Methods for treating advanced heart failure in subjects at high risk for poor outcome - Patents.com

Abstract

The present disclosure relates to methods of treating and / or preventing progressive heart failure in subjects at high risk for poor outcome. Such methods may be used to treat or prevent progressive heart failure in subjects with microvascular and / or macrovascular disease.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the priority benefit of Australian Patent Application No. 2021903706, filed November 17, 2021, and U.S. Patent Application No. 63 / 384,200, filed November 17, 2022, each of which is incorporated by reference in its entirety herein.

[0002] The present disclosure relates to methods of treating and / or preventing advanced heart failure in subjects at high risk for poor outcome. [Background technology]

[0003] Myocardial infarction (MI) remains one of the leading causes of mortality and morbidity in developed countries. A recent review of US Medicare records has been published evaluating data on 350,509 patients over 65 years of age who were hospitalized with acute MI and discharged alive after the event (Schuster et al. (2004) Physiol Heart Circa Physiol., 287(2):525-32). Within one year after the index event, 25.9% of MI patients died and 50.5% were re-admitted. In the month following MI, they were 21 times more likely to die and 12 times more likely to be hospitalized compared to the total Medicare age population.

[0004] Over the past decade, numerous clinical trials evaluating novel pharmacotherapy have been conducted in patients with advanced heart failure (HF). Although progress has been made in reducing morbidity and mortality in HF patients, those with advanced disease continue to experience an unfavorable clinical course characterized by frequent hospitalizations and premature death.

[0005] Clearly, there is a need in the art for treating or preventing progressive heart failure. Summary of the Invention

[0006] The present inventors have surprisingly found that cell therapy is particularly effective in subjects with poor outcomes, such as certain subjects with progressive heart failure, particularly subjects with microvascular disease and / or macrovascular disease.For example, cell therapy is particularly effective in subjects with progressive heart failure in addition to myocardial ischemia and / or diabetes.Thus, in one example, the present disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering a composition comprising mesenchymal precursor cells or stem cells to the subject, the subject having microvascular disease and / or macrovascular disease.

[0007] In another example, the present disclosure relates to a method of reducing the risk of cardiac death, non-fatal myocardial infarction or non-fatal stroke in a subject, the method comprising administering to the subject a composition comprising mesenchymal precursor cells or stem cells, the subject having microvascular and / or macrovascular disease. In one example, the method reduces the non-fatal risk of a non-fatal event, such as a subsequent MI or stroke. Thus, in another example, the present disclosure relates to a method of reducing the risk of myocardial infarction or stroke in a subject, the method comprising administering to the subject a composition comprising mesenchymal precursor cells or stem cells, the subject having microvascular and / or macrovascular disease.

[0008] In another example, the disclosure relates to a method of selecting a heart failure patient for treatment with cell therapy, the method comprising: i) assessing a subject for microvascular and / or macrovascular disease; and ii) selecting a subject with microvascular and / or macrovascular disease for treatment, preferably wherein the treatment comprises administering a composition comprising mesenchymal progenitor or stem cells.

[0009] In these instances, the microvascular and / or macrovascular disease is myocardial ischemia and / or diabetes.

[0010] In one example, the subject's CRP level is 2 mg / L or greater. In another example, the subject's CRP level is less than 5 mg / L. In another example, the subject's CRP level is less than 4 mg / L. In another example, the subject's CRP level is less than 3 mg / L. In another example, the subject's CRP level is between 2-5 mg / L. In another example, the subject's CRP level is between 2-4 mg / L. In another example, the subject's CRP level is between 2-3 mg / L.

[0011] In another example, the subject's LVEF is less than about 45%. In another example, the subject's LVEF is less than 40%. In one example, the LVEF is measured by a two-dimensional echocardiogram. In one example, the subject's LVEF is less than 35% and the LVEF is measured by a multi-gated acquisition scan.

[0012] In one example, the subject has an LVESV greater than 70 ml. In another example, the subject has an LVESV greater than 100 ml. In another example, the subject has an LVESV greater than 130 ml. In another example, the subject has an LVESV between 70 ml and 160 ml. In one example, the subject has class II heart failure according to the New York Heart Association (NYHA) classification scale.

[0013] In another example, the level of N-terminal pro-B-type natriuretic peptide (NT-proBNP) in a subject is ->1000 pg / mL, or -1000pg / ml to 2500pg / ml.

[0014] In one example, the subject had a heart failure hospitalization event over the past 9 months. In another example, the subject had at least one HF outpatient emergency treatment requiring intravenous diuretics and vasodilators. In another example, the subject had received positive inotropic therapy 1-9 months prior to treatment.

[0015] In one example, the subject has persistent left ventricular dysfunction.

[0016] In one example, the subject's risk of cardiac death is reduced following treatment, in this example, the reduced risk can be relative to the risk of cardiac death in a subject not receiving the mesenchymal precursor or stem cells.

[0017] In another example, a subject's risk of ischemic MACE (MI or stroke) is reduced following treatment.

[0018] In one example, the composition is administered transendocardially and / or intravenously. For example, the composition may be administered transendocardially.

[0019] In one example, the mesenchymal precursor or stem cells are mesenchymal precursor cells (MPCs). In one example, MPCs are isolated from bone mononuclear cells using an anti-STRO-3 antibody.

[0020] In another example, the present disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering a composition comprising cells to the subject. In one example, the subject has heart failure of grade II or III according to the New York Heart Association (NYHA) classification scale. In one example, the subject may have heart failure of less than grade III according to the New York Heart Association (NYHA) classification scale. In one example, the subject has heart failure of grade II according to the New York Heart Association (NYHA) classification scale. Thus, in one example, the present disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering a composition comprising cells to the subject, the subject has heart failure of grade II according to the New York Heart Association (NYHA) classification scale.

[0021] In another example, the disclosure relates to a method of reducing progression of heart failure in a subject, the method comprising administering to a subject a composition comprising cells, the subject having stage II heart failure according to the New York Heart Association (NYHA) classification scale.

[0022] In another example, the disclosure relates to a method of reducing cardiac death in a subject having class II heart failure according to the New York Heart Association (NYHA) classification scale, comprising administering to the subject a composition comprising cells.

[0023] In another example, the disclosure relates to a method of selecting a heart failure patient for treatment with cell therapy, the method comprising: i) assessing heart failure according to the New York Heart Association (NYHA) classification scale; and ii) selecting a subject with NYHA class II heart failure. In one example, the method further comprises administering a composition comprising the cells.

[0024] In one example, the cells induce new blood vessel formation in a target tissue. In one example, the cells promote arteriogenesis. In one example, the cells secrete factors that protect at-risk myocardium. Thus, in one example, the disclosure relates to a method of treating or preventing progressive heart failure in a subject, the method comprising administering to the subject a composition comprising cells, the subject having stage II heart failure according to the New York Heart Association (NYHA) classification scale, and the cells induce new blood vessel formation in the target tissue and / or secrete factors that protect at-risk myocardium.

[0025] In one example, the cells are mesenchymal precursor or stem cells (MLPSCs). In one example, the MLPSCs are STRO-1+. In one example, the MLPSCs are mesenchymal stem cells (MSCs). In one example, the MLPSCs are allogeneic. In one example, the cells are culture expanded. In this example, the cells may be TNAP+ prior to being culture expanded. In one example, the cells are cryopreserved.

[0026] In another example, a method of the present disclosure includes: i) selecting a subject having class II heart failure according to the New York Heart Association (NYHA) classification scale; and ii) administering to the subject a composition comprising cells that induce new blood vessel formation in a target tissue.

[0027] In another example, administration of the composition inhibits progression of a subject to NYHA stage III advanced heart failure.

[0028] In one example, the subject has an N-terminal pro-B-type natriuretic peptide (NT-proBNP) level of less than 2200 pg / ml. In another example, the subject has an NT-proBNP level of less than 2000 pg / ml prior to administering the cells. In another example, the subject has an NT-proBNP level of between 1000 pg / ml and 2000 pg / ml prior to administering the cells.

[0029] The inventors have also surprisingly found that cell therapy is particularly effective in subjects in the early stages of advanced heart failure with elevated C-reactive protein (CRP) levels. Thus, in one example, the subject has an elevated CRP level. In one example, the subject has a CRP level greater than 1 mg / L. In one example, the subject has a CRP level greater than 1.5 mg / L. In one example, the subject has a CRP level equal to or greater than 2 mg / L. In one example, the subject has a CRP level greater than 2 mg / L. In another example, the subject has a CRP level between 1.5 and 5 mg / L. In another example, the subject has a C-reactive protein (CRP) level less than 5 mg / L, preferably less than 4 mg / L. In another example, the subject has a CRP level between 1 and 5 mg / L. In another example, the subject has a CRP level between 1.5 and 5 mg / L.

[0030] In another example, the subject has had a heart failure hospitalization event over the past 9 months.

[0031] In another example, the subject has an LVEF of less than about 45%, preferably less than 40%. In another example, the subject has persistent left ventricular dysfunction.

[0032] In another example, the subject's heart failure results from an ischemic event.

[0033] In another example, the subject's heart failure is due to a non-ischemic event.

[0034] In one example, the risk of cardiac death of the subject is reduced after treatment.In one example, the risk reduction is compared with the risk of cardiac death in subjects with NYHA III advanced heart failure.In another example, the risk of ischemic MACE (MI or stroke) of the subject is reduced after treatment.

[0035] In one example, the risk of ischemic MACE (non-fatal MI or non-fatal stroke) is reduced in a subject with stage III heart failure. In another example, the risk of cardiac death is reduced in a subject with stage III heart failure after treatment. In another example, the risk of ischemic MACE and cardiac death is reduced in a subject with stage III heart failure after treatment. In one example, the CRP level of a subject with stage III heart failure is 2 mg / L or higher.

[0036] In one example, the composition is administered transendocardially and / or intravenously. In one example, the composition is administered transendocardially.

[0037] The inventors have also surprisingly identified that cell therapy reduces the risk of ischemic events in subjects with cardiomyopathy.Thus, in one example, the present disclosure also encompasses a method of reducing the risk of cardiac death or non-fatal ischemic events in a subject, the method comprising administering to the subject a composition comprising cells.In one example, the subject has cardiomyopathy.In another example, the subject has microvascular disease and / or macrovascular disease.For example, the subject may have myocardial ischemia and / or diabetes.In one example, the subject has myocardial ischemia.In another example, the subject has diabetes.

