180 days after treatment is initiated.

[0082] In another example, the partial remission is induced after two administrations. In another example, the partial remission is induced after two administrations with once-a-week dosing. In another example, the partial remission is induced after two administrations with once-a-week dosing every two weeks. In another example, the partial remission is induced after three or more administrations. In one example, in the context of GvHD, the partial remission is characterized by one or more or all of the following. - At least a 1-point decrease in the skin %BSA score; - At least a 1-point decrease in the oral score; - At least a 1-point decrease in the eye score; - At least a 1-point decrease in the skin feature score; - At least a 1-point decrease in the gastrointestinal tract score; - At least a 1-point decrease in the liver score; - At least a 1-point decrease in the lung symptom score; - At least a 1-point decrease in the lung FEV1 score; - At least a 1-point decrease in the joint and fascia score; - At least a 1-point decrease in the genital score.

[0083] In one example, partial remission is characterized by at least a 1-point decrease in the skin %BSA score. In another example, partial remission is characterized by at least a 1-point decrease in the oral score. In another example, partial remission is characterized by at least a 1-point decrease in the eye score. In these examples, the scores can be obtained using the NIH Consensus Criteria 2014 for GvHD.

[0084] In another example, partial remission is characterized by one or more or all of the following. - At least a 1-point decrease in the skin %BSA score; - At least a 1-point decrease in the oral score; - At least a 1-point decrease in the eye score.

[0085] There are various classification systems for characterizing GvHD (Lee, S., (2017) Blood., 129(1):30-37). In one example, the NIH Consensus Criteria 2014 can be used to score the outcomes disclosed herein (Jagasia et al., (2015) Biol Blood Marrow Transplant., 21:389-401). The components of the NIH Consensus Criteria 2014 are shown in the table below.

Table 3-1

Table 3-2

[0086] In one example, a partial remission is a decrease of ≥ 1 point in the organ-specific NIH Consensus Criteria 2014 score from the above table. Thus, in one example, the treatment induces a decrease of ≥ 1 point in the skin %BSA score. In another example, the treatment induces a decrease of ≥ 1 point in the oral score. In another example, the treatment induces a decrease of ≥ 1 point in the eye score. In another example, the treatment induces a decrease of ≥ 1 point in the skin feature score. In another example, the treatment induces a decrease of ≥ 1 point in the gastrointestinal tract score. In another example, the treatment induces a decrease of ≥ 1 point in the liver score. In another example, the treatment induces a decrease of ≥ 1 point in the lung symptom score. In another example, the treatment induces a decrease of ≥ 1 point in the lung FEV1 score. In another example, the treatment induces a decrease of ≥ 1 point in the joint and fascia score. In another example, the treatment induces a decrease of ≥ 1 point in the genital score.

[0087] In one example, the treatment induces a complete remission after treatment is initiated. In one example, a complete remission is the complete resolution of GvHD symptoms in all organs. In one example, a complete remission is induced 28 days after treatment is initiated. In one example, a complete remission is induced at least 28 days after treatment is initiated. In one example, a complete remission is induced at least 30 days after treatment is initiated. In one example, a complete remission is induced at least 2 months after treatment is initiated. In another example, a complete remission is induced at least 3 months after treatment is initiated. In another example, a complete remission is induced 28 - 56 days after treatment is initiated. In another example, a complete remission is induced 100 days after treatment is initiated. In another example, a complete remission is induced 160 days after treatment is initiated. In another example, a complete remission is induced 180 days after treatment is initiated.

[0088] In another example, a complete remission is induced after 2 administrations. In another example, a complete remission is induced after 2 administrations with 1 administration per week. In another example, a complete remission is induced after 2 administrations with 1 administration per week every 2 weeks. In another example, a complete remission is induced after 3 or more administrations.

[0089] In another example, again in the context of GvHD, treatment increases the probability of the subject's survival rate. For example, treatment increases the probability that the subject will survive for at least 20 days to 200 days after the start of treatment. In one example, treatment increases the probability that the subject will survive for at least 180 days after the start of treatment. In another example, treatment increases the probability that the subject will survive for at least 100 days. In one example, the improvement in probability is determined compared to a subject not treated with the compositions of the present disclosure. In one example, treatment results in a greater than 60% increase in the 100-day survival rate. In another example, treatment results in a greater than 60% increase in the 100-day survival rate. In another example, treatment results in a greater than 65% increase in the 100-day survival rate. In another example, treatment results in a greater than 68% increase in the 100-day survival rate. In another example, treatment results in a greater than 70% increase in the 100-day survival rate. In another example, treatment results in a greater than 71% increase in the 100-day survival rate. In another example, treatment results in a greater than 72% increase in the 100-day survival rate. In another example, treatment results in a 60% - 75% increase in the 100-day survival rate. In another example, treatment results in a 65% - 75% increase in the 100-day survival rate. In another example, treatment results in a 70% - 75% increase in the 100-day survival rate. In one example, the 100-day survival rate is measured across the entire population of patients, and the average 100-day survival rate observed in that population is provided as a measure of the 100-day survival rate for each patient in the population. In one example, this measure is compared to a subject (or population of subjects) not treated with the compositions of the present disclosure.

[0090] Mesenchymal progenitor cells As used herein, the term "mesenchymal progenitor or stem cells (MLPSCs)" refers to undifferentiated pluripotent cells that have the ability to self-renew while maintaining pluripotency and the ability to differentiate into a number of cell types, either of mesenchymal origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts and tendons, or of non-mesodermal origin, such as hepatocytes, neurons and epithelial cells. To avoid misunderstanding, "mesenchymal progenitor cells" refers to mesenchymal cells such as bone, cartilage, muscle and fat cells, as well as cells that can differentiate into fibrous connective tissue.

[0091] The term "mesenchymal progenitor cells or stem cells" includes both parent cells and their undifferentiated progeny. This term also includes mesenchymal progenitor cells, multipotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal progenitor cells, and their undifferentiated progeny.

[0092] Mesenchymal progenitor cells or stem cells can be autologous, allogeneic, xenogeneic, syngeneic, or isogeneic. Autologous cells are isolated from the same individual into which they are re-transplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of a different species. Syngeneic or isogeneic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred laboratory animal models.

[0093] In one example, mesenchymal progenitor cells or stem cells are allogeneic. In one example, allogeneic mesenchymal progenitor cells or stem cells are cultured and cryopreserved.

[0094] Mesenchymal progenitor cells or stem cells are primarily present in bone marrow, but have also been shown to be present in a variety of host tissues, including, for example, umbilical cord blood and cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp. They are also found in skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis, and periosteum, and can differentiate into germ cell lineages such as mesoderm and / or endoderm and / or ectoderm. Thus, mesenchymal progenitor cells or stem cells can differentiate into a number of cell types, including but not limited to adipose, osseous, cartilaginous, elastic, muscular, and fibrous connective tissues. The specific lineage-commitment and differentiation pathways into which these cells enter depend on mechanical influences and / or endogenous bioactive factors, such as growth factors, cytokines, and / or various influences from local microenvironmental conditions established by the host tissue.

[0095] As used herein, the terms "enriched", "enrichment" or variations thereof are used to describe a population of cells in which the proportion of one particular cell type or the proportion of the number of several particular cell types is increased compared to a population of untreated cells (e.g., cells in their native environment). In one example, a population enriched in mesenchymal progenitor cells or stem cells contains 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 progenitor cells or stem cells. In this regard, the term "population of cells enriched in mesenchymal progenitor cells or stem cells" is construed to provide an explicit underpinning of the term "population of cells containing X% mesenchymal progenitor cells or stem cells", where X% is the percentage described herein. Mesenchymal progenitor cells or stem cells, in some examples, can form clonogenic colonies, e.g., CFU-F (fibroblasts) or a subset thereof (e.g., 50% or 60% or 70% or 70% or 90% or 95%) can have this activity.

[0096] In one example of the present disclosure, the mesenchymal progenitor cells or stem cells are mesenchymal stem cells (MSCs). The MSCs can be a homogeneous composition or a mixed cell population enriched in MSCs. A homogeneous MSC composition can be obtained by culturing adherent bone marrow cells or periosteal cells, and the MSCs can be identified by specific cell surface markers identified by specific monoclonal antibodies. Methods for obtaining a cell population enriched in MSCs are described, for example, in U.S. Patent No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, perichondrium. In one example, the MSCs are allogeneic. In one example, the MSCs are cryopreserved. In one example, the MSCs are cultured and expanded and cryopreserved.

[0097] In another example, the mesenchymal progenitor cells or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs.

[0098] Isolated or enriched mesenchymal progenitor cells or stem cells can be grown in vitro by culture. Isolated or enriched mesenchymal progenitor cells or stem cells can be cryopreserved, thawed, and then grown in vitro by culture.

