Third-line treatment for GVHD

Abstract

This disclosure relates to a mesenchymal lineage precursor or stem cell-mediated method for treating severe graft-versus-host disease (GvHD) in subjects refractory to at least two prior-line therapies.

Description

[Technical Field]

[0001] This disclosure relates to a mesenchymal lineage precursor or stem cell-mediated method for treating severe graft-versus-host disease (GvHD) in subjects refractory to at least two prior-line therapies. [Background technology]

[0002] Acute and chronic graft-versus-host disease (GvHD) is an immunological disorder that is a leading cause of non-relapse mortality (NRM) after allogeneic stem cell transplantation. Acute GvHD affects 40%–60% of patients and targets the skin, liver, and gastrointestinal tract.

[0003] Gravity-versus-host disease (GvHD) remains a significant unmet clinical need, particularly in patients who have become refractory to currently approved therapies. Indeed, these patients experience high mortality rates. Clearly, there is an unmet therapeutic need in the art for treating GvHD, especially in patients at high risk of poor outcomes. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent No. 5,486,359 [Patent Document 2] U.S. Patent No. 6,251,295 [Patent Document 3] International Publication No. 2016139340 [Non-patent literature]

[0005] [Non-Patent Document 1] J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984) [Non-Patent Document 2] J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989) [Non-Patent Document 3] TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Vol. 1 and Vol. 2, IRL Press (1991) [Non-Patent Document 4] DM Glover and BD Hames (eds.), and FM Ausubel et al. (eds.), *Current Protocols in Molecular Biology*, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date). [Non-Patent Document 5] Ed Harlow and David Lane (eds.), *Antibodies: A Laboratory Manual*, *Cold Spring Harbour Laboratory*, (1988). [Non-Patent Document 6] JE Coligan et al. (eds.), *Current Protocols in Immunology*, John Wiley & Sons (including all updates to date). [Non-Patent Document 7] Lee, S., (2017) Blood., 129(1): pp. 30-37. [Non-Patent Document 8] Jagasia et al., (2015) Biol Blood Marrow Transplant., 21:389-401 pp. [Non-Patent Document 9] Remington's Pharmaceutical Sciences, Volume 16, Mac Publishing Company (1980) [Non-Patent Document 10] Jagasia et al., (2020) Blood., 135: pp. 1739-1749 [Non-Patent Document 11] Zeiser et al., (2020) N Engl J Med., 382: pp. 1800-1810 [Non-Patent Document 12] Abedin et al., (2021) British Journal of Haematology., 195: pp. 429-443 [Summary of the Invention] [Problem to be Solved by the Invention]

[0006] The present inventors have surprisingly confirmed that mesoderm lineage precursors or stem cells are particularly effective for treating GvHD in subjects who have failed two prior lines of therapy. In fact, a marked improvement in survival has been observed in GvHD patients administered MSCs as third-line therapy. [Means for Solving the Problem]

[0007] Accordingly, in a first aspect, the present disclosure relates to a method for treating graft-versus-host disease (GvHD) in a human subject in need thereof, comprising the step of administering to the subject a composition comprising mesoderm lineage precursors or stem cells (MLPSCs), wherein the MLPSCs are administered to the subject as third-line therapy.

[0008] In another aspect, the present disclosure relates to a method for treating graft-versus-host disease (GvHD) in a human subject, comprising i) selecting a subject having GvHD that has failed two lines of therapy; ii) administering to the subject a composition comprising mesoderm lineage precursors or stem cells (MLPSCs), wherein the MLPSCs are administered to the subject as third-line therapy A method comprising

[0009] In one example, the subject treated according to the method disclosed herein is refractory to steroids and second-line therapy. In one example, the second-line therapy is ruxolitinib. In another example, the second-line therapy is a biologic. Exemplary biologics include alemtuzumab, basiliximab, or tocilizumab.

[0010] In one example, MLPSCs in the composition inhibit IL-2Rα expression by ≥60%, and the inhibition of IL-2Rα expression is determined by obtaining a population of cells containing cultured, cryopreserved, and thawed MLPSCs; co-culturing MLPSCs in culture medium with a population of cells containing T cells; and determining the level of inhibition of IL-2Rα expression.

[0011] In one example, treatment reduces the risk of death in the subject. For instance, death may be a 6-month non-recurrence mortality (NRM).

[0012] In one example, the target group is children. In another example, the target group is ≥12 years old.

[0013] In another example, the subject has acute graft-versus-host disease (aGvHD). In yet another example, the subject is: - Grade B, C, or D according to the IBMTR Severity Scale; - Grade II GvHD according to the Glucksberg Severity Scale; - Grade III / IV GvHD according to the Glucksberg Severity Scale; - Minnesota High-Risk GvHD It is classified as one or more of the following.

[0014] In one example, the subject has chronic GvHD.

[0015] In one example, the subject has multiple organ lesions.

[0016] In one case, the subject has inflammatory bowel disease (IBD). In another case, the IBD is Crohn's disease or ulcerative colitis. In yet another case, the IBD is Crohn's disease. In yet another case, the Crohn's disease is present in the subject's rectum and / or colon. In yet another case, the subject's IBD is refractory to one or more of adalimumab, certolizumab pegol, vedolizumab, or ustekinumab.

[0017] In one example, the treatment increases the probability of subjects surviving for at least 28 days after the start of treatment. In one example, the treatment increases the probability of subjects surviving for at least 100 days, preferably at least 180 days, after the start of treatment. In one example, the treatment increases the probability of subjects surviving for at least 100 days after the start of treatment. In one example, the probability of survival of subjects is increased compared to subjects who do not receive MLPSC. In one example, the probability of survival of subjects is greater than 40%, greater than 50%, or preferably greater than 60% after the start of treatment. In one example, the probability of survival of subjects is greater than 40% after the start of treatment. In one example, the probability of survival of subjects is greater than 50% after the start of treatment. In one example, the probability of survival of subjects is greater than 60% after the start of treatment. In one example, the probability of survival of subjects is about 30% to about 80% after the start of treatment. In one example, the probability of survival of subjects is about 40% to about 70% after the start of treatment. In one example, the probability of survival of subjects is about 50% to about 65% after the start of treatment. In one example, the probability of survival after the start of treatment is approximately 60% to 70%.

[0018] For example, MLPSCs are cultured and amplified from a population of STRO-1+ MLPSCs. For instance, a population of STRO-1+ MLPSCs may contain approximately 0.1% to 75% STRO-1+ cells.

[0019] For example, MLPSCs are mesenchymal stem cells (MSCs).

[0020] In one example, the composition further comprises Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA). In another example, the composition is 6.68 × 10⁻⁶. 6It contains more than 1 / mL of live cells. In one example, the composition is administered intravenously.

[0021] In one example, the subject receives at least two doses of medication. In another example, the treatment involves administering medication twice a week for four weeks. In yet another example, the treatment involves administering medication twice a week for eight weeks. In one example, the medications are administered at least three days apart. [Brief explanation of the drawing]

[0022] [Figure 1] This figure shows the Kaplan-Meier survival estimates up to day 100 for 71 patients with acute GVHD treated with MSCs as a third-line treatment after failure of second-line agents, including corticosteroids and ruxolitinib. [Figure 2] This figure shows the Kaplan-Meier survival estimates up to day 100 for 71 patients with acute GVHD treated with MSC as a third-line treatment, stratified based on response or non-response at day 28. [Figure 3A-3B] A. This figure shows the Kaplan-Meier survival rate estimate up to day 100 for MSCs provided as a second-line treatment (n=54) in children under 18 years of age with steroid-refractory acute GvHD. B. This figure shows the Kaplan-Meier survival rate estimate up to day 100 for MSCs provided as a third-line treatment (n=35) in children under 18 years of age with steroid-refractory acute GvHD. [Modes for carrying out the invention]

[0023] General techniques and definitions Unless otherwise specifically defined, all technical and scientific terms used herein shall be construed to have the same meaning as those commonly understood by those skilled in the art (e.g., in cell culture, molecular biology, stem cell culture, immunology, clinical research, general medicine, and biochemistry).

[0024] Unless otherwise indicated, the cell culture techniques and assays used in this disclosure are standard procedures well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Vol. 1 and 2, IRL Press (1991), DM Glover and BD Hames (eds.); and FM Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988); and JE Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons. This is described and explained through the source's documentation (including all updates to date).

[0025] The terms "and / or," for example, "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as providing explicit support for both meanings or either meaning.

[0026] As used herein, the term “about” means ±10%, more preferably ±5%, of the specified value, unless otherwise stated.

[0027] The terms “level” and “amount” are used to specify the amount of a particular substance in a cell preparation. For example, the level of a particular substance may be specified using a specific concentration, mass, percentage (e.g., v / v%), or ratio. In one example, the level is expressed in terms of how much of a particular marker is expressed by the cells disclosed herein under culture conditions. In one example, expression refers to 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 into their culture medium under culture conditions.

[0028] In one example, the level of a particular marker is determined under culture conditions. The term "culture conditions" is used to refer to cells growing under culture. In one example, culture conditions refer to a population of actively dividing cells. Such cells may, in one example, be in the exponential growth phase. In another example, the cells may be in the stationary phase.

[0029] For example, the culture conditions include co-culturing the MLPSC population disclosed herein with a second cell population, such as a population containing peripheral blood mononuclear cells (PBMCs). For example, the co-culturing includes culturing the MLPSC population disclosed herein with a population of activated PBMCs. For example, PBMCs may be activated using anti-CD3 and anti-CD28 antibodies before co-culturing with the MLPSC population disclosed herein.

[0030] In one example, the "culture conditions" include co-culturing MLPSCs and T cells in a ratio of approximately 1 MLPSC:2 T cells, or less. For example, 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 approximately 30-84 hours after cell culture under the culture conditions.

[0031] In one example, the level of a particular marker may be determined by taking a sample of cell culture medium and measuring the level of the marker in the sample. In another example, the level of a particular marker may be determined by taking a sample of cells and measuring the level of the marker in the cell lysate. Those skilled in the art will understand that secreted markers can be measured by sampling culture medium, while markers expressed on the surface of cells can be measured by assessing a sample of cell lysate. In one example, the sample is taken when the cells are in the exponential growth phase. In another example, the sample is taken at least 2 or 3 days after being cultured. In yet another example, the sample is taken approximately 30–84 hours after co-culture. In one example, the sample is taken from a co-culture of MLPSCs and activated PBMCs. In this example, the cell sample may be lysed and the level of the marker may be determined. For example, the level of IL2-RA can be determined using various methods, such as a method based on enzyme-linked immunosorbent assay (ELISA). In one example, ELISA is used. (i) Adding sample diluent to each well of a microplate pre-coated with a monoclonal antibody specific to IL2-RA; (ii) Adding the co-culture sample to the wells of a microplate pre-coated with a monoclonal antibody specific to IL2-RA; (iii) Incubating the microplate for a sufficient time to allow the IL2-RA-specific monoclonal antibody to specifically bind to any IL2-RA in the sample; (iv) The process of washing the microplate; (v) Adding IL2-RA conjugate to the wells; (vi) Incubating the microplate for a sufficient time to allow the conjugate to specifically bind to any captured IL2-RA; (vii) The process of washing the microplate; (viii) Adding the substrate solution to the wells; (ix) Incubate the microplate for a sufficient amount of time for color development; (x) Adding stop solution to wells; (xi) A process of reading the optical density with a microplate reader set to 450 nm, with wavelength correction at 570 nm; (xii) Step to determine the concentration of IL2-RA Includes.

[0032] In another example, IL2-RA levels are determined using fluorescence-activated cell sorting (FACS) with a suitable antibody such as anti-CD25. Additional antibodies may be employed if it is necessary to distinguish CD25+ cell types.

[0033] In one example, the level of IL2-RA under co-culture is compared to the level of IL2-RA in a culture population of activated PBMCs. The levels of IL2-RA are then compared to provide the level of IL2-RA inhibition under co-culture. For example, the level of IL2-RA may be inhibited by at least 60% under co-culture (i.e., MLPSC:activated PBMC) compared to single culture (activated PBMC only). Those skilled in the art will understand other suitable methods for determining the level of IL2-RA under culture conditions.

[0034] Culturing and growing cells from cryopreserved intermediates means thawing cells that have been subjected to cryogenic freezing and culturing them in vitro under conditions suitable for cell growth.