[0038] In one example, the ischemic event is the formation of a cerebrovascular occlusion or a cardiac occlusion. In one example, the ischemic event is a stroke or a myocardial infarction. In one example, the subject has a non-ischemic cardiomyopathy. In one example, the cells are administered transendocardially. In one example, the subject has class II or class III heart failure according to the New York Heart Association (NYHA) classification scale. In one example, the subject has active inflammation. In one example, the subject has class II heart failure and "active inflammation." In one example, the active inflammation is characterized by a CRP level greater than 1.5 mg / L. In another example, the active inflammation is characterized by a CRP level greater than 2 mg / L. Exemplary cells are described above and throughout this disclosure. In one example, the cells induce new blood vessel formation in a target tissue. In one example, the cells promote arteriogenesis. In one example, the cells secrete factors that protect at-risk myocardium. In one example, the cells are MLPSCs. In one example, the MLPSCs are STRO-1+. In one example, the MLPSCs are mesenchymal stem cells (MSCs). In one example, the MLPSCs are allogeneic. In one example, the cells are culture-expanded. In this example, the cells may be TNAP+ before being culture-expanded. In one example, the cells are cryopreserved.

[0039] In one example, the subject has an N-terminal pro-B-type natriuretic peptide (NT-proBNP) level between 1000 pg / ml and 2000 pg / ml prior to administration of the cells. In another example, the subject has an elevated C-reactive protein (CRP) level. In another example, the subject has a CRP level of 1 mg / L or greater. In another example, the subject has a CRP level of 2 mg / L or greater. In another example, the subject has a CRP level of 2-5 mg / L. In another example, the subject has a CRP level of 3-5 mg / L.

[0040] In one embodiment of the above example, the method of the present disclosure comprises: 7 ~2×10 8 The method includes administering the cells.

[0041] In another example, the composition administered further comprises Plasma-Lyte A, dimethylsulfoxide (DMSO), and human serum albumin (HSA). In one example, the composition administered further comprises a solution of Plasma-Lyte A (70%), DMSO (10%), and HSA (25%), the HSA solution comprising 5% HSA and 15% buffer. In one example, the composition comprises 6.68x10 6 Contains > 10 viable cells / mL.

[0042] In another example, the composition comprises allogeneic mesenchymal progenitor cells (MPCs) derived from human bone marrow that have been isolated from bone mononuclear cells with anti-STRO-3 antibodies, expanded ex vivo, and cryopreserved.

[0043] In one example, the ischemic event is one or more of a heart attack, stroke, or cardiac death. In one example, the method reduces the risk of a 3-point MACE.

[0044] The inventors have also surprisingly identified that elevated CRP levels are associated with increased risk of cardiac death, myocardial infarction or stroke. Thus, in one example, the disclosure relates to a method for determining elevated risk of one or more of cardiac death, myocardial infarction or stroke in a subject, the method comprising measuring a level of CRP in a sample obtained from the subject, where elevated CRP indicates elevated risk of cardiac death, myocardial infarction or stroke. In one example, the subject has advanced heart failure. In one example, the subject's heart failure is NYHA class II heart failure. In another example, a level of CRP greater than 1 mg / L indicates elevated risk of cardiac death, myocardial infarction or stroke. In another example, a level of CRP greater than 1.5 mg / L indicates elevated risk of cardiac death, myocardial infarction or stroke. In another example, a level of CRP equal to or greater than 2 mg / L indicates elevated risk of cardiac death, myocardial infarction or stroke. In one example, the method determines elevated risk of cardiac death. [Brief description of the drawings]

[0045] [Figure 1] Reduced incidence of ischemic MACE (MI, stroke). [Figure 2A] Reduced incidence of ischemic MACE (MI, stroke); NYHA class II. [Figure 2B] Reduced incidence of ischemic MACE (MI, stroke); NYHA class III. [Figure 3A] Reduced incidence of ischemic MACE (MI, stroke); ischemic. [Figure 3B] Reduced incidence of ischemic MACE (MI, stroke); non-ischemic. [Figure 4A] Cardiac death among all treated patients (n=537). [Figure 4B] Cardiac death in grade II patients (n = 206). [Figure 4C] Cardiac death in grade III patients (n=331). [Figure 4D] Cardiac death in all treated patients (n=537); stage II patients (n=206); stage III patients (n=331). [Figure 5A] Cardiac death in NYHA class II patients; ischemic. [Figure 5B] Cardiac death in NYHA class II patients; nonischemic. [Figure 6] Cardiac death in study patients. [Figure 7A] TTFE composite IMM MACE. [Figure 7B] Rate-normalized composite IMM MACE. [Figure 7C] Curves for all treated patients with baseline CRP ≥ 2mg / L versus CRP ≤ 2mg / ml. [Figure 7D] Curves shown for non-fatal MI or non-fatal stroke. [Figure 7E] 3-point TTFE composite IMM MACE of CV death or nonfatal MI or nonfatal stroke. [Figure 7F] 3-point TTFE composite IMM MACE of CV death or nonfatal MI or nonfatal stroke. [Figure 8A] TTFE irreversible morbidity. [Figure 8B] Rate-normalized irreversible morbidity. [Figure 8C] Curves for irreversible morbidity TTFE MACE (non-fatal MI or non-fatal stroke) for all treated patients (n=537). [Figure 8D] Curves for irreversible morbidity TTFE MACE (non-fatal MI or non-fatal stroke) in grade II patients (n=206). [Figure 8E] Curves for irreversible morbidity TTFE MACE (non-fatal MI or non-fatal stroke) in grade III patients (n=331). [Figure 8F] Irreversible morbidity TTFE MACE (non-fatal MI or non-fatal stroke) in all treated patients (n=537); in stage II patients (n=206) and in stage III patients (n=331). [Figure 9A] Reduction in the incidence of composite cardiac death or ischemic MACE (MI, stroke); all treated patients (n=537). [Figure 9B] Reduced incidence of composite cardiac death or ischemic MACE (MI, stroke); grade II patients (n=206). [Figure 9C] Reduced incidence of composite cardiac death or ischemic MACE (MI, stroke); grade III patients (n=331). [Figure 9D] Reduction in the incidence of composite cardiac death or ischemic MACE (MI, stroke); all treated patients (n=537); stage II patients (n=206); stage III patients (n=331). [Figure 10] Cardiac death in NYHA class II patients--a multi-year follow-up study. [Figure 11A] NYHA class II patients with baseline hsCRP ≥ 2mg / L were at significantly higher risk of progression to cardiac death. [Figure 11B] NYHA class II patients with baseline hsCRP ≥ 2mg / L were at significantly higher risk of progression to cardiac death. [Figure 12A] NYHA class II patients with baseline hsCRP ≥ 2mg / L were at significantly higher risk of 3-point MACE (cardiac death / MI / stroke). [Figure 12B]NYHA class II patients with baseline hsCRP ≥ 2mg / L were at significantly higher risk of 3-point MACE (cardiac death / MI / stroke). [Figure 13] Heart failure patients with myocardial ischemia and / or diabetes have worse outcomes than other heart failure patients as measured by 3-point IMM MACE. [Figure 14A] Cell therapy reduced the risk of 3-point TTFE composite IMM MACE by 36% in high-risk patients with myocardial ischemia and / or diabetes. [Figure 14B] Cell therapy reduced the risk of 3-point TTFE composite IMM MACE by 36% in high-risk patients with myocardial ischemia and / or diabetes. [Figure 15A] Composite 3-point MACE and inflammation: In patients with myocardial ischemia and / or diabetes and hsCRP >2mg / L, cell therapy reduced the risk of TTFE of 3-point MACE by 54%. [Figure 15B] Composite 3-point MACE and inflammation: In patients with myocardial ischemia and / or diabetes and hsCRP >2mg / L, cell therapy reduced the risk of TTFE of 3-point MACE by 54%. [Figure 15C] Composite 3-point MACE and inflammation: In patients with myocardial ischemia and / or diabetes and hsCRP >2mg / L, cell therapy reduced the risk of TTFE of 3-point MACE by 54%. [Figure 16] High risk of triple composite MACE (MI, stroke, or CV death) in patients with ischemic CHF: Cell therapy prevents triple MACE in these subjects with CRP levels ≥ 2 mg / L. [Figure 17] High risk of non-fatal MACE (MI or stroke) in all diabetic CHF patients: Cell therapy prevents non-fatal MACE (MI or stroke) in diabetic CHF patients with CRP levels ≥ 2 mg / L. [Figure 18]High risk of triple-MACE (MI, stroke, or CV death) in diabetic CHF patients: Cell therapy prevents triple-MACE in these subjects with CRP levels ≥ 2 mg / L. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] General Techniques and Definitions Unless otherwise specified, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular biology, stem cell culture, immunology, and biochemistry).

[0047] Unless otherwise indicated, the cell culture techniques and assays utilized 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 Harbor Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), and F. M. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current editions), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J. E. Coligan et al. (editors), Current Protocols in Immunology, John It is described and explained throughout the literature in sources such as Wiley & Sons (including all current editions).

[0048] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing clear support for both meanings or either meaning.

[0049] As used herein, the term "about" refers to + / - 10%, more preferably + / - 5% of the specified value, unless otherwise specified.

[0050] The terms "level" and "amount" are used to define the amount of a particular substance in a sample from a subject or cell culture medium (or a sample therefrom). For example, a particular concentration, weight, percentage (e.g., v / v%) or ratio can be used to define the level of a particular substance in a sample. In one example, the level is expressed in terms of the extent to which a particular marker is expressed by the cells of the present disclosure under culture conditions. In one example, expression represents cell surface expression. In another example, the level is expressed in terms of the amount of a particular marker released from the cells described herein under culture conditions. In one example, a sample is obtained from a patient or subject (e.g., a blood sample) and the level of the substance in the sample is measured to determine the level of the substance in the sample.

[0051] In one example, the levels are expressed in pg / ml. For example, the levels of NT-proBNP can be expressed in pg / ml. In one example, the levels are expressed in mg / L. For example, the levels of CRP can be expressed in mg / L. In another example, the levels are expressed in mg / L. 6 Expressed in pg per piece.

[0052] In one example, the level of a particular marker in a cell culture medium is determined under culture conditions. The term "culture conditions" is used to refer to cells growing in culture. In one example, culture conditions refer to a cell population that is actively dividing. Such cells may be in an exponential growth phase, in one example. For example, the level of a particular marker may be determined by taking a sample of the 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 the cells and measuring the level of the marker in a cell lysate. It will be apparent to one of skill in the art that a secreted marker may be measured by sampling the culture medium, while a marker expressed on the surface of the cells may be measured by evaluating a sample of the cell lysate. In one example, the sample is taken when the cells are in an exponential growth phase. In one example, the sample is taken after at least two days of culture.

[0053] Cultivation and propagation of cells from cryopreserved intermediates involves thawing cryogenically frozen cells and culturing them in vitro under conditions suitable for proliferation of the cells.

