Cryopreserved intermediates and potency assays therefor

Abstract

The present disclosure relates to improved cell intermediates and potency assays for obtaining the improved cell intermediates. Such intermediates and assays can be suitable for providing cellular compositions for the treatment of various inflammatory conditions.

Description

Technical Field

[0001] The present disclosure relates to cell intermediates, potency assays for characterizing the same, and selective methods for culture expansion. Such intermediates, assays, and methods may be useful for providing cell compositions for treating various inflammatory disorders. Background Art

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

[0003] Physicochemical parameters (e.g., characterization of size, morphology, light scattering properties, tensile strength, cell number, confluence, identification of phenotypic markers, secreted substances, genotype, gene expression profiles) are often 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., produces the desired effect). In contrast, biological characterization considers the effect of the product on biological systems modeled in vitro or in vivo in animals and ultimately in the clinic.

[0004] Potency testing must demonstrate one or more relevant biological activities of the product. Potency testing need not reflect all biological functions of the product, but should indicate one or more relevant biological functions. It is expected that for analytical methods used for potency testing, accuracy, sensitivity, specificity, and reproducibility will be established and that they will be appropriately robust.

[0005] There is a need to develop cell products for treating diseases (e.g., diseases requiring immunosuppression). It is also preferred to identify parameters that are critical to the efficacy of cell therapy products and control them (e.g., through potency testing) so that products of consistent quality can be manufactured. Summary of the invention

[0006] Prior efficacy testing focused on characterizing the final cell product for administration. Although such testing is still important, it will be understood by those skilled in the art that the final cell product is cultured and expanded from an intermediate (also referred to as a donor cell bank) stored at low temperatures. The inventor's research has unexpectedly determined the importance of testing the efficacy of such cell intermediates before they are cultured and expanded into final cell products. For example, the inventor has determined certain features of the donor cell bank, which, when met, will provide "memory" for the final product subsequently derived from the donor cell bank. In fact, after controlling the downstream culture expansion process, the inventor unexpectedly found that certain features of the donor cell bank were associated with the final cell product, which could confer improved 100-day survival rates in the context of treating inflammatory diseases GvHD. These findings represent a major advance in the art, because only intermediate cell populations with therapeutic effects can be selected for large-scale expansion in 3D culture. Without these findings, if the resulting cell product does not meet the required treatment criteria for administration, the efforts of large-scale expansion may be wasted.

[0007] Therefore, in certain embodiments, the methods of the present disclosure facilitate the selection of cell intermediates that are most suitable for culture expansion. In a first example, the present disclosure relates to a method for selecting a cell population for culture expansion, the method comprising: (i) obtaining a mesenchymal precursor lineage or stem cell (MLPSC) population; (ii) culturing an MLPSC population in a culture medium; (iii) determining the level of IL2-RA inhibition under culture conditions; and (iv) selecting an MLPSC population that inhibits IL2-RA by at least 55% under culture conditions for further expansion. In another example, the method includes selecting an MLPSC population that inhibits IL2-RA by at least 56% under culture conditions for further expansion. In another example, the method includes selecting an MLPSC population that inhibits IL2-RA by at least 60% under culture conditions for further expansion. In another example, the method includes selecting an MLPSC population that inhibits IL2-RA by at least 70% under culture conditions for further expansion. In another example, the method includes selecting an MLPSC population that inhibits IL2-RA by 55% to 75% under culture conditions for further expansion. In another example, the method includes selecting a MLPSC population that suppresses IL2-RA by 60% to 75% under culture conditions for further expansion. In another example, the MLPSC population selected for further expansion also expresses at least 80pg / ml TNF-R1 under culture conditions. In another example, the MLPSC population selected for further expansion also expresses at least 100pg / ml or at least 105pg / ml 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 involves at least 3 to 5 passages.

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

[0011] The inventors' findings also indicate that administration of cell compositions expanded from cell intermediate cultures selected according to the methods of the present disclosure unexpectedly improves the 100-day survival rate of patients with inflammatory diseases such as GvHD. Improving the 100-day survival rate in the context of GvHD is particularly evident in patients with severe disease (grade D GvHD). Therefore, in another embodiment, the present disclosure encompasses a method for determining the therapeutic efficacy of an intermediate population of a mesenchymal precursor lineage or stem cell (MLPSC), the method comprising: (i) obtaining an MLPSC population; (ii) culturing the cells in a culture medium; and (iii) determining the level of IL2-RA inhibition under culture conditions, wherein at least 55% of IL2-RA inhibition under culture conditions indicates therapeutic efficacy. In one example, IL2-RA inhibition greater than 56% under culture conditions indicates therapeutic efficacy. In one example, IL2-RA inhibition greater than 60% under culture conditions indicates therapeutic efficacy. In one example, IL2-RA inhibition greater than 70% under culture conditions indicates therapeutic efficacy. In another example, IL2-RA inhibition greater than 75% under culture conditions indicates therapeutic efficacy. In another example, IL2-RA inhibition of 55% to 75% under culture conditions indicates therapeutic efficacy. In another example, IL2-RA inhibition greater than 75% under culture conditions indicates therapeutic efficacy. In another example, IL2-RA inhibition of 60% to 75% under culture conditions indicates therapeutic efficacy.

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

[0013] The inventor's discovery also supports a new treatment for inflammatory diseases. Therefore, in one embodiment, the present disclosure encompasses a method for treating a subject with an inflammatory disease, the method comprising administering to a subject in need a composition comprising a culture-expanded mesenchymal lineage precursor or stem cell (MLPSC) population, wherein the MLPSC is culture-expanded from an MLPSC population that suppresses IL2-RA by at least 55% under culture conditions. In one example, the culture-expanded MLPSC comprising the administered composition suppresses IL2-RA by at least 56% under culture conditions. In one example, the culture-expanded MLPSC comprising the administered composition suppresses IL2-RA by at least 60% under culture conditions. In one example, the culture-expanded MLPSC comprising the administered composition suppresses IL2-RA by at least 70% under culture conditions. In another example, the culture-expanded MLPSC comprising the administered composition suppresses IL2-RA by at least 75% under culture conditions. In another example, the culture-expanded MLPSC comprising the administered composition suppresses IL2-RA by 55% to 75% under culture conditions. In another example, the culture-expanded MLPSC containing the administered composition suppresses IL2-RA 55% to 75% under culture conditions. In another example, the culture-expanded MLPSC containing the administered composition suppresses IL2-RA 60% to 75% under culture conditions. In another example, the MLPSC is cultured and expanded from a MLPSC population that also expresses at least 80pg / ml TNF-R1 under culture conditions. In another example, the MLPSC is cultured and expanded from a MLPSC population that also expresses at least 105pg / ml TNF-R1 under culture conditions.

[0014] In another embodiment, the present disclosure also relates to novel cryopreserved cell intermediates. Such intermediates are usually passaged only 2 to 5 times after isolation and then cryopreserved. Therefore, in one example, the present disclosure relates to cryopreserved cell intermediates, which include a culture-expanded mesenchymal lineage precursor or stem cell (MLPSC) population, wherein the MLPSC population is culture-expanded for 2 to 5 passages, and the culture-expanded MLPSC: a) inhibits IL2-RA by at least 55% under culture conditions; and / or b) expresses at least 80pg / ml 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 100pg / ml 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 80pg / ml 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 TNF-R1 or at least 105 pg / ml 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 TNF-R1 or at least 105 pg / ml TNF-R1 under culture conditions. In another example, the cell intermediate: a) inhibits IL2-RA by 55% to 75% under culture conditions; and / or b) expresses 80 pg / ml to 105 pg / ml TNF-R1 under culture conditions.

[0015] In the examples cited above, IL2-RA inhibition was measured by co-culturing MLPCS with activated PBMCs.

[0016] In one example, the cell intermediates of the present disclosure are cultured and expanded in 2D culture. For example, 2D culture may include cell culture performed in a multilayer cell factory. In one example, the cell intermediates of the present disclosure are cultured and expanded in about 5% CO2, for example, 5% + / - 2% CO2. In one example, the cell culture is performed by passive CO2 priming. For example, the cell intermediates of the present disclosure can be cultured and expanded in about 5% CO2, for example, 5% + / - 2% CO2, by passive priming. For the avoidance of doubt, cell culture according to such examples 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, immunosuppressants and / or biologic therapy. In one example, the subject is refractory to a single biologic. In one example, the subject is refractory to multiple biologics.

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

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

[0020] In another example, the present disclosure relates to a method for manufacturing a drug product, the drug product comprising a mesenchymal lineage precursor or stem cell (MLPSC) population. In one example, the method includes: obtaining a determination of whether a test population of MLPSC has a predetermined level of IL2-RA inhibition under culture conditions, and if the test population of MLPSC has at least a predetermined level of IL2-RA inhibition under culture conditions, at least a portion of the test population of MLPSC is cultured and expanded as a drug product, thereby manufacturing a drug product; or if the MLPSC population has less than a predetermined level of IL2-RA inhibition under culture conditions, at least a portion of the test population of MLPSC is discarded. In one example, the predetermined level of IL2-RA inhibition under culture conditions is at least 55%. In one example, the predetermined level of IL2-RA inhibition under culture conditions is at least 60%. In one example, the predetermined level of IL2-RA inhibition under culture conditions is at least 70%. In another example, the method further includes obtaining a determination of whether the test population of MLPSC has a predetermined level of TNF-R1 under culture conditions, and if the test population of MLPSC has at least a predetermined level of IL2-RA inhibition under culture conditions and a predetermined level of TNF-R1 under culture conditions, at least a portion of the test population of MLPSC is cultured and expanded as a drug product, thereby manufacturing a drug product; or if the MLPSC population has less than a predetermined level of IL2-RA inhibition under culture conditions and less than a predetermined level of TNF-R1 under culture conditions, at least a portion of the test population of MLPSC is discarded. In one example, the predetermined level of TNF-R1 is at least 80pg / ml. In another example, the predetermined level of TNF-R1 is at least 100pg / ml. In another example, the predetermined level of TNF-R1 is at least 105pg / ml.

[0021] In one example, at least a portion of the test population of MLPSCs is expanded to provide a drug product with a MLPSC population characterized by greater than 70% inhibition of IL2-RA under culture conditions. In another example, at least a portion of the test population of MLPSCs is expanded to provide a drug product with a MLPSC population characterized by greater than 75% inhibition of IL2-RA under culture conditions.

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

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

[0024] In one example, the methods of the present disclosure encompass administration of 1×10 7 Up to 2×108 cells. For example, 1×10 7 Up to 2×10 8 Multiple doses of about 1×10 cells can be administered at regular intervals, for example, on day 0, day 30, day 60, and day 90. In one example, the methods of the present disclosure encompass administration of about 1×10 8 cells / dose.

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

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

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

[0028] Figure 1 : Changes in donor cell bank (DCB) preparation improved DCB potency (measured as % inhibition of IL2-RA).

[0029] Figure 2 : Changes in donor cell bank (DCB) preparation increased TNF-R1 expression.

[0030] Figure 3 : The survival rate of aGVHD patients who received stem cell products prepared from DCB2 was significantly improved compared with that of aGVHD patients who received stem cell products prepared from DCB1.

[0031] Figure 4 : Controlling downstream culture expansion of DCB1 and DCB2, the survival rate of aGVHD patients receiving stem cell products prepared by DCB2 was still significantly improved compared with aGVHD patients receiving stem cell products prepared by DCB1.

[0032] Figure 5 : Controlling downstream culture expansion of DCB1 and DCB2 further improved the beneficial effect on survival of patients with grade D aGVHD who received stem cell products prepared from DCB2 compared to those who received stem cell products prepared from DCB1. DETAILED DESCRIPTION

[0033] Throughout this specification, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter shall be considered to cover both one and more of those steps, compositions of matter, groups of steps, or groups of compositions of matter (i.e., one or more).

[0034] It will be appreciated by those skilled in the art that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the disclosure includes all such variations and modifications. The disclosure also includes all steps, features, compositions and compounds mentioned or indicated in this specification, either individually or collectively, and any and all combinations or any two or more of the steps or features.

[0035] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for illustrative purposes only. As described herein, functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0036] Unless expressly stated otherwise, any example disclosed herein should be considered applicable to any other example mutatis mutandis.

[0037] Unless specifically defined otherwise, all technical and scientific terms used herein should be considered to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, stem cell therapy, immunology, immunohistochemistry, protein chemistry, biochemistry).

[0038] Unless otherwise indicated, the surgical techniques used in this disclosure are standard procedures well known to those skilled in the art.