[0035] In one example, the "level" or "amount" of a specific marker, such as IL2-RA or TNF-R1, is determined before the cells are cryopreserved. For example, the level may be determined after the first 2-5 passages of the cells. In another example, the level or amount of a specific marker is determined after the first cryopreservation of the cells. In yet another example, the level is determined after the second cryopreservation of the cells. For example, cells may be cultured to provide intermediates, cryopreserved, thawed, then re-seeded under culture conditions so that the level of a specific marker can be determined under culture conditions, and cryopreserved again.

[0036] "Isolated" or "purified" means cells separated from at least some components of their natural environment. This term includes the overall physical separation of cells from their natural environment (e.g., removal from a donor). The term "isolated" includes alterations in the relationship between a cell and its directly adjacent cells, such as by dissociation. The term "isolated" does not refer to cells within a tissue section. When used to refer to a population of cells, the term "isolated" includes the population of cells resulting from the proliferation of isolated cells as described in this disclosure.

[0037] The terms “passaging,” “passaging,” or “subculturing” are used in the context of this disclosure to refer to known cell culture techniques used to keep cells viable and growing under culture conditions for extended periods so that the cell number can continuously increase. The degree to which a cell line has been subculturished is often expressed as “passage number,” which is generally used to refer to the number of times the cells have been subculturished. In one example, one passaging includes the steps of removing non-adherent cells and leaving adherent mesenchymal lineage precursors or stem cells. Such mesenchymal lineage precursors or stem cells may then be dissociated from the substrate or flask (e.g., by using a protease such as trypsin or collagenase), culture medium may be added, washing may be optionally performed (e.g., by centrifugation), and then the mesenchymal lineage precursors or stem cells may be re-seed or re-seeded into one or more culture vessels containing a larger overall surface area. The mesenchymal lineage precursors or stem cells may then continue to grow under culture. In another example, the method for removing non-adherent cells includes a non-enzymatic treatment step (e.g., using EDTA). In one example, mesenchymal lineage precursors or stem cells are passaged at or near the confluence point (e.g., about 75% to about 95% confluence). In another example, mesenchymal lineage precursors or stem cells are seeded at a concentration of about 10%, about 15%, or about 20% cells / ml of the culture medium.

[0038] As used in the context of this disclosure, the terms “medium” or “media” include components of the environment surrounding cells under culture. The medium is intended to contribute to and / or provide suitable conditions for cell growth. The medium may be solid, liquid, gaseous, or a mixture of phases and materials. The medium may include liquid growth media, as well as liquid media that do not sustain cell growth. An example of a gaseous medium includes a gaseous phase to which cells growing on a petri dish or other solid or semi-solid support are exposed.

[0039] In one example, this disclosure includes selecting a particular subject having GvHD for treatment with the MLPSC composition disclosed herein. In one example, a subject having GvHD that has failed two lines of therapy is selected for treatment. For example, a GvHD subject that is refractory to steroids and second-line therapy is selected for treatment. In one example, a GvHD subject that is refractory to steroids and ruxolitinib is selected for treatment.

[0040] In one example, a subject experiences a reduced risk of death after treatment with MLPSC as a third-line therapy. In another example, the risk reduction may be compared to the risk of death in a subject who did not receive MLPSC. In yet another example, the risk reduction is compared to the risk of death in the same subject before receiving MLPSC.

[0041] In one example, death is defined as a "non-relapse death." The term "non-relapse death" refers to death without relapse or progressive disease. In one example, the subject has a reduced risk of non-relapse death for six months. In another example, the subject has a reduced risk of non-relapse death for one year.

[0042] In one example, treatment increases the overall survival rate of the subject. In another example, treatment increases the probability that a subject will survive for at least 100 days after the start of treatment. In yet another example, treatment increases the probability that a subject will survive for at least 180 days after the start of treatment. In one example, the increase in probability is determined compared to a subject who does not receive treatment for MLPSC.

[0043] As used herein, the term “subject” refers to a human subject. For example, a subject may be an adult. In another example, a subject may be a child. In yet another example, a subject may be a young adult. In one example, a subject may be a pediatric subject. Pediatrics is a branch of medicine that deals with the care of infants, children, and adolescents. In one example, a subject is under 25 years of age. In another example, a subject is under 21 years of age. In yet another example, a subject is under 18 years of age. In one example, a pediatric subject may range in age from birth to 17 years of age.

[0044] Terms such as "subject," "patient," or "individual" may be used interchangeably in this disclosure in context.

[0045] Subjects treated according to the methods disclosed herein are refractory to a particular therapy. The term “refractory” is used in the context herein to refer to GvHD that has ceased to respond to a particular therapy. For example, a subject with steroid-refractory GvHD has GvHD that has ceased to respond to steroid treatment. In one example, a subject is considered refractory to a series of therapies if their GvHD worsens within three days of therapy. In another example, a subject is considered refractory to a series of therapies if their GvHD worsens within seven days of therapy.

[0046] Subjects may be refractory to multiple lines of therapy. For example, a GvHD subject may be refractory to steroid therapy and second-line therapy. "Line of therapy" is used in the context of this disclosure (and clinically) to describe the sequence in which various therapies are given to a patient with GvHD as their disease progresses. For example, subjects treated herein may be refractory to first-line steroid therapy and second-line therapy. In one example, a subject's GvHD may become refractory to steroids before second-line therapy is administered. In another example, a subject may be treated with steroids in combination with second-line therapy and become refractory to both lines of therapy before being treated with an MLPSC disclosed herein.

[0047] In the context of this disclosure, “therapeutic efficacy” is used to mean MLPSCs and compositions disclosed herein that can treat, inhibit, and / or block a disease. For example, therapeutically effective MLPSCs and compositions disclosed herein can treat, inhibit, and / or block GvHD in patients who have failed two prior lines of therapy. In one example, therapeutically effective MLPSCs and compositions disclosed herein increase 100-day survival in subjects with GvHD who have failed two prior lines of therapy.

[0048] The term “clinically proven” (used independently or to modify the term “effective”) means that efficacy has been demonstrated by a clinical trial, and that the clinical trial meets the approval criteria of the U.S. Food and Drug Administration, EMEA, or the regulatory body of the equivalent country. For example, a clinical study could be a randomized, double-blind study of appropriate size used to clinically demonstrate the effect of a composition. In one example, a clinically proven effective dose is the dose demonstrated by a clinical trial to meet a specified endpoint. In one example, the endpoint is protection from death. In other words, the endpoint increases survival. For example, 100-day survival may increase when the treatment is administered in accordance with this disclosure.

[0049] Accordingly, the terms “clinically proven efficacy” and “clinically proven effectiveness” may be used in the context of this disclosure to refer to the doses, dosage regimens, treatments, or methods disclosed herein. Efficacy may be measured based on changes in the course of the disease in response to administration of the compositions disclosed herein. For example, the compositions disclosed herein are administered to a subject in an amount and over time sufficient to induce improvement, preferably sustained improvement, in at least one indicator reflecting the severity of GvHD. Various indicators reflecting the severity of the disease may be assessed to determine whether the amount and time of treatment are sufficient. Such indicators include, for example, clinically recognized indicators of disease severity or symptoms. In one example, the degree of improvement is determined by a physician capable of making this determination based on signs, symptoms, or other test results (e.g., MAP; Reg3α and ST2 levels; skin % BSA; oral score; eye score of at least 1 point; skin characteristic score; gastrointestinal score; liver score; pulmonary symptom score; pulmonary FEV1 score; joint and fascia score; and / or reproductive tract score).

[0050] In one example, a clinically proven effective dose improves patient survival. In another example, a clinically proven effective dose reduces the risk of death in the subject, e.g., non-recurrence death. In yet another example, a clinically proven effective dose increases 100-day survival. In one example, the method of this disclosure administers a clinically proven effective dose of the composition disclosed herein. In one example, a clinically proven effective dose is characterized by an overall survival rate of >40% 100 days after treatment. In one example, a clinically proven effective dose is characterized by an overall survival rate of >50% 100 days after treatment. In one example, a clinically proven effective dose is characterized by an overall survival rate of >55% 100 days after treatment. In one example, a clinically proven effective dose is characterized by an overall survival rate of 40% to 80% 100 days after treatment. In one example, a clinically proven effective dose is characterized by an overall response rate of >40%. In one example, a clinically proven effective dose is characterized by an overall response rate of >50%.

[0051] For example, the compositions of this disclosure include genetically unmodified mesenchymal precursor lines or stem cells. As used herein, the term “genetically unmodified” refers to cells that have not been modified by nucleic acid transfection. To avoid misunderstanding, in the context of this disclosure, mesenchymal precursor lines or stem cells transfected with protein-encoding nucleic acids will be considered genetically modified.

[0052] The term “total dose” is used in the context of this disclosure to refer to the total number of cells received by a subject treated in accordance with this disclosure. In one example, the total dose consists of one administration of cells. In another example, the total dose consists of two administrations of cells. In yet another example, the total dose consists of three administrations of cells. In yet another example, the total dose consists of four or more administrations of cells. For example, the total dose may consist of two to four administrations of cells.

[0053] The term “conditioned medium” is used in the context of this disclosure to refer to a medium obtained from MLPSCs under culture conditions. Such a medium contains MLPSC secretomes, proteins detached from the surface of MLPSCs, and other particles such as extracellular vesicles. The conditioned medium of this disclosure contains extracellular vesicles and / or secreted metabolites such as prostaglandin E2. In certain examples, this disclosure relates to the administration of extracellular vesicles such as exomes obtained from conditioned medium obtained from MLPSCs under culture conditions. In one example, the conditioned medium is obtained when the MLPSCs are in the exponential growth phase. In another example, the conditioned medium is obtained at least 2 or 3 days after culturing. In yet another example, the conditioned medium is obtained approximately 30–84 hours after culturing.

[0054] Throughout this specification, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of the specified element, integer, or process, or group of elements, integers, or processes, but not the exclusion of any other element, integer, or process, or group of elements, integers, or processes.

[0055] Throughout this specification, unless otherwise specifically stated or contextually required, any reference to a single process, composition of a substance, group of processes, or group of compositions of a substance shall be construed as encompassing one or more (i.e., one or more) of those processes, compositions of a substance, groups of processes, or groups of compositions of a substance.

[0056] Those skilled in the art will understand that the disclosures described herein are open to variations and modifications other than those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure includes all the processes, properties, compositions, and compounds mentioned or shown herein, individually or collectively, as well as any combination or any two or more of such processes or properties.

[0057] This disclosure should not be limited in scope by the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure, as described herein.

[0058] Any example disclosed herein shall be construed as to apply mutatis mutandis to any other example unless otherwise specifically noted.

[0059] Graft-versus-host disease The methods described herein encompass the treatment of graft-versus-host disease (GvHD). GvHD is an immunological disorder that is a major limiting factor in the success and availability of allogeneic bone marrow or stem cell transplantation. GvHD occurs in acute (aGvHD) or chronic (cGvHD) forms. Acute GvHD usually appears within 100 days after bone marrow or stem cell transplantation. Chronic GvHD generally appears later than aGvHD (>100 days after transplantation) and has some characteristics of an autoimmune disease. It may develop anew after resolution of aGvHD or as an extension of aGvHD. Chronic GvHD can cause several often debilitating symptoms, including widespread skin rashes, painful stomatitis, shortness of breath, and pain in the limbs and joints.

[0060] The inventors have confirmed that the MLPSC composition of this disclosure is remarkably effective as a third-line therapy for GvHD. The remarkable nature of our findings stems, at least in part, from the very poor prognosis in patients with GvHD who are refractory to two prior-line therapies. Therefore, the method of this disclosure is effective in a subset of patients with GvHD. These subjects are refractory to two prior-line therapies. In one example, these subjects have steroid-refractory GvHD. For example, a GvHD subject may be refractory to steroids and second-line therapy. In one example, the subject is refractory to steroids and ruxolitinib. In another example, the subject is refractory to steroids and biologics. In one example, the biologic is alemtuzumab, basiliximab, or tocilizumab. In one example, the steroid is a corticosteroid. In another example, the steroid is a glucocorticoid. In yet another example, the steroid is prednisone. In one case, the patient with GvHD is refractory to corticosteroids and second-line therapies such as ruxolitinib. In another case, the patient with GvHD is refractory to prednisone and second-line therapies such as ruxolitinib. In yet another case, the patient has GvHD that is refractory to steroid therapy and is intolerant to ruxolitinib.

[0061] In one example, this disclosure encompasses the treatment of a patient with acute GvHD. In another example, this disclosure encompasses the treatment of a patient with chronic GvHD. In one example, GvHD is present in the stomach and / or intestines of the subject as damage to these organs. In this example, inflammation at various sites may occur along the stomach and / or intestines, and in certain specific examples, it may be preferable to directly administer treatment to one or more of the inflammations at these sites.