[0054] In one example, the "level" or "amount" of a particular marker is determined after the cells are cryopreserved and then reseeded in culture. For example, the level is determined after the first cryopreservation of the cells. In another example, the level is determined after the second cryopreservation of the cells. For example, the cells can be culture-expanded from the cryopreserved intermediate and cryopreserved again before being reseeded in culture so that the level of the particular marker can be determined under culture conditions.

[0055] As used herein, the terms "treating," "treat," "treatment," and "reducing progression" include administering a population of mesenchymal stem cells or progenitor cells, and / or their progeny, and / or soluble factors derived therefrom, and / or extracellular vesicles derived therefrom, thereby reducing or eliminating at least one symptom of progressive heart failure, or, in the context of reducing progression, delaying the onset of progressive heart failure.

[0056] In one example, the disclosure encompasses selecting a particular subject with advanced heart failure for treatment with the cell composition disclosed herein. In one example, a subject with grade II heart failure is selected for treatment. In another example, a subject with active inflammation is selected for treatment. For example, a subject with a CRP level of 2 mg / L or greater is selected for treatment. In another example, a subject with microvascular disease and / or macrovascular disease is selected for treatment. For example, a subject with myocardial ischemia and / or diabetes is selected for treatment. In one example, a subject with active inflammation and either grade II heart failure and / or microvascular disease and / or macrovascular disease is selected for treatment.

[0057] The term "subject" as used herein refers to a human subject. For example, the subject may be an adult. In another example, the subject may be a child. In another example, the subject may be an adolescent. Terms such as "subject", "patient" or "individual" are terms that can be used interchangeably in this disclosure in context. Subjects in need of treatment include those who already have progressive heart failure, as well as those whose progressive heart failure should be prevented, delayed or stopped.

[0058] In one example, the composition of the present disclosure comprises mesenchymal precursor or stem cells that are not genetically modified. As used herein, the term "non-genetically modified" refers to cells that are not modified by transfection with a nucleic acid. For the avoidance of doubt, in the context of the present disclosure, mesenchymal precursor or stem cells that are transfected with a nucleic acid that codes for a protein are considered to be genetically modified.

[0059] As used herein, the term "sample" refers to an extract from a subject that can measure CRP levels. "Sample" includes the extract and / or derivative and / or fraction of the sample. In the present disclosure, any biological material can be used as the sample, as long as it can be taken from a subject and assayed to measure the level of CRP in the subject. In one example, the sample is a blood sample. In one example, the blood sample is obtained from a subject with NYHA II heart failure.

[0060] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" will be understood to be implied as including a stated element, integer or step, or group of elements, integers or steps, but not as excluding any other element, integer or step, or group of elements, integers or steps.

[0061] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition in question, group of steps or group of compositions in question should be interpreted as encompassing one and more (i.e., one or more) of that step, composition in question, group of steps or group of compositions in question.

[0062] Those skilled in the art will understand that the disclosure described herein is capable of variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all of the steps, features, compositions and compounds referred to or shown herein, individually or collectively, and any combination or any two or more of the above steps or features.

[0063] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are for illustrative purposes only, and functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure, as described herein.

[0064] Any example disclosed herein is deemed to apply mutatis mutandis to any other example, unless expressly stated otherwise.

[0065] progressive heart failure Cardiomyopathy is a disease of the heart muscle that makes it difficult for the heart to pump blood to the rest of the body. When the heart cannot pump enough to keep blood flowing to meet the body's demands, heart failure can occur. Cardiomyopathy can occur after ischemic or non-ischemic events. One cause of ischemic heart failure is contractile dysfunction after a myocardial infarction (MI). An MI occurs when blood no longer flows properly to a part of the heart. The lack of blood supply results in a localized area of ​​myocardial necrosis called an infarct or infarction. The infarcted heart cannot pump enough to keep blood flowing to meet the body's demands, leading to multiple pathophysiological responses and ultimately heart failure. Non-ischemic cardiomyopathy is not associated with known coronary artery disease. One example is dilated cardiomyopathy (DCM), in which the left ventricle, the main pumping chamber of the heart, becomes enlarged, dilated, and weakened, reducing the heart's ability to pump blood.

[0066] When the heart is no longer able to pump sufficiently to maintain blood flow to meet the body's demands, a series of compensatory mechanisms are initiated that contribute to mitigating the decline in cardiac output and help maintain sufficient blood pressure to perfuse vital organs. As a result, patients with heart failure may not progress for long periods of time. However, compensatory mechanisms eventually fail to compensate for the damaged heart, resulting in a progressive decline in cardiac output, referred to as "progressive heart failure." In the context of this disclosure, the terms chronic heart failure, congestive heart failure, congestive cardiac failure, systolic dysfunction, and progressive heart failure can be used interchangeably with "progressive heart failure."

[0067] The method of the present disclosure can be used to treat progressive heart failure in certain populations of MI subjects.Subjects in need of treatment include those who already have progressive heart failure, as well as those whose progressive heart failure should be prevented, delayed or stopped.In these examples, subject can have progressive heart failure of NYHA grade II or III.For example, subject can have progressive heart failure of NYHA grade II.

[0068] In a first example, the present disclosure relates to treating a subject defined according to the New York Heart Association (NYHA) classification scale. In one example, the subject has advanced heart failure less than grade III. In one example, the subject has grade II heart failure. In one example, the NYHA classification is assigned based on the subject's symptoms. For example, the NYHA classification can be assigned based on the following table: [Table 1]

[0069] In one example, the subject's heart failure is caused by an ischemic event. In one example, the subject's heart failure is caused by myocardial infarction (MI). For example, the subject can be an MI subject. The term "myocardial infarction (MI) subject" is used to define a subject who has had a myocardial infarction. In one example, the subject's heart failure is caused by non-ischemic cardiomyopathy.

[0070] In a second example, the disclosure relates to the treatment of subjects with advanced heart failure and active inflammation. "Active inflammation" is defined by elevated C-reactive protein levels. In one example, active inflammation is characterized by CRP levels of 2 mg / L or higher. Thus, in this second example, the disclosure relates to the treatment of subjects with advanced heart failure and CRP levels of 2 mg / L or higher. In one example, these subjects may have advanced heart failure of grade II or III according to the NYHA. In another example, these subjects have microvascular disease and / or macrovascular disease. For example, these subjects may have ischemia and / or diabetes. Thus, in one example, the subject may have advanced heart failure, CRP levels of 2 mg / L or higher, advanced heart failure of grade II or III, and microvascular disease and / or macrovascular disease. In another example, the subject may have advanced heart failure, CRP levels of 2 mg / L or higher, advanced heart failure of grade II or III, and ischemia and / or diabetes. In another example, the subject may have advanced heart failure, a CRP level of 2 mg / L or more, advanced heart failure of grade II or III, and ischemia. In another example, the subject may have advanced heart failure, a CRP level of 2 mg / L or more, advanced heart failure of grade II or III, and diabetes. In another example, the subject may have advanced heart failure, a CRP level of 2 mg / L or more, advanced heart failure of grade II, and ischemia. In another example, the subject may have advanced heart failure, a CRP level of 2 mg / L or more, advanced heart failure of grade II, and diabetes.

[0071] "C-reactive protein" or "CRP" is an inflammatory mediator whose levels are elevated under conditions of acute inflammatory flare-ups and rapidly normalize when inflammation subsides. In one example, a subject treated according to the present disclosure has an elevated risk or cardiac death. In one example, a subject treated according to the present disclosure may have elevated CRP. The term "elevated CRP" is used in the context of the present disclosure to refer to an increased CRP level compared to baseline CRP levels. In one example, a CRP level of 1 mg / L or greater is elevated. In another example, a CRP level of 1.5 mg / L or greater is elevated. In another example, a CRP level of 2 mg / L or greater is elevated.

[0072] In a third example, the disclosure relates to the treatment of subjects with advanced heart failure and microvascular and / or macrovascular disease. "Microvascular disease" (sometimes called small artery disease or small vessel disease) is a heart disease that affects the walls and intima of the small coronary vessels that branch off from the larger coronary arteries. In coronary MVD, the coronary vessels of the heart do not necessarily have plaque, but rather may have damage to the inner walls of the vessels, which can lead to spasms and reduced blood flow to the heart muscle. In one example, microvascular disease is "myocardial ischemia," a condition characterized by obstruction of blood flow to the myocardium (myocardium) due to partial or complete occlusion of a coronary artery. Examples of myocardial ischemia include ischemic heart failure, angina pectoris, and stroke. "Major vessel disease" is characterized by the process of atherosclerosis, which leads to narrowing of the arterial walls of the coronary vasculature. Atherosclerosis is believed to result from chronic inflammation in the coronary vasculature and damage to the arterial wall(s). Diabetes dramatically accelerates atherosclerosis by causing inflammation and slowing blood flow, and thus represents an example of macrovascular disease. In one example, the diabetes is type I diabetes or type II diabetes. In one example, the diabetes is type II diabetes.

[0073] Thus, in one example, the subject may have progressive heart failure and microvascular disease and / or macrovascular disease. In another example, the subject may have progressive heart failure and ischemia and / or diabetes. In another example, the subject may have progressive heart failure and ischemia. In another example, the subject may have progressive heart failure and diabetes. In these examples, the subject may also have active inflammation. For example, the subject may have progressive heart failure, a CRP level of 2 mg / L or more, and microvascular disease and / or macrovascular disease. In another example, the subject may have progressive heart failure, a CRP level of 2 mg / L or more, and ischemia and / or diabetes. In another example, the subject may have progressive heart failure, a CRP level of 2 mg / L or more, and ischemia. In another example, the subject may have progressive heart failure, a CRP level of 2 mg / L or more, and ischemia. In another example, the subject may have progressive heart failure, a CRP level of 2 mg / L or more, and diabetes.

[0074] The subjects of the first, second, and third examples above can be further characterized as follows: In one example, a subject treated according to the present disclosure has an initial CRP level of 2 mg / L or greater. For example, a subject can have stage II or III heart failure and an initial CRP level of 2 mg / L or greater. In another example, a subject can have stage II heart failure and an initial CRP level of 2 mg / L or greater. In one example, a subject treated according to the present disclosure has an initial CRP level of less than 5 mg / L. In another example, a subject has an initial CRP level of less than 4 mg / L. In another example, a subject has an initial CRP level of 2-6 mg / L. In another example, a subject has an initial CRP level of 3-6 mg / L. In another example, a subject has an initial CRP level of 4-5 mg / L.

[0075] There are various assays available for measuring CRP levels, such as antibody-based immunoassays. For example, CRP levels can be measured in blood samples using enzyme-linked immunosorbent (ELISA) assays. In one example, a blood sample is obtained from a patient and purified before contacting with an anti-CRP antibody. The degree of antibody binding is used to quantify the level of CRP in the blood sample (e.g., mg / L).