[0039] Methods for obtaining and enriching populations of mesenchymal lineage stem cells or precursor cells are known in the art. For example, enriched populations of mesenchymal lineage stem cells or precursor cells can be obtained by using flow cytometry and cell sorting procedures based on the use of cell surface markers expressed on mesenchymal lineage stem cells or precursor cells.

[0040] All documents cited or referenced herein, and all documents cited or referenced in documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference in their entirety.

[0041] Selected Definitions

[0042] The term "intermediate" used in the context of the present disclosure refers to an intermediate population of freshly isolated and amplified (e.g., about 2 passages) of MLPSC in early culture. For example, the generation of an intermediate population of MLPSC may include separation of nucleated bone marrow cells (NBMC), cell culture, and passage twice from a bone marrow aspirate (BMA) to amplify related cells. In one example, cells are then harvested and prepared for analysis according to the methods described herein. In one example, the harvested cells are cryopreserved. Therefore, the generation of intermediates disclosed herein is the first stage of the process of producing a composition for administration to a subject. Although the examples cited above refer to the separation of NBMC from BMA, it will be understood by those skilled in the art that related cells can be obtained from other suitable sources (e.g., adipose tissue) discussed herein, and the same early culture amplification is performed to provide intermediates encompassed by the present disclosure.

[0043] In certain examples disclosed herein, the intermediates of the present disclosure are cultured and expanded to provide a MLPSC population for administration. In one example, such a population may be referred to as a pharmaceutical composition or a pharmaceutical product (DP). For example, the culture expansion of an intermediate involves cell culture and more passages (e.g., more than three passages) for culture expansion. The culture expansion of an intermediate will be discussed further below.

[0044] The cryopreserved intermediate according to the present disclosure may also be referred to as a donor cell bank (DCB).

[0045] The terms "level" and "amount" are used to define the amount of a particular substance in a cell preparation. For example, a specific concentration, weight, percentage (e.g., v / v%), or ratio can be used to define the level of a particular substance. In one example, the level is expressed in terms of the amount of a particular marker expressed by the cells disclosed herein under culture conditions. In one example, expression represents cell surface expression. In another example, the level is expressed in terms of how much of a particular marker is released from the cells described herein to their culture medium under culture conditions.

[0046] In one example, the level is expressed in pg / ml. In another example, the level is expressed in pg / 10 6 If necessary, the levels in pg / ml can be converted to pg / 10 6 For example, in the context of TNF-R1, in one example, 80 pg / ml TNF-R1 is equivalent to about 9.4 pg TNF-R1 / 10 6 In one example, in the context of TNF-R1, in one example, 100 pg / ml TNF-R1 is equivalent to about 11.5 pg TNF-R1 / 10 6In one example, 105 pg / ml TNF-R1 is equivalent to about 12.5 pg TNF-R1 / 10 6 In another example, 150 pg / ml TNF-R1 is equivalent to about 17.5 pg TNF-R1 / 10 6 In another example, 200 pg / ml TNF-R1 is equivalent to about 23.5 pg TNF-R1 / 10 6 Cells, etc.

[0047] In one example, the level of a particular marker is determined under culture conditions. The term "culture conditions" is used to refer to cells grown in culture. In one example, culture conditions refer to a population of cells that are actively dividing. In one example, such cells may be in an exponential growth phase. In one example, the cells may be in a stationary phase.

[0048] In one example, the culture conditions include co-cultivation of MLPSC colonies disclosed herein with a second cell colony, such as a colony comprising peripheral blood mononuclear cells (PBMCs). In one example, co-cultivation includes culturing MLPSC colonies disclosed herein and activated PBMC colonies. For example, PBMCs can be activated using anti-CD3 and anti-CD28 antibodies before co-cultivation with MLSPC colonies disclosed herein.

[0049] In one example, "culture conditions" include co-culturing MLPSCs and T cells at a ratio of about 1 MLPSC: 2 T cells or less. For example, co-culturing MLPSCs and T cells at a ratio of 1: 3, 1: 4, 1: 5, 1: 10, 1: 20, 1: 30, 1: 40, 1: 50, 1: 60, 1: 70, 1: 80, 1: 90 or 1 MLPSC: 100 T cells or less. In this example, the level of IL2-RA inhibition is determined after cell culture for about 30 to 84 hours under culture conditions.

[0050] In one example, the level of a specific marker can be determined by obtaining a cell culture medium sample and measuring the level of the marker in the sample. In another example, the level of a specific marker can be determined by obtaining a cell sample and measuring the level of the marker in a cell lysate. It will be appreciated by those skilled in the art that secreted markers can be measured by sampling the culture medium, while markers expressed on the cell surface can be measured by evaluating a sample of a cell lysate. In one example, a sample is obtained when the cell is in an exponential growth phase. In one example, a sample is obtained after culturing for at least two or three days. In another example, the sample is collected after about 30 to 84 hours of co-culture. In one example, a sample is collected from a co-culture of MLPSC and activated PBMC. In this example, a cell sample can be lysed and the level of the marker can be determined. For example, various methods, such as an enzyme-linked immunosorbent assay (ELISA)-based method can be used to determine the level of IL2-RA. In one example, ELISA includes:

[0051] (i) adding sample diluent to each well of a microplate pre-coated with a monoclonal antibody specific for IL2-RA;

[0052] (ii) adding the co-cultured samples to the wells of a microtiter plate pre-coated with a monoclonal antibody specific for IL2-RA;

[0053] (iii) incubating the microplate for a sufficient time to allow the monoclonal antibody specific for IL2-RA to specifically bind to any IL2-RA in the sample;

[0054] (iv) washing the microplate;

[0055] (v) adding IL2-RA conjugate to the wells;

[0056] (vi) incubating the microplate for a time sufficient to allow the conjugate to bind specifically to any captured IL2-RA;

[0057] (vii) washing the microplate;

[0058] (viii) adding substrate solution to the wells;

[0059] (ix) incubating the microplate for a sufficient time to allow color development;

[0060] (x) adding stop solution to the wells;

[0061] (xi) reading the optical density on a microplate reader set to 450 nm and calibrated at 570 nm;

[0062] (xii) Determine the concentration of IL2-RA.

[0063] In another example, fluorescence activated cell sorting (FACS) is used to determine the level of IL2-RA with appropriate antibodies, such as anti-CD25. Additional antibodies may also be used if differentiation of CD25+ cell types is desired.

[0064] In one example, the IL2-RA level in the co-culture is compared with the IL2-RA level in the culture group of activated PBMC. The level of IL2-RA is then compared to provide the level of IL2-RA inhibition in the co-culture. For example, relative to a single culture (PBMC activated alone), the level of IL2-RA in the co-culture (i.e., MLPSC: activated PBMC) can be inhibited by at least 60%. Other suitable methods for determining the IL2-RA level under culture conditions will be understood by those skilled in the art.

[0065] Culturing and expanding cells from a cryopreserved intermediate means thawing the cells that have been subjected to cryopreservation and culturing them in vitro under conditions suitable for the growth of the cells.

[0066] 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 can 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 cells are cryopreserved for the first time. In another example, the level is determined after the cells are cryopreserved for the second time. For example, cells can be cultured and expanded to provide intermediates, cryopreserved, thawed, and then re-inoculated into culture so that the level of a particular marker can be determined under culture conditions and then cryopreserved for the second time.

[0067] "Isolated" or "purified" means a cell that has been separated from at least some components of its natural environment. The term includes the gross physical separation of a cell from its natural environment (e.g., removal from a donor). The term "isolated" includes altering the relationship of a cell to its directly associated neighboring cells, such as by dissociation. The term "isolated" does not refer to cells in a tissue section. When used to refer to a cell population, the term "isolated" includes a cell population produced by proliferation of an isolated cell of the disclosure.

[0068] The term "passage (passage, passaging)" or "passage culture" is used in the context of the present disclosure to refer to the known cell culture techniques for keeping cells alive and growing for a long time under culture conditions so that the number of cells is constantly increased. The degree of passage culture cell line is usually expressed as "passage number" and is usually used to refer to the number of times that cells have been passaged. In one example, a passage includes removing non-adherent cells and leaving adherent mesenchymal lineage precursors or stem cells. Such mesenchymal lineage precursor cells or stem cells can then be dissociated from matrix or flask (for example, by using proteases, such as trypsin or collagenase), medium can be added, optional washing (for example, by centrifugation), and then mesenchymal lineage precursor cells or stem cells can be re-plated or re-inoculated into one or more culture containers with a larger total surface area. Then, mesenchymal lineage precursor cells or stem cells can continue to expand in culture. In another example, the method for removing non-adherent cells includes the step of non-enzymatic treatment (for example, with EDTA). In one example, mesenchymal lineage precursor cells or stem cells are passaged at or near confluence (e.g., about 75% to about 95% confluence). In one example, mesenchymal lineage precursor cells or stem cells are seeded at a concentration of about 10%, about 15%, or about 20% cells / ml culture medium.

[0069] The terms "culture medium" or "medium" used in the context of the present disclosure include components of the environment surrounding cells in culture. It is contemplated that the culture medium facilitates and / or provides conditions suitable for cell growth. The culture medium can be solid, liquid, gaseous, or a mixture of phases and materials. The culture medium can include liquid growth medium as well as liquid medium that does not sustain cell growth. Exemplary gaseous culture media include gas phases to which cells grown on culture dishes or other solid or semisolid supports are exposed.

[0070] As used herein, the term "treating," "treat," or "treatment" includes administering a population of mesenchymal lineage stem cells or precursor cells and / or their progeny and / or soluble factors derived therefrom to reduce or eliminate at least one symptom of an inflammatory disease. In one example, treatment includes administering a population of culture-expanded mesenchymal lineage stem cells or precursor cells disclosed herein. In one example, the therapeutic response is determined relative to a baseline. In one example, the therapeutic response is determined relative to a control patient population.

[0071] In one example, the method of the present disclosure inhibits the progression of disease or disease complications in the subject. "Inhibiting" the progression of disease or disease complications in the subject means preventing or reducing the progression of disease and / or disease complications in the subject. Therefore, in one example, the method of the present disclosure inhibits the progression of disease severity. For example, such methods can inhibit the progression of GvHD severity (i.e., inhibit the progression of grading).

[0072] As used herein, the terms "prevent" or "preventing" include administering a population of mesenchymal lineage stem or precursor cells and / or progeny thereof and / or soluble factors derived therefrom to prevent or inhibit the development of at least one symptom of an inflammatory disease.

[0073] The term "subject" as used herein 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.

[0074] As used herein, the term "non-genetically modified" refers to cells that have not been modified by transfection with a nucleic acid. For the avoidance of doubt, in the context of the present disclosure, a mesenchymal lineage precursor or stem cell transfected with a nucleic acid encoding Ang1 will be considered genetically modified.

[0075] 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 treated according to the present disclosure. In one example, the total dose consists of one cell administration. In another example, the total dose consists of two cell administrations. In another example, the total dose consists of three cell administrations. In another example, the total dose consists of four or more cell administrations. For example, the total dose can consist of two to four cell administrations.

[0076] The term "clinically proven" (independent use or modified term "effective") means that efficacy has been demonstrated by clinical trials, wherein the clinical trials have met the approval criteria of the U.S. Food and Drug Administration, EMEA or corresponding national regulatory agencies. For example, the clinical study can be a sufficiently large, randomized, double-blind study for clinically demonstrating the effect of the composition. In one example, the clinically proven effective amount is the amount that the clinical trial shows meets the specified endpoint. In one example, the endpoint is protection from death. In other words, the endpoint increases the survival rate. For example, the 100-day survival rate can be increased when the therapeutic agent is administered according to the present disclosure.

[0077] Therefore, the terms "clinically demonstrated efficacy" and "clinically proven effective" can be used in the context of the present disclosure to refer to dosage, dosage regimen, treatment or method disclosed herein. Efficacy can be measured based on changes in the disease process in response to the administration of the composition disclosed herein. For example, the composition of the present disclosure is administered to the subject in an amount and duration sufficient to cause improvement, preferably sustained improvement, in at least one indicator reflecting the severity of the inflammatory disease. Various indicators reflecting the severity of the disease can be evaluated to determine whether the amount and time of treatment are sufficient. Such indicators include, for example, clinically recognized indicators of the severity of the disease or symptoms. In one example, the degree of improvement is determined by a doctor, who can make this determination based on signs, symptoms or other test results. In one example, the clinically proven effective amount increases patient survival. In another example, the clinically proven effective amount reduces the risk of death in the subject. In another example, the clinically proven effective amount increases the 100-day survival rate. In one example, the method of the present disclosure administers a clinically proven effective amount of the composition disclosed herein.