[0062] In one example, this disclosure encompasses the use of MLPSC as a third-line therapy for treating subjects with severe GvHD. In one example, severe GvHD is determined based on the subject's MAGIC algorithm probability (MAP). MAP is a validated analysis that combines serum concentrations of two biomarkers, regenerated islet protein 3-alpha (Reg3α) and soluble interleukin 1 receptor-like 1 (ST2), into a single value that predicts the response to therapy at 28 days and long-term outcomes such as 6-month relapse-free mortality (NRM). MAP is,

[0063]

number

[0064] It is calculated by, in the formula,

[0065]

number

[0066] is the predicted probability of NRM over 6 months (Hartwell et al.; Major-Monfried et al.). The more severe the GvHD, the higher the MAP score. Therefore, in one example, “severe GvHD” as used in this disclosure is defined based on MAP. In one example, a subject has a MAP greater than 0.16, preferably greater than 0.2, and more preferably greater than 0.25. A MAP score of ≥0.291 is a validated threshold for identifying GvHD patients with the most severe disease (Hartwell et al.; Major-Monfried et al.). For example, patients with a MAP score of ≥0.291 are at high risk of non-response to treatment and death. Therefore, in one example, a subject has a MAP ≥0.29. In another example, a subject has a MAP ≥0.291. In yet another example, a subject has a MAP between 0.15 and 0.8. In yet another example, a subject has a MAP between 0.16 and 0.7. In yet another example, a subject has a MAP between 0.2 and 0.7. In another example, the target has MAPs ranging from 0.29 to 0.6.

[0067] In one example, subjects with severe GvHD, as determined by a high MAP, have a low probability of achieving a clinical response to first-line therapy within 28 days and / or a high risk of 6-month relapse-free mortality. Thus, in one example, the subject has a 70% 6-month relapse-free mortality rate. In another example, the subject has a 80% 6-month relapse-free mortality rate. In yet another example, the subject has a 90% 6-month relapse-free mortality rate. In these examples, the subject may have a MAP ≥ 0.29.

[0068] A high MAP indicates severe gastrointestinal (GI) foveal injury. GI foveals are glands found between the villi in the inner wall of the intestinal epithelium of the small and large intestines. GI foveal cells provide stem cells for the regeneration of the intestinal epithelium. For example, subjects with a MAP of ≥0.29 generally have severe GI foveal injury. In some cases, foveal injury is determined by histological assessment. For example, a biopsy of gastrointestinal tissue is taken during endoscopy or colonoscopy and then histologically assessed for cellular damage and inflammation. Thus, in some cases, subjects treated according to this disclosure may have severe GI foveal injury.

[0069] As outlined above, MAP is determined based on Reg3α and ST2 levels. Therefore, in one example, serum concentrations of Reg3α and ST2 are determined in the subject. In another example, the levels of ST2 and Reg3α are quantified in the same sample. In yet another example, the levels of ST2 and Reg3α are quantified in separate samples. In one example, multiple samples are acquired periodically, and ST2 and Reg3α are quantified in each sample, thereby monitoring the subject's MAP over time.

[0070] The severity of GvHD can also be graded according to the pattern of organ lesions and the state of clinical outcomes. Multi-organ lesions include skin rashes, liver lesions, and / or gastrointestinal (GI) lesions. Examples of skin rashes, liver lesions, and GI lesions are provided in Table 1 and Table 2. In one example, the subject has GvHD with multi-organ lesions. In another example, severe GvHD is graded according to the Glucksberg scale (Glucksberg et al., 1974; Thomas et al., 1975) (Table 1). For example, the subject may have grade II GvHD or grade III / IV GvHD according to the Glucksberg scale. In one example, the subject has grade II GvHD. In another example, the subject has grade III / IV GvHD.

[0071] In another example, severe GvHD is graded according to the IBMTR severity index (Table 2) (Rowlings et al., 1997). In one example, the subjects have GvHD grade B, grade C, or grade D according to the IBMTR severity scale.

[0072] In another example, the subject has Minnesota high-risk GvHD. Minnesota high-risk acute GvHD is defined as either cutaneous stage 4; lower GI stage 3-4 or liver stage 3-4; or cutaneous stage 3+, and either lower GI stage 2-4 or liver stage 2-4 GvHD (MacMillan et al., 2015). In each of these examples, the subject may also have a high MAP score. For example, the subject may have a MAP score ≥ 0.29.

[0073] [Table 1]

[0074] [Table 2]

[0075] In one example, a patient with GvHD treated in accordance with this disclosure has inflammatory bowel disease (IBD). For example, GvHD may be associated with Crohn's disease or ulcerative colitis. In one example, GvHD may be associated with Crohn's disease. In one example, Crohn's disease is present in the rectum and / or colon of the patient. In one example, the IBD of the patient is refractory to one or more lines of therapy. In one example, the IBD of the patient is refractory to treatment with a biologic. In one example, the IBD of the patient is refractory to treatment with adalimumab, certolizumab pegol, vedolizumab, or ustekinumab.

[0076] Select a patient In one example, the disclosure includes selecting a specific subject having GvHD for treatment with MLPSC. In one example, a subject having GvHD that has failed two lines of therapy is selected for treatment. For example, a method for selecting a subject for treatment includes the step of i) selecting a subject having GvHD that has failed two lines of therapy for treatment. In one example, the selection method includes the step of 1) selecting a subject having GvHD that is refractory to steroids and second-line therapy. In one example, the selection method includes the step of 1) selecting a subject having GvHD that is refractory to steroids and ruxolitinib. In one example, the selection method includes the step of 1) selecting a subject having GvHD that is refractory to prednisone and ruxolitinib.

[0077] Treatment response The methods of this disclosure relate to third-line treatment of GvHD. As used herein, the terms “to treat,” “to cure,” “to treat,” and “to reduce progression” include administering a population of MLPSCs and / or their offspring and / or soluble factors and / or extracellular vesicles derived therefrom to reduce or eliminate at least one symptom of GvHD.

[0078] In one example, the treatment reduces the subject's ST2 and / or Reg3α levels. For example, the subject's ST2 and / or Reg3α levels are reduced compared to the subject's baseline levels. In another example, the treatment reduces the subject's ST2 level. For example, the subject's ST2 level is reduced compared to the subject's baseline ST2 level. In yet another example, the treatment reduces the subject's Reg3α level. For example, the subject's Reg3α level is reduced compared to the subject's baseline Reg3α level. In yet another example, the treatment reduces the subject's MAP. For example, the subject's MAP is reduced compared to the subject's baseline level. In one example, the subject's MAP is reduced to <0.29. In yet another example, the subject's MAP is reduced to <0.291. In these examples, the treatment reduces the subject's MAP by day 28. In yet another example, the treatment reduces the subject's MAP by day 100. In yet another example, the treatment reduces the subject's MAP by day 160. In yet another example, the treatment reduces the subject's MAP by day 180. In one example, the reduction in MAP persists for at least one month. In another example, the decline in MAP persists for at least three months.

[0079] In one example, treatment reduces the levels of one or more inflammatory biomarkers, such as ELAFIN, sIL2-ra, TNFR1, IL-8, and HGF. For example, treatment may reduce ELAFIN levels. In another example, treatment may reduce sIL2-ra levels. In yet another example, treatment may reduce TNFR1 levels. In yet another example, treatment may reduce IL-8 levels. In one example, the levels of inflammatory biomarkers are reduced between 50 and 200 days after treatment. In one example, the levels of inflammatory biomarkers are reduced between 100 and 200 days after treatment. In yet another example, the levels of inflammatory biomarkers are reduced by 100 days after treatment. In yet another example, the levels of inflammatory biomarkers are reduced by 200 days after treatment.

[0080] In another example, the reduction in the target inflammatory biomarker persists at days 100, 160, and 180. For instance, a reduction in one or more of the following may be observed at day 100 and persist at day 180: ELAFIN, sIL2-ra, TNFR1, IL-8, and HGF.

[0081] For example, a decrease in one or more levels of ELAFIN, sIL2-ra, TNFR1, IL-8, and HGF is associated with a decrease in the target MAP score.

[0082] In one example, treatment reduces GI crypt injury in the subject. For instance, the GI crypt injury in the subject is reduced compared to the subject's baseline level. In these examples, a reduction in MAP indicates a reduction in crypt injury. In another example, a reduction in crypt injury is determined by histological assessment of gastrointestinal tissue biopsies obtained by endoscopy or colonoscopy. For example, treatment reduces histological signs of cellular damage and inflammation in the gastrointestinal crypts.

[0083] In one case, treatment is observed on day 28.

[0084] As used herein, the term “response” means a response to therapy. In one example, a subject is considered to have responded if they have improvement in at least one organ without progression in any other organ, and no additional therapy is required. In another example, a subject is considered to have not responded if they have stable or progressive GvHD, or if further lines of therapy are required. In this example, a subject who does not respond is a non-responder.

[0085] In one example, treatment induces a partial response. In another example, the partial response is induced at least 28 days after treatment is initiated. In yet another example, the partial response is induced at least 30 days after treatment is initiated. In yet another example, the partial response is induced at least 2 months after treatment is initiated. In yet another example, the partial response is induced at least 3 months after treatment is initiated. In yet another example, the partial response is induced within 3 months. In yet another example, the partial response is induced between 28 and 56 days after treatment is initiated. In yet another example, the partial response is induced 100 days after treatment is initiated. In yet another example, the partial response is induced 160 days after treatment is initiated. In yet another example, the partial response is induced 180 days after treatment is initiated.

[0086] In another example, a partial response is induced after 8 doses. In yet another example, a partial response is induced after administering the medication twice a week for 4 weeks. In yet another example, a partial response is induced after 3 or more doses. In one example, the partial response is as follows: - A reduction of at least 1 point in the skin % BSA score; - A reduction of at least 1 point in the oral score; - A reduction of at least 1 point in the score; - A reduction of at least 1 point in the skin characteristic score; - A reduction of at least 1 point in the gastrointestinal score; - A reduction of at least 1 point in the liver score; - A reduction of at least 1 point in the lung symptom score; - A reduction of at least 1 point in the lung FEV1 score; - A reduction of at least 1 point in joint and fascia scores; - Reduce the genital score by at least 1 point. Characterized by one, more, or all of the following.

[0087] In one example, a partial response is characterized by a reduction of at least 1 point in the skin % BSA score. In another example, a partial response is characterized by a reduction of at least 1 point in the oral score. In yet another example, a partial response is characterized by a reduction of at least 1 point in the eye score. In these examples, the scores may be obtained using the NIH Consensus Criterion 2014 for GvHD.

[0088] In another example, the partial response is as follows: - A reduction of at least 1 point in the skin % BSA score; - A reduction of at least 1 point in the oral score; - Reduce the score by at least 1 point Characterized by one, more, or all of the following.

[0089] Various classification systems exist for characterizing GvHD (Lee, S., (2017) Blood., 129(1): pp. 30-37). For example, the NIH Consensus Criteria 2014 can be used to score the outcomes disclosed herein (Jagasia et al., (2015) Biol Blood Marrow Transplant., 21: pp. 389-401). The components of the NIH Consensus Criteria 2014 are shown in the table below:

[0090] [Table 3A]

[0091] [Table 3B]

[0092] [Table 3C]

[0093] In one example, a partial response is a decrease of ≥1 point in the organ-specific NIH Consensus Criteria 2014 score from the table above. Thus, in one example, treatment induces a decrease of ≥1 point in the skin % BSA score. In another example, treatment induces a decrease of ≥1 point in the oral score. In yet another example, treatment induces a decrease of ≥1 point in the eye score. In yet another example, treatment induces a decrease of ≥1 point in the skin characteristic score. In yet another example, treatment induces a decrease of ≥1 point in the gastrointestinal score. In yet another example, treatment induces a decrease of ≥1 point in the liver score. In yet another example, treatment induces a decrease of ≥1 point in the pulmonary symptom score. In yet another example, treatment induces a decrease of ≥1 point in the pulmonary FEV1 score. In yet another example, treatment induces a decrease of ≥1 point in the joint and fascia score. In yet another example, treatment induces a decrease of ≥1 point in the reproductive tract score.

[0094] In one case, treatment induces a complete response after initiation. In another case, a complete response is the complete resolution of GvHD symptoms in all organs. In one case, a complete response is induced 28 days after initiation of treatment. In another case, a complete response is induced at least 28 days after initiation of treatment. In another case, a complete response is induced at least 30 days after initiation of treatment. In one case, a complete response is induced at least 2 months after initiation of treatment. In yet another case, a complete response is induced at least 3 months after initiation of treatment. In yet another case, a complete response is induced 28–56 days after initiation of treatment. In yet another case, a complete response is induced 100 days after initiation of treatment. In yet another case, a complete response is induced 160 days after initiation of treatment. In yet another case, a complete response is induced 180 days after initiation of treatment.