[0076] B-type natriuretic peptide (BNP) is a hormone produced by the heart. N-terminal (NT) prohormone BNP (NT-proBNP) is an inactive prohormone released from the same molecule that produces BNP. Both BNP and NT-proBNP are released in response to changes in pressure within the heart. These changes can be associated with heart failure and other heart problems. Levels increase when heart failure develops or worsens, and levels decrease when heart failure stabilizes. Thus, BNP is a useful marker of the progression of heart failure. In one example, the subject's NT-proBNP level before administering the composition of the present disclosure is less than 2500 pg / ml. In another example, the subject's NT-proBNP level before administering the composition of the present disclosure is less than 2400 pg / ml. In another example, the subject's NT-proBNP level before administering the composition of the present disclosure is less than 2000 pg / ml. In another example, the subject's NT-proBNP level prior to administration of the composition of the present disclosure is less than 1900 pg / ml. In another example, the subject's NT-proBNP level prior to administration of the composition of the present disclosure is between 2200 pg / ml and 1000 pg / ml. In another example, the subject's NT-proBNP level prior to administration of the composition of the present disclosure is between 2200 pg / ml and 1100 pg / ml. In another example, the subject's NT-proBNP level prior to administration of the composition of the present disclosure is between 2100 pg / ml and 1200 pg / ml. In another example, the subject's NT-proBNP level prior to administration of the composition of the present disclosure is between 2000 pg / ml and 1500 pg / ml.

[0077] In another example, prior to administration of the compositions disclosed herein, the subject has had a heart failure hospitalization event over the past 12 months. In another example, prior to administration of the compositions disclosed herein, the subject has had a heart failure hospitalization event over the past 9 months. In another example, prior to administration of the compositions disclosed herein, the subject has had a heart failure hospitalization event over the past 6-12 months. In one example, the heart failure hospitalization event is worsening signs and symptoms of heart failure. In another example, the heart failure hospitalization event

[0078] In another example, the subject can walk at least 320 meters in 6 minutes before administering the composition of the present disclosure. In another example, the subject can walk at least 330 meters in 6 minutes before administering the composition of the present disclosure. In another example, the subject can walk at least 340 meters in 6 minutes before administering the composition of the present disclosure. In another example, the subject can walk at least 350 meters in 6 minutes before administering the composition of the present disclosure.

[0079] In one example, the subject may have persistent left ventricular dysfunction. Left ventricular dysfunction is characterized by a decrease in myocardial contractility. The decrease in myocardial contractility in the left ventricle causes a decrease in left ventricular ejection fraction (LVEF). Thus, LVEF provides one way to determine left ventricular dysfunction. Another parameter of left ventricular function is left ventricular end systolic volume (LVESV), which is a measurement of the adequacy of cardiac ejection, related to contractile function. LVEF and LVESV provide an assessment of left ventricular contractile performance, and are often used together to characterize persistent left ventricular dysfunction.

[0080] LVEF and LVESV can be measured by several methods known in the art, such as echocardiogram (e.g., two-dimensional echocardiogram), single photon emission computed tomography (SPECT), cardiac magnetic resonance imaging (cMRI), or multi-gated acquisition scan.

[0081] In one example, a subject with an LVEF of less than 45% has left ventricular dysfunction. In another example, a subject with an LVEF of less than about 44%, 43%, 42%, 41% has left ventricular dysfunction. In another example, a subject with an LVEF of less than about 40% has left ventricular dysfunction. In another example, a subject with an LVEF of less than about 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30% has left ventricular dysfunction.

[0082] In the context of the present disclosure, the term "persistent left ventricular dysfunction" is used to define left ventricular dysfunction that persists over a period of time or series of measurements. For example, "persistent left ventricular dysfunction" can include left ventricular dysfunction that persists for about 1 day to about 14 days or longer.

[0083] In one example, the subject has an LVEF of less than 45%. In another example, the subject has an LVEF of less than 40%. In other examples, the subject has an LVEF of less than 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%.

[0084] In one example, the subject's LVESV is greater than 70ml. In another example, the subject's LVESV is greater than 100ml. In another example, the subject's LVESV is greater than 130ml. In another example, the subject's LVESV is between 70ml and 160ml. In these examples, the subject can also have an LVEF as described above. For example, the subject can have an LVESV of greater than 70ml and an LVEF of less than 45%.

[0085] In one example, the subject's heart failure results from an ischemic event or a non-ischemic event. In one example, the subject's heart failure results from an ischemic event as disclosed below.

[0086] The methods of the present disclosure relate to treating the progressive decline in cardiac output that is characteristic of progressive heart failure. Thus, "treat" and "treatment" in the context of this disclosure refer to both therapeutic treatment and prophylactic or preventative measures.

[0087] In one example, the treatment comprises administering a composition of the present disclosure. In one example, the method of the present disclosure reduces or inhibits the progression of advanced heart failure. In one example, the treatment inhibits the progression of the subject to NYHA stage III advanced heart failure. In another example, the treatment reduces the risk of cardiac death. In one example, the reduced risk of cardiac death is compared to the risk of cardiac death in subjects with NYHA stage III advanced heart failure. In another example, the risk of ischemic MACE (MI or stroke) is reduced after treatment. In one example, the risk of ischemic MACE (MI or stroke) is reduced by at least 50% compared to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 55% compared to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 60% compared to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 65% compared to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 70% compared to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 50%-70% compared to baseline.

[0088] In another example, the risk of 3-point MACE (cardiac death / MI / stroke) is reduced after treatment. In the context of this disclosure, "3-point MACE" is used to refer to the composite of cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke (cardiac death / MI / stroke). In one example, the risk of 3-point MACE is reduced by at least 30% compared to baseline. In another example, the risk of 3-point MACE is reduced by at least 40% compared to baseline. In another example, the risk of 3-point MACE is reduced by at least 45% compared to baseline. In another example, the risk of 3-point MACE is reduced by at least 50% compared to baseline. In another example, the risk of 3-point MACE is reduced by at least 30%-50% compared to baseline.

[0089] In one example, the treatment improves the survival rate of the patient. In one example, the treatment improves the probability that the subject will survive for at least 1000 days after the start of the treatment. In another example, the treatment improves the probability that the subject will survive for at least 2000 days after the start of the treatment. In one example, the improved probability is determined compared to a subject not treated with the composition of the present disclosure. In one example, the improved probability is determined compared to a subject with heart failure of degree III.

[0090] In one example, the treatment reduces the likelihood or risk of heart failure-related major adverse cardiovascular events (HF-MACE), defined as a composite of cardiac-related death or resuscitated cardiac death, or non-fatal decompensated heart failure events. In one example, the likelihood or risk of HF-MACE is reduced for at least 6 months, at least 12 months, at least 24 months, or at least 36 months after administration of the compositions disclosed herein. In one example, the treatment reduces the likelihood or risk of all-cause mortality.

[0091] Ischemic events In one example, the present disclosure relates to a method for reducing the risk or incidence of ischemic events in a subject, particularly a subject with cardiomyopathy. In one example, the present disclosure relates to a method for reducing the risk or incidence of ischemic events in a subject with cardiomyopathy and elevated CRP. In one example, the risk or incidence is reduced compared to a subject not administered the composition of the present disclosure. For example, the risk or incidence can be reduced compared to an untreated subject. In one example, the ischemic event is caused by the formation of an occlusion. In one example, the occlusion is an arterial occlusion. In one example, the ischemic event is the formation of a cerebrovascular occlusion. In another example, the ischemic event is the formation of a cardiac occlusion. For example, the occlusion can occur in a coronary artery.

[0092] Examples of ischemic events caused by the formation of a blockage include heart attack and stroke. Thus, in one example, the present disclosure relates to a method of reducing the risk or incidence of a heart attack or stroke in a subject with a cardiomyopathy.

[0093] The risk or incidence of ischemic events in subjects with cardiomyopathy is reduced by administering a cell therapy, such as a composition of the present disclosure.

[0094] In one example, the ischemic event is non-fatal. In one example, the ischemic event is fatal, and in this example, the method of the present disclosure reduces the risk of cardiac death due to the ischemic event. Thus, in one example, the method of the present disclosure encompasses a method of reducing the risk of cardiac death or non-fatal ischemic event in a subject, the method comprising administering to the subject a composition comprising mesenchymal precursor or stem cells. In one example, the subject has one or more or all of the following: - Grade II heart failure - Microvascular and / or macrovascular disease; -Active inflammation.

[0095] For example, the disclosure encompasses a method of reducing the risk of cardiac death or a non-fatal ischemic event in a subject, the method comprising administering to a subject a composition comprising mesenchymal precursor or stem cells, the subject having stage II heart failure.

[0096] In another example, the disclosure encompasses a method of reducing the risk of cardiac death or a non-fatal ischemic event in a subject, the method comprising administering to a subject a composition comprising mesenchymal precursor or stem cells, the subject having microvascular and / or macrovascular disease.

[0097] In another example, the disclosure encompasses a method of reducing a risk of cardiac death or a non-fatal ischemic event in a subject, the method comprising administering to a subject a composition comprising mesenchymal precursor or stem cells, the subject having active inflammation.

[0098] In one example, the subject has non-ischemic cardiomyopathy.For example, the subject's cardiomyopathy can be caused by an enlarged left ventricle (dilated cardiomyopathy).In another example, the cardiomyopathy is caused by a viral infection.

[0099] In another example, the subject has class II or class III heart failure according to the New York Heart Association (NYHA) classification scale.

[0100] In another example, the subject has an N-terminal pro-B-type natriuretic peptide (NT-proBNP) level between 1000 pg / ml and 2000 pg / ml prior to administration of the cells. In another example, the subject has an elevated C-reactive protein (CRP) level. In another example, the subject has a CRP level of 1.5 mg / L or greater. In another example, the subject has a CRP level of 2 mg / L or greater. In another example, the subject has a CRP level between 1-5 mg / L. In another example, the subject has a CRP level between 3-5 mg / L.

[0101] In one example, the cells are administered transendocardially.

[0102] In one example, the risk reduction is a 3-year risk reduction. In another example, the risk reduction is a 5-year risk reduction. In these examples, the risk of an ischemic event is reduced over a defined period of time.

[0103] Mesenchymal progenitor cells The term "mesenchymal precursor or stem cell (MLPSC)" as used herein refers to an undifferentiated multipotent cell that has the ability to self-renew while maintaining pluripotency and to differentiate into a multitude of cell types, either of mesenchymal origin, e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts and tendons, or of non-mesodermal origin, e.g., hepatocytes, neural cells and epithelial cells. For the avoidance of doubt, "mesenchymal 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.

[0104] The term "mesenchymal precursor or stem cells" includes both parental cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal precursor cells and their undifferentiated progeny.