[0078] "Therapeutic efficacy" is used in the context of the present disclosure to refer to MLPSCs and compositions disclosed herein that can treat, inhibit and / or prevent disease. For example, MLPSCs and compositions disclosed herein with therapeutic effects can treat, inhibit and / or prevent inflammatory diseases (such as GvHD). In one example, MLPSCs and compositions disclosed herein with therapeutic effects increase the 100-day survival rate of subjects with GvHD.

[0079] The term "and / or", such as "X and / or Y", should be understood to mean "X and Y" or "X or Y", and should be used to provide clear support for both meanings or either meaning.

[0080] Unless stated to the contrary, the term "about" as used herein means + / - 10%, more preferably + / - 5% of the specified value.

[0081] Throughout this specification, the word "comprise" or variations such as "comprising" or "including" will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0082] As used herein, the singular form "a," "an," and "the" include singular and plural references unless the context dictates otherwise.

[0083] Inflammatory diseases

[0084] As used herein, the term "inflammatory disease" should be understood to encompass diseases including, but not limited to, graft versus host disease (GvHD), pruritus, skin inflammation, psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, Hashimoto's thyroidis, myasthenia gravis, type I or type II diabetes, diabetic nephropathy, asthma, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, seborrheic dermatitis, Sjoegren's syndrome, keratoconjunctivitis, uveitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory diseases of the joints, skin or muscles, acute or chronic idiopathic inflammatory arthritis, myositis, demyelinating diseases, chronic obstructive pulmonary disease, interstitial lung disease, interstitial nephritis, chronic active hepatitis.

[0085] In one example, the inflammatory disease is mediated by T cell activation. Examples of such diseases include GvHD, rheumatoid arthritis, and diabetes. In one example, the inflammatory disease mediated by T cell activation is refractory to at least one biological therapy.

[0086] In one example, inflammatory disease is excessive inflammation. The term "excessive inflammation" used in the context of the present disclosure refers to a serious and persistent inflammatory process in the body. For example, excessive inflammation can refer to a serious and persistent inflammatory process in the airways and / or lungs, kidneys or liver. Therefore, excessive inflammation can affect multiple organs and their vascular systems in the body. In one example, excessive inflammation is associated with cytokine storm or cytokine release syndrome (CRS). In one example, cytokine storm or CRS involve a large amount of release of inflammatory cytokines (such as IL-6).

[0087] In one example, excessive inflammation leads to secondary (or acquired) hemophagocytic lymphohistiocytosis (sHLH). Thus, in one example, the methods of the present disclosure encompass the treatment of hemophagocytic lymphohistiocytosis (HLH).

[0088] In one example, the hyperinflammation is caused by a viral infection. For example, the hyperinflammation can be caused by rhinovirus, influenza virus, respiratory syncytial virus (RSV) or coronavirus. In one example, the hyperinflammation can be caused by coronavirus. For example, the coronavirus can be coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) or COVID-19. In one example, the hyperinflammation is caused by Epstein-Barr virus (EBV) or herpes simplex virus (HSV).

[0089] In one example, the inflammatory disease is multisystem inflammatory syndrome (MIS).

[0090] 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.

[0091] In one example, inflammatory diseases are graft-versus-host disease (GvHD). "Graft-versus-host disease (GvHD)" is an immune disorder, which is a major factor limiting the success and availability of allogeneic bone marrow or stem cell transplantation. GvHD occurs in acute (aGvHD) or chronic (cGvHD) forms. Acute GvHD usually manifests within 100 days after bone marrow or stem cell transplantation. Chronic GvHD usually manifests later than aGvHD (>100 days after transplantation), and has some characteristics of autoimmune diseases. It can develop from scratch, after aGvHD subsides, or as an extension of aGvHD. Chronic GvHD can cause a variety of symptoms that often make people weak, including large-area rashes, painful oral ulcers, shortness of breath, and limb and joint pain. For example, the CD5+B cell reconstruction of patients with cGvHD is impaired. In one example, cGvHD is steroid therapy refractory. In one example, cGvHD is biological therapy refractory. In one example, cGvHD is refractory to steroid therapy and biologic therapy.

[0092] The severity of GvHD can be graded according to the organ involvement pattern and clinical performance status. Multi-organ involvement includes rash, liver involvement and / or gastrointestinal (GI) involvement. Tables 1 and 2 provide examples of rash, liver involvement and gastrointestinal involvement. In one example, the subject suffers from GvHD with multi-organ involvement. In one example, the subject treated according to the present disclosure suffers from severe GvHD. In one example, severe GvHD is graded according to the Glucksberg scale (Glucksberg et al., 1974; Thomas et al., 1975) (Table 1). For example, according to the Glucksberg scale, the subject can suffer from grade II GvHD or grade III / IV GvHD. In one example, the subject suffers from grade II GvHD. In another example, the subject suffers from grade III / IV GvHD.

[0093] In another example, severe GvHD is graded according to the IBMTR severity index (Table 2) (Rowlings et al., 1997). In one example, the subject has grade B, C or D GvHD according to the IBMTR severity scale. In one example, the subject has grade D GvHD.

[0094] In another example, the subject has Minnesota high-risk GvHD. Minnesota high-risk acute GvHD is defined as skin stage 4; lower gastrointestinal (GI) stage 3-4 or liver stage 3-4; or skin stage 3+ and lower gastrointestinal stage 2-4 or liver stage 2-4 GvHD (MacMillan et al., 2015). In each of these examples, the subject can also have a high MAP score. For example, the subject can have a MAP score of ≥ 0.29.

[0095] Table 1: Glucksberg acute GvHD clinical stages and grades (Rowlings et al., 1997).

[0096]

[0097] Table 2: IBMTR Severity Index criteria for acute GvHD (Rowlings et al., 1997).

[0098]

[0099] *Index is assigned based on the maximum value of individual organ systems

[0100] In one example, a subject with severe GvHD is unresponsive to treatment with primary therapy. In one example, the subject deteriorates within 3 days of primary therapy. In this example, if the severity of the subject's GvHD increases, it indicates worsening of the condition. For example, according to MAP, the severity of the subject's GvHD has increased. In another example, according to the Glucksberg scale, the severity of the subject's GvHD has increased. In another example, according to the IMBTR scale, the severity of the subject's GvHD has increased. In another example, according to organ involvement, the severity of the subject's GvHD has increased.

[0101] In another example, the subject is unresponsive within 7 days of the primary therapy. For example, the subject is refractory to the primary therapy. In one example, the primary therapy is systemic steroids. In one example, the subject suffers from severe GvHD and is steroid refractory. In one example, the steroid is a corticosteroid. In another example, the steroid is a glucocorticoid. In another example, the steroid is prednisone. In another example, the subject suffers from severe GvHD and is steroid and second-line therapy refractory. For example, second-line therapy can include extracorporeal photopheresis, etanercept, infliximab, ruxolitinib, antithymocyte globulin, mycophenolic acid, alemtuzumab, basiliximab or tocilizumab.

[0102] Response to treatment

[0103] The methods disclosed herein relate to the treatment of inflammatory diseases such as GvHD. As used herein, "treating", "treat", "treatment", "slowing down the progression" include administering a mesenchymal lineage stem or precursor cell population and / or its progeny and / or soluble factors derived therefrom and / or extracellular vesicles derived therefrom, thereby reducing or eliminating at least one symptom of an inflammatory disease.

[0104] As used herein, the term "response" means a response to a therapy. In one example, a subject is considered to have responded if at least one of the subject's organs has improved without progression in any other organ, and if no additional therapy is required. In another example, a subject is considered to have not responded if the subject has stable or progressive GvHD or if a secondary therapy is subsequently required. In this example, a subject who does not respond is a non-responder.

[0105] In one example, the treatment causes a partial response. In one example, the partial response is caused at least 28 days after the start of treatment. In one example, the partial response is caused 28 days after the start of treatment. In one example, the partial response is caused at least 30 days after the start of treatment. In one example, the partial response is caused at least 2 months after the start of treatment. In another example, the partial response is caused at least 3 months after the start of treatment. In another example, the partial response is caused within 3 months. In another example, the partial response is caused 28 to 56 days after the start of treatment. In another example, the partial response is caused 100 days after the start of treatment. In another example, the partial response is caused 160 days after the start of treatment. In another example, the partial response is caused 180 days after the start of treatment.

[0106] In another example, a partial response is elicited after two doses. In another example, a partial response is elicited after two doses administered once a week. In another example, a partial response is elicited after two doses administered once a week for two weeks. In another example, a partial response is elicited after three or more doses. In one example, in the context of GvHD, a partial response is characterized by one or more or all of the following:

[0107] - a decrease in skin %BSA score of at least one point;

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

[0109] - Eye score decreased by at least one point;

[0110] - a decrease in skin feature score of at least one point;

[0111] - A decrease in GI score of at least one point;

[0112] - Decrease in liver score by at least one point;

[0113] - A decrease in the pulmonary symptom score of at least one point;

[0114] - A decrease in lung FEV1 score of at least one point;

[0115] - Joint and fascia scores decreased by at least one point;

[0116] - A decrease in the reproductive tract score of at least one point.

[0117] In one example, a partial response is characterized by a decrease in the skin % BSA score of at least one point. In another example, a partial response is characterized by a decrease in the oral score of at least one point. In another example, a partial response is characterized by a decrease in the eye score of at least one point. In these examples, the score can be obtained using the NIH Consensus Criteria 2014 for GvHD.

[0118] In another example, the partial response is characterized by one or more or all of the following:

[0119] - a decrease in skin %BSA score of at least one point;

[0120] - Oral score decreased by at least one point;

[0121] - Eye score decreased by at least one point.

[0122] There are multiple classification systems for characterizing GvHD (Lee, S., (2017) Blood., 129(1): 30–37). In one example, the NIH consensus criteria 2014 can be used to score the results disclosed herein (Jagasia et al., (2015) Biol Blood Marrow Transplant., 21: 389-401). The components of the NIH consensus criteria 2014 are shown in the following table:

[0123] Table 3: Organ scores for GvHD

[0124]

[0125]

[0126] In one example, a partial response is a decrease of ≥ 1 point in the organ-specific NIH Consensus Criteria 2014 score in the table above. Thus, in one example, treatment results in a decrease in skin % BSA score of > In another example, the treatment results in a decrease in the oral score > In another example, treatment results in a decrease in eye score > In another example, the treatment results in a decrease in the skin characteristic score > In another example, treatment results in a decrease in the gastrointestinal score > In another example, treatment results in a decrease in liver score > In another example, treatment results in a decrease in pulmonary symptom score > In another example, treatment results in a decrease in pulmonary FEV1 score > In another example, treatment results in a decrease in joint and fascia scores > In another example, treatment results in a decrease in reproductive tract score > 1 point.

[0127] In one example, after treatment begins, treatment causes a complete response. In one example, a complete response is complete resolution of GvHD symptoms in all organs. In one example, a complete response is caused 28 days after treatment begins. In one example, a complete response is caused at least 28 after treatment begins. In one example, a complete response is caused at least 30 after treatment begins. In one example, a complete response is caused at least 2 months after treatment begins. In another example, a complete response is caused at least 3 months after treatment begins. In another example, a complete response is caused 28 to 56 days after treatment begins. In another example, a complete response is caused 100 days after treatment begins. In another example, a complete response is caused 160 days after treatment begins. In another example, a complete response is caused 180 days after treatment begins.

[0128] In another example, a complete response is elicited after two doses. In another example, a complete response is elicited after two doses administered once a week. In another example, a complete response is elicited after two doses administered once a week for two weeks. In another example, a complete response is elicited after three or more doses.

[0129] In another example, also in the context of GvHD, treatment increases the probability of survival of the subject. For example, treatment increases the probability that the subject survives at least 20 days to 200 days after the start of treatment. In one example, treatment increases the probability that the subject survives at least 180 days after the start of treatment. In another example, treatment increases the probability that the subject survives at least 100 days. In one example, the increase in probability is determined relative to a subject not treated with the composition of the present disclosure. In one example, treatment increases the 100-day survival rate by more than 60%. In another example, treatment increases the 100-day survival rate by more than 60%. In another example, treatment increases the 100-day survival rate by more than 65%. In another example, treatment increases the 100-day survival rate by more than 68%. In another example, treatment increases the 100-day survival rate by more than 70%. In another example, treatment increases the 100-day survival rate by more than 71%. In another example, treatment increases the 100-day survival rate by more than 72%. In another example, treatment increases the 100-day survival rate by 60% to 75%. In another example, treatment increases the 100-day survival rate by 65% ​​to 75%. In another example, treatment increases the 100-day survival rate by 70% to 75%. In one example, the 100-day survival rate of a patient population is determined, and the average 100-day survival rate observed in the population provides a measure of the 100-day survival rate of each patient in the population. In one example, the measure is compared with a subject (or subject population) not treated with a composition of the present disclosure.