[0095] In another example, a complete response is induced after two doses. In yet another example, a complete response is induced after administering the medication twice a week for four weeks. In yet another example, a complete response is induced after three or more doses.

[0096] In another example, the treatment increases the probability of survival of the subject. For example, the treatment increases the probability of a subject surviving for at least 20 to 200 days after the start of treatment. In one example, the treatment increases the probability of a subject surviving for at least 180 days after the start of treatment. In another example, the treatment increases the probability of a subject surviving for at least 100 days. In one example, the increase in probability is determined compared to a subject not treated with the composition of this disclosure.

[0097] In another example, treatment reduces the subject's risk of relapse-free death over six months. For example, the subject's risk of relapse-free death over six months is reduced by 20% to 80%. In one example, the subject's risk is reduced by at least 70%. In another example, the subject's risk is reduced by at least 60%. In yet another example, the subject's risk is reduced by at least 50%. In yet another example, the subject's risk is reduced by at least 40%. In yet another example, the subject's risk is reduced by at least 30%.

[0098] In another example, treatment reduces the subject's risk of one-year relapse-free death. For example, the subject's risk of one-year relapse-free death is reduced by 20% to 80%. In one example, the subject's risk is reduced by at least 70%. In another example, the subject's risk is reduced by at least 60%. In yet another example, the subject's risk is reduced by at least 50%. In yet another example, the subject's risk is reduced by at least 40%. In yet another example, the subject's risk is reduced by at least 30%.

[0099] In one example, the treatment provides an overall response rate of >40%. In another example, the treatment provides an overall response rate of >50%.

[0100] Mesenchymal lineage precursor or stem cell As used herein, the term “mesenchymal lineage precursor or stem cell (MLPSC)” refers to an undifferentiated pluripotent cell that has the ability to regenerate while maintaining pluripotency and to differentiate into several cell types of mesenchymal origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons, or several cell types of non-mesoderm origin, such as hepatocytes, nerve cells, and epithelial cells. To avoid misunderstanding, “mesenchymal lineage precursor cell” refers to a cell that can differentiate into mesenchymal cells such as bone, cartilage, muscle, and adipocytes, as well as fibrous connective tissue.

[0101] The term “mesenchymal lineage precursor or stem cell” includes both parent cells and their undifferentiated offspring. The term also includes mesenchymal progenitor cells (MPCs), pluripotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal progenitor cells, and their undifferentiated offspring.

[0102] MLPSCs can be autologous, allogeneic, allogeneic, syngeneic, or homogeneous. Autologous cells are isolated from the same individual from which they will be re-transplanted. Allogeneic cells are isolated from a donor of the same species. Allogeneic cells are isolated from a donor of a different species. Syngeneic or homogeneous cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0103] In one example, MLPSCs are of the same species. In another example, the same MLPSCs are cultured, enlarged, and cryopreserved.

[0104] MLPSCs are primarily found in bone marrow, but have also been shown to be present in a variety of host tissues, including, for example, umbilical cord blood and cord, adult peripheral blood, adipose tissue, trabeculae, and dental pulp. They are also found in the skin, spleen, pancreas, brain, kidney, liver, heart, retina, hair follicles, intestines, lungs, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis, and periosteum; and can differentiate into germline cells such as mesoderm and / or endoderm and / or ectoderm. Therefore, MLPSCs can differentiate into a multitude of cell types, including but not limited to adipose, ossicular, cartilaginous, elastic, muscular, and fibrous connective tissue. The specific lineage constraints and differentiation pathways through which these cells enter depend on mechanical influences and / or various influences from endogenous bioactive factors such as growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue.

[0105] The terms “enriched,” “enriched,” or variations thereof are used herein to describe a population of cells in which the proportion of one particular cell type or several particular cell types is increased compared to an untreated population of cells (e.g., cells in their natural environment). In one example, an MLPSC-enriched population contains at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% MLPSCs. In this regard, the term “MLPSC-enriched population of cells” is considered to provide explicit support for the term “a population of cells containing X% MLPSCs,” where X% is the percentage stated herein. MLPSCs may, in some examples, form clonal colonies, for example, CFU-F (fibroblasts) or a subset thereof (e.g., 50%, 60%, 70%, 70%, 90%, or 95%) may have this activity.

[0106] In one example, the MLPSCs of this disclosure are cultured and grown from a population of MLPSCs that are STRO-1+. In another example, the MLPSCs are cultured and grown from a population of MLPSCs containing approximately 0.1% to 75% STRO-1+ cells. In yet another example, the MLPSCs are cultured and grown from a population of MLPSCs containing approximately 0.5% to 75% STRO-1+ cells. In yet another example, the MLPSCs are cultured and grown from a population of MLPSCs containing approximately 0.1% to 75% STRO-1+ cells. In yet another example, the MLPSCs are cultured and grown from a population of MLPSCs containing approximately 0.1% to 75% STRO-1+ cells. In yet another example, the MLPSCs are cultured and grown from a population of MLPSCs containing approximately 1% to 75% STRO-1+ cells. In yet another example, the MLPSCs are cultured and grown from a population of MLPSCs containing approximately 0.1% to 75% STRO-1+ cells. In another example, MLPSCs are cultured and augmented from a population of MLPSCs containing approximately 10% to 75% STRO-1+ cells.

[0107] In one example of this disclosure, MLPSCs are mesenchymal stem cells (MSCs). MSCs may be a homogeneous composition or a mixed cell population enriched with MSCs. A homogeneous MSC composition may be obtained by culturing adherent bone marrow or periosteal cells, and MSCs may be identified by specific cell surface markers identified by specific monoclonal antibodies. A method for obtaining an MSC-enriched cell population is described, for example, in U.S. Patent No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, the MSCs are homogeneous. In one example, the MSCs are cryopreserved. In another example, the MSCs are cultured, augmented, and cryopreserved.

[0108] In another example, MLPSCs are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, and MHC1+ MSCs.

[0109] Isolated or enriched MLPSCs can be amplified in vitro by culture. Isolated or enriched MLPSCs can be cryopreserved, thawed, and then amplified in vitro by culture.

[0110] In one example, isolated or enriched MLPSCs were cultured in culture medium (serum-free or serum-supplemented), such as alpha minimal essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, at a concentration of 50,000 viable cells / cm³. 2 Seeds are seeded and allowed to adhere to the culture vessel overnight at 37°C and 20% O2. Then, the culture medium is replaced and / or changed as needed, and the cells are cultured for a further 68-72 hours at 37°C and 5% O2.

[0111] As will be understood by those skilled in the art, cultured MLPSCs 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 MLPSCs are also biologically different from in vivo cells and have a higher growth rate compared to most non-periodic (quiescent) cells in vivo.

[0112] In one example, a population of cells is enriched from a cell preparation containing selectable forms of STRO-1+ cells. In this context, the term “selectable forms” would be understood to mean that the cells express a marker (e.g., a cell surface marker) that enables the selection of STRO-1+ cells. The marker may be STRO-1, but does not have to be STRO-1. For example, cells expressing STRO-2 and / or STRO-3(TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 (e.g., mesenchymal progenitor cells (MPCs)) also express STRO-1 (and may be STRO-1bright), as described and / or illustrated herein. Thus, the indicator that a cell is STRO-1+ does not mean that the cell is selected solely 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, MPC can be isolated from bone mononuclear cells using an anti-STRO-3 antibody.

[0113] References to the selection of cells or populations thereof do not necessarily require selection from a specific tissue source. As described herein, STRO-1+ cells can be selected, isolated, or enriched from a wide variety of sources. Nevertheless, in some examples, these terms provide support for selection from any tissue containing STRO-1+ cells (e.g., mesenchymal progenitor cells), or angiogenic tissue, or pericytes (e.g., STRO-1+ pericytes), or any one or more of the tissues enumerated herein.

[0114] In one example, the cells used in this disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+(HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0115] "Individually" means that this disclosure separately encompasses the enumerated markers or groups of markers, and that even though individual markers or groups of markers may not be separately listed herein, the appended claims may define such markers or groups of markers separately and divisibly from one another.

[0116] "Collectively" means that this disclosure encompasses any number or combination of enumerated markers or groups of markers, and that even though such a number or combination of markers or groups of markers may not be specifically mentioned herein, the appended claims may define such combination or subcombination separately and divisibly from any other combination of markers or groups of markers.

[0117] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue-nonspecific alkaline phosphatase. For example, the term includes the liver isoform (LAP), the bone isoform (BAP), and the kidney isoform (KAP). In one example, TNAP is BAP. In one example, as used herein, TNAP refers to a molecule capable of binding to a STRO-3 antibody produced by a hybridoma cell line deposited with ATCC on December 19, 2005, under the provisions of the Budapest Convention, under deposit accession number PTA-7282.

[0118] Furthermore, in one example, STRO-1+ cells can induce clonal CFU-F.

[0119] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germline cells. Non-limiting examples of lineages to which STRO-1+ cells can be restricted include bone progenitor cells; hepatocyte precursors that are pluripotent for cholangioepithelial cells and hepatocytes; neuron-restricting cells that can produce glial cell precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; precursors for cardiomyocytes and cardiomyocytes, including glucose-responsive insulin-secreting pancreatic beta cell lines. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as progenitor cells of the following: retinal pigment epithelial cells, fibroblasts, keratinocytes and other skin cells, dendritic cells, hair follicle cells, renal duct epithelial cells, smooth muscle and skeletal muscle cells, testicular precursors, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes, and oligodendrocyte cells.

[0120] In one example, MLPSCs are obtained from a single or multiple donors, and the donor samples or MLPSCs are then pooled and subsequently cultured and augmented.

[0121] MLPSCs encompassed by this disclosure may also be cryopreserved before administration to a subject. In one example, MLPSCs are cultured and then cryopreserved before administration to a subject.

[0122] In one example, the disclosure includes MLPSCs and their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the disclosure includes MLPSCs and extracellular vesicles isolated therefrom. For example, the MLPSCs of the disclosure can be cultured and augmented for a period and under conditions appropriate for the secretion of extracellular vesicles into cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

[0123] As used herein, the term “extracellular vesicles” refers to lipid particles spontaneously released from cells, ranging in size from approximately 30 nm to 10 microns, although typically they are less than 200 nm in size. They are released from cells (e.g., mesenchymal stem cells; STRO-1 + It may contain proteins, nucleic acids, lipids, metabolites, or organelles derived from cells.

[0124] As used herein, the term “exosome” generally refers to a type of extracellular vesicle, typically ranging in size from approximately 30 nm to 150 nm, originating from the endosomal compartment of mammalian cells, from which it is transported to and released from the cell membrane. They may contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolites, and function in intercellular communication by being secreted from one cell and taken up by another cell to deliver their cargo.

[0125] Culture growth "Cultured" MLPSCs are distinguished from newly isolated cells in that they have been cultured and subcultured in cell culture medium (i.e., subcultured).

[0126] In one example, newly isolated cells are cultured and grown over approximately one or two passages to provide an intermediate population. In another example, newly isolated cells are cultured and grown over two passages to provide an intermediate population. In yet another example, newly isolated cells are cultured and grown over approximately one to three passages to provide an intermediate population. In one example, the newly isolated cells are STRO-1+. For example, a population of STRO-1+ MLPSCs may contain approximately 0.1% to 75% STRO-1+ cells.

[0127] In one example, related cells are isolated and cultured to increase their size over two passages to provide an intermediate MLPSC population. The intermediate MLPSC population is then cultured to increase its size to provide a drug product (DP). For example, the DP composition of this disclosure is produced by culturing cells from an intermediate cryopreserved MLPSC population, or in other words, from a cryopreserved intermediate. In one example, the intermediate cell population may be cultured for a further three passages (i.e., a total of five passages) to provide the DP.

[0128] In one example, MLPSCs are cultured and grown over approximately 4 to 10 passages. In another example, MLPSCs are cultured and grown over at least 5, 6, 7, 8, 9, and 10 passages. For example, MLPSCs can be cultured and grown over at least 5 passages. In one example, MLPSCs can be cultured and grown over at least 5 to 10 passages. In another example, MLPSCs can be cultured and grown over at least 5 to 8 passages. In another example, MLPSCs can be cultured and grown over at least 5 to 7 passages. In another example, MLPSCs can be cultured and grown over more than 7 passages. In these examples, MLPSCs can be cultured and grown before cryopreservation to provide an intermediate cryopreservation population of MLPSCs, which can then be subjected to further culture and growth.