[0099] In one example, isolated or enriched mesenchymal progenitor cells or stem cells are seeded at 50,000 viable cells per cm2 into a culture medium (serum-free or serum-supplemented), such as alpha minimum essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, and allowed to attach to the culture vessel overnight at 37°C and 20% O2. Thereafter, the culture medium is changed and / or modified as needed, and the cells are cultured for an additional 68 - 72 hours at 37°C and 5% O2.

[0100] As will be appreciated by those skilled in the art, cultured mesenchymal progenitor cells or stem cells have a different phenotype from in vivo cells. For example, in one embodiment, they express one or more markers of CD44, NG2, DC146, and CD140b. Cultured mesenchymal progenitor cells or stem cells are also biologically different from in vivo cells and have a higher growth rate than most of the in vivo quiescent (resting) cells.

[0101] In one example, the population of cells 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 cell expresses a marker (e.g., a cell surface marker) that enables the selection of STRO-1+ cells. The marker can be STRO-1, but it need not be. For example, as described and / or exemplified herein, cells that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 (e.g., mesenchymal progenitor cells) also express STRO-1 (which can be STRO-1bright). Thus, the designation that a cell is STRO-1+ does not mean that the cell is selected by STRO-1 expression alone. In one example, the cells are selected based at least on STRO-3 expression, e.g., they are STRO-3+ (TNAP+). For example, MPCs can be isolated from bone mononuclear cells using an anti-STRO-3 antibody.

[0102] References to the selection of a cell or population thereof do not necessarily require selection from a specific tissue source. As described herein, STRO-1+ cells can be selected, isolated, or enriched from a variety of sources. That being said, in some examples, these terms provide assistance to the selection from any tissue comprising STRO-1+ cells (e.g., mesenchymal progenitor cells), or vascular tissue, or tissue comprising pericyte cells (e.g., STRO-1+ pericytes), or any one or more of the tissues enumerated herein.

[0103] In one example, the cells used in the present disclosure are selected individually or collectively from one or more markers consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0104] "Individually" means that the present disclosure encompasses the listed markers or marker groups separately, and even if the individual markers or marker groups cannot be separately enumerated herein, the appended claims can define such markers or marker groups separately from each other and divisibly.

[0105] "Collectively" means that the present disclosure encompasses any number or combination of the listed markers or marker groups, and even if the number or combination of such markers or marker groups cannot be specifically enumerated herein, the appended claims can define such a combination or sub - combination separately and divisibly from any other combination of markers or marker groups.

[0106] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue - nonspecific alkaline phosphatase. For example, this term encompasses liver isoform (LAP), bone isoform (BAP), and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP as used herein refers to a molecule that can bind to the STRO - 3 antibody produced by a hybridoma cell line deposited with the ATCC on December 19, 2005, under the terms of the Budapest Treaty with the deposit accession number PTA - 7282.

[0107] Furthermore, in one example, STRO - 1+ cells can give rise to clonogenic CFU - F.

[0108] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germ cell lines. Non-limiting examples of lineages to which STRO-1+ cells can skew include: bone progenitors; hepatocyte precursors that are pluripotent for bile duct epithelial cells and hepatocytes; neural restricted cells that can give rise to glial cell precursors that develop into oligodendrocytes and astrocytes; neuron precursors that develop into neurons; precursors of cardiomyocytes and cardiomyocytes, glucose-responsive insulin-secreting pancreatic beta cell lines. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells and chondrocytes, and the following: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal tubular epithelial cells, smooth muscle and skeletal muscle cells, testicular precursors, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiomyocytes, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes and oligodendrocyte precursor cells.

[0109] In one example, mesenchymal progenitor cells or stem cells are obtained from a single donor or multiple donors, the donor samples or mesenchymal progenitor cells or stem cells are then pooled, and then cultured and expanded.

[0110] The mesenchymal progenitor cells or stem cells encompassed by the present disclosure can also be cryopreserved prior to administration to a subject. In one example, the mesenchymal progenitor cells or stem cells are cultured and expanded and cryopreserved prior to administration to a subject.

[0111] In one example, the present disclosure encompasses mesenchymal progenitor cells or stem cells, and their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal progenitor cells or stem cells, and extracellular vesicles isolated therefrom. For example, the mesenchymal progenitor lineage cells or stem cells of the present disclosure can be cultured and expanded for a period and under conditions suitable for secreting extracellular vesicles into a cell culture medium. Subsequently, the secreted extracellular vesicles can be obtained from the culture medium for use in therapy.

[0112] 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 about 10 microns, although typically they are less than 200 nm in size. They can contain proteins, nucleic acids, lipids, metabolites, or organelles from the releasing cell (e.g., mesenchymal stem cells; STRO-1 + cells).

[0113] As used herein, the term "exosome" generally refers to a type of extracellular vesicle that ranges in size from about 30 nm to about 150 nm and is derived from the endosomal compartment of mammalian cells, from which they are sent to and released from the cell membrane. They can contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolites and can function in cell - cell communication by being secreted from one cell and taken up by other cells to deliver their cargo.

[0114] In one example, the compositions of the present disclosure include cells that induce new blood vessel formation in a target tissue. In one example, the target tissue is the heart. In another example, the cells secrete factors that protect at - risk or damaged myocardium. In one example, the at - risk or damaged myocardium is exposed to insufficient blood flow due to an ischemic event. In one example, the cells secrete factors that decrease apoptosis of cardiomyocytes.

[0115] Cell culture expansion In one example, mesenchymal progenitor cells or stem cells are cultured and expanded. "Cultured and expanded" mesenchymal progenitor cells or stem cells are distinguished from freshly isolated cells in that they have been cultured and passaged (i.e., subcultured) in a cell culture medium. In one example, freshly isolated cells are cultured and passaged about 1 or 2 times to provide an intermediate population. In one example, freshly isolated cells are passaged 2 times to provide an intermediate population. In another example, freshly isolated cells are cultured and passaged about 1 - 3 times to provide an intermediate population.

[0116] Thus, in one example, relevant cells are isolated and passaged twice to provide an intermediate MLPSC population. Next, the intermediate MLPSC population is cultured and expanded to provide DPs. For example, the intermediate cell population can be further passaged three times (i.e., a total of five passages) to provide DPs.

[0117] In one example, mesenchymal progenitor cells or stem cells are passaged and expanded about 4 to 10 times. In one example, mesenchymal progenitor cells or stem cells are passaged and expanded at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 times. For example, mesenchymal progenitor cells or stem cells can be passaged and expanded at least 5 times. In one example, mesenchymal progenitor cells or stem cells can be passaged and expanded at least 5 to 10 times. In one example, mesenchymal progenitor cells or stem cells can be passaged and expanded at least 5 to 8 times. In one example, mesenchymal progenitor cells or stem cells can be passaged and expanded at least 5 to 7 times. In one example, mesenchymal progenitor cells or stem cells can be passaged and expanded more than 7 times. In these examples, MLPSCs are cultured and expanded before cryopreservation to provide an intermediate cryopreserved MLPSC population, which can then be further subjected to culture and expansion.

[0118] In one example, by the methods of the present disclosure, an intermediate population (e.g., cryopreserved intermediate) is selected for further culture and expansion based on certain criteria such as the level of IL2-RA inhibition. For example, a cryopreserved intermediate can be selected for culture and expansion if the MLPSC population inhibits IL2-RA by at least 55% under culture conditions. In another example, a cryopreserved intermediate can be selected for culture and expansion if the MLPSC population inhibits IL2-RA by at least 60% under culture conditions.

[0119] In other examples, the methods of the present disclosure include evaluating the therapeutic efficacy of a cryopreserved intermediate population of MLPSCs.

[0120] In one example, the DP composition of the present disclosure is made by culturing cells from an intermediate cryopreserved MLPSC population, in other words, a cryopreserved intermediate.

[0121] In one example, the compositions of the present disclosure include mesenchymal progenitor cells or stem cells cultured and expanded from cryopreserved intermediates. In one example, the cells cultured and expanded from cryopreserved intermediates are subcultured and expanded at least 3, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 times. For example, mesenchymal progenitor cells or stem cells can be subcultured and expanded at least 3 times. In one example, mesenchymal progenitor cells or stem cells can be subcultured and expanded at least 3 - 10 times. In one example, mesenchymal progenitor cells or stem cells can be subcultured and expanded at least 3 - 8 times. In one example, mesenchymal progenitor cells or stem cells can be subcultured and expanded at least 3 - 7 times.

[0122] In one example, the inhibition of IL2 - RA by mesenchymal progenitor cells or stem cells cultured and expanded from cryopreserved intermediates disclosed herein is evaluated under culture conditions (e.g., co - culture with T cells). In one example, these MLPSCs inhibit IL2 - RA by more than 70% under culture conditions. In another example, these MLPSCs inhibit IL2 - RA by more than 72% under culture conditions. In another example, these MLPSCs inhibit IL2 - RA by 70% - 80% under culture conditions.