[0105] Mesenchymal progenitor or stem cells can be autologous, allogeneic, xenogeneic, syngenic or allogeneic. Autologous cells are isolated from the same individual in which they are to be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngenic or allogeneic cells are isolated from genetically identical organisms, such as twins, clones or highly inbred research animal models.

[0106] In one example, the mesenchymal progenitor or stem cells are allogeneic. In one example, the allogeneic mesenchymal progenitor or stem cells are expanded in culture and cryopreserved.

[0107] Mesenchymal progenitor or stem cells are primarily present in bone marrow, but have also been shown to be present in a variety of host tissues, including, for example, umbilical cord blood and umbilical cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp. They are also found in the skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis, and periosteum, and can differentiate into germline cells such as mesoderm and / or endoderm and / or ectoderm. Thus, mesenchymal progenitor or stem cells can differentiate into a number of cell types, including, but not limited to, adipose, osseous, cartilaginous, elastic, muscular, and fibrous connective tissues. The particular lineage-commitment and differentiation pathways that these cells enter depend on various influences from mechanical influences and / or endogenous bioactive factors, such as growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue.

[0108] As used herein, the terms "enriched", "enriched" or variations thereof are used to describe a population of cells in which the percentage of one particular cell type or the number of several particular cell types is increased compared to a population of untreated cells (e.g., cells in their native environment). In one example, a population enriched for mesenchymal progenitor or stem cells contains at least about 0.1%, or 0.5%, or 1%, or 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 50%, or 75% mesenchymal progenitor or stem cells. In this regard, the term "a population of cells enriched for mesenchymal progenitor or stem cells" is interpreted as providing explicit support for the term "a population of cells containing X% mesenchymal progenitor or stem cells", where X% is a percentage as described herein. Mesenchymal progenitor or stem cells, in some instances, can form clonogenic colonies, e.g., CFU-F (fibroblasts) or a subset thereof (e.g., 50% or 60% or 70% or 70% or 90% or 95%) can have this activity.

[0109] In one example of the present disclosure, the mesenchymal progenitor or stem cells are mesenchymal stem cells (MSCs). The MSCs can be a homogenous composition or a mixed cell population enriched in MSCs. A homogenous MSC composition can be obtained by culturing adherent bone marrow or periosteal cells, and the MSCs can be identified by specific cell surface markers identified by unique monoclonal antibodies. Methods for obtaining cell populations enriched in MSCs are described, for example, in U.S. Pat. No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, the MSCs are allogeneic. In one example, the MSCs are cryopreserved. In one example, the MSCs are culture-expanded and cryopreserved.

[0110] In another example, the mesenchymal progenitor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs.

[0111] The isolated or enriched mesenchymal progenitor or stem cells can be expanded in vitro by culture. The isolated or enriched mesenchymal progenitor or stem cells can be cryopreserved, thawed, and then expanded in vitro by culture.

[0112] In one example, isolated or enriched mesenchymal progenitor or stem cells are 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 1 cm. 2 Seed at 50,000 viable cells per well and allow them to attach to the culture vessel overnight at 37 °C and 20% O. Then, replace and / or change the culture medium as needed and culture the cells for an additional 68-72 h at 37 °C and 5% O.

[0113] As will be understood by those skilled in the art, cultured mesenchymal precursor or stem cells are phenotypically distinct from in vivo cells. For example, in one embodiment, they express one or more markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal precursor or stem cells are also biologically distinct from in vivo cells, having a higher proliferation rate than most non-cycling (quiescent) cells in vivo.

[0114] In one example, a population of cells is enriched from a cell preparation that contains STRO-1+ cells in a selectable form. In this regard, the term "selectable form" is understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for the selection of STRO-1+ cells. The marker may be, but need not be, STRO-1. For example, as described and / or exemplified herein, cells (e.g., mesenchymal progenitor cells) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (which may be STRO-1 bright). Thus, the indication that a cell is STRO-1+ does not mean that the cell is selected solely by STRO-1 expression. In one example, the cells are selected based on at least STRO-3 expression, e.g., they are STRO-3+ (TNAP+). For example, MPCs can be isolated from bone mononuclear cells using anti-STRO-3 antibodies.

[0115] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells can be selected, isolated, or enriched from a wide variety of sources. Although, in some instances, these terms provide assistance in selecting from any tissue that contains STRO-1+ cells (e.g., mesenchymal progenitor cells), or vascular tissue, or tissue that contains pericytes (e.g., STRO-1+ pericytes), or any one or more of the tissues listed herein.

[0116] In one example, the cells used in the present disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+(HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0117] "Individually" means that the disclosure encompasses the recited markers or groups of markers separately, and that even if individual markers or groups of markers cannot be separately recited herein, the appended claims may define such markers or groups of markers separately and divisibly from one another.

[0118] "Collectively" means that the disclosure encompasses any number or combination of the recited markers or markers, and that notwithstanding that such number or combination of markers or markers may be specifically recited herein, the appended claims may define such combination or subcombination separately and divisibly from any other combination of markers or markers.

[0119] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, the term encompasses liver isoform (LAP), bone isoform (BAP) and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP as used herein refers to a molecule capable of binding to STRO-3 antibody produced by the hybridoma cell line deposited with ATCC on December 19, 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0120] Furthermore, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0121] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germ cell lineages. Non-limiting examples of lineages that STRO-1+ cells can commit to include bone precursor cells; hepatocyte precursors that are multipotent into bile duct epithelial cells and hepatocytes; neural restricted cells that can produce glial precursors that develop into oligodendrocytes and astrocytes; neuronal precursors that develop into neurons; cardiac muscle and cardiac muscle cell precursors, 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 the following: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular precursors, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes and oligodendrocytes progenitors.

[0122] In one example, mesenchymal precursor or stem cells are obtained from a single donor or multiple donors, and the donor samples or mesenchymal precursor or stem cells are subsequently pooled and then expanded in culture.

[0123] The mesenchymal precursor or stem cells encompassed by the present disclosure can also be cryopreserved prior to administration to a subject. In one example, the mesenchymal precursor or stem cells are expanded in culture and cryopreserved prior to administration to a subject.

[0124] In one example, the present disclosure encompasses mesenchymal precursor or stem cells, as well as their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal precursor or stem cells, as well as extracellular vesicles isolated therefrom. For example, the mesenchymal precursor or stem cells of the present disclosure can be cultured and grown for a period of time and under conditions suitable for secreting extracellular vesicles into cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

[0125] As used herein, the term "extracellular vesicles" refers to lipid particles that are naturally released from cells and range in size from about 30 nm to about 10 microns, but typically they are less than 200 nm in size. They are released naturally from the releasing cells (e.g., mesenchymal stem cells; STRO-1 + The nucleic acid may contain proteins, nucleic acids, lipids, metabolic products or organelles from a cell.

[0126] The term "exosomes" as used herein refers to a type of extracellular vesicles that generally range in size from about 30 nm to about 150 nm and originate from the endosomal compartment of mammalian cells, from where they are delivered to and released from the cell membrane. They may contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolic products, and may function in intercellular communication by being secreted from one cell and taken up by other cells to deliver their cargo.

[0127] In one example, the composition of the present disclosure includes cells that induce new blood vessel formation in a target tissue. In one example, the target tissue is the heart. In another example, the cells secrete factors that protect at-risk or damaged myocardium. In one example, the at-risk or damaged myocardium is exposed to insufficient blood flow due to an ischemic event. In one example, the cells secrete factors that reduce apoptosis of cardiomyocytes.

[0128] Cell culture growth In one example, the mesenchymal progenitor or stem cells are culture expanded. "Culture expanded" mesenchymal progenitor or stem cell cultures are distinct from freshly isolated cells in that they have been cultured and passaged (i.e., subcultured) in cell culture medium. In one example, the culture expanded mesenchymal progenitor or stem cells are cultured for about 4-10 passages. In one example, the mesenchymal progenitor or stem cells are cultured for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 passages. For example, the mesenchymal progenitor or stem cells can be culture expanded for at least 5 passages. In one example, the mesenchymal progenitor or stem cells can be culture expanded for at least 5-10 passages. In one example, the mesenchymal progenitor or stem cells can be culture expanded for at least 5-8 passages. In one example, the mesenchymal progenitor or stem cells can be culture expanded for at least 5-7 passages. In one example, the mesenchymal progenitor or stem cells can be culture expanded for more than 10 passages. In another example, mesenchymal progenitor or stem cells can be culture-expanded for more than 7 passages. In these examples, stem cells can be culture-expanded before cryopreservation to provide intermediate cryopreserved MLPSC populations. In one example, the composition of the present disclosure is made by culturing intermediate cryopreserved MLPSC populations, in other words, cells from cryopreserved intermediates.

[0129] In one example, the composition of the present disclosure includes mesenchymal precursor cells or stem cells cultured and expanded from a cryopreserved intermediate. In one example, the cells cultured and expanded from a cryopreserved intermediate are cultured and expanded for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 passages. For example, the mesenchymal precursor cells or stem cells can be cultured and expanded for at least 5 passages. In one example, the mesenchymal precursor cells or stem cells can be cultured and expanded for at least 5-10 passages. In one example, the mesenchymal precursor cells or stem cells can be cultured and expanded for at least 5-8 passages. In one example, the mesenchymal precursor cells or stem cells can be cultured and expanded for at least 5-7 passages. In one example, the mesenchymal precursor cells or stem cells can be cultured and expanded for more than 10 passages. In another example, the mesenchymal precursor cells or stem cells can be cultured and expanded for more than 7 passages.

[0130] In one example, mesenchymal progenitor or stem cells cultured and expanded from cryopreserved intermediates can be cultured and expanded in a medium that does not contain animal proteins. In one example, mesenchymal progenitor or stem cells cultured and expanded from cryopreserved intermediates can be cultured and expanded in a xeno-free medium. In one example, mesenchymal progenitor or stem cells cultured and expanded from cryopreserved intermediates can be cultured and expanded in a medium that does not contain fetal bovine serum.

[0131] In one embodiment, the mesenchymal precursor or stem cells can be obtained from a single donor or multiple donors, and the donor samples or mesenchymal precursor or stem cells are then pooled and then culture expanded. In one example, the culture expansion process includes: i. increasing the number of viable cells by serial propagation, comprising establishing a primary culture of the isolated mesenchymal progenitor or stem cells, and then serially establishing an initial non-primary (P1) culture of the isolated mesenchymal progenitor or stem cells from the previous culture, to provide a preparation of at least about 1 billion viable cells; ii. expanding the P1 culture of isolated mesenchymal progenitor or stem cells by passaging into a second, non-primary (P2) culture of mesenchymal progenitor or stem cells; and iii. Preparing and cryopreserving an in-process intermediate mesenchymal progenitor or stem cell preparation obtained from a P2 culture of mesenchymal progenitor or stem cells; and iv. Thawing the cryopreserved in-process intermediate mesenchymal precursor cell or stem cell preparation and expanding the in-process intermediate mesenchymal precursor cell or stem cell preparation by serial propagation.