[0129] In one example, the composition of the present disclosure comprises MLPSCs cultured from a cryopreserved intermediate. In one example, the cells cultured from the cryopreserved intermediate are cultured for at least 3, at least 5, at least 6, at least 7, at least 8, at least 9, and at least 10 passages. For example, MLPSCs may be cultured for at least 3 passages. In one example, MLPSCs may be cultured for at least 3 to 10 passages. In one example, MLPSCs may be cultured for at least 3 to 8 passages. In one example, MLPSCs may be cultured for at least 3 to 7 passages.

[0130] In one example, MLPSCs may be obtained from a single donor or multiple donors, and the donor samples or MLPSCs are then pooled and subsequently cultured and augmented as needed. In one example, the culture augmentation process is as follows: i. A step of increasing the number of viable cells by passage to provide a preparation of at least about 1 billion viable cells, wherein passage includes establishing a primary culture of isolated MLPSCs and then successively establishing a first non-primary (P1) culture of MLPSCs isolated from the previous culture; ii. The process of increasing the P1 culture of isolated MLPSCs to a second non-primary (P2) culture of MLPSCs by subculturing; and iii. A step of preparing and cryopreserving an ongoing intermediate MLPSC preparation obtained from the P2 culture of MLPSC; and optionally, iv. Thawing the frozen, ongoing MLPSC intermediate preparation and increasing the amount of the ongoing MLPSC intermediate preparation by passaging. Includes.

[0131] In one example, the increased MLPSC preparation was i. Less than approximately 0.75% of CD45+ cells; ii. At least approximately 95% CD105+ cells; iii. At least approximately 95% of CD166+ cells It has a profile that includes this.

[0132] The processes of MLPSC isolation and ex vivo augmentation can be carried out using any equipment and cell handling methods known in the art. Various culture augmentation embodiments of this disclosure employ steps that require cell manipulation, such as seeding, nutrient supplementation, debonding of adherent cultures, or washing. Any step that manipulates cells has the potential to damage them. While MLPSCs can generally withstand a certain amount of damage during preparation, cells are preferably manipulated by handling procedures and / or equipment that properly carry out the prescribed steps while minimizing damage to the cells.

[0133] In one example, MLPSCs are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculation wash bag, a rotating membrane filter with inlet and outlet ports, a filtrate bag, a mixing zone, a final product bag for washed cells, and appropriate tubing, as described in, for example, U.S. Patent No. 6,251,295 incorporated herein by reference.

[0134] In one example, MLPSC compositions cultured according to this disclosure are 95% homogeneous in terms of being CD105-positive and CD166-positive and CD45-negative. In one example, this homogeneity persists through ex vivo growth; i.e., through multiple population doublings.

[0135] In one example, the MLPSCs of this disclosure are cultured and augmented in 2D culture. For example, the MLPSCs of this disclosure may be cultured and augmented in a cell factory. In a particular example, 3D culture of the intermediates disclosed herein may be subsequently carried out using, for example, a bioreactor. In one example, the MLPSCs of this disclosure are first cultured and augmented in 2D culture before being further augmented in 3D culture. In one example, the intermediate cell population of this disclosure is not cultured and augmented in 3D culture.

[0136] In one example, the MLPSCs of this disclosure are cultured and amplified from an intermediate population. In another example, the MLPSCs of this disclosure are cultured and amplified from an intermediate in a 2D culture before being seeded into a 3D culture.

[0137] In the context of both intermediate populations and the therapeutic compositions derived therefrom, in one example, the MLPSCs of the Disclosure are cultured and augmented in 2D culture for at least 3 days before seeding in a further culture system such as a cell factory or 3D culture in a bioreactor. In one example, the MLPSCs of the Disclosure are cultured and augmented in 2D culture for at least 4 days before seeding in a further culture system. In one example, the MLPSCs of the Disclosure are cultured and augmented in 2D culture for 3 to 5 days before seeding in a further culture system. In these examples, 2D culture may 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 may be carried out in various bioreactor types such as agitated tank type, wave bag type, and vertical wheel type.

[0138] In one example, CO2 is provided during the culture and growth of MLPSCs. In another example, MLPSCs are cultured and grown in less than 9% CO2. In yet another example, MLPSCs are cultured and grown in less than 8% CO2. In yet another example, MLPSCs are cultured and grown in 5% CO2. For example, MLPSCs may be cultured and grown in 5% ± 2% CO2. In yet another example, MLPSCs are cultured and grown with passive CO2 priming. For example, cell factories may be passively primed with 5% CO2.

[0139] Priming a cell factory involves maintaining CO2 tension between the cell factory and the incubator and stabilizing the pH level of the growth medium. Active priming involves actively passing CO2 gas through a bacterial vent air filter into each culture vessel (e.g., cell factory) for a specified period (e.g., approximately 10 minutes). However, active priming has the potential to introduce contaminants into the culture because it requires an open port to provide the gas. Passive priming involves placing a closed culture system in an incubator with an appropriate CO2 concentration before cell seeding (e.g., approximately 12–72 hours).

[0140] In one example, the cells of this disclosure are STRO-3+ before being cultured and augmented to provide an intermediate cell population.

[0141] Cell culture medium The MLPSCs disclosed herein can be cultured and grown in a variety of suitable growth media.

[0142] As used in the context of this disclosure, the terms “medium” or “media” include components of the environment surrounding cells. The medium contributes to and / or provides suitable conditions for cell growth. The medium may be solid, liquid, gaseous, or a mixture of phases and materials. The medium may include liquid growth media, as well as liquid media that do not sustain cell growth. The medium also includes gelatinous media such as agar, agarose, gelatin, and collagen matrix. An example gaseous medium includes a gaseous phase to which cells growing on a petri dish or other solid or semi-solid support are exposed.

[0143] Cell culture media used for cell growth contain all essential amino acids and may also contain non-essential amino acids. Generally, amino acids are classified into essential amino acids (Thr, Met, Val, Leu, Ile, Phe, Trp, Lys, His) and non-essential amino acids (Gly, Ala, Ser, Cys, Gln, Asn, Asp, Tyr, Arg, Pro).

[0144] Those skilled in the art will understand that, for optimal results, the basal medium must be appropriate for the cell line of interest. For example, if it is found that this energy source is depleted and therefore growth is limited, it may be necessary to increase the level of glucose (or other energy source) in the basal medium or to add glucose (or other energy source) during the course of culture. In one example, the dissolved oxygen (DO) level may also be controlled.

[0145] In one example, the cell culture medium contains human-derived additives. For instance, human serum and human platelet cell lysates may be added to the cell culture medium.

[0146] In one example, the cell culture medium contains only human-derived additives. Therefore, in one example, the cell culture medium is heterogeneous. To avoid misunderstanding, in these examples, the culture medium is free of animal proteins. In one example, the cell culture medium used in the method of this disclosure is free of animal components.

[0147] In one example, the culture medium contains serum. In another example, the culture medium is a fetal bovine serum-free culture medium containing growth factors that promote MLPSC proliferation. In one embodiment, the culture medium is a serum-free stem cell culture medium. In one example, the cell culture medium is Basic culture medium; Platelet-derived growth factor (PDGF); Fibroblast growth factor 2 (FGF2) Includes.

[0148] In one example, the culture medium contains platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2), with FGF2 levels less than approximately 6 ng / ml. For example, FGF2 levels may be less than approximately 5 ng / ml, less than approximately 4 ng / ml, less than approximately 3 ng / ml, less than approximately 2 ng / ml, or less than approximately 1 ng / ml. In other examples, FGF2 levels may be less than approximately 0.9 ng / ml, less than approximately 0.8 ng / ml, less than approximately 0.7 ng / ml, less than approximately 0.6 ng / ml, less than approximately 0.5 ng / ml, less than approximately 0.4 ng / ml, less than approximately 0.3 ng / ml, or less than approximately 0.2 ng / ml.

[0149] In another example, the FGF2 level is approximately 1 pg / ml to 100 pg / ml. In yet another example, the FGF2 level is approximately 5 pg / ml to 80 pg / ml. In one example, the FGF2 level is approximately 1 ng / ml.

[0150] In one example, PDGF is PDGF-BB. In another example, the level of PDGF-BB is approximately 1 ng / ml to 150 ng / ml. In yet another example, the level of PDGF-BB is approximately 7.5 ng / ml to 120 ng / ml. In yet another example, the level of PDGF-BB is approximately 15 ng / ml to 60 ng / ml. In yet another example, the level of PDGF-BB is at least approximately 10 ng / ml. In yet another example, the level of PDGF-BB is at least approximately 15 ng / ml. In yet another example, the level of PDGF-BB is at least approximately 20 ng / ml. In yet another example, the level of PDGF-BB is at least approximately 21 ng / ml. In yet another example, the level of PDGF-BB is at least approximately 22 ng / ml. In yet another example, the level of PDGF-BB is at least approximately 23 ng / ml. In yet another example, the level of PDGF-BB is at least approximately 24 ng / ml. In yet another example, the level of PDGF-BB is at least approximately 25 ng / ml.

[0151] In another example, PDGF is PDGF-AB. In one example, the level of PDGF-AB is approximately 1 ng / ml to 150 ng / ml. In another example, the level of PDGF-AB is approximately 7.5 ng / ml to 120 ng / ml. In another example, the level of PDGF-AB is approximately 15 ng / ml to 60 ng / ml. In another example, the level of PDGF-AB is at least approximately 10 ng / ml. In another example, the level of PDGF-AB is at least approximately 15 ng / ml. In another example, the level of PDGF-AB is at least approximately 20 ng / ml. In another example, the level of PDGF-AB is at least approximately 21 ng / ml. In another example, the level of PDGF-AB is at least approximately 22 ng / ml. In another example, the level of PDGF-AB is at least approximately 23 ng / ml. In another example, the level of PDGF-AB is at least approximately 24 ng / ml. In another example, the level of PDGF-AB is at least approximately 25 ng / ml.

[0152] In other examples, additional factors may be added to the cell culture medium. In one example, the culture medium further contains EGF. EGF is a growth factor that stimulates cell proliferation by binding to its receptor, EGFR. In one example, the method of this disclosure includes the step of culturing a population of stem cells in a fetal bovine serum-free cell culture medium further containing EGF. In one example, the level of EGF is approximately 0.1–7 ng / ml. For example, the level of EGF may be at least approximately 5 ng / ml.

[0153] In another example, the EGF level is approximately 0.2 ng / ml to 3.2 ng / ml. In yet another example, the EGF level is approximately 0.4 ng / ml to 1.6 ng / ml. In yet another example, the EGF level is approximately 0.2 ng / ml. In yet another example, the EGF level is at least approximately 0.3 ng / ml. In yet another example, the EGF level is at least approximately 0.4 ng / ml. In yet another example, the EGF level is at least approximately 0.5 ng / ml. In yet another example, the EGF level is at least approximately 0.6 ng / ml. In yet another example, the EGF level is at least approximately 0.7 ng / ml. In yet another example, the EGF level is at least approximately 0.8 ng / ml. In yet another example, the EGF level is at least approximately 0.9 ng / ml. In yet another example, the EGF level is at least approximately 1.0 ng / ml.

[0154] In the above examples, basal media such as Alpha MEM or StemSpan® may be supplemented with reference amounts of growth factors. In one example, the culture medium includes Alpha MEM or StemSpan® supplemented with 32 ng / ml PDGF-BB, 0.8 ng / ml EGF, and 0.02 ng / ml FGF2. In another example, the culture medium includes Alpha MEM or StemSpan® supplemented with 10 ng / ml PDGF-BB, 5 ng / ml EGF, and 1 ng / ml FGF2.

[0155] In other examples, additional factors may be added to the cell culture medium. For example, the cell culture medium may be supplemented with one or more stimulating factors selected from the group consisting of epidermal growth factor (EGF), 1α,25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and stromal-derived factor 1α (SDF-1α). In another embodiment, cells may also be cultured in the presence of at least one cytokine in an amount sufficient to support cell growth. In yet another embodiment, cells may be cultured in the presence of heparin or a derivative thereof. For example, the cell culture medium may contain about 50 ng / ml of heparin. In other examples, the cell culture medium contains approximately 60 ng / ml of heparin, approximately 70 ng / ml of heparin, approximately 80 ng / ml of heparin, approximately 90 ng / ml of heparin, approximately 100 ng / ml of heparin, approximately 110 ng / ml of heparin, approximately 110 ng / ml of heparin, approximately 120 ng / ml of heparin, approximately 130 ng / ml of heparin, approximately 140 ng / ml of heparin, approximately 150 ng / ml of heparin, or a derivative thereof. 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. Thus, exemplary heparin sulfates include male liver heparin sulfate (MML HS) and female liver heparin sulfate (FML HS).