[0130] Mesenchymal lineage precursor cells

[0131] As used herein, the term "mesenchymal lineage precursor or stem cell (MLPSC)" refers to an undifferentiated pluripotent cell that has the ability to self-renew while maintaining pluripotency and the ability to differentiate into a number of cell types of mesenchymal origin (e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons) or non-mesodermal origin (e.g., hepatocytes, neural cells, and epithelial cells). For the avoidance of doubt, "mesenchymal lineage precursor cells" refer to cells that can differentiate into mesenchymal cells (such as bone, cartilage, muscle, and adipocytes) and fibrous connective tissue.

[0132] The term "mesenchymal lineage precursor or stem cell" includes both parental cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSC), perivascular mesenchymal precursor cells, and their undifferentiated progeny.

[0133] Mesenchymal lineage precursors or stem cells can be autologous, allogeneic, xenogeneic, syngeneic or isogenic. Autologous cells are isolated from the same individual into which they are to be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0134] In one example, the mesenchymal lineage precursors or stem cells are allogeneic. In one example, the allogeneic mesenchymal lineage precursors or stem cells are culture expanded and cryopreserved.

[0135] Mesenchymal lineage precursors or stem cells mainly reside in the bone marrow, but have also been shown to be present in a variety of host tissues, including, for example, umbilical cord blood and umbilical cord, adult peripheral blood, adipose tissue, trabecular bone and dental pulp. They are also present in skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestines, lungs, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis and periosteum; and can be differentiated into reproductive system such as mesoderm and / or endoderm and / or ectoderm. Therefore, mesenchymal lineage precursors or stem cells can be differentiated into a large number of cell types, including but not limited to adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue and fibrous connective tissue. The specific lineage-stereotype and differentiation pathways that these cells enter depend on the various effects from mechanical influences and / or endogenous bioactive factors (such as growth factors, cytokines and / or local microenvironment conditions established by host tissues).

[0136] The term "enriched", "enrichment" or its variant is used herein to describe such a cell colony, in which the ratio of a specific cell type or the ratio of many specific cell types increases when compared to an untreated cell colony (e.g., cells in their native environment). In one example, a colony enriched for mesenchymal lineage precursors or stem cells comprises at least about 0.1%, or 0.5%, or 1%, or 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 50%, or 75% of mesenchymal lineage precursors or stem cells. In this regard, the term "cell colony enriched for mesenchymal lineage precursors or stem cells" will be considered to provide clear support for the term "cell colony comprising X% mesenchymal lineage precursors or stem cells", wherein X% is a percentage as listed herein. In some examples, mesenchymal lineage precursors or stem cells can form clonogenic colonies, such as CFU-F (fibroblasts) or a subset thereof (e.g., 50%, or 60%, or 70%, or 70%, or 90%, or 95%) can have such activity.

[0137] In one example of the present disclosure, the mesenchymal lineage precursor or stem cell is a mesenchymal stem cell (MSC). MSC can be a homogenous composition or can be a mixed cell population enriched with MSC. Homogeneous MSC compositions can be obtained by culturing adherent bone marrow or periosteal cells, and MSC can be identified by specific cell surface markers identified with unique monoclonal antibodies. The method for obtaining a cell population enriched with MSC is described in, for example, U.S. Patent No. 5,486,359. Alternative sources of MSC include, but are not limited to, blood, skin, cord blood, muscle, fat, bone, and perichondrium. In one example, MSC is allogenic. In one example, MSC is cryopreserved. In one example, MSC is cultured, amplified, and cryopreserved.

[0138] In another example, the mesenchymal lineage precursor or stem cell is a CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSC.

[0139] The isolated or enriched mesenchymal lineage precursors or stem cells can be expanded in vitro by culture.The isolated or enriched mesenchymal lineage precursors or stem cells can be cryopreserved, thawed and subsequently expanded in vitro by culture.

[0140] In one example, the isolated or enriched mesenchymal lineage precursors or stem cells are cultured at 50,000 viable cells / cm 2The cells are plated in culture medium (serum-free or serum-supplemented), for example, alpha minimum essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, and allowed to adhere to the culture vessels overnight at 37° C., 20% O 2. The culture medium is then replaced and / or changed as needed, and the cells are cultured at 37° C., 5% O 2 for an additional 68 to 72 hours.

[0141] As will be appreciated by those skilled in the art, cultured mesenchymal lineage precursors or stem cells are phenotypically different from in vivo cells. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal lineage precursors or stem cells are also biologically different from in vivo cells, having a higher proliferation rate than most non-circulating (quiescent) cells in vivo.

[0142] In one example, a cell population is enriched from a cell preparation containing 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 allows the selection of STRO-1+ cells. The marker may be STRO-1, but not necessarily. For example, as described and / or exemplified herein, cells (e.g., mesenchymal precursor cells) expressing STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (and may be STRO-1bright). Therefore, the indication that the cell is STRO-1+ does not mean that the cell is selected only by STRO-1 expression. In one example, cells are selected based on at least STRO-3 expression, for example, they are STRO-3+ (TNAP+). For example, MPCs can be separated from bone mononuclear cells with anti-STRO-3 antibodies.

[0143] Reference to the selection of cells or their populations does not necessarily require selection from a specific tissue source. As described herein, STRO-1+ cells can be selected from or isolated from or enriched from a variety of sources. That is, in some instances, these terms provide support for selection from any tissue or vascularized tissue containing STRO-1+ cells (e.g., mesenchymal precursor cells) or tissue containing pericytes (e.g., STRO-1+ pericytes) or any one or more of the tissues listed herein.

[0144] In one example, the cells used in the present disclosure express one or more markers, alone or collectively, selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0145] "Individually" means that the disclosure individually encompasses the recited marker or marker groups, and although individual markers or marker groups may not be individually listed herein, the appended claims may define such markers or marker groups individually and separately from each other.

[0146] "Collectively" means that the present disclosure encompasses any number or combination of the listed markers or marker groups, and although such number or combination of markers or marker groups may not be specifically listed herein, the appended claims may define such combinations or sub-combinations individually and separately from any other combination of markers or marker groups.

[0147] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue nonspecific alkaline phosphatase. For example, the term encompasses liver isoforms (LAP), bone isoforms (BAP), and kidney isoforms (KAP). In one example, TNAP is BAP. In one example, TNAP as used herein refers to a molecule that can bind to a STRO-3 antibody produced by a hybridoma cell line deposited with the ATCC on December 19, 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0148] Furthermore, in one example, STRO-1+ cells were able to generate clonogenic CFU-F.

[0149] In one example, a significant proportion of STRO-1+ cells are able to differentiate into at least two different germ lineages. Non-limiting examples of lineages to which STRO-1+ cells can be committed include bone precursor cells; hepatocyte progenitor cells, which are multipotent for bile duct epithelial cells and hepatocytes; neural restricted cells, which can generate glial precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; myocardium and precursors of cardiomyocytes; pancreatic beta cell lineage that secretes glucose-responsive insulin. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as precursors of the following cells: retinal pigment epithelial cells, fibroblasts, skin cells (such as keratinocytes), dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiomyocytes, smooth muscle cells, skeletal muscle cells, pericytes, vascular cells, epithelial cells, glial cells, neurons, astrocytes, and oligodendrocytes.

[0150] In one example, mesenchymal lineage precursors or stem cells are obtained from a single donor or multiple donors, wherein the donor samples or mesenchymal lineage precursors or stem cells are subsequently pooled and then expanded in culture.

[0151] Mesenchymal lineage precursors or stem cells encompassed by the present disclosure may also be cryopreserved prior to administration to a subject. In one example, the mesenchymal lineage precursors or stem cells are culture expanded and cryopreserved prior to administration to a subject.

[0152] In one example, the present disclosure encompasses mesenchymal lineage precursors or stem cells and their progeny, soluble factors derived therefrom and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal lineage precursors or stem cells and extracellular vesicles isolated therefrom. For example, the mesenchymal lineage precursors or stem cells of the present disclosure can be cultured and expanded for a period of time under conditions suitable for secretion of extracellular vesicles into cell culture medium. Secreted extracellular vesicles can then be obtained from the culture medium for therapy.

[0153] As used herein, the term "extracellular vesicle" refers to lipid particles naturally released from cells and ranging in size from about 30 nm to as large as 10 microns, but the size of the lipid particles is generally less than 200 nm. The lipid particles may contain extracellular vesicles from releasing cells (e.g., mesenchymal stem cells; STRO-1 + cells) proteins, nucleic acids, lipids, metabolites or organelles.

[0154] As used herein, the term "exosome" refers to a type of extracellular vesicle, the size of which is generally in the range of about 30 nm to about 150 nm and which originates from the endosomal compartment of mammalian cells, from which it is transported to the cell membrane and released. The extracellular vesicle may contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolites, and plays a role in intercellular communication by being secreted from one cell and taken up by other cells to deliver their cargo.

[0155] In one example, the composition of the present disclosure comprises cells that induce neovascularization in a target tissue. In one example, the target tissue is the heart. In another example, the cell secretes a factor that protects the at-risk or damaged myocardium. In one example, the at-risk or damaged myocardium has been subjected to a lack of blood flow caused by an ischemic event. In one example, the cell secretes a factor that reduces cardiomyocyte apoptosis.

[0156] Cell culture and expansion

[0157] In one example, mesenchymal lineage precursors or stem cells are cultured and expanded. The difference between "culture-expanded" mesenchymal lineage precursors or stem cells and freshly separated cells is that they have been cultured and passed down (i.e., subculture) in cell culture medium. In one example, freshly separated cell culture expansion is about 1 or 2 times passed down to provide an intermediate colony. In one example, freshly separated cell culture expansion is 2 times passed down to provide an intermediate colony. In another example, freshly separated cell culture expansion is about 1 to 3 times passed down to provide an intermediate colony.

[0158] Therefore, in one example, the related cells are isolated and the related cells are cultured and expanded for 2 passages to provide an intermediate MLPSC population. The intermediate MLPSC population is then cultured and expanded to provide DP. For example, the intermediate cell population can be cultured for three more passages (i.e., a total of 5 passages) to provide DP.

[0159] In one example, the mesenchymal lineage precursor cells or stem cells are cultured and expanded for about 4-10 generations. In one example, the mesenchymal lineage precursors or stem cells are cultured and expanded for at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages. For example, the mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5 passages. In one example, the mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-10 passages. In one example, the mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-8 passages. In one example, the mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-7 passages. In one example, the mesenchymal lineage precursors or stem cells can be cultured and expanded for more than 7 passages. In these examples, MLPSCs can be cultured and expanded before cryopreservation to provide a cryopreserved intermediate MLPSC population, and then the cryopreserved intermediate MLPSC population is further cultured and expanded.

[0160] In one example, the method of the present disclosure selects an intermediate population (e.g., a cryopreserved intermediate) for further culture expansion based on certain criteria (such as the level of IL2-RA inhibition). For example, if the MLPSC population inhibits IL2-RA by at least 55% under culture conditions, cryopreserved intermediates can be selected for culture expansion. In another example, if the MLPSC population inhibits IL2-RA by at least 60% under culture conditions, cryopreserved intermediates can be selected for culture expansion.

[0161] In other examples, the methods of the present disclosure encompass assessing the therapeutic efficacy of an intermediate population of cryopreserved MLPSCs.

[0162] In one example, the DP composition of the present disclosure is produced by culturing cells from a cryopreserved intermediate MLPSC population, or in other words, a cryopreserved intermediate.

[0163] In one example, the compositions of the present disclosure include mesenchymal lineage precursors or stem cells cultured and amplified from cryopreserved intermediates. In one example, cells cultured and amplified from cryopreserved intermediates are cultured and amplified for at least 3 generations, at least 5 generations, at least 6 generations, at least 7 generations, at least 8 generations, at least 9 generations, at least 10 generations. For example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 3 generations. In one example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 3-10 generations. In one example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 3-8 generations. In one example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 3-7 generations.

[0164] In one example, the effect of IL2-RA inhibition by mesenchymal lineage precursors or stem cells expanded from cryopreserved intermediate cultures disclosed herein is evaluated under culture conditions (e.g., co-cultured 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% to 80% under culture conditions.