[0123] In one example, the cultured and expanded mesenchymal progenitor cells or stem cells can be cultured and expanded in a medium without animal proteins (e.g., in a medium without xenogeneic components). In one example, mesenchymal progenitor cells or stem cells can be cultured and expanded in a medium without fetal bovine serum.

[0124] In one embodiment, mesenchymal progenitor cells or stem cells can be obtained from a single donor or multiple donors, and the donor samples or mesenchymal progenitor cells or stem cells are then pooled and then cultured and expanded as needed. In one example, the culture and expansion process includes the following: i. Increasing the number of viable cells by subculturing to provide a preparation of at least about 1 billion viable cells, wherein the subculturing includes establishing a primary culture of isolated mesenchymal progenitor cells or stem cells and then successively establishing a first non-primary (P1) culture of mesenchymal progenitor cells or stem cells isolated from the previous culture; ii. Propagating a P1 culture of isolated mesenchymal progenitor cells or stem cells by subculturing to a second non-primary (P2) culture of mesenchymal progenitor cells or stem cells; and iii. Preparing and cryopreserving an intermediate mesenchymal progenitor cell or stem cell preparation in the process obtained from the P2 culture of mesenchymal progenitor cells or stem cells; and optionally iv. Thawing the cryopreserved intermediate mesenchymal progenitor cell or stem cell preparation in the process and propagating the intermediate mesenchymal progenitor cell or stem cell preparation in the process by subculturing.

[0125] In the context of the present disclosure, a particular assay may be performed between steps iii and iv. For example, IL2-RA inhibition may be determined under culture conditions after step iii. In one example, step iv is only performed when a desired level of IL2-RA inhibition is observed under culture conditions. In this example, the cell population is selected for culture expansion based on IL2-RA inhibition under culture conditions.

[0126] In one example, the propagated mesenchymal progenitor cell or stem cell preparation has an antigen profile and an activity profile including: i. Less than about 0.75% CD45+ cells; ii. At least about 95% CD105+ cells; iii. At least about 95% CD166+ cells.

[0127] In one example, the expanded mesenchymal progenitor or stem cell population is an intermediate population that can inhibit IL2-RA expression by CD3 / CD28-activated PBMCs by at least 55% compared to a control. In another example, the expanded mesenchymal progenitor or stem cell population is an intermediate population that can inhibit IL2-RA expression by CD3 / CD28-activated PBMCs by at least 60% compared to a control. In another example, the expanded mesenchymal progenitor or stem cell population is an intermediate population that can inhibit IL2-RA expression by CD3 / CD28-activated PBMCs by at least 65% compared to a control. In another example, the expanded mesenchymal progenitor or stem cell population is an intermediate population that can inhibit IL2-RA expression by CD3 / CD28-activated PBMCs by at least about 70% compared to a control. In another example, the expanded mesenchymal progenitor or stem cell population is an intermediate population that can inhibit IL2-RA expression by CD3 / CD28-activated PBMCs by 55-70% compared to a control. In another example, the expanded mesenchymal progenitor or stem cell population is an intermediate population that can inhibit IL2-RA expression by CD3 / CD28-activated PBMCs by 60-70% compared to a control.

[0128] In one example, the expanded mesenchymal progenitor or stem cell population is expanded from an intermediate MLPSC population and can inhibit IL2-RA expression by CD3 / CD28-activated PBMCs by at least 70% compared to a control. In another example, the expanded mesenchymal progenitor or stem cell population is expanded from an intermediate MLPSC population and can inhibit IL2-RA expression by CD3 / CD28-activated PBMCs by 70%-75% compared to a control.

[0129] The process of isolating and ex vivo expanding mesenchymal progenitor cells or stem cells can be carried out using any device and cell handling method known in the art. The various culture expansion embodiments of the present disclosure use steps that require cell manipulation, such as seeding, feeding, dissociation of adherent cultures, or washing steps. Any step of manipulating cells has the potential to damage the cells. Mesenchymal progenitor cells or stem cells can generally withstand a certain degree of damage during preparation, but it is preferred to manipulate the cells with handling procedures and / or devices that appropriately perform the predetermined step(s) while minimizing damage to the cells.

[0130] In one example, mesenchymal progenitor cells or stem cells are washed in a device comprising a cell source bag, a wash solution bag, a recirculating wash bag, a rotating membrane filter having an inlet and an outlet port, a filtrate bag, a mixing zone, a final product bag for the washed cells, and appropriate tubing, as described, for example, in U.S. Patent No. 6,251,295, which is incorporated herein by reference.

[0131] In one example, the mesenchymal progenitor cell 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, i.e., through multiple population doublings.

[0132] In one example, the mesenchymal progenitor cells or stem cells of the present disclosure are cultured and expanded in 2D culture. For example, the mesenchymal progenitor cells or stem cells of the present disclosure can be cultured and expanded in a cell factory. In certain examples, the 3D culture of the intermediates disclosed herein may be subsequently carried out, for example, using a bioreactor. In one example, the mesenchymal progenitor cells or stem cells of the present disclosure are first cultured and expanded in 2D culture before being further expanded in 3D culture. In one example, the intermediate cell population of the present disclosure is not cultured and expanded in 3D culture. In one example, IL2-RA inhibition is evaluated prior to subsequent culture expansion in a cell factory or 3D culture.

[0133] In one example, the mesenchymal progenitor cells or stem cells of the present disclosure are cultured and expanded from an intermediate population. In one example, the mesenchymal progenitor cells or stem cells of the present disclosure are cultured and expanded from an intermediate in 2D culture prior to seeding in 3D culture.

[0134] In the context of both the intermediate population and the therapeutic composition expanded from the intermediate population, in one example, the mesenchymal progenitor cells or stem cells of the present disclosure are cultured and expanded in 2D culture for at least 3 days prior to seeding in a further culture system such as 3D culture in a cell factory or bioreactor. In one example, the mesenchymal progenitor cells or stem cells of the present disclosure are cultured and expanded in 2D culture for at least 4 days prior to seeding in a further culture system. In one example, the mesenchymal progenitor cells or stem cells of the present disclosure are cultured and expanded in 2D culture for 3 - 5 days prior to seeding in a further culture system. In these examples, the 2D culture can be performed in a cell factory. Various cell factory products are commercially available (e.g., Thermofisher, Sigma, Corning). In one example, the cell factory has at least 5 layers. In one example, the cell factory has at least 10 layers. In one example, the cell factory has at least 20 layers. The 3D culture can be carried out in various bioreactor types such as stirred tanks, wave bags, and vertical wheels.

[0135] In one example, CO2 is provided during the culture and 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 using passive priming of CO2. For example, the cell factory can be passively primed using 5% CO2.

[0136] The priming cell factory maintains the CO2 tension between the cell factory and the incubator and stabilizes the pH level of the growth medium. Active priming involves actively passing CO2 gas through a bacterial vent air filter into each culture vessel (e.g., cell factory) for a defined period of time (e.g., about 10 minutes). However, active priming requires an open port to supply gas and may introduce contaminants into the culture. Passive priming involves placing a closed culture system in an incubator at an appropriate CO2 concentration prior to cell seeding (e.g., about 12 - 72 hours).

[0137] In one example, the cells of the present disclosure are STRO-3+ and they are later cultured and expanded to provide an intermediate cell population.

[0138] Cell culture medium The mesenchymal progenitor cells or stem cells disclosed herein can be cultured and expanded in a variety of suitable growth media.

[0139] As used in the context of the present disclosure, the term "medium" or "media" includes the components of the environment surrounding the cells. The medium contributes to and / or provides conditions suitable for growing the cells. The medium can be solid, liquid, gaseous, or a mixture of these phases and materials. The medium can include a liquid growth medium as well as a liquid medium that does not support cell growth. The medium also includes gelatinous media such as agar, agarose, gelatin, and collagen matrices. An exemplary gaseous medium includes the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed.

[0140] The cell culture medium used for culturing and expanding contains 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).

[0141] One of ordinary skill in the art will understand that the basal medium must be appropriate for the cell line of interest for optimal results. For example, increasing the level of glucose (or other energy source) in the basal medium, or adding glucose (or other energy source) during the culture process, may be necessary if it is found that this energy source is depleted and thus limits growth. In one example, the dissolved oxygen (DO) level can also be controlled.

[0142] In one example, the cell culture medium contains human-derived additives. For example, human serum and human platelet cell lysate can be added to the cell culture medium.

[0143] In one example, the cell culture medium contains only human-derived additives. Thus, in one example, the cell culture medium is xeno-free. To avoid misunderstanding, in these examples, the culture medium does not contain animal proteins. In one example, the cell culture medium used in the methods of the present disclosure does not contain animal components.