[0156] In another example, the cell culture medium of this disclosure promotes stem cell proliferation while maintaining stem cells in an undifferentiated state. Stem cells are considered undifferentiated if they are not constrained to a specific differentiation lineage. As discussed above, stem cells exhibit morphological features that distinguish them from differentiated cells. Furthermore, undifferentiated stem cells express genes that can be used as markers for detecting differentiation status. Polypeptide products can also be used as markers for detecting differentiation status. Thus, those skilled in the art can easily determine whether the method of this disclosure maintains stem cells in an undifferentiated state using routine morphological, genetic, and / or proteomics analyses.

[0157] Culture medium supplement In certain cases, the MLPSCs of this disclosure are cultured and amplified in a medium supplemented with pro-inflammatory cytokines and / or non-fetal serum. In one example, the cell culture medium contains IFN-gamma and / or TNF-alpha. In one example, the cell culture medium contains IFN-gamma. For example, the level of IFN-gamma may be less than 1 ng / ml. In one example, the level of IFN-gamma is less than 500 pg / ml or less than 100 pg / ml. In one example, the cell culture medium contains TNF-alpha. For example, the level of TNF-alpha may be less than 1 ng / ml. In one example, the level of TNF-alpha is less than 750 pg / ml or less than 400 pg / ml. In one example, the cell culture medium contains IFN-gamma and / or TNF-alpha, and the levels of both are less than 1 ng / ml.

[0158] In one example, the cell culture medium contains one or more pro-inflammatory cytokines that can bind to receptors on the surface of MLPSCs.

[0159] For example, the cell culture medium contains one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-alpha, MIP-1-beta, and IP-10. For instance, the cell culture medium may contain IL-8.

[0160] In one example, the cell culture medium contains IFN-gamma and / or TNF-alpha, and one or more pro-inflammatory cytokines selected from the group consisting of IL-6; IL-8; IL-17A; MCP-1; MIP-1-alpha; MIP-1-beta; and IP-10. In one example, the levels of IFN-gamma and / or TNF-alpha are less than 1 ng / ml.

[0161] For example, the cell culture medium is as follows: i. IFN-gamma levels exceeding 1 pg / ml; ii. TNF-alpha levels exceeding 2 pg / ml; iii. IL-6 levels exceeding 3 pg / ml; IV. IL-8 levels exceeding 500 pg / ml; IL-17A levels above 0.2 pg / ml; vi. MCP-1 levels above 3 pg / ml; vii. MIP-1-alpha levels exceeding 0.5 pg / ml; viii. MIP-1-beta levels exceeding 3 pg / ml; ix. IP-10 levels exceeding 500 pg / ml Characterized by one, more, or all of the following.

[0162] In another example, the culture medium is as follows: i. IFN-gamma levels exceeding 10 pg / ml; ii. TNF-alpha levels exceeding 20 pg / ml; iii. IL-6 levels exceeding 30 pg / ml; IV. IL-8 levels exceeding 5,000 pg / ml; IL-17A levels above 2 pg / ml; vi. MCP-1 levels exceeding 30 pg / ml; vii. MIP-1-alpha levels exceeding 50 pg / ml; viii. MIP-1-beta levels exceeding 30 pg / ml; ix. IP-10 levels exceeding 5,000 pg / ml It contains serum characterized by one, more, or all of the following.

[0163] In one example, the culture medium contains IL-10. In another example, the culture medium contains IL-36RA. In yet another example, the culture medium contains both IL-10 and IL-36RA. In one example, the level of IL-10 is above 0.3 pg / ml. For example, the level of IL-10 may be above 30 pg / ml. In one example, the level of IL-10 is above 400 pg / ml. In one example, the level of IL-36RA is above 50 pg / ml.

[0164] In another example, the method of this disclosure encompasses culture amplification in a cell culture medium containing neonatal serum. Various examples of appropriate serum (and its levels) are disclosed herein.

[0165] serum "Neonatal serum" refers to serum acquired after birth. For example, the culture medium may be supplemented with mammalian neonatal serum (e.g., bovine). In one example, the culture medium may be supplemented with animal neonatal serum. In another example, the culture medium may be supplemented with human neonatal serum.

[0166] In one example, the cell culture medium is supplemented with neonatal serum at least approximately 1% v / v, at least approximately 2% v / v, at least approximately 3% v / v, at least approximately 4% v / v, at least approximately 5% v / v, at least approximately 6% v / v, at least approximately 7% v / v, at least approximately 8% v / v, at least approximately 9%, at least approximately 10%, at least approximately 11%, at least approximately 12%, at least approximately 13%, at least approximately 14%, at least approximately 15%, at least approximately 16%, at least approximately 17%, at least approximately 18%, at least approximately 19%, at least approximately 20%, at least approximately 21%, at least approximately 22%, at least approximately 23%, at least approximately 24%, and at least approximately 25% v / v. In one example, the cell culture medium is supplemented with neonatal serum at approximately 1% v / v to approximately 15% v / v. In another example, the cell culture medium is supplemented with neonatal serum at approximately 1% v / v to approximately 10% v / v. In one example, the cell culture medium is supplemented with approximately 5% v / v to 10% v / v neonatal serum.

[0167] In one example, neonatal serum contains at least one inflammatory cytokine. As used herein, the term “inflammatory cytokine” refers to a signaling molecule that promotes inflammation. In the example, one or more cytokines are selected from the group including IL-1β, IL-6, TNF-α, IFN-γ, and / or IL-1ra.

[0168] In one example, the neonatal serum contains IFN-gamma. In another example, the neonatal serum contains TNF-alpha. In yet another example, the neonatal serum contains IFN-gamma and TNF-alpha. In yet another example, the neonatal serum contains one or more pro-inflammatory cytokines selected from the group consisting of IL-6; IL-8; IL-17A; MCP-1; MIP-1-alpha; MIP-1-beta; and IP-10. For example, the neonatal serum may contain IL-8. In one example, the neonatal serum contains IFN-gamma and / or TNF-alpha, as well as one or more pro-inflammatory cytokines selected from the group consisting of IL-6; IL-8; IL-17A; MCP-1; MIP-1-alpha; MIP-1-beta; and IP-10. In another example, neonatal serum contains IFN-gamma and TNF-alpha, as well as one or more pro-inflammatory cytokines selected from the group consisting of IL-6; IL-8; IL-17A; MCP-1; MIP-1-alpha; MIP-1-beta; and IP-10. In one example, the level of IFN-gamma is less than 1 ng / ml. In another example, the level of TNF-alpha is less than 1 ng / ml. In yet another example, the levels of both IFN-gamma and TNF-alpha are less than 1 ng / ml. For example, the level of IFN-gamma may be less than 500 pg / ml or less than 100 pg / ml. In one example, the level of TNF-alpha is less than 750 pg / ml or less than 400 pg / ml.

[0169] Methods for detecting the presence of cytokines in serum are known in the art, including, for example, enzyme-linked immunosorbent assay (ELISA). In another example, the presence of cytokines in serum is detected by measuring cytokine mRNA using polymerase chain reaction (PCR) techniques, such as reverse transcription PCR.

[0170] In one example, neonatal serum may be neonatal calf serum (NBCS). In one example, NBCS is obtained from neonatal calves that have been fed colostrum. In one example, NBCS contains elevated levels of at least one inflammatory cytokine compared to NBCS obtained from calves that have not been fed colostrum. In one example, NBCS contains elevated levels of at least one inflammatory cytokine compared to fetal serum such as FCS.

[0171] In one example, NBCS is obtained within 4 weeks after the calf's birth. In another example, NBCS is obtained within 21 days after the calf's birth. For example, NBCS is obtained ≤ 21 days after the calf's birth. In one example, NBCS is obtained between the calf's birth date and 21 days after birth. In one example, NBCS is obtained between the calf's birth date and 14 days after birth. In one example, NBCS is obtained between the calf's birth date and 10 days after birth. In one example, NBCS is obtained between the calf's birth date and 7 days after birth. In one example, NBCS is obtained between 6 hours and 72 hours after birth. In one example, NBCS is obtained between 6 hours and 48 hours after birth. In one example, NBCS is obtained between 6 hours and 24 hours after birth. For example, NBCS (Nursing, Nucleotide, and Childhood Surgery) is acquired between 12 and 24 hours after birth.

[0172] In one example, the cell culture medium is supplemented with at least approximately 1% v / v, at least approximately 2% v / v, at least approximately 3% v / v, at least approximately 4% v / v, at least approximately 5% v / v, at least approximately 6% v / v, at least approximately 7% v / v, at least approximately 8% v / v, at least approximately 9%, at least approximately 10%, at least approximately 11%, at least approximately 12%, at least approximately 13%, at least approximately 14%, at least approximately 15%, at least approximately 16%, at least approximately 17%, at least approximately 18%, at least approximately 19%, at least approximately 20%, at least approximately 21%, at least approximately 22%, at least approximately 23%, at least approximately 24%, and at least approximately 25% v / v NBCS. In one example, the cell culture medium is supplemented with approximately 1% v / v to approximately 15% v / v NBCS. In another example, the cell culture medium is supplemented with approximately 5% v / v to approximately 10% v / v NBCS. In one example, the cell culture medium is supplemented with at least approximately 5% v / v NBCS.

[0173] In one example, the culture medium is also supplemented with fetal serum. In one example, the fetal serum is fetal calf serum (FCS). The terms fetal calf serum (FCS) and fetal bovine serum (FBS) are intended to be used interchangeably in the context of this disclosure. In one example, the cell culture medium is supplemented with less than 10% v / v FCS. In another example, the cell culture medium is supplemented with approximately 5% v / v FCS.

[0174] In one example, the cell culture medium does not contain fetal serum.

[0175] In one example, the cell culture medium does not contain FCS.

[0176] In one example, the culture medium is supplied with a mixture of FCS and NBCS. In another example, the cell culture medium is supplied with approximately 5% v / v FCS and approximately 5% v / v NBCS (i.e., a 1:1 ratio of FCS to NBCS). In another example, the culture medium may be supplied with a mixture of FCS and NBCS such that the FCS:NBCS ratio is at least approximately 0.4:1, at least approximately 0.5:1, at least approximately 0.6:1, at least approximately 0.7:1, at least approximately 0.8:1, at least approximately 0.9:1, at least approximately 1:1, at least approximately 1.5:1, or at least approximately 2:1. In one example, the FCS:NBCS ratio is approximately 0.5:1 to approximately 2:1. In another example, the FCS:NBCS ratio is approximately 0.8:1 to approximately 1.5:1. In another example, the FCS:NBCS ratio is approximately 0.8:1 to approximately 1.2:1. In yet another example, the FCS:NBCS ratio is approximately 1:1.

[0177] For example, a mixture of FCS and NBCS may constitute at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, and at least about 25% v / v of the cell culture medium. For example, a mixture of FCS and NBCS may constitute about 1% v / v to about 15% v / v of the cell culture medium. In one example, a mixture of FCS and NBCS may constitute approximately 2% v / v to approximately 12% v / v of the cell culture medium. In another example, a mixture of FCS and NBCS may constitute approximately 5% v / v to approximately 12% v / v of the cell culture medium. In yet another example, a mixture of FCS and NBCS may constitute approximately 8% v / v to approximately 12% v / v of the cell culture medium. In yet another example, a mixture of FCS and NBCS may constitute approximately 10% v / v of the cell culture medium. However, in this example, the cell culture medium is supplemented with FCS at least approximately 1% v / v, at least approximately 2% v / v, at least approximately 3% v / v, at least approximately 4% v / v, at least approximately 5% v / v, at least approximately 6% v / v, at least approximately 7% v / v, at least approximately 8% v / v, and at least approximately 9% v / v, but less than 10% v / v. In one example, the cell culture medium is supplemented with approximately 1% v / v to 9% v / v of FCS. In another example, the cell culture medium is supplemented with approximately 3% v / v to 8% v / v of FCS. In yet another example, the cell culture medium is supplemented with approximately 3% v / v to 6% v / v of FCS. In yet another example, the cell culture medium is supplemented with approximately 5% v / v of FCS.