[0132] In one example, the expanded mesenchymal progenitor or stem cell preparation has an antigenic and activity profile that includes: i. CD45+ cells less than approximately 0.75%; ii. at least about 95% CD105+ cells; iii. At least approximately 95% CD166+ cells.

[0133] In one example, the expanded mesenchymal progenitor or stem cell preparation is capable of inhibiting IL2-Rα expression by CD3 / CD28-activated PBMCs by at least about 30% compared to a control.

[0134] In one example, the cultured and expanded mesenchymal progenitor or stem cells are cultured and expanded for about 4-10 passages, the mesenchymal progenitor or stem cells are cryopreserved after at least 2 or 3 passages, and then further cultured and expanded. In one example, the mesenchymal progenitor or stem cells are cultured and expanded for at least 1, at least 2, at least 3, at least 4, or at least 5 passages, cryopreserved, and then further cultured and expanded for at least 1, at least 2, at least 3, at least 4, or at least 5 passages before being cultured according to the methods of the present disclosure.

[0135] The process of isolation and ex vivo expansion of mesenchymal precursor or stem cells can be carried out using any device and cell handling method known in the art. Various culture expansion embodiments of the present disclosure use steps that require cell manipulation, such as seeding, feeding, dissociation of adherent cultures, or washing steps. Any step that manipulates cells has the potential to damage the cells. Although mesenchymal precursor or stem cells can generally tolerate some damage during preparation, it is preferable to manipulate the cells with handling procedures and / or devices that adequately perform a given step(s) while minimizing damage to the cells.

[0136] In one example, mesenchymal progenitor or stem cells are washed in an apparatus that includes a cell source bag, a wash solution bag, a recirculating wash bag, a spinning membrane filter with inlet and outlet ports, a filtrate bag, a mixing zone, a final product bag for the washed cells, and appropriate tubing, for example, as described in U.S. Pat. No. 6,251,295, which is incorporated herein by reference.

[0137] In one example, mesenchymal progenitor or stem cell compositions cultured according to the present disclosure are 95% homogeneous for being CD105 positive, CD166 positive and CD45 negative. In one example, this homogeneity persists through ex vivo expansion, i.e., multiple population doublings.

[0138] In one example, the mesenchymal precursor or stem cells of the present disclosure are cultured and expanded in 3D culture. For example, the mesenchymal precursor or stem cells of the present disclosure can be cultured and expanded in a bioreactor. In one example, the mesenchymal precursor or stem cells of the present disclosure are cultured and expanded first in 2D culture before being further expanded in 3D culture. In one example, the mesenchymal precursor or stem cells of the present disclosure are cultured and expanded from a master cell bank. In one example, the mesenchymal precursor or stem cells of the present disclosure are cultured and expanded from a master cell bank in 2D culture before being seeded in 3D culture. In one example, the mesenchymal precursor or stem cells of the present disclosure are cultured and expanded from a master cell bank in 2D culture for at least 3 days before being seeded in 3D culture in a bioreactor. In one example, the mesenchymal precursor or stem cells of the present disclosure are cultured and expanded from a master cell bank in 2D culture for at least 4 days before being seeded in 3D culture in a bioreactor. In one example, mesenchymal progenitor or stem cells of the present disclosure are culture expanded from a master cell bank in 2D culture for 3-5 days before being seeded into 3D culture in a bioreactor. In these examples, the 2D culture can be performed in a cell factory. Various cell factory products are commercially available (e.g., Thermofisher, Sigma).

[0139] In one example, the cells of the present disclosure are STRO-3+, which are subsequently expanded in culture to provide a cryopreserved intermediate, which in this example can then be thawed and subjected to further expansion in culture (e.g., to a pharmaceutical product).

[0140] Cell culture media The mesenchymal precursor or stem cells disclosed herein can be cultured and grown in a variety of suitable growth media.

[0141] The term "medium" or "media" as used in the context of this disclosure includes components of the environment surrounding cells. A medium contributes to and / or provides suitable conditions for growing cells. A medium can be a solid, liquid, gas, or a mixture of phases and materials. A medium can include liquid growth media as well as liquid media that do not support cell growth. Media also include gelatin media such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous media include the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed.

[0142] The cell culture medium used for culture growth contains all essential and may also contain non-essential amino acids. Generally, amino acids are classified as essential (Thr, Met, Val, Leu, Ile, Phe, Trp, Lys, His) and non-essential (Gly, Ala, Ser, Cys, Gln, Asn, Asp, Tyr, Arg, Pro).

[0143] 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, it may be necessary to increase the level of glucose (or other energy source) in the basal medium or add glucose (or other energy source) during the culture process if this energy source is found to be depleted and thus limiting growth. In one example, dissolved oxygen (DO) levels can also be controlled.

[0144] In one example, the cell culture medium includes human-derived additives, for example, human serum and human platelet cell lysates can be added to the cell culture medium.

[0145] In one example, the cell culture medium contains only human-derived additives. Thus, in one example, the cell culture medium is xeno-free. For the avoidance of doubt, in these examples, the culture medium does not contain animal protein. In one example, the cell culture medium used in the method of the present disclosure does not contain animal components.

[0146] In one example, the culture medium comprises serum. In another example, the culture medium is a culture medium that does not contain fetal bovine serum and that contains growth factors that promote proliferation of mesenchymal progenitor or stem cells. In one embodiment, the culture medium is a serum-free stem cell culture medium. In one example, the cell culture medium comprises: Basal medium; Platelet-derived growth factor (PDGF); Fibroblast growth factor 2 (FGF2).

[0147] In one example, the culture medium includes platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2), and the level of FGF2 is less than about 6 ng / ml. For example, the FGF2 level can be less than about 5 ng / ml, less than about 4 ng / ml, less than about 3 ng / ml, less than about 2 ng / ml, or less than about 1 ng / ml. In other examples, the FGF2 level is less than about 0.9 ng / ml, less than about 0.8 ng / ml, less than about 0.7 ng / ml, less than about 0.6 ng / ml, less than about 0.5 ng / ml, less than about 0.4 ng / ml, less than about 0.3 ng / ml, or less than about 0.2 ng / ml.

[0148] In another example, the level of FGF2 is about 1 pg / ml to 100 pg / ml, hi another example, the level of FGF2 is about 5 pg / ml to 80 pg / ml.

[0149] In one example, the PDGF is PDGF-BB. In one example, the level of PDGF-BB is about 1 ng / ml to 150 ng / ml. In another example, the level of PDGF-BB is about 7.5 ng / ml to 120 ng / ml. In another example, the level of PDGF-BB is about 15 ng / ml to 60 ng / ml. In another example, the level of PDGF-BB is at least about 10 ng / ml. In another example, the level of PDGF-BB is at least about 15 ng / ml. In another example, the level of PDGF-BB is at least about 20 ng / ml. In another example, the level of PDGF-BB is at least about 21 ng / ml. In another example, the level of PDGF-BB is at least about 22 ng / ml. In another example, the level of PDGF-BB is at least about 23 ng / ml. In another example, the level of PDGF-BB is at least about 24 ng / ml. In another example, the level of PDGF-BB is at least about 25 ng / ml.

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

[0151] In other examples, additional factors can be added to the cell culture medium. In one example, the culture medium further comprises EGF. EGF is a growth factor that stimulates cell proliferation by binding to its receptor EGFR. In one example, the method of the disclosure comprises culturing a population of stem cells in a fetal bovine serum-free cell culture medium further comprising EGF. In one example, the level of EGF is about 0.1-7 ng / ml. For example, the level of EGF can be at least about 5 ng / ml.

[0152] In another example, the level of EGF is between about 0.2 ng / ml and 3.2 ng / ml. In another example, the level of EGF is between about 0.4 ng / ml and 1.6 ng / ml. In another example, the level of EGF is between about 0.2 ng / ml. In another example, the level of EGF is at least about 0.3 ng / ml. In another example, the level of EGF is at least about 0.4 ng / ml. In another example, the level of EGF is at least about 0.5 ng / ml. In another example, the level of EGF is at least about 0.6 ng / ml. In another example, the level of EGF is at least about 0.7 ng / ml. In another example, the level of EGF is at least about 0.8 ng / ml. In another example, the level of EGF is at least about 0.9 ng / ml. In another example, the level of EGF is at least about 1.0 ng / ml.

[0153] In the above example, a basal medium such as Alpha MEM or StemSpan™ can be supplemented with the reference amounts of growth factors. In one example, the culture medium comprises Alpha MEM or StemSpan™ supplemented with 32 ng / ml PDGF-BB, 0.8 ng / ml EGF, and 0.02 ng / ml FGF.

[0154] In other examples, additional factors can be added to the cell culture medium. For example, the cell culture medium can be supplemented with one or more stimulatory factors selected from the group consisting of epidermal growth factor (EGF), lα, 25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor alpha (TNF-α), interleukin-lβ (IL-lβ), and stromal derived factor lα (SDF-lα). In another embodiment, the cells can be cultured in the presence of at least one cytokine in an amount sufficient to support the growth of the cells. In another embodiment, the cells can be cultured in the presence of heparin or a derivative thereof. For example, the cell culture medium can include about 50 ng / ml of heparin. In other examples, the cell culture medium comprises about 60 ng / ml heparin, about 70 ng / ml heparin, about 80 ng / ml heparin, about 90 ng / ml heparin, about 100 ng / ml heparin, about 110 ng / ml heparin, about 110 ng / ml heparin, about 120 ng / ml heparin, about 130 ng / ml heparin, about 140 ng / ml heparin, about 150 ng / ml heparin or a derivative thereof. In one example, the heparin derivative is a sulfate. Various forms of heparin sulfate are known in the art, including heparin sulfate 2 (HS2). HS2 can be derived from a variety of sources, including, for example, the liver of a male and / or female mammal. Thus, exemplary heparin sulfates include male liver heparin sulfate (MML HS) and female liver heparin sulfate (FML HS).

[0155] In another example, the cell culture medium of the present disclosure promotes the proliferation of stem cells while maintaining them in an undifferentiated state. Stem cells are considered undifferentiated when they are not committed to a specific lineage of differentiation. As described above, stem cells exhibit morphological characteristics that distinguish them from differentiated cells. In addition, undifferentiated stem cells express genes that can be used as markers to detect the differentiation state. Polypeptide products can also be used as markers to detect the differentiation state. Thus, those skilled in the art can easily determine whether the method of the present disclosure maintains stem cells in an undifferentiated state using routine morphological, genetic and / or proteomic analysis.