[0144] In one example, the culture medium contains 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 growth of mesenchymal progenitor cells or stem cells. In one embodiment, the culture medium is a serum-free stem cell culture medium. In one example, the cell culture medium includes the following: A basal medium; Platelet-derived growth factor (PDGF); Fibroblast growth factor 2 (FGF2).

[0145] In one example, the culture medium contains platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2), and the level of FGF2 is less than about 6 ng / ml. For example, the FGF2 level can be less than about 5 ng / ml, less than about 4 ng / ml, less than about 3 ng / ml, less than about 2 ng / ml, less than about 1 ng / ml. In one example, the FGF2 level is about 1 ng / ml.

[0146] In one example, the PDGF is PDGF-BB. In one example, the level of PDGF-BB is from about 1 ng / ml to 150 ng / ml. In another example, the level of PDGF-BB is from about 7.5 ng / ml to 120 ng / ml. In another example, the level of PDGF-BB is from about 15 ng / ml to 60 ng / ml. In another example, the level of PDGF-BB is about 10 ng / ml. In another example, the level of PDGF-BB is at least about 10 ng / ml or less.

[0147] In other examples, additional factors can be added to the cell culture medium. In one example, the culture medium further contains 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 population of stem cells in a fetal bovine serum-free cell culture medium that further contains EGF. In one example, the level of EGF is from about 0.1 to 7 ng / ml. For example, the level of EGF can be at least about 5 ng / ml.

[0148] In another example, the level of EGF is from about 1 ng / ml to 8 ng / ml. In another example, the level of EGF is from about 3 ng / ml to 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.

[0149] In the above examples, a reference amount of growth factor can be supplemented to a basal medium such as Alpha MEM or StemSpan™. In one example, the culture medium contains Alpha MEM or StemSpan™ supplemented with 10 ng / ml of PDGF-BB, 5 ng / ml of EGF, and 1 ng / ml of FGF.

[0150] In other examples, additional factors can be added to the cell culture medium. For example, the cell culture medium can be supplemented with one or more stimulatory factors selected from the group consisting of epidermal growth factor (EGF), lα,25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor α (TNF-α), interleukin-lβ (IL-lβ), and stromal-derived factor lα (SDF-lα). In another embodiment, the cells can also be cultured in the presence of at least one cytokine in an amount sufficient to support cell growth. In another embodiment, the cells can be cultured in the presence of heparin or a derivative thereof. In one example, the heparin derivative is a 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. Thus, exemplary heparin sulfates include male liver heparin sulfate (MML HS) and female liver heparin sulfate (FML HS).

[0151] In another example, the cell culture medium of the present disclosure maintains stem cells in an undifferentiated state. Stem cells are considered undifferentiated if they do not tend towards a particular differentiation lineage. As described above, stem cells exhibit morphological characteristics that distinguish them from differentiated cells. Additionally, undifferentiated stem cells express genes that can be used as markers for detecting the differentiated state. The polypeptide product can also be used as a marker for detecting the differentiated state. Thus, one of ordinary skill in the art can readily determine whether the method of the present disclosure maintains stem cells in an undifferentiated state using conventional morphological analysis, genetic analysis, and / or proteomics analysis.

[0152] Co-culture of mesenchymal progenitor cells or stem cells with stimulated or activated T cells Interleukin-2 receptor alpha chain (「IL2-RA」; also called 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 IL2-RA expression after co-culture of activated PBMCs with mesenchymal progenitor cells or stem cells. Inhibition of IL2-RA expression is associated with an inhibitory effect on T cell activation.

[0153] In one example, activated PBMCs are co-cultured with mesenchymal progenitor cells or stem cells in a culture medium containing at least one T cell stimulant at a concentration capable of stimulating and / or activating T cells. In another embodiment, the T cells are first stimulated and / or activated prior to co-culture with mesenchymal progenitor cells or stem cells.

[0154] In one example, mesenchymal progenitor cells or stem cells are co-cultured with PBMCs in a culture medium containing agents capable of stimulating CD3 on T cells, such as an antibody against CD3 and an antibody against CD28, and agents capable of stimulating CD28, such as mouse anti-human CD3 and mouse anti-human CD28. In one example, the antibody against CD3 and / or the antibody against CD28 are added to the culture medium in soluble form at a concentration of about 2 μg / ml each.

[0155] In one embodiment, PBMCs are co-cultured with mesenchymal progenitor cells or stem cells at a ratio of PBMC:mesenchymal progenitor cells or stem cells of 5:1. For example, 1×10 6 PBMCs may be co-cultured with 2×10 5 MLPSCs. In one example, the cells may be co-cultured at a final volume of 1 ml.

[0156] Determination of Therapeutic Efficacy In one embodiment, the present disclosure relates to a method for determining the therapeutic efficacy of an intermediate population of mesenchymal progenitor lineage cells or stem cells (MLPSCs). In certain instances, such a method may be useful for establishing a "memory" in the intermediate cell population towards subsequent end products derived therefrom. In other words, such a method demonstrates the therapeutic efficacy in the intermediate cell population and the product(s) cultured and grown therefrom as a result. The therapeutic efficacy of the resulting product (or selected product) may, in certain instances, be characterized using additional assays as needed.

[0157] In one example, a method for determining the therapeutic efficacy of an intermediate population of mesenchymal progenitor lineage cells or stem cells (MLPSCs) includes: (i) obtaining a population of MLPSCs; (ii) culturing the cells in a culture medium; and (iii) determining the level of IL2-RA inhibition under the culture conditions, wherein at least 55% IL2-RA inhibition under the culture conditions indicates therapeutic efficacy. In another example, greater than 56% IL2-RA inhibition indicates therapeutic efficacy. In another example, greater than 60% IL2-RA inhibition indicates therapeutic efficacy. In another example, greater than 70% IL2-RA inhibition indicates therapeutic efficacy.

[0158] In other examples, the method for determining therapeutic efficacy further includes determining the level of TNF-R1 expression under the culture conditions, wherein an expression of at least 100 pg / ml of TNF-R1 indicates therapeutic efficacy. In another example, an expression of at least 105 pg / ml of TNF-R1 indicates therapeutic efficacy.

[0159] By practicing the method for determining therapeutic efficacy disclosed herein, an intermediate suitable for culturing and expanding into a therapeutically effective cell composition can be provided. In one example, such a cultured and expanded composition is characterized by a threshold level of IL2-RA inhibition. In one example, the level of IL2-RA inhibition is higher than the level observed in a previous intermediate population. In one example, the level of IL2-RA inhibition is greater than 70% under culture conditions. In another example, the level of IL2-RA inhibition is greater than 75% under culture conditions. In another example, the level of IL2-RA inhibition is between 65% and 75% under culture conditions. In another example, the level of IL2-RA inhibition is between 70% and 75% under culture conditions.

[0160] Determination of the amount of IL2-RA level The present disclosure contemplates any form of assay for determining the level of IL2-RA, including, for example, Western blot, enzyme-linked immunosorbent assay (ELISA), fluorescence-linked immunosorbent assay (FLISA), competitive assay, radioimmunoassay, lateral flow immunoassay, flow-through immunoassay, electrochemiluminescence assay, turbidimetry-based assay, nephelometry titration-based assay, fluorescence-activated cell sorting (FACS)-based assay.

[0161] After co-culture of MLPSC and T cells, the cells can be collected and lysed using methods well known in the art. The cell lysate can then be evaluated for the presence of IL2-RA, for example, by ELISA or FLISA. Alternatively, the level of IL2-RA expression may be determined by evaluating intact cells, for example, by flow cytometry.

[0162] In one example, inhibition of IL2-RA expression is measured by comparing the level of IL2-RA expression in a population of cells containing T cells with the level of IL2-RA in a population of cells after co-culture of a population of cells containing T cells with a population of cells containing mesenchymal progenitor cells or stem cells, and the difference is expressed as "percentage inhibition".

[0163] The above assay is easily modified using chemiluminescence or electrochemiluminescence as the detection criterion.

[0164] As will be apparent to those skilled in the art, other detection methods based on immunosorbent assays are useful in practicing the present disclosure. For example, radiolabels for detection, or gold labels (e.g., colloidal gold) for detection, or liposomes, e.g., immunosorbent methods based on encapsulation of NAD+ for detection, or acridinium-conjugated immunosorbent assays, and the like.

[0165] In some examples of the present disclosure, the level of IL2-RA is determined using a surface plasmon resonance detector (e.g., BIAcore™, GE Healthcare, Piscataway, N.J.), a flow-through device (e.g., as described in U.S. Patent No. 7,205,159), a micro or nanoimmunoassay device (e.g., as described in U.S. Patent No. 7,271,007), a lateral flow device (e.g., as described in U.S. Publication No. 2004 / 0228761 or U.S. Publication No. 2004 / 0265926), a fluorescence polarization immunoassay (FPIA, e.g., as described in U.S. Patent No. 4,593,089 or U.S. Patent No. 4,751,190), or an immunoturbidimetric assay (e.g., as described in U.S. Patent No. 5,571,728 or U.S. Patent No. 6,248,597).