[0178] Ascorbic acid In one example, the cell culture medium is supplemented with a short-acting ascorbic acid derivative. The term "short-acting" encompasses ascorbic acid derivatives that are approximately 80-90% oxidized after 24 hours of cell culture under neutral pH and 37°C conditions. In one example, the short-acting L-ascorbic acid derivative is an L-ascorbate, e.g., sodium L-ascorbate. In one example, the cell culture medium may contain at least about 0.005 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may contain at least about 0.01 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may contain at least about 0.02 g / L of the short-acting ascorbic acid derivative. In yet another example, the cell culture medium may contain at least about 0.03 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may contain at least about 0.04 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may contain at least about 0.05 g / L of a short-acting ascorbic acid derivative. In yet another example, the cell culture medium may contain at least about 0.06 g / L of a short-acting ascorbic acid derivative.

[0179] In another example, the cell culture medium may contain a short-acting ascorbic acid derivative, but not a substantial amount of a long-acting ascorbic acid derivative. For example, the cell culture medium may contain a short-acting ascorbic acid derivative, but can only contain at most 0.04 g / L of a long-acting ascorbic acid derivative. In yet another example, the cell culture medium may contain a short-acting ascorbic acid derivative, but can only contain at most 0.03 g / L of a long-acting ascorbic acid derivative. In yet another example, the cell culture medium may contain a short-acting ascorbic acid derivative, but can only contain at most 0.02 g / L of a long-acting ascorbic acid derivative. In yet another example, the cell culture medium may contain a short-acting ascorbic acid derivative, but can only contain at most 0.01 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative, but may contain no more than 0.005 g / L of a long-acting ascorbic acid derivative. In yet another example, the cell culture medium may contain a short-acting ascorbic acid derivative, but may not contain a long-acting ascorbic acid derivative. In yet another example, the cell culture medium may contain L-ascorbate sodium salt, but may not contain a substantial amount of L-ascorbic acid-2-phosphate.

[0180] Other additives In one example, the cell culture medium contains human-derived additives. For example, human serum and human platelet cell lysates may be added to the cell culture medium. In another example, additional factors may be added to the cell culture medium. For example, the cell culture medium may be supplemented with one or more stimulating factors selected from the group consisting of platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), 1α,25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and stromal-derived factor 1α (SDF-1α). In another embodiment, cells may also be cultured in the presence of at least one cytokine in an amount sufficient to support cell growth. In yet another embodiment, cells may be cultured in the presence of heparin or a derivative thereof.

[0181] In the above example, the basal medium, such as Alpha MEM or StemSpan®, may be supplemented with a reference amount of serum and, in certain cases, other additives. Further examples of suitable culture media for culturing stem cells can be found, for example, in International Publication No. WO2016139340.

[0182] Cell modification The MLPSCs disclosed herein may be modified to inhibit cell lysis upon administration. Antigen modification may induce immunological nonresponsiveness or resistance, thereby preventing the induction of effector stages of the immune response (e.g., cytotoxic T cell generation, antibody production, etc.) that ultimately contribute to the rejection of foreign cells in a normal immune response. Antigens that may be modified to achieve this goal include, for example, MHC class I antigens, MHC class II antigens, LFA-3, and ICAM-1.

[0183] MLPSCs can also be genetically modified to express proteins important for the differentiation and / or maintenance of rhabdomyoskeletal muscle cells. Exemplary proteins include growth factors (TGF-β, insulin-like growth factor 1 (IGF-1), FGF), myogenic factors (e.g., myoD, myogenin, myogenic factor 5 (Myf5), myogenic regulator (MRF)), transcription factors (e.g., GATA-4), cytokines (e.g., cardiotropin-1), members of the neuregulin family (e.g., neuregulin 1, 2, and 3), and homeobox genes (e.g., Csx, tinman, and NKx families).

[0184] composition The MLPSCs disclosed herein can be cultured and amplified from cryopreserved intermediates to produce a preparation containing at least one therapeutic dose.

[0185] For example, the composition of the present disclosure is 10 × 10 cells. 6 pieces ~ cells 35×10 6 It contains 20 × 10 cells. In another example, the composition contains 20 × 10 6 pieces ~ cells 30×106 cells. In other examples, the composition comprises at least 100×10 6 cells. In another example, the composition comprises 50×10 6 cells to 500×10 6 cells. In other examples, the composition of the present disclosure comprises 150 million cells. In another example, the composition of the present disclosure comprises 20 million to 100 million cells. In one example, the composition of the present disclosure comprises 6 million to 7 million cells / ml of cells in 3.8 ml.

[0186] In one example, the composition of the present disclosure comprises a conditioned medium obtained from the MLPSCs disclosed herein.

[0187] In one example, the composition of the present disclosure comprises a pharmaceutically acceptable carrier and / or excipient. The terms "carrier" and "excipient" refer to compositions of materials conventionally used in the art to facilitate storage, administration, and / or biological activity of the active compound (see, for example, Remington's Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980)). A carrier may also reduce any undesirable side effects of the active compound. Suitable carriers are, for example, stable, e.g., incapable of reacting with other components in the carrier. In one example, the carrier does not cause significant local or systemic adverse effects in a recipient at the dosage and concentration employed for treatment.

[0188] Suitable carriers for the present disclosure include those conventionally used, such as water, physiological saline, aqueous dextrose, lactose, Ringer's solution, buffered solutions, hyaluronan, and glycol, which are exemplary liquid carriers for solutions, particularly when isotonic. Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

[0189] In another example, the carrier is, for example, a culture medium composition on which cells grow or are suspended. Such a culture medium composition does not induce any adverse effects in the subject to which it is administered. The exemplary carriers and excipients do not adversely affect the viability of cells and / or the ability of cells to treat or prevent disease.

[0190] In one example, a carrier or excipient provides buffering activity to maintain cells and / or soluble factors at an appropriate pH, thereby allowing them to exert biological activity, for example, a carrier or excipient is phosphate-buffered saline (PBS). PBS is an attractive carrier or excipient because it interacts minimally with cells and factors and allows for the rapid release of cells and factors, in which case the compositions of the present disclosure may be produced as a liquid for direct application, for example, by injection, into the bloodstream or into tissue or surrounding or adjacent areas.

[0191] The compositions of this disclosure can be cryopreserved. Cryopreservation of mesenchymal lineage precursors or stem cells can be carried out using slow-freezing methods or “fast” freezing protocols known in the art. Preferably, the cryopreservation method maintains similar phenotypes, cell surface markers, and growth rates of the cryopreserved cells compared to unfreezed cells.

[0192] The cryopreserved composition may contain a cryopreservation solution. The pH of the cryopreservation solution is typically 6.5 to 8, preferably 7.4.

[0193] The cryopreservation solution may include a sterile, non-pyrogenic isotonic solution, such as PlasmaLyte ATM. 100 mL of PlasmaLyte ATM contains 526 mg of sodium chloride, USP(NaCl); 502 mg of sodium gluconate (C6H11NaO7); 368 mg of sodium acetate trihydrate, USP(C2H3NaO2·3H2O); 37 mg of potassium chloride, USP(KCl); and 30 mg of magnesium chloride, USP(MgCl2·6H2O). It does not contain antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5-8.0).

[0194] The cryopreservation solution may contain Profreeze®. The cryopreservation solution may additionally or alternatively contain a culture medium, such as αMEM.

[0195] To facilitate freezing, cryoprotective agents, such as dimethyl sulfoxide (DMSO), are typically added to the cryopreservation solution. Ideally, the cryoprotective agent should be non-toxic to cells and patients, non-antigenic, chemically inactive, provide high viability after thawing, and allow transplantation without washing. However, DMSO, the most commonly used cryoprotective agent, exhibits some cytotoxicity. To reduce the cytotoxicity of the cryopreservation solution, hydroxyethyl starch (HES) may be used as a substitute for DMSO or in combination with DMSO.

[0196] The cryopreservation solution may contain one or more of DMSO, hydroxyethyl starch, human serum components, and other protein extenders. In one example, the cryopreservation solution may contain a solution of Plasma-Lyte A (70%), DMSO (10%), and HSA (25%), where the HSA solution contains 5% HSA and 15% buffer.

[0197] For example, the cryopreservation solution may further contain one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0198] The cryopreserved composition may be thawed and administered directly to the subject, or added to another solution, for example, hyaluronic acid. Alternatively, the cryopreserved composition may be thawed, and the mesenchymal lineage precursor or stem cells may be resuspended in an alternative carrier before administration.

[0199] The compositions described herein may be administered alone or as mixtures with other cells. Different types of cells may be mixed with the compositions disclosed herein immediately before or shortly before administration, or they may be co-cultured together over a period prior to administration.

[0200] In one example, the composition contains an effective amount or a therapeutically or prophylactically effective amount of mesenchymal lineage precursors or stem cells and / or their offspring and / or soluble factors derived therefrom. For example, the composition contains about 1 × 10⁻⁶ 5 Individual stem cells ~ approx. 1×10 9 Individual stem cells, or approximately 1.25 × 10⁻¹⁴ cells. 3 Individual stem cells ~ approx. 1.25×10 7 This includes individual stem cells / kg (for an 80kg subject). The exact amount of cells to be administered depends on a variety of factors, including the subject's age, weight, and sex, as well as the degree and severity of the disorder being treated.

[0201] Regardless of the number of cells provided in the composition, one example is 1 × 10 cells. 6 cells / kg~cells 5×10 6 Cells / kg are administered. In one example, 2 × 10 cells 6 Cells / kg are administered. In one example, 2 × 10 cells 6 The medication is administered in two doses at a dose of 1 / kg.

[0202] In one example, cells 50 × 10 6 ~200×10 7 Individuals are administered. In other examples, 60 × 10 cells are used. 6 ~200×10 6 Individual or cell 75 × 10 6 ~150×10 6 Individual cells are administered. In one example, 75 × 10⁶ cells are administered. 6 Individuals are administered. In another example, 150 × 10 cells 6 Individuals are administered. In another example, 1 × 10 cells 7 ~2×10 8 Individual doses are administered.

[0203] For example, the composition is 5.00 × 10 6It contains more than 5.50 × 10⁶ live cells / mL. In another example, the composition contains 5.50 × 10⁶ cells / mL. 6 It contains more than 10 cells / mL of live cells. In another example, the composition contains 6.00 × 10 6 It contains more than 10 cells / mL of live cells. In another example, the composition contains 6.50 × 10 6 It contains more than 10 cells / mL of live cells. In another example, the composition contains 6.68 × 10⁶ cells. 6 It contains more than 10 cells / mL of live cells. In another example, the composition contains 6.68 × 10⁶ cells in 3.8 ml. 6 Contains 10¹ / mL. In another example, the composition contains 6.68 × 10¹ in 3.8 ml. 6 Contains 1 / mL of live cells.

[0204] For example, mesenchymal lineage precursors or stem cells constitute at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, and at least about 99% of the cell population of the composition.

[0205] In one example, culture-enhanced MLPSCs in the administered composition inhibit IL2-RA by at least 56% under culture conditions. In another example, culture-enhanced MLPSCs in the administered composition inhibit IL2-RA by at least 60% under culture conditions. In yet another example, culture-enhanced MLPSCs in the administered composition inhibit IL2-RA by at least 70% under culture conditions. In yet another example, culture-enhanced MLPSCs in the administered composition inhibit IL2-RA by at least 75% under culture conditions. In yet another example, culture-enhanced MLPSCs in the administered composition inhibit IL2-RA by 55% to 75% under culture conditions. In yet another example, culture-enhanced MLPSCs in the administered composition inhibit IL2-RA by 55% to 75% under culture conditions. In yet another example, culture-enhanced MLPSCs in the administered composition inhibit IL2-RA by 60% to 75% under culture conditions.

[0206] In one example, the composition may be optionally packaged in a suitable container along with written instructions for use for the desired purpose.

[0207] In one example, MLPSC may be administered to the wall of the target gastrointestinal tract. In another example, MLPSC may be administered to the site of inflammation in the wall of the target gastrointestinal tract. For example, MLPSC may be administered within the site of inflammation in the wall of the target gastrointestinal tract. In these examples, the site of inflammation may be confirmed endoscopically before administration. For example, endoscopic confirmation may be based on visual examination and / or histological analysis of an endoscopic biopsy by a trained physician. In yet another example, the composition of this disclosure is administered intravenously.

[0208] In one example, the compositions described herein may be administered as a single dose.

[0209] In some examples, the compositions described herein may be administered in multiple doses. For example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least fourteen, or at least sixteen doses. In one example, eight doses are administered. In another example, sixteen doses are administered.