[0156] Cellular modification The mesenchymal precursor or stem cells disclosed herein can be modified to inhibit cell lysis upon administration. Modification of antigens can induce immunological unresponsiveness or tolerance, thereby preventing induction of effector steps of the immune response (e.g., production of cytotoxic T cells, antibody production, etc.) that ultimately play a role in rejection of foreign cells in normal immune responses. Antigens that can be modified to achieve this goal include, for example, MHC class I antigens, MHC class II antigens, LFA-3, and ICAM-1.

[0157] Mesenchymal progenitor or stem cells may also be genetically modified to express proteins important for the differentiation and / or maintenance of striated skeletal 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 regulatory factors (MRFs)), transcription factors (e.g., GATA-4), cytokines (e.g., cardiotropin-1), members of the neuregulin family (e.g., neuregulins 1, 2, and 3), homeobox genes (e.g., Csx, tinman, and NKx families).

[0158] composition The mesenchymal or stem cells disclosed herein can be expanded in culture from a cryopreserved intermediate to produce a preparation comprising at least one therapeutic dose.

[0159] In one example, the composition of the present disclosure is 10×10 6 cells ~35×10 6 In another example, the composition comprises 20x10 6 cells ~30x10 6 In other examples, the composition comprises at least 100×10 6 In another example, the composition comprises 50x10 6 cells ~500x10 6In another example, the composition of the disclosure comprises 150 million cells.

[0160] In one example, the disclosed compositions include a pharma- ceutically acceptable carrier and / or excipient. The terms "carrier" and "excipient" refer to a composition of matter conventionally used in the art to facilitate the preservation, administration, and / or biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980)). A carrier may also reduce undesirable side effects of an active compound. A suitable carrier is, for example, stable and, for example, incapable of reacting with other components in the carrier. In one example, a carrier does not cause significant local or systemic adverse effects in a recipient at the dosages and concentrations used in therapy.

[0161] Carriers suitable for the present disclosure include those conventionally used, for example, water, saline, aqueous dextrose, lactose, Ringer's solution, buffer, hyaluronan and glycols are exemplary liquid carriers, particularly for solutions (when isotonic). Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

[0162] In another example, the carrier is, for example, a media composition in which cells are grown or suspended. Such media composition does not induce any adverse effects in the subject to which it is administered. Exemplary carriers and excipients do not adversely affect the viability of cells and / or the ability of cells to treat or prevent disease.

[0163] In one example, the carrier or excipient provides buffering activity to maintain the cells and / or soluble factors at the appropriate pH, thereby exerting biological activity, e.g., the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it interacts minimally with the cells and factors, allowing for rapid release of the cells and factors; in such cases, the compositions of the present disclosure can be prepared as liquids for direct application, e.g., by injection, to the bloodstream or to tissues or areas surrounding or adjacent to tissues.

[0164] The compositions of the present disclosure can be cryopreserved. Cryopreservation of mesenchymal progenitor or stem cells can be performed using slow cooling or "fast" freezing protocols known in the art. Preferably, the cryopreservation method maintains the similar phenotype, cell surface markers and proliferation rate of cryopreserved cells compared to non-frozen cells.

[0165] The cryopreservation composition may contain a cryopreservation solution, the pH of which is typically 6.5 to 8, and preferably 7.4.

[0166] Cryopreservation solutions may include sterile, non-pyrogenic, isotonic solutions, such as PlasmaLyte ATM. 100 mL of PlasmaLyte ATM contains 526 mg Sodium Chloride, USP (NaCl); 502 mg Sodium Gluconate (C6H11NaO7); 368 mg Sodium Acetate Trihydrate, USP (C2H3NaO2·3H2O), 37 mg Potassium Chloride, USP (KCl), and 30 mg Magnesium Chloride, USP (MgCl2·6H2O). No antimicrobial agents are included. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5-8.0).

[0167] The cryopreservation solution may include Profreeze™. The cryopreservation solution may additionally or alternatively include culture medium, such as αMEM.

[0168] To facilitate freezing, cryoprotectants, such as dimethyl sulfoxide (DMSO), are usually added to the cryopreservation solution. Ideally, the cryoprotectant should be non-toxic to cells and patients, non-antigenic, chemically inert, provide high survival rates after thawing, and allow for wash-free transplantation. However, DMSO, the most commonly used cryoprotectant, exhibits some cytotoxicity. Hydroxyethyl starch (HES) can be used as an alternative or in combination with DMSO to reduce the cytotoxicity of the cryopreservation solution.

[0169] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulking agents. In one example, the cryopreservation solution includes a Plasma-Lyte A (70%), DMSO (10%), HSA (25%) solution, where the HSA solution includes 5% HSA and 15% buffer.

[0170] In one example, the cryopreservation solution may further comprise one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0171] The cryopreserved composition may be thawed and administered directly to a subject or may be added to another solution, for example, containing hyaluronic acid. Alternatively, the cryopreserved composition may be thawed and the mesenchymal precursor or stem cells resuspended in an alternative carrier prior to administration.

[0172] The compositions described herein can be administered alone or as a mixture with other cells. Different types of cells can be mixed with the compositions of the present disclosure immediately or immediately prior to administration, or can be co-cultured together for a period of time prior to administration.

[0173] In one example, the composition comprises an effective amount, or a therapeutically or prophylactically effective amount, of mesenchymal precursor or stem cells and / or their progeny and / or soluble factors derived therefrom. For example, the composition comprises about 1×10 5 ~Approx. 1×10 9 Stem cells, or approximately 1.25 × 103 ~Approx. 1.25×10 7 Stem cells / kg (80 kg subject). The exact amount of cells administered will depend on a variety of factors, including the age, weight and sex of the subject, as well as the extent and severity of the disorder being treated.

[0174] Regardless of the number of cells provided in the composition, in one example, 50×10 6 ~200×10 7 In another example, 60×10 cells are administered. 6 ~200×10 6 cells or 75 x 10 6 ~150×10 6 Cells are administered. In one example, 75x10 6 In another example, 150x10 6 Cells are administered.

[0175] In one example, the composition is 5.00x10 6 In another example, the composition comprises more than 5.50x10 viable cells / mL. 6 In another example, the composition comprises more than 6.00x10 viable cells / mL. 6 In another example, the composition comprises more than 6.50x10 viable cells / mL. 6 In another example, the composition comprises more than 6.68×10 viable cells / mL. 6 Contains more than viable cells / mL.

[0176] In one example, the mesenchymal precursor or stem cells comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of the cell population of the composition.

[0177] In one example, the composition may be packaged in a suitable container, optionally together with written instructions for a desired purpose.

[0178] The compositions of the present disclosure may be administered systemically, such as by intravenous administration. In one example, the compositions are administered transendocardially.

[0179] Risk of cardiac death, myocardial infarction or stroke In one example, the method of the present disclosure is a method for assessing the risk of one or more of cardiac death, myocardial infarction, or stroke based on the level of CRP of a subject. For example, the method of the present disclosure is a method for assessing the risk of cardiac death based on the level of CRP of a subject. In one example, the present disclosure encompasses a method for determining the elevated risk of one or more of cardiac death, myocardial infarction, or stroke in a subject, the method comprising measuring the level of CRP in a sample obtained from the subject, where elevated CRP indicates elevated risk of cardiac death, myocardial infarction, or stroke. In one example, the subject has advanced heart failure. For example, the subject may have advanced heart failure of NYHA grade II. Thus, in one example, the sample is obtained from a subject with advanced heart failure of NYHA grade II. In one example, the sample is a blood sample.

[0180] In one example, a level of CRP greater than 1 mg / L indicates an increased risk of cardiac death, myocardial infarction or stroke. In one example, a level of CRP greater than 1.5 mg / L indicates an increased risk of cardiac death, myocardial infarction or stroke. In one example, a level of CRP equal to or greater than 2 mg / L indicates an increased risk of cardiac death, myocardial infarction or stroke. In one example, a level of CRP greater than 2 mg / L to 5 mg / L indicates an increased risk of cardiac death, myocardial infarction or stroke. In one example, the level of CRP is measured after an ischemic event. In one example, the ischemic event is a myocardial infarction.

[0181] In one example, a composition comprising cells that induce new blood vessel formation in target tissue is administered to a subject that is assessed to be at high risk of cardiac death, myocardial infarction or stroke.Thus, in one example, the present disclosure relates to a method for treating advanced heart failure, the method comprising:i) selecting a subject with heart failure of grade II according to the New York Heart Association (NYHA) classification scale and elevated CRP level; andii) administering a composition according to the present disclosure to the subject.In one example, the subject's CRP level is 2mg / ml or more.

[0182] It will be appreciated by those skilled in the art that many variations and / or modifications may be made to the invention as illustrated in the specific embodiments without departing from the spirit and scope of the invention as broadly described, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0183] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0184] This application claims priority to Australian Patent No. 2021903706, filed November 17, 2021, and U.S. Patent No. 63 / 384,200, filed November 17, 2022, the disclosures of which are incorporated herein by reference.

[0185] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the specification is solely for the purpose of providing context to the present invention and is not to be construed as an admission that any or all of such matters was part of the prior art or common general knowledge in the fields relevant to this invention existing prior to the priority date of each claim in this application. EXAMPLES

[0186] composition The composition consists of allogeneic MPCs derived from human bone marrow isolated from bone mononuclear cells by anti-STRO-3 antibodies, expanded ex vivo and cryopreserved.

[0187] patient Baseline data All patients (aged 18-80 years) had heart failure with reduced ejection fraction (HFrEF), a progressive disease that leads to repeated hospitalizations and an increased risk of death. [Table 2]

[0188] Key inclusion criteria included a left ventricular ejection fraction of 40% or less by two-dimensional echocardiogram or 35% or less by multi-gated acquisition scan. Additionally, the study population was enriched at enrollment to include patients with more severe disease by the presence of one or more of the following: (a) at least one HF hospitalization in the 1-9 months prior to screening, and / or (b) at least one outpatient emergency HF care visit requiring intravenous diuretics, vasodilators, and / or positive inotropic therapy in the 1-9 months prior to screening, and / or (c) for patients with atrial fibrillation, plasma NT-proBNP levels of >1000 pg / mL or >1200 pg / mL.

[0189] The primary and secondary outcome measures were: 1) Non-fatal major adverse cardiovascular events (MACE): - Heart failure MACE (recurrent decompensated heart failure events or high-grade arrhythmias); - Ischemic MACE (heart attack or stroke).