[0166] Determination of the amount of TNFR1 level In a particular example, the method of the present disclosure also includes determining the expression of TNFR1 by mesenchymal progenitor cells or stem cells.

[0167] Those skilled in the art will understand that the above-described methods for detecting IL2-RA expression are also available for detecting TNFR1 expression. In a preferred embodiment, the cell lysate is evaluated by ELISA or FLISA.

[0168] In one form, such an assay involves immobilizing the TNFR1 binding protein on a solid matrix. Next, a test sample is brought into direct contact with the TNFR1 binding protein to bind or capture TNFR1 in the sample. After washing, any unbound protein in the sample is removed, and a protein that binds to TNFR1 at a distinct epitope is brought into direct contact with the captured TNFR1. This detector protein is generally labeled as described above. Alternatively, a second labeled protein that binds to the detector protein can also be used. After washing to remove any unbound protein, in the case of ELISA described above, a detectable reporter molecule is detected by addition of a substrate. Next, the level of TNFR1 in the sample is determined using a standard curve generated using a known amount of the marker or by comparison with a control sample.

[0169] Selection of cells for culture expansion In one embodiment, the present disclosure encompasses selecting cells for culture expansion based on certain criteria. In an example, the cells include an intermediate MLPSC population. In one example, the MLPSC population is evaluated with respect to the required criteria, and if the criteria are met, the population is selected for culture expansion.

[0170] In one example, the MLPSC population is selected for further expansion when it inhibits IL2-RA by at least 55% under culture conditions. In one example, the MLPSC population is an intermediate population. In one example, the intermediate MLPSC population is selected for further expansion when it inhibits IL2-RA by at least 55% under culture conditions. In another example, the intermediate MLPSC population is selected for further expansion when it inhibits IL2-RA by at least 56% under culture conditions. In another example, the intermediate MLPSC population is selected for further expansion when it inhibits IL2-RA by at least 60% under culture conditions. In another example, the intermediate MLPSC population is selected for further expansion when it inhibits IL2-RA by at least 70% under culture conditions. In another example, the intermediate MLPSC population is selected for further expansion when it inhibits IL2-RA by 55-70% under culture conditions. In another example, the intermediate MLPSC population is selected for further expansion when it inhibits IL2-RA by 60-70% under culture conditions. In another example, the intermediate MLPSC population selected for further expansion also expresses at least 80 pg / ml of TNF-R1 under culture conditions. In another example, the intermediate MLPSC population selected for further expansion also expresses at least 105 pg / ml of TNF-R1 under culture conditions. In another example, the intermediate MLPSC population selected for further expansion also expresses at least 120 pg / ml of TNF-R1 under culture conditions. In another example, the intermediate MLPSC population selected for further expansion also expresses 80 pg / ml of TNF-R1 to 120 pg / ml of TNFR1 under culture conditions.

[0171] The selection process is not particularly limited as long as it can select a cell population characterized by relevant criteria such as the % of IL2-RA inhibition. In one example, a series of intermediate MLPSC populations are evaluated for IL2-RA inhibition under culture conditions, and the population that inhibits IL2-RA by at least 55% under culture conditions is selected for further expansion. In one example, a series of intermediate MLPSC populations are evaluated for IL2-RA inhibition under culture conditions, and the population that inhibits IL2-RA by at least 60% under culture conditions is selected for further expansion. In one example, those intermediate MLPSC populations that meet the relevant threshold level of IL2-RA inhibition are pooled prior to further expansion.

[0172] It should be understood that the selection process does not require immediate culture expansion. Rather, the "selected" population can be cryopreserved and cultured and expanded at a later stage. In one example, a portion of the intermediate cell population is cultured and expanded, and the rest of the population is cryopreserved for later stage culture and expansion.

[0173] In one example, the selected cell population is immediately cultured and expanded. In another example, the selected cell population is cryopreserved to enable later stage culture and expansion.

[0174] In one example, the selected cell population is cultured and expanded to provide a pharmaceutical composition. In one example, the pharmaceutical composition is characterized by certain criteria such as the level of IL2-RA inhibition. In one example, the culture expansion of the selected MLPSC population provides an MLPSC population characterized by more than 60% IL2-RA inhibition. In another example, the culture expansion of the selected MLPSC population provides an MLPSC population characterized by more than 70% IL2-RA inhibition under culture conditions. In another example, the culture expansion of the selected MLPSC population provides an MLPSC population characterized by more than 75% IL2-RA inhibition under culture conditions. In another example, the culture expansion of the selected MLPSC population provides an MLPSC population characterized by 60% - 80% IL2-RA inhibition under culture conditions.

[0175] Formulations and Methods for Their Manufacture The inventors further defined a method of manufacturing a formulation by selecting a population of MLPSCs for culture expansion based on a predetermined level of IL2-RA inhibition under culture conditions. Thus, in one example, the present disclosure provides a method of manufacturing a formulation comprising a population of MLPSCs, the method comprising obtaining a determination as to whether a test population of MLPSCs has a predetermined level of IL2-RA inhibition under culture conditions, and culturing and expanding at least a portion of the test population of MLPSCs to a formulation if the test population of MLPSCs has at least the predetermined level of IL2-RA inhibition under culture conditions, thereby manufacturing the formulation, or discarding at least a portion of the test population of MLPSCs if the population of MLPSCs has less than the predetermined level of IL2-RA inhibition in culture.

[0176] In one example, the method further comprises obtaining a determination as to whether a test population of MLPSCs has a predetermined level of TNF-R1 under culture conditions, and culturing and expanding at least a portion of the test population of MLPSCs to a formulation if the test population of MLPSCs has at least the predetermined level of IL2-RA inhibition and the predetermined level of TNF-R1 under culture conditions, thereby manufacturing the formulation, or discarding at least a portion of the test population of MLPSCs if the population of MLPSCs has less than the predetermined level of IL2-RA inhibition and less than the predetermined level of TNF-R1 under culture conditions.

[0177] In one example, the test population is obtained from a population of MLPSCs during 3D culture. For example, the MLPSCs can be present during bioreactor culture. In one example, the test population is obtained from a cryopreserved population of MLPSCs. In one example, the test population is representative of a larger population of MLPSCs, such as multiple cryopreserved populations of MLPSCs.

[0178] As used herein, the term "predetermined level" refers to the level of a marker indicating the therapeutic efficacy of a culture growth preparation. In one example, the predetermined level is the level of IL-2RA inhibition indicating the therapeutic efficacy of a culture growth preparation. In one example, the predetermined level of IL-2RA inhibition is at least 55% under culture conditions. In one example, the predetermined level of IL-2RA inhibition is at least 56% under culture conditions. In one example, the predetermined level of IL-2RA inhibition is at least 60% under culture conditions. In another example, the predetermined level of IL-2RA inhibition is at least 70% under culture conditions. In one example, the predetermined level of IL-2RA inhibition is 55% - 80% under culture conditions. In another example, the predetermined level of IL-2RA inhibition is 55% - 75% under culture conditions. In another example, the predetermined level of IL-2RA inhibition is 60% - 80% under culture conditions. In another example, the predetermined level of IL-2RA inhibition is 60% - 75% under culture conditions.

[0179] In one example, the predetermined level is the level of TNF-R1 under culture conditions. In one example, the predetermined level of TNF-R1 is at least 80 pg / ml under culture conditions. In another example, the predetermined level of TNF-R1 is at least 100 pg / ml under culture conditions. In another example, the predetermined level of TNF-R1 is at least 105 pg / ml under culture conditions.

[0180] In one example, the predetermined level is a clinically proven effective predetermined level. In one example, the level is clinically proven to be effective in the treatment of inflammatory diseases such as GvHD. In another example, the predetermined level is predetermined by a regulatory authority such as the US Food and Drug Administration (FDA). In one example, the predetermined level corresponds to an increase in the survival rate of GvHD patients.

[0181] In one example, culturing and expanding at least a portion of the test population of MLPSCs to a formulation provides an MLPSC population characterized by greater than 70% IL2-RA inhibition under culture conditions. In another example, culturing and expanding at least a portion of the test population of MLPSCs to a formulation provides an MLPSC population characterized by greater than 75% IL2-RA inhibition under culture conditions.