[0165] In one example, the culture-expanded mesenchymal lineage precursors or stem cells can be cultured and expanded in a medium without animal protein (e.g., a medium without exogenous components). In one example, the mesenchymal lineage precursors or stem cells can be cultured and expanded in a medium without fetal bovine serum.

[0166] In one embodiment, mesenchymal lineage precursors or stem cells can be obtained from a single donor or multiple donors, wherein the donor samples or mesenchymal lineage precursors or stem cells are subsequently pooled and then cultured and expanded as needed. In one example, the culture expansion process includes:

[0167] i. expanding said number of viable cells by passage expansion to provide a preparation of at least about 1 billion viable cells, wherein the passage expansion comprises establishing a primary culture of the isolated mesenchymal lineage precursors or stem cells, and then successively establishing a first non-primary (P1) culture of the isolated mesenchymal lineage precursors or stem cells from the previous culture;

[0168] ii. expanding the isolated P1 culture of mesenchymal lineage precursors or stem cells into a second non-primary (P2) culture of mesenchymal lineage precursors or stem cells by subculture expansion; and,

[0169] iii. preparing and cryopreserving an in-process intermediate mesenchymal lineage precursor or stem cell preparation obtained from a P2 culture of mesenchymal lineage precursors or stem cells; and, optionally

[0170] iv. thawing the cryopreserved in-process intermediate mesenchymal lineage precursor or stem cell preparation and expanding the in-process intermediate mesenchymal lineage precursor or stem cell preparation by passage expansion.

[0171] In the context of the present disclosure, certain determinations can be performed between steps iii and iv. For example, IL2-RA inhibition can be determined under culture conditions after step iii. In one example, step iv is only performed if the desired level of IL2-RA inhibition is observed under culture conditions. In this example, a cell population is selected for culture expansion based on IL2-RA inhibition under culture conditions.

[0172] In one example, the expanded mesenchymal lineage precursor or stem cell preparation has an antigenic profile and an activity profile comprising:

[0173] i. less than about 0.75% CD45+ cells;

[0174] ii. at least about 95% CD105+ cells;

[0175] iii. At least about 95% CD166+ cells.

[0176] In one example, the expanded mesenchymal lineage precursor or stem cell population is an intermediate population that can inhibit the expression of IL2-RA by CD3 / CD28 activated PBMC by at least 55% relative to the control. In another example, the expanded mesenchymal lineage precursor or stem cell population is an intermediate population that can inhibit the expression of IL2-RA by CD3 / CD28 activated PBMC by at least 60% relative to the control. In another example, the expanded mesenchymal lineage precursor or stem cell population is an intermediate population that can inhibit the expression of IL2-RA by CD3 / CD28 activated PBMC by at least about 65% relative to the control. In another example, the expanded mesenchymal lineage precursor or stem cell population is an intermediate population that can inhibit the expression of IL2-RA by CD3 / CD28 activated PBMC by at least about 70% relative to the control. In another example, the expanded mesenchymal lineage precursor or stem cell population is an intermediate population that can inhibit IL2-RA expression by CD3 / CD28 activated PBMCs by 55% to 70% relative to a control. In another example, the expanded mesenchymal lineage precursor or stem cell population is an intermediate population that can inhibit IL2-RA expression by CD3 / CD28 activated PBMCs by 60% to 70% relative to a control.

[0177] In one example, the expanded mesenchymal lineage precursor or stem cell population is expanded from an intermediate MLPSC population and is capable of inhibiting IL2-RA expression by CD3 / CD28 activated PBMCs by at least 70% relative to a control. In another example, the expanded mesenchymal lineage precursor or stem cell population is expanded from an intermediate MLPSC population and is capable of inhibiting IL2-RA expression by CD3 / CD28 activated PBMCs by 70% to 75% relative to a control.

[0178] Any equipment and cell processing methods known in the art can be used to separate and amplify mesenchymal lineage precursors or stem cells in vitro. Various culture expansion embodiments disclosed herein use steps that require manipulation of cells, such as inoculation, feeding, dissociation of adherent cultures, or washing steps. Any step of manipulating cells is likely to damage the cells. Although mesenchymal lineage precursors or stem cells can generally tolerate a certain amount of damage during preparation, it is preferred to manipulate cells by fully performing one or more given steps of the operating procedures and / or equipment while minimizing damage to the cells.

[0179] In one example, mesenchymal lineage precursors or stem cells are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculating wash bag, a rotating membrane filter having an inlet port and an outlet port, a filtrate bag, a mixing zone, a final product bag for the washed cells, and appropriate tubing, e.g., as described in US 6,251,295, which is hereby incorporated by reference.

[0180] In one example, the mesenchymal lineage precursor or stem cell composition cultured according to the present disclosure is 95% homogeneous in terms of being CD105 positive and CD166 positive and CD45 negative. In one example, this homogeneity persists through ex vivo expansion (ie, through multiple population doublings).

[0181] In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded in 2D culture. For example, the mesenchymal lineage precursors or stem cells of the present disclosure can be cultured and expanded in a cell factory. In certain examples, the 3D culture of the intermediates disclosed herein can be carried out using, for example, a bioreactor. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are initially cultured and expanded in 2D culture, and then further expanded in 3D culture. In one example, the intermediate cell population of the present disclosure has not been cultured and expanded in 3D culture. In one example, IL2-RA inhibition is evaluated before subsequent culture expansion in a cell factory or 3D culture.

[0182] In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are culture-expanded from an intermediate population. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are culture-expanded from an intermediate in 2D culture prior to seeding in 3D culture.

[0183] In the context of the intermediate population and the therapeutic composition expanded therefrom, in one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded in 2D culture for at least 3 days, and then inoculated in another culture system (such as a cell factory or 3D culture in a bioreactor). In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded in 2D culture for at least 4 days, and then inoculated in another culture system. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded in 2D culture for 3 to 5 days, and then inoculated in another culture system. In these examples, 2D culture can be carried out in a cell factory. Various cell factory products are commercially available (e.g., Thermofisher, Sigma, Corning). In one example, the cell factory has at least 5 layers. In one example, the cell factory has at least 10 layers. In one example, the cell factory has at least 20 layers. 3D culture can be carried out in various types of bioreactors (such as stirred tanks, wave bags, and vertical wheels).

[0184] In one example, CO2 is provided during the culture expansion of MLPSC. In one example, MLPSC is cultured and expanded in less than 9% CO2. In one example, MLPSC is cultured and expanded in less than 8% CO2. In one example, MLPSC is cultured and expanded in 5% CO2. For example, MLPSC can be cultured and expanded in 5% + / - 2% CO2. In one example, MLPSC is cultured and expanded under passive pretreatment of CO2. For example, the cell factory can be passively pretreated with 5% CO2.

[0185] Pretreatment of the cell factory can maintain the CO2 tension between the cell factory and the incubator and stabilize the pH level of the growth medium. Active pretreatment involves actively passing CO2 gas through a bacterial ventilation filter into each culture dish (e.g., a cell factory) for a specified period of time (e.g., about 10 minutes). However, active pretreatment may introduce contamination into the culture because it requires an open port to provide gas. Passive pretreatment involves placing the closed culture system in an incubator with an appropriate CO2 concentration and then inoculating the cells (e.g., about 12 to 72 hours).

[0186] In one example, the cells of the present disclosure are STRO-3+ prior to expansion in culture to provide an intermediate cell population.

[0187] Cell culture medium

[0188] The mesenchymal lineage precursors or stem cells disclosed herein can be cultured and expanded in a variety of suitable growth media.

[0189] The term "medium" or "media" as used in the context of the present disclosure includes components of the environment surrounding cells. The medium helps and / or provides conditions suitable for allowing cell growth. The medium can be solid, liquid, gas, or a mixture of phases and materials. The medium can include liquid growth medium and liquid medium that does not maintain cell growth. The medium also includes gelatinous medium such as agar, agarose, gelatin and collagen matrix. Exemplary gaseous medium includes the gas phase to which cells grown on a culture dish or other solid or semi-solid support are exposed.

[0190] The cell culture medium used for culture expansion 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).

[0191] Those skilled in the art will appreciate that, in order to obtain optimal results, the basal medium must be appropriate for the cell line of interest. For example, if it is found that the glucose (or other energy source) in the basal medium is depleted and therefore restricts growth, it may be necessary to increase the glucose (or other energy source) level in the basal medium, or to add glucose (or other energy source) during the culture process. In one example, the dissolved oxygen (DO) level may also be controlled.

[0192] In one example, the cell culture medium contains additives of human origin. For example, human serum and human platelet cell lysate can be added to the cell culture medium.

[0193] In one example, the cell culture medium contains only additives of human origin. Thus, in one example, the cell culture medium is free of exogenous components. For the avoidance of doubt, in these examples, the culture medium does not contain animal proteins. In one example, the cell culture medium used in the methods of the present disclosure does not contain animal components.

[0194] In one example, the culture medium comprises serum. In other examples, the culture medium is a culture medium without fetal bovine serum that contains growth factors that promote proliferation of mesenchymal lineage precursors or stem cells. In one embodiment, the culture medium is a serum-free stem cell culture medium. In one example, the cell culture medium comprises:

[0195] Basal culture medium;

[0196] Platelet-derived growth factor (PDGF);

[0197] Fibroblast growth factor 2 (FGF2).

[0198] In one example, the culture medium comprises platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2), wherein the level of FGF2 is less than about 6 ng / ml. For example, the level of FGF2 can be less than about 5 ng / ml, less than about 4 ng / ml, less than about 3 ng / ml, less than about 2 ng / ml, less than about 1 ng / ml. In one example, the level of FGF2 is about 1 ng / ml.

[0199] In one example, PDGF is PDGF-BB. In one example, the level of PDGF-BB is between about 1 ng / ml and 150 ng / ml. In another example, the level of PDGF-BB is between about 7.5 ng / ml and 120 ng / ml. In another example, the level of PDGF-BB is between about 15 ng / ml and 60 ng / ml. In another example, the level of PDGF-BB is about 10 ng / ml. In another example, the level of PDGF-BB is at least about 10 ng / ml or less.

[0200] In other examples, additional factors can be added to the cell culture medium. In one example, the culture medium further comprises EGF. EGF is a growth factor that stimulates cell proliferation by binding to its receptor EGFR. In one example, the method of the present disclosure includes culturing a stem cell colony in a cell culture medium without fetal bovine serum that further comprises EGF. In one example, the level of EGF is between about 0.1ng / ml and 7ng / ml. For example, the level of EGF can be at least about 5ng / ml.

[0201] In another example, the level of EGF is about 1 ng / ml to 8 ng / ml. In another example, the level of EGF is between about 3 ng / ml and 6 ng / ml. In another example, the level of EGF is about 5 ng / ml. In another example, the level of EGF is about 5 ng / ml or less.

[0202] In the above examples, a basal medium such as αMEM or StemSpan TM In one example, the culture medium comprises AlphaMEM or StemSpan supplemented with 10 ng / ml PDGF-BB, 5 ng / ml EGF, and 1 ng / ml FGF. TM .

[0203] In other examples, additional factors can be added to the cell culture medium. For example, one or more stimulatory factors selected from the group consisting of: epidermal growth factor (EGF), lα, 25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and matrix-derived factor 1α (SDF-1α) can be supplemented into the cell culture medium. In another embodiment, cells can also be cultured in the presence of at least one cytokine in an amount sufficient to support cell growth. In another embodiment, cells can be cultured in the presence of heparin or its derivatives. In one example, the heparin derivative is sulfate. Various forms of heparin sulfate are known in the art and include heparin sulfate 2 (HS2). HS2 can be derived from various sources, including, for example, the liver of male and / or female mammals. Therefore, exemplary heparin sulfates include male liver heparin sulfate (MML HS) and female liver heparin sulfate (FML HS).

[0204] In another example, the cell culture medium of the present disclosure maintains stem cells in an undifferentiated state. When stem cells have not yet been stereotyped into a specific differentiation lineage, they are considered to be undifferentiated. As discussed above, stem cells exhibit morphological characteristics that distinguish them from differentiated cells. In addition, undifferentiated stem cells express genes that can be used as markers for detecting differentiation states. Polypeptide products can also be used as markers for detecting differentiation states. Therefore, those skilled in the art can easily determine whether the methods of the present disclosure maintain stem cells in an undifferentiated state using conventional morphological analysis, genetic analysis, and / or proteomic analysis.