[0210] In one example, MLPSCs may be administered intravenously twice a week for four weeks. In another example, MLPSCs are administered once a week. For example, MLPSCs may be administered once a week every two weeks. In one example, MLPSCs may be administered once a month. In one example, 2 × 10 cells 6 Cells / kg are administered twice a week for four consecutive weeks. In another example, 2 × 10 cells 6 The dose is administered twice a week for eight consecutive weeks at a dose of 1 / kg. In this example, additional medication is needed depending on whether the GvHD symptoms remain unresolved.

[0211] Those skilled in the art will understand that numerous variations and / or modifications can be made to the present invention, as shown in the specific embodiments, without departing from the broad spirit or scope of the invention. Therefore, these embodiments should be considered in all respects as illustrative and non-limiting.

[0212] This application claims priority from US63 / 579,213, filed on 28 August 2023, the disclosure of which is incorporated herein by reference.

[0213] All publications discussed and / or referenced herein are incorporated herein by reference as a whole.

[0214] Any statements relating to documents, actions, materials, devices, articles, etc., contained herein are for the sole purpose of providing context for the present invention. None of these should be construed as an acknowledgment that any or all of these matters form part of the foundation of the prior art or were common knowledge in the art relating to the present invention that existed prior to the priority date of each claim of this application. [Examples]

[0215] composition The composition contains ex vivo cultured adult human mesenchymal stromal cells (MSCs) derived from bone marrow aspirate. The composition is cryopreserved in Plasma-Lyte® A supplemented with human serum albumin (5%) and dimethyl sulfoxide (10%). The composition is stored in vials and dispensed (25 × 10⁴ 6 pieces MSC[cells 6.68×10 6 pieces / mL]).

[0216] MLPSC as a third-line therapy for GvHD Acute GVHD that does not respond to both steroid therapy and second-line therapy (in adults, ruxolitinib is the only approved second-line therapy) remains a significant unmet clinical need. As demonstrated by the crucial REACH 1 trial data supporting FDA approval for ruxolitinib in individuals 12 years of age and older, such patients, referred to herein as SR+ acute GVHD, account for 45% of all adults receiving ruxolitinib as second-line therapy for steroid-refractory acute GVHD (Jagasia et al., (2020) Blood., 135:1739-1749). In addition, as demonstrated in the REACH 2 trial, as many as 61% of patients failed to maintain a therapeutic response by 56 days after initiation of ruxolitinib as second-line therapy, and 32% of patients were intolerant to the drug due to treatment ineffectiveness or toxicity (Zeiser et al., (2020) N Engl J Med., 382:1800-1810). Most importantly, the survival rate in these ruxolitinib-resistant patients was only about 30% at 3 months after initiating ruxolitinib (Jagasia et al., 2020). Recent results from a retrospective multicenter study on real-world experience with ruxolitinib as a second-line treatment for steroid-refractory acute GVHD found a median survival of only 21 days for 48 patients resistant to ruxolitinib and 50 days for an additional 16 patients intolerant to ruxolitinib. The median survival time for this group of 64 patients resistant to and / or intolerant to ruxolitinib was 28 days (interquartile 7–90), with an overall survival rate of only 25% up to 3 months (Abedin et al., (2021) British Journal of Haematology, 195:429–43). For the 33 patients in this group who received third-line treatment with the best available therapy, the overall response rate (ORR) at day 28 was 36%, and the median survival time was 28 days (Abedin et al., 2021). These data highlight and emphasize the clear need for safe and effective therapies that will improve response and survival rates in this patient population.

[0217] Seventy-one patients aged 12 years or older with acute GvHD who had not responded to treatment with both steroids and at least one other GvHD treatment received mesenchymal stem cells as third-line treatment under a prospectively defined protocol.

[0218] The 71 patients came from two patient cohorts. The first cohort (Cohort 1) consisted of patients aged 12 years or older and comprised 49 patients treated with MSCs. The second cohort (Cohort 2) also included 22 patients (children and adults) treated with MSCs. Overall response rate (ORR) at day 28 and overall survival (OS) over 100 days were assessed in both cohorts. Despite refractory to two prior lines of therapy, administration of MSCs as a third-line therapy resulted in a 28-day ORR of 59% and an OS over 100 days of 63%.

[0219] As shown in Figure 1, the 100-day survival rates were 61% for Cohort 1 (without EAP / RUX), 69% (9 / 13) for 13 patients from Cohort 2 (without EIND / RUX) who did not receive ruxolitinib as a second line, and 67% (6 / 9) for 9 patients from Cohort 2 (without EIND / RUX) who did receive ruxolitinib as a second line. Therefore, a 100-day overall survival rate of 63% (45 / 71) was observed in patients who received MSC as third-line treatment under a prospectively defined protocol.

[0220] Similarly, the 59% ORR at day 28 for 71 patients reflected the 57%, 62%, and 67% rates for the three subgroups, all substantially higher than the 36% reported for the best available third-line therapy in ruxolitinib-resistant patients (Abedin et al., 2021). Among patients treated with such MSCs who achieved a day 28 ORR, 88% (37 / 42) survived at day 100 (p<0.0001), compared to only 28% (8 / 29) of those who did not achieve a day 28 ORR (Figure 2). A landmark survival analysis of day 100 survival rates for only those patients who were still alive at day 28 demonstrated that the day 100 survival rate was 88% for those with a day 28 ORR and 47% for those who did not respond (log-rank p=0.003), supporting the prediction of survival benefit in this patient population treated with MSCs based on the day 28 ORR intermediate endpoint.

[0221] These results suggest that MSCs may be a highly effective third-line treatment for patients with steroid-refractory GVHD, and that they can significantly improve response and survival rates in these patients who are at very high risk of premature death.

[0222] Comparison with second-line treatment Table 4 shows the primary endpoint of overall response at day 28 in children aged 18 years or younger with SR-aGVHD who were treated with MSCs as a second-line treatment after failure of corticosteroids alone (n=54) or as a third-line treatment after failure of corticosteroids and ruxolitinib or any other active agent (n=35).

[0223] [Table 4]

[0224] For children who received MSC as third-line therapy, the overall response rate at day 28 was 63% (22 / 35), with 95% CI: 46.3 and 76.9. This was comparable to the 69% overall response rate (OR) at day 28 (95% CI 55.9 and 79.8) in children who received MSC as second-line therapy. Of the children in third-line therapy who achieved a day 28 response using MSC, 20 / 22 (91%) were alive at day 100 (p=0.0004).

[0225] The 46.3% lower limit of the 95% confidence interval for MSCs as a third-line therapy excluded the pre-specified null hypothesis of 45% for determining the efficacy of MSC products as second-line therapy in children under 18 years of age (Table 1). The 63% OR at day 28 was comparable to the 36% reported in Abedin et al.'s article (discussed above) for other treatments used as third-line therapy in ruxolitinib-refractory patients.

[0226] Figure 3 shows the 100-day overall survival rate in children aged 18 years or younger with SR-aGVHD who were treated with MSCs as a second-line treatment after failure with corticosteroids alone (n=54), or as a third-line treatment after failure with corticosteroids and either ruxolitinib or another active agent (n=35).

[0227] The 100-day overall survival rate for MSCs was 78% when used as a second-line treatment and 69% when used as a third-line treatment. In the third-line study, the rate of GVHD-related disease or treatment-related death (death due to aGVHD, infection, or tumor recurrence) at 100 days was 31% (11 / 35) for those who received MSCs as the third-line active agent, compared to 73% for the same mortality analysis in ruxolitinib non-responders in the paper by Abedin et al.

[0228] Therefore, in two single-arm studies involving children under 18 years of age with SR-aGVHD, treatment with MSCs resulted in achieving a complete response on day 28 that rejected the null hypothesis in each trial, and resulted in equally high day-100 survival rates when MSCs were used as second-line and third-line therapy. (References) Hartwell et al., (2017) JCI Insight.; 2(3):e89798. Glucksberg et al., (1974); Transplantation; 18, 295-304. MacMillan et al., (2015) Biol Blood Marrow Transplant; 21(4): 761-767. Major-Monfried et al., (2018) Blood; 131(25):2846-2855. Rowlings et al., (1997) British Journal of Haematology; 97, 855-864. Thomas et al., (1975) New England Journal of Medicine; 292, 895-902.

Claims

1. A method for treating graft-versus-host disease (GvHD) in a human subject requiring such treatment, comprising the step of administering a composition comprising mesenchymal lineage precursors or stem cells (MLPSCs) to the subject, wherein the MLPSCs are administered to the subject as a third-line therapy.

2. A method for treating graft-versus-host disease (GvHD) in human subjects, i) A step of selecting subjects with GvHD who have failed two lines of therapy; ii) A step of administering a composition containing mesenchymal lineage precursors or stem cells (MLPSCs) to a subject, wherein the MLPSCs are administered to the subject as a third-line therapy. A method that includes this.

3. The subject is the method according to claim 1 or 2, which is refractory to steroids and second-line therapy.

4. The method according to claim 3, wherein the second-line therapy is ruxolitinib.

5. The method according to claim 3, wherein the second-line therapy is a biological agent.

6. The method according to claim 5, wherein the biological agent is alemtuzumab, basiliximab, or tocilizumab.

7. The method according to any one of claims 1 to 6, wherein the MLPSCs in the composition inhibit IL-2Rα expression by ≥60%, and the inhibition of IL-2Rα expression is determined by obtaining a population of cells containing MLPSCs that have been cultured, cryopreserved, and thawed; co-culturing the MLPSCs in culture medium with a population of cells containing T cells; and determining the level of inhibition of IL-2Rα expression.

8. The treatment according to any one of claims 1 to 7, wherein the treatment reduces the risk of death in the subject.

9. The method according to claim 8, wherein the death is a non-recurrence-free death (NRM) within six months.

10. The method according to any one of claims 1 to 9, wherein the target is children.

11. The method according to any one of claims 1 to 9, wherein the subject is ≥12 years of age.

12. The method according to any one of claims 1 to 11, relating to acute graft-versus-host disease (aGvHD).

13. The targets are as follows: - Grade B, C, or D according to the IBMTR Severity Scale; - Grade II GvHD according to the Glucksberg Severity Scale; - Grade III / IV GvHD according to the Glucksberg Severity Scale; - Minnesota High-Risk GvHD The method according to any one of claims 1 to 12, which is classified as one or more of the above.

14. The method according to any one of claims 1 to 13, relating to a patient having chronic GvHD.

15. The method according to any one of claims 1 to 14, relating to a patient with multi-organ lesions.

16. The method according to any one of claims 1 to 15, relating to a person having inflammatory bowel disease (IBD).

17. The method according to claim 16, wherein IBD is Crohn's disease or ulcerative colitis.

18. The method according to claim 16, wherein IBD is Crohn's disease.

19. The method according to claim 18, wherein Crohn's disease is present in the rectum and / or colon of the subject.

20. The method according to any one of claims 16 to 19, wherein the target IBD is refractory to one or more of adalimumab, certolizumab pegol, vedolizumab, or ustekinumab.

21. The method according to any one of claims 1 to 20, wherein the treatment increases the probability of a subject surviving for at least 28 days after the start of treatment.

22. The method according to any one of claims 1 to 20, wherein the treatment increases the probability of a subject surviving for at least 100 days, preferably at least 180 days, after the start of treatment.

23. The method according to claim 21 or 22, wherein the probability of survival of a subject after the start of treatment is greater than 40%, greater than 50%, or preferably greater than 60%.

24. The method according to any one of claims 21 to 23, wherein the probability of survival of the subject is increased compared to a subject that does not receive MLPSC.

25. The method according to any one of claims 1 to 24, wherein the MLPSCs are cultured and augmented from a population of MLPSCs that are STRO-1+.

26. The method according to claim 25, wherein the population of MLPSCs that are STRO-1+ contains approximately 0.1% to 75% STRO-1+ cells.

27. The method according to any one of claims 1 to 26, wherein MLPSC is a mesenchymal stem cell (MSC).

28. cells 2×10 6 The method according to any one of claims 1 to 27, comprising the step of administering a dose of 1 / kg.

29. cells 1×10 7 ~2 x 10 8 The method according to any one of claims 1 to 27, comprising the step of administering an individual.

30. The method according to any one of claims 1 to 29, wherein the composition further comprises Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA).

31. The composition is 6.68 × 10 6 The method according to any one of claims 1 to 30, comprising more than 1 cells / mL of live cells.

32. The method according to any one of claims 1 to 31, wherein the composition is administered intravenously.

33. The method according to any one of claims 1 to 32, wherein the subject receives at least two doses of medication.

34. The method according to claim 33, wherein the first two doses are administered twice a week over a period of four weeks.

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