[0182] In one example, the present disclosure provides a method of manufacturing an MLPSC formulation, the method comprising: a first step of providing (e.g., culturing and expanding (e.g., in small or large scale cell culture) or manufacturing) or obtaining (e.g., receiving and / or purchasing from a third party (including a third party with a contractual relationship or an independent third party)) a test MLPSC population (e.g., a sample of a test MLPSC population); a second step of obtaining (e.g., detecting, measuring, receiving, or acquiring) a value of at least one of the MLPSC parameters listed in Table A for the test MLPSC population; and a third step of culturing and expanding at least a portion of the test MLPSC population to an MLPSC formulation (e.g., in a form or package for administration as described hereinbelow, optionally cryopreserved) when at least one value for the test MLPSC population meets the reference criteria shown in Table A of the parameter, thereby manufacturing an MSC formulation. In one example, the value(s) includes parameter number 1. In another example, the value(s) includes parameter number 2. In another example, the value(s) includes parameter numbers 1 and 2.

[0183] In one example, such a method includes a second step of obtaining a value of the level of DP IL2-RA inhibition under culture conditions, and the third step of such a method includes releasing at least a portion of the DP (i.e., the release criterion) when the value of the level of IL2-RA inhibition under culture conditions exceeds 55%. In another example, the third step of such a method includes releasing at least a portion of the DP when the value of the level of IL2-RA inhibition under culture conditions exceeds 56%. In another example, the third step of such a method includes releasing at least a portion of the DP when the value of the level of IL2-RA inhibition under culture conditions exceeds 60%. In another example, the third step of such a method includes releasing at least a portion of the DP when the value of the level of IL2-RA inhibition under culture conditions exceeds 65%. In another example, the third step of such a method includes releasing at least a portion of the DP when the value of the level of IL2-RA inhibition under culture conditions exceeds 70%. In another example, the third step of such a method includes releasing at least a portion of the DP when the value of the level of IL2-RA inhibition under culture conditions exceeds 75%. [Table 4]

[0184] The compositions of the present disclosure In one example, the present disclosure includes an intermediate MLPSC population having a memory for a subsequent final product derived from the intermediate. Such intermediates are distinguished from freshly isolated cells in that they are cultured and expanded from their populations to provide the intermediate. They are also distinguished from such final compositions for administration in that they need to be further cultured and expanded to provide the final composition. Thus, in one example, the intermediate MLPSC population of the present disclosure has been passaged at least twice or at least three times. In one example, the intermediate cell population is passaged 1 to 4 times. In one example, the intermediate cell population is cryopreserved.

[0185] In one example, the intermediate cell population is characterized by a threshold level of IL2-RA inhibition. In another example, the intermediate cell population is characterized by a threshold level of TNF-R1 expression. Thus, in one example, the present disclosure relates to a cryopreserved cell intermediate comprising a population of cultured and expanded mesenchymal progenitor cells or stem cells (MLPSCs), wherein the population of MLPSCs is cultured and expanded for 2 to 5 passages, and the cultured and expanded MLPSCs: a) inhibit IL2-RA by at least 55% under culture conditions, and / or b) express at least 80 pg / ml of TNF-R1 under culture conditions. In one example, the MLPSCs in the cell intermediate: a) inhibit IL2-RA by at least 60% under culture conditions, and / or b) express at least 80 pg / ml of TNF-R1 under culture conditions. In one example, the MLPSCs in the cell intermediate: a) inhibit IL2-RA by at least 70% under culture conditions, and / or b) express at least 100 pg / ml of TNF-R1 under culture conditions. In one example, the MLPSCs express at least 105 pg / ml of TNF-R1 under culture conditions.

[0186] In one example of the present disclosure, mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom are provided and / or administered in the form of a composition. Such a composition is cultured and expanded from the intermediate MLPSC population disclosed herein. In one example, such a composition comprises a pharmaceutically acceptable carrier and / or excipient. Thus, in one example, the composition of the present disclosure may comprise mesenchymal progenitor cells or stem cells cultured and expanded from the intermediate population disclosed herein. In one example, such a composition may be a pharmaceutical composition.

[0187] The terms "carrier" and "excipient" refer to compositions of substances conventionally used in the art to facilitate the storage, administration, and / or biological activity of the active compound (see, e.g., Remington’s Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980)). The carrier can also reduce the undesirable side effects of the active compound. A suitable carrier is, for example, stable and, for example, cannot react with other components in the carrier. In one example, the carrier does not cause significant local or systemic adverse effects to the recipient at the dosage and concentration used for treatment.

[0188] Carriers suitable for the present disclosure include those conventionally used, for example, water, physiological saline, aqueous dextrose, lactose, Ringer's solution, buffer solutions, hyaluronic acid, and glycols are exemplary liquid carriers for solutions, particularly (in the case of isotonicity). Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, corn, wheat flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

[0189] In another example, the carrier is, for example, a culture medium composition in which cells are grown or suspended. For example, such a culture medium composition does not induce any adverse effects on the subject to which it is administered.

[0190] Exemplary carriers and excipients do not adversely affect the viability of cells and / or the ability of cells to reduce, prevent, or delay metabolic syndrome and / or obesity.

[0191] In one example, the carrier or excipient provides buffering activity that maintains cells and / or soluble factors at an appropriate pH, thereby exerting biological activity. For example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it minimally interacts with cells and factors and allows for rapid release of cells and factors. In such cases, the compositions of the present disclosure can be manufactured as a liquid for direct application, e.g., by injection, into the bloodstream or into or around or adjacent to a tissue.

[0192] In one example, the composition comprises an effective amount, or a therapeutically or prophylactically effective amount, of mesenchymal progenitor cells or stem cells and / or their progeny and / or soluble factors derived therefrom. For example, the composition comprises from about 1×10 5 to about 1×10 9 stem cells, or from about 1.25×10 3 to about 1.25×10 7 stem cells / kg (for an 80 kg subject). In one example, the composition comprises 2×10 6 cells / kg. The exact amount of cells to be administered depends on various factors including the age, weight and gender of the subject, and the degree and severity of the disorder being treated.

[0193] In one example, 50×10 6 to 200×10 7 cells are administered. In other examples, 60×10 6 to 200×10 6 cells or 75×10 6 to 150×10 6 cells are administered. In one example, 75×10 6 cells are administered. In another example, 150×10 6 cells are administered.

[0194] In one example, the composition comprises viable cells at greater than 5.00×10 6 cells / mL. In another example, the composition comprises viable cells at greater than 5.50×10 6 cells / mL. In another example, the composition comprises viable cells at greater than 6.00×10 6contains viable cells in excess of 100,000 cells / mL. In another example, the composition contains viable cells in excess of 6.50×10 6 cells / mL. In another example, the composition contains viable cells in excess of 6.68×10 6 cells / mL.

[0195] In one example, the methods of the disclosure include administering a total dose of 600 million cells. For example, a subject being treated according to the disclosure can receive multiple doses of the composition described above so long as the total dose of cells does not exceed 600 million cells. For example, a subject can receive 3 doses of 200 million cells. In one example, the total dose of cells is 500 million cells. In one example, the total dose of cells is 400 million cells. For example, a subject can receive 4 doses of 100 million cells. In one example, a subject receives 1 dose of 100 million cells at baseline, followed by 3 doses of 100 million cells once per month over 3 months. In one example, the dose is 2×10 6 cells / kg. In one example, the dose is 2×10 6 cells / kg and the subject receives 2 or 3 doses. In one example, the dose is 2×10 6 cells / kg and the subject receives more than 3 doses.

[0196] In one example, the mesenchymal progenitor or stem cells constitute at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of the cell population of the composition.

[0197] The compositions of the present disclosure can be cryopreserved. Cryopreservation of mesenchymal progenitor cells or stem cells can be carried out using slow cooling methods or "rapid" freezing protocols known in the art. Preferably, the cryopreservation method maintains a similar phenotype, cell surface markers, and growth rate of the cryopreserved cells compared to non-cryopreserved cells.

[0198] The cryopreserved composition may contain a cryopreservation solution. The pH of the cryopreservation solution is typically 6.5 - 8, preferably 7.4.

[0199] The cryopreservation solution may contain a sterile non-pyrogenic isotonic solution such as PlasmaLyte A™. 100 mL of PlasmaLyte A™ contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H 11 NaO7); 368 mg of sodium acetate trihydrate, USP (C2H3NaO2·3H2O); 37 mg of potassium chloride, USP (KCl); and 30 mg of magnesium chloride, USP (MgCl2·6H2O). It does not contain an antibacterial agent. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 - 8.0).

[0200] The cryopreservation solution may contain Profreeze TM and may additionally or alternatively contain a culture medium such as αMEM.

[0201] To facilitate freezing, a cryoprotectant such as dimethyl sulfoxide (DMSO) is usually added to the cryopreservation solution. Ideally, the cryoprotectant should be non-toxic to cells and patients, non-antigenic, chemically inert, provide a high survival rate after thawing, and enable transplantation without washing. However, DMSO, the most commonly used cryoprotectant, 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.