[0205] Co-culture of mesenchymal lineage precursors or stem cells with stimulated or activated T cells

[0206] Interleukin-2 receptor alpha chain ("IL2-RA"; also known as CD25) is a validated marker of T cell activation. In certain embodiments, the methods of the present disclosure measure inhibition of T cell activation. In one example, T cell activation is determined based on the level of T cell IL2-RA expression after co-culture of activated PBMCs with mesenchymal lineage precursors or stem cells. Inhibition of IL2-RA expression is associated with inhibition of T cell activation.

[0207] In one example, activated PBMCs are co-cultured with mesenchymal lineage precursors or stem cells in a culture medium comprising at least one T cell stimulator, the concentration of which is preferably capable of stimulating and / or activating T cells. In another embodiment, T cells are first stimulated and / or activated before being co-cultured with mesenchymal lineage precursors or stem cells.

[0208] In one example, mesenchymal lineage precursors or stem cells are co-cultured with PBMC in a culture medium containing an agent that can stimulate CD3 and an agent that can stimulate CD28 on T cells, such as an antibody to CD3 and an antibody to CD28, e.g., mouse anti-human CD3 and mouse anti-human CD28. In one example, the antibody to CD3 and / or the antibody to CD28 are added to the culture medium in a soluble form, with each antibody at a concentration of about 2 μg / ml.

[0209] In one embodiment, PBMCs are co-cultured with mesenchymal precursors or stem cells at a ratio of 5 PBMCs: 1 mesenchymal precursor or stem cell. 6 PBMCs can be used with 2 × 10 5 In one example, the cells can be co-cultured in a final volume of 1 ml.

[0210] Determining treatment efficacy

[0211] In one embodiment, the present disclosure relates to a method for determining the therapeutic efficacy of an intermediate population of a mesenchymal precursor lineage or stem cell (MLPSC). In some instances, such methods can be used to establish "memory" in an intermediate cell population so that a final product can be subsequently derived therefrom. In other words, such methods demonstrate the therapeutic efficacy of an intermediate cell population and the resulting product of the culture amplification. If desired, additional assays can be used in some instances to characterize the therapeutic efficacy of the resulting product (or selected product).

[0212] In one example, a method for determining the therapeutic efficacy of an intermediate population of a mesenchymal precursor lineage or stem cell (MLPSC) comprises: (i) obtaining a MLPSC population; (ii) culturing the cells in a culture medium; and (iii) determining the level of IL2-RA inhibition under culture conditions, wherein at least 55% IL2-RA inhibition under culture conditions indicates therapeutic efficacy. In another example, IL2-RA inhibition greater than 56% indicates therapeutic efficacy. In another example, IL2-RA inhibition greater than 60% indicates therapeutic efficacy. In another example, IL2-RA inhibition greater than 70% indicates therapeutic efficacy.

[0213] In other examples, the method of determining therapeutic efficacy further comprises determining the level of TNF-R1 expression under culture conditions, wherein expression of at least 100 pg / ml TNF-R1 indicates therapeutic efficacy. In another example, expression of at least 105 pg / ml TNF-R1 indicates therapeutic efficacy.

[0214] The method for determining the therapeutic efficacy disclosed herein can provide suitable intermediates for culture expansion into a cell composition with a therapeutic effect. In one example, such culture expansion composition is characterized by a threshold level of IL2-RA inhibition. In one example, the level of IL2-RA inhibition is greater than the level observed in the aforementioned 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 65% to 75% under culture conditions. In another example, the level of IL2-RA inhibition is 70% to 75% under culture conditions.

[0215] Determination of IL2-RA levels

[0216] 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), competition assay, radioimmunoassay, lateral flow immunoassay, flow-through immunoassay, electrochemiluminescence assay, turbidimetric-based assay, nephelometry-based assay, fluorescence-activated cell sorting (FACS)-based assay.

[0217] After co-culture of MLPSC and T cells, cells can be collected and lysed using methods well known in the art. Cell lysates can then be assayed for the presence of IL2-RA by, for example, ELISA or FLISA. Alternatively, the level of IL2-RA expression can be determined by, for example, flow cytometry of intact cells.

[0218] In one example, inhibition of IL2-RA expression is measured by comparing the level of IL2-RA expression of a cell population comprising T cells to the level of IL2-RA of a cell population after co-culture of a cell population comprising T cells and a cell population comprising mesenchymal lineage precursors or stem cells, and expressing the difference as "percent inhibition."

[0219] The assay described above can be easily modified to use chemiluminescence or electrochemiluminescence as the basis for detection.

[0220] It will be appreciated by the skilled person that other detection methods based on immunosorbent assays can also be used to perform the present disclosure. For example, based on the immunosorbent method described above, detection is performed using a radioactive label, or detection is performed using a gold label (e.g., colloidal gold), or detection is performed using a liposome (e.g., encapsulating NAD+), or using an acridine-linked immunosorbent assay.

[0221] In some examples of the present disclosure, the level of IL2-RA is measured using a surface plasmon resonance detector (e.g., BIAcore TM , GE Healthcare, Piscataway, NJ), flow-through devices (e.g., as described in U.S. Pat. No. 7,205,159), micro- or nano-immunoassay devices (e.g., as described in U.S. Pat. No. 7,271,007), lateral flow devices (e.g., as described in U.S. Publication No. 20040228761 or U.S. Publication No. 20040265926), fluorescence polarization immunoassay (FPIA, e.g., as described in U.S. Pat. No. 4,593,089 or U.S. Pat. No. 4,751,190), or immunoturbidimetric assays (e.g., as described in U.S. Pat. No. 5,571,728 or U.S. Pat. No. 6,248,597).

[0222] Determination of TNFR1 levels

[0223] In certain examples, the methods of the present disclosure further comprise the step of determining TNFR1 expression by mesenchymal lineage precursors or stem cells.

[0224] The skilled artisan will appreciate that the methods described above for detecting IL2-RA expression can also be used to detect TNFR1 expression. In a preferred embodiment, the cell lysate is assayed by ELISA or FLISA.

[0225] In one form, such an assay involves immobilizing a TNFR1 binding protein on a solid substrate. The test sample is then brought into direct contact with the TNFR1 binding protein, and the TNFR1 in the sample is bound or captured. After washing to remove any unbound protein in the sample, a protein that binds to TNFR1 at a different epitope is brought into direct contact with the captured TNFR1. The detection protein is typically labeled as described above. Alternatively, a second labeled protein that binds to the detection protein may be used. After washing to remove any unbound protein, a detectable reporter molecule may be detected by adding a substrate as described above in an ELISA. The level of TNFR1 in the sample is then determined using a standard curve generated by a known amount of marker or by comparison with a control sample.

[0226] Selecting cells for culture expansion

[0227] In one embodiment, the disclosure encompasses cells selected for culture expansion according to certain criteria. In an example, the cells include an intermediate MLPSC population. In an example, whether the MLPSC population meets the necessary criteria is evaluated, and when the criteria are met, the population is selected for culture expansion.

[0228] In one example, if the MLPSC population suppresses IL2-RA by at least 55% under culture conditions, the intermediate MLPSC population is selected for further expansion. In one example, the MLPSC population is an intermediate population. In one example, if the intermediate MLPSC population suppresses IL2-RA by at least 55% under culture conditions, the intermediate MLPSC population is selected for further expansion. In another example, if the intermediate MLPSC population suppresses IL2-RA by at least 56% under culture conditions, the intermediate MLPSC population is selected for further expansion. In another example, if the intermediate MLPSC population suppresses IL2-RA by at least 60% under culture conditions, the intermediate MLPSC population is selected for further expansion. In another example, if the intermediate MLPSC population suppresses IL2-RA by at least 70% under culture conditions, the intermediate MLPSC population is selected for further expansion. In another example, if the intermediate MLPSC population suppresses IL2-RA by 55% to 70% under culture conditions, the intermediate MLPSC population is selected for further expansion. In another example, if the intermediate MLPSC population suppresses IL2-RA by 60% to 70% under culture conditions, the intermediate MLPSC population is selected for further expansion. In another example, the intermediate MLPSC population selected for further expansion also expresses at least 80pg / ml TNF-R1 under culture conditions. In another example, the intermediate MLPSC population selected for further expansion also expresses at least 105pg / ml TNF-R1 under culture conditions. In another example, the intermediate MLPSC population selected for further expansion also expresses at least 120pg / ml TNF-R1 under culture conditions. In another example, the intermediate MLPSC population selected for further expansion also expresses 80pg / ml TNF-R1 and 120pg / ml TNFR1 under culture conditions.

[0229] The selection process is not particularly limited as long as they can select cell populations characterized by relevant criteria (such as IL2-RA inhibition%). In one example, a series of intermediate MLPSC populations are evaluated for IL2-RA inhibition under culture conditions, and a population that inhibits IL2-RA inhibition 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 a population that inhibits IL2-RA inhibition by at least 60% under culture conditions is selected for further expansion. In one example, intermediate MLPSC populations that meet the relevant threshold level of IL2-RA inhibition are combined before further expansion.

[0230] It should be understood that the selection process does not require immediate culture expansion. Instead, the "selected" population can be cryopreserved and culture expanded at a later stage. In one example, a portion of the intermediate cell population is culture expanded, and the remainder of the population is cryopreserved to be culture expanded at a later stage.

[0231] In one example, the selected cell population is immediately cultured and expanded. In another example, the selected cell population is cryopreserved for culture expansion at a later time.

[0232] 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 IL2-RA inhibition greater than 60%. In another example, the culture expansion of the selected MLPSC population provides an MLPSC population characterized by IL2-RA inhibition greater than 70% under culture conditions. In another example, the culture expansion of the selected MLPSC population provides an MLPSC population characterized by IL2-RA inhibition greater than 75% under culture conditions. In another example, the culture expansion of the selected MLPSC population provides an MLPSC population characterized by IL2-RA inhibition of 60% to 80% under culture conditions.

[0233] Pharmaceutical products and methods of manufacturing the same

[0234] The inventors have also identified a method for manufacturing a drug product by selecting a MLPSC population for culture expansion based on a predetermined level of IL2-RA inhibition under culture conditions. Therefore, in one example, the present disclosure provides a method for manufacturing a drug product comprising a MLPSC population, the method comprising: obtaining a determination of whether a test population of MLPSC has a predetermined level of IL2-RA inhibition under culture conditions, and if the test population of MLPSC has at least a predetermined level of IL2-RA inhibition under culture conditions, then culturing and expanding at least a portion of the test population of MLPSC into a drug product, thereby manufacturing a drug product; or if the MLPSC population has less than a predetermined level of IL2-RA inhibition under culture conditions, then discarding at least a portion of the test population of MLPSC.

[0235] In one example, the method further includes obtaining a determination of whether the test population of MLPSCs has a predetermined level of TNF-R1 under culture conditions, and if the test population of MLPSCs has at least a predetermined level of IL2-RA inhibition under culture conditions and a predetermined level of TNF-R1 under culture conditions, culturing and expanding at least a portion of the test population of MLPSCs into a drug product to manufacture a drug product; or if the MLPSC population has less than a predetermined level of IL2-RA inhibition under culture conditions and less than a predetermined level of TNF-R1 under culture conditions, discarding at least a portion of the test population of MLPSCs.

[0236] In one example, the test population is obtained from a MLPSC population in 3D culture. For example, MLPSC can be cultured in a bioreactor. In one example, the test population is obtained from a MLPSC population preserved at low temperatures. In one example, the test population represents a larger MLPSC population, such as a plurality of MLPSC populations preserved at low temperatures.

[0237] As used herein, the term "predetermined level" refers to the level of a marker that indicates the therapeutic efficacy of a culture-expanded drug product. In one example, the predetermined level is a level of IL-2RA inhibition that indicates the therapeutic efficacy of a culture-expanded drug product. In one example, the predetermined level of IL-2RA inhibition under culture conditions is at least 55%. In one example, the predetermined level of IL-2RA inhibition under culture conditions is at least 56%. In one example, the predetermined level of IL-2RA inhibition under culture conditions is at least 60%. In another example, the predetermined level of IL-2RA inhibition under culture conditions is at least 70%. In one example, the predetermined level of IL-2RA inhibition under culture conditions is 55% to 80%. In another example, the predetermined level of IL-2RA inhibition under culture conditions is 55% to 75%. In another example, the predetermined level of IL-2RA inhibition under culture conditions is 60% to 80%. In another example, the predetermined level of IL-2RA inhibition under culture conditions is 60% to 75%.