[0190] 2) Death from cardiac causes Cell therapy unexpectedly reduced the incidence of ischemic MACE (MI, stroke) by 60% compared to controls (N=537 patients; p=0.002; Figure 1). Figure 2 shows the significant reduction in the incidence of ischemic MACE (MI, stroke) observed in NYHA classes II and III compared to controls. These data suggest that cell therapy may reduce the risk of ischemic events in patients with cardiomyopathy. Figure 3 shows that a significant reduction in the incidence of ischemic MACE (MI, stroke) was observed in both ischemic and non-ischemic patients. Patients with cardiomyopathy are at risk for obstructive plaque development due to ongoing inflammatory processes in these patients. These problematic processes appear to be inhibited by cell therapy, given the general reduction in ischemic MACE observed in both ischemic and non-ischemic cardiomyopathy patients. Thus, the present data appear to support the general concept that cell therapy can be administered to lower the risk of ischemic event(s) in patients suffering from cardiomyopathy.

[0191] Surprisingly, cell therapy reduced cardiac death in NYHA stage II patients but not in NYHA stage III patients (Figures 4 and 6). This result was surprising because it suggested that a threshold level of viable myocardium was required for cell therapy to reduce cardiac death. In other words, patients with stage III heart failure may have progressed too far in the disease continuum for cell therapy to improve survival. These findings suggest that cell therapy may be particularly effective in NYHA stage II patients. Given the ability of administered cells to induce new blood vessel formation in target tissues, our findings suggest a general concept for reducing cardiac death in patients with grades lower than NYHA stage III by administering cell therapy. In further support of this concept, we noted that the reduction in cardiac death observed in NYHA stage II patients was maintained despite the cause of the cardiomyopathy, and a reduction in cardiac death was observed in ischemic and non-ischemic NYHA stage II patients (Figure 5).

[0192] 3) Improved outcomes 3-point MACE Subsequent analysis revealed that a single injection of cell therapy significantly reduced the risk of the composite outcome of 3-point irreversible morbidity or mortality MACE compared with controls across all 537 patients treated. The risk reduction was even more evident in subjects with CRP ≥ 2 mg / ml. The risk of 3-point MACE was reduced by 33% using time to first event [HR 0.667; 95% CI (0.472, 0.941); P = 0.021; Figure 7A] and by 35% in recurrent event rate analysis normalized to time of follow-up (i.e., events per 100 patient-years) [HR 0.646; 95% CI (0.466, 0.895); P = 0.009; Figure 7B]. Kaplan-Meier curves for this composite outcome in patients with plasma hsCRP levels > 2 mg / L or < 2 mg / L are shown in Figure 7C. As shown in FIG. 7C, in all treated patients with CRP above 2 mg / L, cell therapy Non-fatal MI and non-fatal stroke (Figure 7C1); and The risk of cardiac death or ischemic MACE (MI or stroke) composite (Figure 7C2) was significantly reduced.

[0193] Ischemic MACE In all 537 treated patients, a single injection of cell therapy reduced the risk of occurrence of irreversible morbidity (non-fatal MI or non-fatal stroke) compared to controls by 65% ​​using time to first event (TTFE) analysis [HR 0.346; 95% CI (0.180, 0.664); P = 0.001; Figure 8A] and by 69% using recurrent event rate normalization [HR 0.306; 95% CI (0.162, 0.579); P < 0.001; Figure 8B].

[0194] A prespecified subgroup analysis was performed for all treated patients based on the presence or absence of inflammation at the time of treatment. A single injection of cell therapy in 301 patients with inflammation (CRP ≥ 2 mg / L) reduced the risk of non-fatal MI or non-fatal stroke by 79% using TTFE [HR 0.206; 95% CI (0.070, 0.611); P = 0.004; Figure 8A] and by 83% using recurrent event rate normalization [HR 0.170; 95% CI (0.059, 0.492); P = 0.001; Figure 8B]. Considered together with the 3-point MACE analysis described above, these data support the rationale for selecting and treating patients with heart failure, active inflammation, preferably defined by CRP ≥ 2 mg / L.

[0195] Cell therapy significantly reduced the composite of cardiac death or ischemic MACE (MI or stroke) by 33% in all patients (Figure 9). Further analysis of patient groups then revealed that cell therapy significantly reduced the composite of cardiac death or ischemic MACE (MI or stroke) by 60% in NYHA stage II patients compared to controls, further supporting the rationale for selectively treating patients with NYHA stage II heart failure (Figure 9). It was also noted that cell therapy prevented the progression of cardiac death in NYHA stage II patients over multiple years of follow-up (Figure 10).

[0196] Cell therapy unexpectedly reduced the risk of cardiac death (Figure 11) and 3-point MACE (cardiac death / MI / stroke, Figure 12) in patients with elevated CRP levels, particularly NYHA class II patients with CRP levels ≥2mg / L. These beneficial effects were not evident in patients with baseline CRP <2mg / L, suggesting that cell therapy may be particularly beneficial in the presence of active inflammation.

[0197] Further data analysis revealed that CRP was a significant marker of cardiac death. As shown in FIG. 11, patients with elevated CRP levels (above 2 mg / L) had a significantly increased risk of cardiac death. These data further support the treatment of NYHA stage II patients with cell therapy, especially when those patients have elevated CRP levels, e.g., CRP above 2 mg / L. These data also support the usefulness of CRP levels as an indicator or patients at risk of cardiac death.

[0198] Myocardial ischemia and / or diabetes HF patients with microvascular or macrovascular disease (myocardial ischemia or diabetes) represent some of the most difficult to treat in terms of poor outcomes (highest hazard ratio for treated patients (Table 1)). Indeed, analysis of this patient population revealed that untreated control HF patients with myocardial ischemia and / or diabetes (n=276) were significantly associated with worse outcomes as measured by 3-point IMM MACE than other HF control patients (n=84). Notably, untreated control HF patients with myocardial ischemia and / or diabetes had significantly increased incidence of cardiovascular death or nonfatal MI or nonfatal stroke (Figure 13). However, cell therapy was surprisingly effective in these patients with myocardial ischemia and / or diabetes (n=385; Table 1), lowering the risk of 3-point composite IMM MACE by 36% (Figure 14). These data suggest that cell therapy may be particularly beneficial in HF patients with micro- or macrovascular disease, particularly myocardial ischemia and / or diabetes, supporting the rationale for selecting these patients for treatment. [Table 3]

[0199] As shown in FIG. 15, the above therapeutic efficacy was also more pronounced in HF patients with active inflammation (i.e., CRP levels of 2 mg / L or higher) in addition to microvascular disease (myocardial ischemia; FIG. 16) or macrovascular disease (diabetes; FIG. 17 and FIG. 18). These data provide further support for the therapeutic efficacy of cell therapy in patients with active inflammation. Moreover, they suggest that cell therapy is particularly beneficial in HF patients with either active inflammatory micro- and small-vascular disease or macrovascular disease, particularly myocardial ischemia or diabetes, also supporting the rationale for selecting these patients for treatment.

[0200] Comparison Data Heart failure with reduced ejection fraction (HFrEF) is a progressive disease leading to repeated hospitalizations and increased risk of death. Treatments that reduce irreversible morbidity (non-fatal myocardial infarction (MI) or non-fatal stroke) and mortality remain unclear. It is therefore particularly surprising that cell therapy was superior to other investigational agents evaluated for cardiovascular risk reduction using the 3-point IMM MACE (TTFE composite for cardiovascular death or non-fatal MI or non-fatal stroke) (Table 2). [Table 4]

Claims

1. A composition for use in treating or preventing progressive heart failure in a subject, the composition comprising mesenchymal progenitor cells or stem cells, wherein the subject has microvascular disease and / or macrovascular disease.

2. A composition comprising mesenchymal progenitor cells or stem cells for use in reducing the risk of cardiac death or a non-fatal ischemic event in a subject, wherein the subject has microvascular disease and / or macrovascular disease.

3. The composition of claim 1 or 2, wherein the subject has myocardial ischemia.

4. The composition described in claim 1 or 2, wherein the subject has diabetes.

5. The composition described in claim 1 or 2, wherein the subject has active inflammation.

6. The composition of claim 5, wherein the subject's CRP level is 2 mg / L or greater.

7. The composition of claim 1 or 2, - the subject has a left ventricular ejection fraction (LVEF) of less than about 45%, preferably less than 40%; - the subject has a left ventricular end-systolic volume (LVESV) greater than 70 ml, and / or - the subject has grade II heart failure according to the New York Heart Association (NYHA) classification scale, composition.

8. 8. The composition of claim 7, wherein the subject's LVESV is between 70 ml and 160 ml.

9. The composition described in claim 1 or claim 2, wherein the use comprises: i) selecting a subject having microvascular disease and / or macrovascular disease for treatment; and ii) administering to the subject a composition comprising mesenchymal progenitor cells or stem cells.

10. The composition of claim 1 or 2, - the subject has a level of N-terminal pro-B-type natriuretic peptide (NT-proBNP) greater than 1000 pg / mL or between 1000 pg / ml and 2500 pg / ml; - The subject has had a heart failure hospitalization event over the past 9 months, - the subject has persistent left ventricular dysfunction, and / or - the subject's heart failure is due to an ischemic or non-ischemic event, composition.

11. 6. The composition of claim 5, wherein the subject has a C-reactive protein (CRP) level of less than 5 mg / L, less than 4 mg / L, or less than 3 mg / L.

12. 3. The composition of claim 1 or 2, wherein the subject's risk of cardiac death is reduced after treatment, wherein the reduced risk is relative to the risk of cardiac death in a subject not administered the mesenchymal progenitor or stem cells, and / or the subject's risk of ischemic MACE (MI or stroke) is reduced after treatment.

13. The composition of claim 1 or 2, wherein the composition is administered transendocardially and / or intravenously.

14. The composition of claim 1 or 2, wherein the mesenchymal precursor cells or stem cells are mesenchymal precursor cells (MPCs) or mesenchymal stem cells (MSCs).

15. The composition of claim 1 or 2, - the cells are allogeneic, - the cells are grown in culture, - the cells are STRO-3+ before they are expanded in culture, and / or - the cells are cryopreserved, composition.

16. 1 x 10 ７ ~2 x 10 ８ The composition of claim 1 or 2, comprising cells.

17. The composition of claim 1 or 2, further comprising Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA).

18. 6.68×10 ６ 3. The composition of claim 1 or 2, comprising more than viable cells / mL.

19. The composition of claim 1 or 2, comprising allogeneic mesenchymal progenitor cells (MPCs) derived from human bone marrow that have been isolated from bone mononuclear cells using an anti-STRO-3 antibody, expanded ex vivo, and cryopreserved.

20. The composition of claim 2, - the ischemic event is the formation of an arterial occlusion, - the ischemic event is the formation of a cerebrovascular or cardiac occlusion, or - the ischemic event is a stroke or a myocardial infarction, composition.

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