[0202] The cryopreservation solution may contain one or more of DMSO, hydroxyethyl starch, human serum components, and other protein extenders. In one example, the cryopreservation solution contains about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further contain one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0203] In one embodiment, the cells are suspended in 42.5% Profreeze™ / 50% αMEM / 7.5% DMSO and cooled in a controlled rate freezer.

[0204] The cryopreserved composition may be thawed and administered directly to a subject or added to another solution containing, for example, HA. Alternatively, the cryopreserved composition may be thawed and mesenchymal progenitor cells or stem cells may be resuspended in an alternative carrier prior to administration.

[0205] In one example, the cell composition of the present disclosure may contain Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA). For example, the composition of the present disclosure may contain a Plasma-Lyte A (70%), DMSO (10%), HSA (25%) solution, and the HSA solution contains 5% HSA and 15% buffer.

[0206] In one example, the composition described herein may be administered in a single dose.

[0207] In some examples, the composition described herein may be administered in multiple doses. For example, at least 2, at least 3, at least 4 doses. In other examples, the composition described herein may be administered over at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 doses.

[0208] In one example, the mesenchymal progenitor cells or stem cells may be administered systemically. In another example, the composition of the present disclosure is administered intravenously.

[0209] In one example, the mesenchymal progenitor cells or stem cells are administered once a week. For example, the mesenchymal progenitor cells or stem cells can be administered once a week every two weeks. In another example, the mesenchymal progenitor cells or stem cells are administered twice a week. In one example, the mesenchymal progenitor cells or stem cells are administered once a month. In one example, two doses of the mesenchymal progenitor cells or stem cells are administered once a week for two weeks. In another example, two doses of the mesenchymal progenitor cells or stem cells are administered once a week every two weeks. In another example, four doses of the mesenchymal progenitor cells or stem cells are administered over two weeks, and then subsequent doses are administered monthly. In one example, two doses of the mesenchymal progenitor cells or stem cells are administered once a week every two weeks, and then subsequent doses are administered monthly. In one example, four doses are administered monthly.

[0210] In one example, the compositions of the present disclosure comprise a “clinically proven effective” amount of MLPSC. In one example, the compositions of the present disclosure comprise a “clinically proven effective” amount of MSC.

[0211] It will be understood by those skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure. Accordingly, the present embodiments are to be considered in all respects as illustrative and not restrictive.

[0212] The following specific examples are to be construed as merely illustrative and in no way limiting of the remainder of the present disclosure. Those skilled in the art will appreciate that the present invention can be utilized to its fullest extent based on the above description without further elaboration.

Examples

[0213] Intermediate Stem Cell Populations and Their Ex Vivo Culture Expansion for the Treatment of Inflammatory Diseases Donor Cell Bank Generation and Analysis Carbon dioxide (CO2) in the culture medium of cultured and expanded mesenchymal stem cells (ce-MSCs) is necessary to maintain a buffer between carbonic acid and bicarbonate and ensure that the culture medium is maintained at the physiological pH (7.2 - 7.4) required for optimal cell growth.

[0214] A donor cell bank (DCB) containing ce-MSCs was previously generated by culturing and expanding the MSC population in a 10-layer cell factory (CF10) using active priming with 10 ± 2% CO2. However, if the CO2 concentration is too high, the intracellular pH becomes extremely low, negatively affecting the growth rate and changing the metabolism, productivity, functionality, and glycosylation profile of mammalian cells. Therefore, the method for manufacturing DCB was changed to require seeding MSCs in CF10 and culturing the cells using passive 5 ± 2% CO2 priming. In other words, the passive 5 ± 2% CO2 priming (referred to herein as the "DCB2" method) represents a deviation from the previous manufacturing method involving active priming with 10 ± 2% CO2 (referred to herein as the "DCB1" method).

[0215] The % inhibition of IL2-RA was measured after co-culture of T cells and mesenchymal stem cells, and the inhibition of IL2-RA expression is related to the inhibitory effect on T cell activation, and an effective measure of the therapeutic efficacy of inflammatory diseases such as GvHD (e.g., a 100-day life extension effect) can be obtained. Surprisingly, the inventors found that the potency increased when measuring the % IL2-RA inhibition in the cell population generated using the DCB2 method (Figure 1). In the cell population generated using the DCB2 method, the level of TNFR-1 expression also increased (Figure 2).

[0216] Generation and analysis of donor products Even more surprisingly, when the lots of DCB1 and DCB2 were further expanded to the clinical formulation (DP), a significant increase in the 100-day survival rate (74%) was observed in the patients administered with the DP derived from DCB2 as compared to the survival rate (56%) of the patients administered with the DP derived from DCB1. The increase in the 100-day survival rate was even more prominent in the patients with severe grade D disease, being 72% in the patients administered with the DP derived from DP2 as compared to 44% in the patients administered with the DP derived from DP1. The results are shown in Table 1; the life-prolonging effect is also graphically shown in Figure 3.

Table 5

[0217] Next, the inventors identified that the life-prolonging effect can be predicted and thus the DCB population can be selected for expansion based on the IL2-RA inhibition%. Interestingly, when the lots of DCB1 and DCB2 were further expanded to the clinical DP, the average% of IL2-RA expression was 76.5% in the DP derived from DCB2 as compared to 63.7% in the DP derived from DCB1. This is a significant finding as the patients administered with the DP grown from the DCB1 intermediate, characterized by an average% of IL2-RA of 63.7%, had a 100-day survival rate of 56% and decreased to 44% in the patients with severe grade D disease. In contrast, the patients administered with the DP grown from the DCB2 intermediate, characterized by an average% of IL2-RA of 76.5%, had a 100-day survival rate of 74% and decreased only to 72% in the patients with severe grade D disease (Figure 4; p = 0.019). Indeed, the life-prolonging effect was most prominent in the patients with severe grade D disease, recording a 100-day survival rate of 74% in the patients administered with the DP grown from the DCB2 intermediate and decreasing to 46% in the patients administered with the DP grown from the DCB1 intermediate (Figure 5; p = 0.012).

[0218] Since the DPs from both DCB1 and DCB2 were prepared in the same way, this controls any possibility of downstream processes affecting the final DP characteristics. Thus, the inventors' discovery supports the importance of characterizing cell intermediates prior to expansion into DPs. In particular, in the context of GvHD treatment, failure to do so can significantly impact overall patient survival. The inventors' discovery also supports a marked improvement in the method of selecting cell intermediates for large-scale culture expansion into formulations, reducing the risk of consuming resources to expand cell populations that do not meet the required potency criteria.

[0219] Summary The donor cell bank manufacturing process provides "memory" to subsequent final products derived from that DCB. Changes in DCB manufacturing are associated with higher % inhibition of IL2-RA in both DCB and DP. An increase in % inhibition of IL2-RA is associated with a marked improvement in survival, particularly in grade D patients. These findings support a method of selecting and analyzing DCBs for use in therapy, particularly in the treatment of inflammatory disorders such as GvHD.

[0220] Inhibition of IL-2RA on peripheral blood mononuclear cells (PBMCs) by mesenchymal or progenitor stem cells (MLPSCs) Preparation of MLPSCs Thaw cryopreserved human MLPSCs and resuspend them in IL-2Ra MLPSC culture medium at a concentration of 4 x 10 5 viable MLPSCs / mL. Perform viability assessment using trypan blue. Next, seed the MLPSCs in a 24-well plate. Add 500 μL of 4 x 10 5 viable MLPSCs / mL to each well. Incubate the cells at 37 °C ± 2 °C, 5% ± 2% CO2 for within 24 ± 1 hours. Prior to co-culture with PMBCs, confirm cell attachment to the plate and ensure that the cell morphology is long and flat, "like fibroblasts".

[0221] Preparation of PMBCs Thaw the PMBCs and resuspend them in co-culture medium (50% IL-2RA MLPSC culture medium and 50% DMEM) at a concentration of 2 x 10 6 viable cells / mL. Viability assessment is performed using trypan blue.

[0222] Co-culture of MLPSC and PBMC Stimulated PMBCs are prepared by adding CD3 and CD28 antibodies (final concentration 4 μg / ml) to a portion of 2 x 10 6 viable PMBCs. A portion of viable PMBCs not stimulated with CD3 and CD28 antibodies is kept as a negative control. Next, 500 μL of the stimulated PMBCs are added to test wells containing Ce-MLPSC (either the intermediate population or the formulation).

[0223] Stimulated PMBCs alone are also used as a positive control, and unstimulated PMBCs are used as a negative control. 500 μL of each control sample and 500 μL of co-culture medium are added to the control wells of a culture plate.

[0224] Next, the MLPSC and PMBC are cultured in an incubator at 37 °C ± 2 °C, 5% ± 2% CO2 for 72 - 74 hours. After incubation, the PMBC samples are gently collected, centrifuged, and resuspended in lysis buffer to prepare cell lysates. The lysates are then incubated on wet ice for 15 minutes.