[0238] In one example, the predetermined level is the level of TNF-R1 under culture conditions. In one example, the predetermined level of TNF-R1 under culture conditions is at least 80 pg / ml. In another example, the predetermined level of TNF-R1 under culture conditions is at least 100 pg / ml. In another example, the predetermined level of TNF-R1 under culture conditions is at least 105 pg / ml.

[0239] 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 treating inflammatory diseases, such as GvHD. In another example, the predetermined level is predetermined by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In one example, the predetermined level corresponds to an improved survival rate in patients with GvHD.

[0240] In one example, at least a portion of the test population of MLPSCs is cultured and expanded to provide a drug product with a MLPSC population characterized by greater than 70% inhibition of IL2-RA under culture conditions. In another example, at least a portion of the test population of MLPSCs is cultured and expanded to provide a drug product with a MLPSC population characterized by greater than 75% inhibition of IL2-RA under culture conditions.

[0241] In one example, the present disclosure provides a method for manufacturing a MLPSC drug product, such method comprising a first step of providing (e.g., culture expansion (e.g., small-scale or large-scale cell culture) or manufacturing) or obtaining (e.g., receiving and / or purchasing from a third party (including a contract-related third party or a non-contract-related (e.g., 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 obtaining) at least one value of a MLPSC parameter for the test MLPSC population listed in Table A, and a third step of culture expansion (e.g., a portion of a manufacturing batch, culture or run, an entire manufacturing batch, culture or run, or multiple manufacturing batches, cultures or runs) of at least a portion of the test MLPSC population into a MLPSC drug product (e.g., a form or packaging intended for administration described later herein; optionally a cryopreserved MLPSC drug product), thereby manufacturing a MSC drug product. In one example, the value comprises parameter number 1. In another example, the value comprises parameter number 2. In another example, the value includes parameter numbers 1 and 2.

[0242] In one example, such a method includes a second step, which includes obtaining a value of the DP IL2-RA inhibition level under culture conditions, and the third step of such a method includes releasing at least a portion of the DP (i.e., the release standard) if the value of the IL2-RA inhibition level under culture conditions is greater than 55%. In another example, the third step of such a method includes releasing at least a portion of the DP if the value of the IL2-RA inhibition level under culture conditions is greater than 56%. In another example, the third step of such a method includes releasing at least a portion of the DP if the value of the IL2-RA inhibition level under culture conditions is greater than 60%. In another example, the third step of such a method includes releasing at least a portion of the DP if the value of the IL2-RA inhibition level under culture conditions is greater than 65%. In another example, the third step of such a method includes releasing at least a portion of the DP if the value of the IL2-RA inhibition level under culture conditions is greater than 70%. In another example, the third step of such a method includes releasing at least a portion of the DP if the value of the IL2-RA inhibition level under culture conditions is greater than 75%.

[0243] Table A

[0244]

[0245] Compositions of the present disclosure

[0246] In one example, the present disclosure encompasses intermediate MLPSC populations, and the memory of subsequent final products derived from the intermediates. Such intermediates differ from freshly isolated cells in that they are obtained by culture expansion from a population of freshly isolated cells. They also differ from the final composition for administration in that they require further culture expansion to obtain such a composition. Therefore, in one example, the intermediate MLPSC population of the present disclosure has been passaged at least 2 times or at least 3 times. In one example, the intermediate cell population is passaged 1 to 4 times. In one example, the intermediate cell population is cryopreserved.

[0247] 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. Therefore, in one example, the present disclosure relates to a cryopreserved cell intermediate, which includes a culture-expanded mesenchymal lineage precursor or stem cell (MLPSC) population, wherein the MLPSC population is culture-expanded for 2 to 5 passages, and the culture-expanded MLPSC: a) inhibits IL2-RA by at least 55% under culture conditions; and / or b) expresses at least 80pg / ml TNF-R1 under culture conditions. In one example, the MLPSC in the cell intermediate: a) inhibits IL2-RA by at least 60% under culture conditions; and / or b) expresses at least 80pg / ml TNF-R1 under culture conditions. In one example, the MLPSC in the cell intermediate: a) inhibits IL2-RA by at least 70% under culture conditions; and / or b) expresses at least 100pg / ml TNF-R1 under culture conditions. In one example, the MLPSCs express at least 105 pg / ml TNF-R1 under culture conditions.

[0248] In an example of the present disclosure, mesenchymal lineage precursor cells or stem cells and / or their progeny and / or soluble factors derived therefrom are provided and / or applied in the form of a composition. Such compositions are cultured and expanded from intermediate MLPSC populations disclosed herein. In an example, such compositions include pharmaceutically acceptable carriers and / or excipients. Therefore, in an example, the compositions of the present disclosure may include mesenchymal lineage precursors or stem cells cultured and expanded from intermediate populations disclosed herein. In an example, such compositions may be pharmaceutical compositions.

[0249] The terms "carrier" and "excipient" refer to compositions of matter that are routinely used in the art to facilitate storage, administration, and / or biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th edition, Mac Publishing Company (1980)). Carriers can also reduce any undesirable side effects of the active compound. Suitable carriers are, for example, stable, e.g., unable to react with other ingredients in the carrier. In one example, the carrier does not produce significant local or systemic side effects in the recipient at the dose and concentration used for treatment.

[0250] Suitable carriers for the present disclosure include those conventionally used, for example, water, saline, aqueous dextrose, lactose, Ringer's solution, buffered solutions, hyaluronic acid and glycols are exemplary liquid carriers, particularly (when isotonic) for solutions. Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, etc.

[0251] In another example, the carrier is a medium composition, such as a medium in which the cells are grown or suspended. For example, such a medium composition does not cause any side effects in the subject to which it is administered.

[0252] Exemplary carriers and excipients do not adversely affect the viability of the cells and / or the ability of the cells to reduce, prevent or delay metabolic syndrome and / or obesity.

[0253] In one example, the carrier or excipient provides buffering activity to maintain cells and / or soluble factors at a suitable pH to exert biological activity, for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it has minimal interaction with cells and factors and allows rapid release of cells and factors, in which case the compositions of the present disclosure can be produced as liquids for direct application to the bloodstream, for example, by injection, or to tissues or areas surrounding or adjacent to tissues.

[0254] In one example, the composition comprises an effective amount or a therapeutically or preventively effective amount of mesenchymal lineage precursors or stem cells and / or their progeny and / or soluble factors derived therefrom. For example, the composition comprises about 1×10 5 stem cells is about 1×10 9 stem cells or about 1.25×10 3 stem cells is about 1.25×10 7 stem cells / kg (80 kg subject). In one example, the composition comprises 2×10 6 The exact amount of cells to be administered depends on a variety of factors, including the age, weight, and sex of the subject, and the extent and severity of the condition being treated.

[0255] In one example, 50×10 6 Up to 200×10 7 In other examples, 60×10 6 Up to 200×10 6 cells or 75×10 6 Up to 150×10 6 In one example, 75×106 In another example, 150×10 6 cells.

[0256] In one embodiment, the composition comprises greater than 5.00×10 6 In another example, the composition comprises greater than 5.50×10 6 In another example, the composition comprises greater than 6.00×10 6 In another example, the composition comprises greater than 6.50×10 6 In another example, the composition comprises greater than 6.68×10 6 viable cells / mL.

[0257] In one example, the methods of the present disclosure encompass administration of a total dose of 600 million cells. For example, a subject treated according to the present disclosure may receive multiple doses of the above composition, as long as the total dose of cells does not exceed 600 million cells. For example, a subject may 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 may receive 4 doses of 100 million cells. In one example, a subject receives 1 dose of 100 million cells at baseline, followed by three doses of 100 million cells administered once a month for three months. In one example, the dose is 2×10 6 In one example, the dose is 2×10 6 cells / kg, and the subject receives 2 doses or 3 doses. In one example, the dose is 2×10 6 cells / kg, and subjects received more than 3 doses.

[0258] In one example, mesenchymal lineage precursors or stem cells comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of the cell population of the composition.

[0259] The compositions of the present disclosure can be cryopreserved. The cryopreservation of mesenchymal lineage precursor cells or stem cells can be performed using slow freezing methods or rapid freezing protocols known in the art. Preferably, the cryopreservation method maintains similar phenotypes, cell surface markers and growth rates of cryopreserved cells compared to unfrozen cells.

[0260] The cryopreserved composition may comprise a cryopreservation solution. The pH of the cryopreservation solution is generally 6.5 to 8, preferably 7.4.

[0261] The cryopreservation solution may comprise a sterile, pyrogen-free, isotonic solution such as, for example, PlasmaLyte A TM 100mLPlasmaLyte A TM Contains 526 mg sodium chloride, USP (NaCl); 502 mg sodium gluconate (C6H 11 NaO7); 368 mg sodium acetate trihydrate, USP (C2H3NaO2·3H2O); 37 mg potassium chloride, USP (KCl); and 30 mg magnesium chloride, USP (MgCl2·6H2O). It does not contain an antimicrobial agent. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).

[0262] Cryopreservation solutions may include Profreeze TM The cryopreservation solution may additionally or alternatively comprise a culture medium, such as αMEM.

[0263] In order to facilitate freezing, cryoprotectants such as dimethyl sulfoxide (DMSO) are usually added to the cryopreservation solution. Ideally, the cryoprotectant should be non-toxic to cells and patients, non-antigenic, chemically inert, provide high survival rates after thawing and allow transplantation without washing. However, the most commonly used cryoprotectant DMSO shows some cytotoxicity. Hydroxyethyl starch (HES) can be used as a substitute or in combination with DMSO to reduce the cytotoxicity of the cryopreservation solution.

[0264] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein fillers. In one example, the cryopreserved solution includes about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further include one or more of methylcellulose, polyvinyl pyrrolidone (PVP), and trehalose.

[0265] In one embodiment, cells are suspended in 42.5% Profreeze TM / 50% αMEM / 7.5% DMSO and cooled in a controlled-rate refrigerator.

[0266] The cryopreserved composition can be thawed and administered directly to a subject or added to another solution, e.g., a solution comprising HA. Alternatively, the cryopreserved composition can be thawed and the mesenchymal lineage precursors or stem cells resuspended in an alternative carrier prior to administration.

[0267] In one example, the cell composition of the present disclosure may include Plasma-Lyte A, dimethyl sulfoxide (DMSO) and human serum albumin (HSA). For example, the composition of the present disclosure may include Plasma-Lyte A (70%), DMSO (10%), HSA (25%) solution, HSA solution including 5% HSA and 15% buffer.

[0268] In one example, the compositions described herein can be administered as a single dose.

[0269] In some examples, the compositions described herein can be administered in multiple doses. For example, at least 2, at least 3, at least 4 doses. In other examples, the compositions described herein can be administered in at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 doses.

[0270] In one example, the mesenchymal lineage precursor cells or stem cells can be administered systemically. In another example, the compositions of the present disclosure can be administered intravenously.

[0271] In one example, mesenchymal lineage precursor cells or stem cells are administered once a week. For example, mesenchymal lineage precursor cells or stem cells may be administered once a week every two weeks. In another example, mesenchymal lineage precursor cells or stem cells are administered twice a week. In one example, mesenchymal lineage precursor cells or stem cells may be administered once a month. In one example, two doses of mesenchymal lineage precursor cells or stem cells are administered once a week within two weeks. In another example, two doses of mesenchymal lineage precursor cells or stem cells are administered once a week every two weeks. In another example, four doses of mesenchymal lineage precursor cells or stem cells are administered within two weeks, followed by subsequent doses administered monthly. In one example, two doses of mesenchymal lineage precursor cells or stem cells are administered once a week every two weeks, followed by subsequent doses administered monthly. In one example, four doses are administered monthly.

[0272] In one example, the composition of the present disclosure comprises a "clinically proven effective" amount of MLPSCs. In one example, the composition of the present disclosure comprises a "clinically proven effective" amount of MSCs.

[0273] It will be appreciated by those skilled in the art that many changes and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure. Therefore, the embodiments of the present invention are to be considered in all aspects as illustrative and not restrictive.

[0274] The following specific examples should be construed as merely illustrative, and not limiting in any way the remainder of the disclosure. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent.

[0275] Example

[0276] Intermediate stem cell populations and their in vitro expansion for the treatment of inflammatory diseases.

[0277] Donor cell bank generation and analysis

[0278] Carbon dioxide (CO2) is required in culture media for expanded mesenchymal stem cells (ce-MSCs) to maintain a buffer between carbonate and bicarbonate to ensure that the medium is maintained at the physiological pH (7.2-7.4) required for optimal cell growth.