[0225] For example, Ce-MSC (either the intermediate population or the formulation) is co-cultured with unstimulated or stimulated (CD3; CD28 antibody) peripheral blood mononuclear cells (PBMCs) as shown below. These conditions typically had a stem cell:PMBC ratio of approximately 1:5.

Table 6

[0226] The plates were incubated at 37 °C for 60 - 84 hours. Subsequently, the cells were collected, lysed, and the IL2-RA levels were evaluated using a commercially available ELISA kit according to the manufacturer's (R&D systems) instructions.

[0227] IL-2RA ELISA Prepare IL-2RA standards from 2500 - 78 pg / ml and add them to the ELISA plates together with the control and co-culture lysate samples. Next, add an anti-lL-2RA antibody that forms a complex with HRP to all wells. Incubate the plates at ambient temperature for approximately 3 hours with gentle shaking at 200 rpm on an orbital shaker. After incubation, add the substrate to each well. Incubate the plates again in the dark at ambient temperature for 20 minutes, then add the stop solution to stop the reaction. Read the absorbance of the wells at 570 nm wavelength corrected and 450 nm within 30 minutes.

[0228] To calculate the amount of IL-2RA, calculate the A450nm - 570nm value for each well using standard analysis software (e.g., BioTek). Subtract the average absorbance of the negative control at 0 pg / mL from the A450nm - 570nm value. Interpolate the concentration of IL-2RA in the test sample from the standard curve of the IL-R2A standard. The % inhibition of IL-2RA on PBMC by MLPSC is calculated using the following formula: % Inhibition = [[1 - (co-culture MLPSC and PBMC / stimulated PBMC)] × 100].

[0229] Those skilled in the art will understand that many variations and / or modifications may be added to the present invention without departing from the spirit and scope of the present invention as broadly described. Accordingly, this embodiment should be considered illustrative in all respects and not restrictive.

[0230] This application claims the priority of AU2022901890 filed on July 5, 2022, and this disclosure is incorporated herein by reference.

[0231] All of the publications discussed above are hereby incorporated by reference in their entirety.

[0232] Any discussion of documents, acts, materials, devices, articles, etc. included in this specification is for the sole purpose of providing context for the present invention. It is not to be regarded as an admission that any part or all of these matters form part of the prior art base or 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

**Claim 1** A method of selecting a cell population for culturing and proliferation, comprising: (i) obtaining a population of mesenchymal progenitor lineage cells or stem cells (MLPSCs); (ii) culturing said MLPSC population in a culture medium; (iii) determining the level of IL2-RA inhibition under culture conditions; and (iv) selecting, for further proliferation, an MLPSC population that inhibits IL2-RA by at least 55% under culture conditions. **Claim 2** The method according to claim 1, further comprising selecting, for further proliferation, an MLPSC population that inhibits IL2-RA by at least 60% under culture conditions. **Claim 3** The method according to claim 1 or 2, wherein the MLPSC population selected for further proliferation also expresses at least 80 pg / ml of TNF-R1 under culture conditions. **Claim 4** The method according to any one of claims 1 to 3, further comprising culturing and proliferating the selected MLPSC population to provide a pharmaceutical composition. **Claim 5** The method according to any one of claims 1 to 4, wherein the culturing and proliferation of the selected MLPSC population provides an MLPSC population characterized by more than 60% and more than 70% inhibition of IL2-RA under culture conditions. **Claim 6** A method for determining the therapeutic effectiveness of an intermediate population of mesenchymal progenitor lineage cells or stem cells (MLPSCs), comprising: (i) obtaining a population of MLPSCs; (ii) culturing said cells in a culture medium; and (iii) determining the level of IL2-RA inhibition under culture conditions, wherein at least 55% inhibition of IL2-RA under culture conditions indicates therapeutic effectiveness. **Claim 7** The method according to claim 6, wherein more than 60% and more than 70% inhibition of IL2-RA under culture conditions indicates therapeutic effectiveness. **Claim 8** The method according to claim 6 or 7, further comprising determining the level of TNF-R1 expression under culture conditions, wherein expression of at least 80 pg / ml of TNF-R1 under culture conditions indicates therapeutic effectiveness. **Claim 9** The method according to any one of claims 6 to 8, wherein, depending on the determination of therapeutic effectiveness, the intermediate population of MLPSCs is cultured and proliferated to provide a pharmaceutical composition. **Claim 10** The method according to claim 9, wherein the pharmaceutical composition comprises an MLPSC population characterized by 60% to 80% inhibition of IL2-RA under culture conditions. **Claim 11** A method for treating a subject suffering from an inflammatory disease, comprising administering to a subject in need thereof a composition comprising a cultured and expanded population of mesenchymal progenitor or stem cells (MLPSCs), wherein said MLPSCs are cultured and expanded from a population of MLPSCs that inhibit IL2-RA by at least 55% under culture conditions, said method.

12. The method according to claim 11, wherein the cultured and expanded MLPSCs comprised in the administered composition inhibit IL2-RA by at least 56% and at least 60% under culture conditions.

13. The method according to claim 11 or claim 12, wherein said MLPSCs are cultured and expanded from a population of MLPSCs that also express at least 80 pg / ml of TNF-R1 under culture conditions.

14. A cryopreserved cell intermediate comprising a population of cultured and expanded mesenchymal progenitor or stem cells (MLPSCs), wherein the population of said MLPSCs has been cultured and expanded for 2 to 5 passages, and wherein said cultured and expanded MLPSCs a) inhibit IL2-RA by at least 55% under culture conditions, and / or b) express at least 80 pg / ml of TNF-R1 under culture conditions, said cell intermediate.

15. The method according to any one of claims 11 to 13, or the cell intermediate according to claim 14, wherein said MLPSCs a) inhibit IL2-RA by at least 60% and at least 70% under culture conditions, and / or b) express at least 100 pg / ml of TNF-R1 and at least 105 pg / ml of TNF-R1 under culture conditions.

16. The method or cell intermediate according to any one of claims 1 to 15, wherein said MLPSCs are cultured and expanded in 2D culture.

17. The MLPSC is cultured and proliferated with 5% ± 2% CO ２ and is the method or cell intermediate according to any one of claims 1 to 16.

18. The MLPSC is cultured and grown in 5% + / − 2% CO ２ using passive priming, according to the method or cell intermediate of claim 17.

19. The method according to any one of claims 11 to 13, wherein said inflammatory disease is graft-versus-host disease (GvHD).

20. The method according to claim 19, wherein said GvHD is grade D.

21. The method according to claim 19, wherein said GvHD is chronic GvHD.

22. The method according to any one of claims 11 to 21, wherein treatment increases the 100-day survival rate to more than 60% and up to more than 70%.

23. The method according to any one of claims 11 to 22, wherein said subject is refractory to steroid immunosuppressants and / or biological therapies.

24. The subject is the method according to any one of claims 11 to 23, which receives at least two dosages of the composition.

25. The method or cell intermediate according to any one of claims 1 to 24, wherein the MLPSC is a mesenchymal stem cell.

26. A method for manufacturing a formulation comprising a population of mesenchymal progenitor cells or stem cells (MLPSCs), comprising obtaining a determination as to whether a test population of MLPSCs has a predetermined level of IL2-RA inhibition under culture conditions; and when the test population of MLPSCs has at least the predetermined level of IL2-RA inhibition under culture conditions, culturing and expanding at least a part of the test population of MLPSCs into a formulation, thereby manufacturing the formulation; or when the population of MLPSCs has less than the predetermined level of IL2-RA inhibition under culture conditions, discarding at least a part of the test population of MLPSCs.

27. The method according to claim 26, wherein the predetermined level of IL2-RA inhibition is at least 55% under culture conditions.

28. The method according to claim 26, wherein the predetermined level of IL2-RA inhibition is at least 60% under culture conditions.

29. The method further comprises obtaining a determination as to whether a test population of MLPSCs has a predetermined level of TNF-R1 under culture conditions; and when the test population of MLPSCs has at least the predetermined level of IL2-RA inhibition and the predetermined level of TNF-R1 under culture conditions, culturing and expanding at least a part of the test population of MLPSCs into a formulation, thereby manufacturing the formulation; or when the population of MLPSCs has less than the predetermined level of IL2-RA inhibition and less than the predetermined level of TNF-R1 under culture conditions, discarding at least a part of the test population of MLPSCs. The method according to any one of claims 26 to 28.

30. The method according to claim 29, wherein the predetermined level of TNF-R1 is at least 80 pg / ml, at least 100 pg / ml, at least 105 pg / ml.

31. Culturing and expanding at least a part of the test population of MLPSCs into a formulation provides a population of MLPSCs characterized by more than 60%, more than 70% IL2-RA inhibition under culture conditions. The method according to any one of claims 26 to 30.

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