[0279] Donor cell banks (DCBs) containing ce-MSCs were previously produced by expanding MSC populations in a ten-layer cell factory (CF10) with active pretreatment culture at 10±2% CO2. However, when CO2 concentrations are too high, intracellular pH decreases excessively, adversely affecting growth rates and altering the metabolism, productivity, function, and glycosylation profile of mammalian cells. Therefore, the DCB manufacturing method was modified to require MSCs to be seeded in CF10 and the cells to be cultured under passive 5%±2% CO2 pretreatment. In other words, the passive 5%±2% CO2 pretreatment (referred to herein as the "DCB2" method) is different from the previous manufacturing method (referred to herein as the "DCB1" method), which involved active pretreatment with 10%±2% CO2.

[0280] The % inhibition of IL2-RA was measured after co-culture of T cells with mesenchymal stem cells, and inhibition of IL2-RA expression correlates with inhibition of T cell activation, providing an effective measure of therapeutic efficacy (e.g., 100-day survival benefit) for inflammatory diseases such as GvHD. Unexpectedly, the inventors found that cell populations generated using the DCB2 method had increased potency, measured as % inhibition of IL2-RA ( Figure 1 The cell populations generated using the DCB2 method also showed increased expression of TNFR-1 ( Figure 2 ).

[0281] Donor product generation and analysis

[0282] More surprisingly, when the DCB1 and DCB2 batches were further expanded into clinical drug products (DPs), the 100-day survival observed in patients administered the DCB2-derived DP (74%) was significantly higher than the 100-day survival observed in patients administered the DCB1-derived DP (56%). The improvement in 100-day survival was even more pronounced for patients with severe grade D disease; 72% for patients administered the DP2-derived DP and 44% for patients administered the DP1-derived DP. The results are shown in Table 1; the survival benefit is also presented graphically in Figure 3 middle.

[0283] Table 1 - Improved survival for all patients with acute GVHD

[0284]

[0285] The inventors subsequently discovered that a beneficial effect on survival could be predicted and therefore a DCB population could be selected for expansion based on IL2-RA inhibition %. Interestingly, when the DCB1 and DCB2 batches were further expanded into clinical DPs, the average IL2RA expression % for the DP derived from DCB2 was 76.5%, while the average IL2RA expression % for the DP derived from DCB1 was 63.7%. This is an important finding because patients administered with a DP expanded from a DCB1 intermediate and characterized by an average IL2-RA% of 63.7% were associated with a 100-day survival rate of 56%, while for patients with severe grade D disease, this survival rate was reduced to 44%. In contrast, patients administered with a DP expanded from a DCB2 intermediate and characterized by an average IL2-RA% of 76.5% were associated with a 100-day survival rate of 74%, while for patients with severe grade D disease, this survival rate was reduced to 72% ( Figure 4 ; p=0.019). Indeed, the beneficial effect on survival was most pronounced for patients with severe grade D disease, with a 100-day survival rate of 74% recorded for patients administered DP expanded from DCB2 intermediates, whereas this rate decreased to 46% for patients treated with DP expanded from DCB1 intermediates ( Figure 5 ; p=0.012).

[0286] Since the DP of DCB1 and DCB2 are generated in the same manner, thereby controlling for any possibility of downstream processes affecting the final DP characteristics, the inventors' findings highlight the importance of characterizing cell intermediates before they are expanded into DP, especially in the context of GvHD therapy, as failure to do so can have a significant impact on the overall survival of patients. The inventors' findings also significantly improve the methods of selecting cell intermediates for large-scale culture expansion into drug products, thereby reducing the risk of wasting resources by expanding cell populations that do not meet the necessary potency standards.

[0287] Overview

[0288] The donor cell bank manufacturing process provides a "memory" for subsequent final products derived from the DCB. Changes in DCB manufacturing are associated with higher IL2-RA inhibition% in both DCB and DP. The increase in IL2-RA inhibition% was accompanied by a significant improvement in survival, especially in grade D patients. These findings lay the foundation for methods of selecting and analyzing DCBs for therapy, especially the treatment of inflammatory conditions such as GvHD.

[0289] Suppression of IL-2RA on peripheral blood mononuclear cells (PBMCs) by mesenchymal lineage or precursor stem cells (MLPSCs)

[0290] Preparation of MLPSCs

[0291] Cryopreserved human MLPSCs were thawed and resuspended in IL-2Ra MLPSC medium at a concentration of 4.00E+05 viable MLPSCs / mL. Viability was assessed using trypan blue. MLPSCs were then seeded in 24-well plates. 500 μL of 4.00E+05 viable MLPSCs / mL were added to each well. Cells were incubated at 37°C±2°C, 5%±2% CO2 for no more than 24±1 hours. Before co-culturing with PMBCs, it is necessary to check whether the cells are attached to the plate and ensure that the cell morphology is "fibroblast-like" long and flat.

[0292] Preparation of PMBCs

[0293] PMBCs were thawed and resuspended in co-culture medium (50% IL-2R AMLPSC medium and 50% DMEM) at a concentration of 2.00E+06 viable cells / mL. Viability assessment was performed using trypan blue.

[0294] Co-culture of MLPSCs and PBMCs

[0295] Stimulated PMBCs are prepared by adding CD3 and CD28 antibodies (final concentration is 4 μg / ml) to a portion of 2.00E+06 live PMBCs. A portion of live PMBCs not stimulated with CD3 and CD28 antibodies is kept as a negative control. Then 500 μL of stimulated PMBCs are added to the test wells comprising Ce-MLPSCs (intermediate colonies or drug products).

[0296] PMBC stimulated alone were used as positive control, and unstimulated PMBC were used as negative control. 500 μL of the corresponding control sample and 500 μL of co-culture medium were added to the control wells of the culture plate.

[0297] MLPSCs and PMBCs were then placed in an incubator at 37°C ± 2°C, 5% ± 2% CO2 for 72 to 74 hours. After incubation, PMBC samples were slowly harvested, centrifuged, and resuspended in lysis buffer to prepare cell lysates. The lysates were then placed on wet ice and incubated for 15 minutes.

[0298] For example, Ce-MSCs (intermediate population or drug product) were co-cultured with unstimulated or stimulated (CD3; CD28 antibodies) peripheral blood mononuclear cells (PBMCs) as shown below. These conditions represent a ratio of approximately 1 stem cell: 5 PBMCs:

[0299]

[0300] The plates were incubated at 37°C for 60-84 hours, then cells were harvested, lysed and IL2-RA levels were assessed using a commercially available ELISA kit according to the manufacturer's instructions (R&D systems).

[0301] IL-2RA ELISA

[0302] IL-2RA standards at 2500 to 78 pg / ml were prepared and added to the ELISA plate along with control and co-culture lysate samples. Anti-IL-2RA antibody conjugated to HRP was then added to all wells. The plate was incubated at ambient temperature for approximately 3 hours with slow shaking at 200 rpm on an orbital shaker. After incubation, substrate was added to each well. The plate was incubated again at ambient temperature in the dark for 20 minutes before adding stop solution to stop the reaction. The absorbance in the wells was read at 450 nm within 30 minutes with wavelength correction at 570 nm.

[0303] To calculate the amount of IL-2RA, standard analysis software (e.g., BioTek) was used to calculate the A450nm-570nm value for each well. The average absorbance of the 0 pg / ml negative control was subtracted from the A450nm-570nm value. The IL-2RA concentration in the test sample was obtained by interpolation calculation based on the standard curve of the IL-R2A standard. The % inhibition of IL-2RA on PBMC by MLPSC was calculated using the following formula: % inhibition = [[1-(co-cultured MLPSC PBMC / stimulated PBMC)] × 100].

[0304] It will be appreciated by those skilled in the art that many changes and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Therefore, the embodiments of the present invention are considered to be illustrative and not restrictive in all aspects.

[0305] This application claims priority to AU2022901890 filed on July 5, 2022, the disclosure of which is incorporated herein by reference.

[0306] All publications discussed above are incorporated herein in their entirety.

[0307] Any discussion of documents, acts, materials, devices, articles or the like has been included in this specification solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Claims

1. A method for selecting a cell population for culture expansion, the method comprising: (i) obtaining a population of mesenchymal precursor lineage or stem cells (MLPSC); (ii) culturing the MLPSC population in a culture medium; (iii) determining the level of IL2-RA inhibition under the culture conditions; and (iv) selecting a population of MLPSCs that inhibit IL2-RA by at least 55% under the culture conditions for further expansion.

2. The method of claim 1, comprising selecting a population of MLPSCs that suppress IL2-RA by at least 60% under culture conditions for further expansion.

3. The method of claim 1 or claim 2, wherein the MLPSC population selected for further expansion also expresses at least 80 pg / ml TNF-R1 under culture conditions.

4. The method of any one of claims 1 to 3, further comprising culturing and expanding the selected MLPSC population to provide a pharmaceutical composition.

5. The method of any one of claims 1 to 4, wherein culture expansion of the selected MLPSC population provides a MLPSC population characterized by greater than 60%, greater than 70% inhibition of IL2-RA under culture conditions.

6. A method for determining therapeutic efficacy of an intermediate population of mesenchymal precursor lineage or stem cells (MLPSC): (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% IL2-RA inhibition under culture conditions indicates therapeutic efficacy.

7. The method of claim 6, wherein inhibition of IL2-RA greater than 60%, greater than 70% under culture conditions indicates therapeutic efficacy.

8. The method of claim 6 or claim 7, further comprising determining the level of TNF-R1 expression under culture conditions, wherein expression of at least 80 pg / ml TNF-R1 under culture conditions is indicative of therapeutic efficacy.

9. The method of any one of claims 6 to 8, wherein after determining the efficacy of the treatment, the intermediate population of MLPSCs is culture-expanded to provide a pharmaceutical composition.

10. The method of claim 9, wherein the pharmaceutical composition comprises a population of MLPSCs characterized by 60% to 80% inhibition of IL2-RA under culture conditions.

11. A method for treating a subject having an inflammatory disease, the method comprising administering to a subject in need thereof a composition comprising a culture-expanded population of mesenchymal lineage precursor or stem cells (MLPSCs), wherein the MLPSCs are culture-expanded from a population of MLPSCs that inhibit IL2-RA by at least 55% under culture conditions.

12. The method of claim 11, wherein culture-expanded MLPSCs comprising the administered composition suppress IL2-RA by at least 56%, at least 60% under culture conditions.

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

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

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

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

17. The method or cell intermediate of any one of claims 1 to 16, wherein the MLPSCs are cultured and expanded in 5% + / - 2% CO2.

18. The method or cell intermediate of claim 17, wherein the MLPSCs are cultured and expanded by passive preconditioning in 5% + / - 2% CO2.

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

20. The method of claim 19, wherein the GvHD is grade D.

21. The method of claim 19, wherein the GvHD is chronic GvHD.

22. The method of any one of claims 11 to 21, wherein treatment improves 100-day survival to greater than 60%, greater than 70%.

23. The method of any one of claims 11 to 22, wherein the subject is refractory to steroid immunosuppressants and / or biologic therapy.

24. The method of any one of claims 11 to 23, wherein the subject receives at least two doses of the composition.

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

26. A method of manufacturing a pharmaceutical product comprising a population of mesenchymal lineage precursor or stem cells (MLPSCs), the method comprising: A determination is obtained as to whether a test population of MLPSCs has a predetermined level of IL2-RA inhibition under culture conditions, and if the test population of MLPSCs has at least the predetermined level of IL2-RA inhibition under culture conditions, at least a portion of the test population of MLPSCs is cultured and expanded as a drug product to manufacture the drug product; or if the test population of MLPSCs has less than the predetermined level of IL2-RA inhibition under culture conditions, at least a portion of the test population of MLPSCs is discarded.

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

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

29. The method of any one of claims 26 to 28, further comprising obtaining a determination of whether a test population of MLPSCs has a predetermined level of TNF-R1 under culture conditions, and if the test population of MLPSCs has at least the predetermined level of IL2-RA inhibition under culture conditions and has the predetermined level of TNF-R1 under culture conditions, culturing and expanding at least a portion of the test population of MLPSCs into a drug product to manufacture the drug product; or if the MLPSC population has less than the predetermined level of IL2-RA inhibition under culture conditions and has less than the predetermined level of TNF-R1 under culture conditions, discarding at least a portion of the test population of MLPSCs.

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

31. The method of any one of claims 26 to 30, wherein culturing at least a portion of the test population of MLPSCs provides a population of MLPSCs characterized by greater than 60%, greater than 70% inhibition of IL2-RA under culture conditions for a drug product.

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