Methods of Treating Advanced Heart Failure in a Subject With Class II Heart Failure - Patent application

Abstract

The present disclosure relates to a method for treating and / or preventing progressive heart failure in a subject with an early stage of heart failure. Such a method can be used to treat or prevent progressive heart failure in a subject with class II heart failure according to the New York Heart Association (NYHA) classification scale.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Australian Application No. 2020 / 904675, filed December 15, 2020, Australian Application No. 2021 / 900059, filed January 12, 2021, Australian Application No. 2021 / 902941, filed September 10, 2021, Australian Application No. 2021 / 903365, filed October 20, 2021, and U.S. Application No. 63 / 289,868, filed December 15, 2021, each of which is incorporated by reference in its entirety herein.

[0002] The present disclosure relates to a method for treating and / or preventing progressive heart failure in a subject with an early stage of heart failure. Such a method can be used to treat or prevent progressive heart failure in a subject with class II heart failure according to the New York Heart Association (NYHA) classification scale. [Background technology]

[0003] Myocardial infarction (MI) remains one of the leading causes of mortality and morbidity in the developed world. An update of the US Medicare records was published evaluating data including 350,509 acute MI hospitalizations in patients over 65 years of age who survived and were discharged from the hospital after the event (Schuster et al. (2004) Physiol Heart Circa Physiol.,287(2):525-32). Within the first year after the index event, 25.9% of MI patients died and 50.5% were re-admitted. In the month following the MI, they were 21 times more likely to die and 12 times more likely to be hospitalized than the general Medicare age group.

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

[0005] [Patent Document 1] Australian Application No. 2020 / 904675 [Patent Document 2] Australian Application No. 2021 / 900059 [Patent Document 3] Australian Application No. 2021 / 902941 [Patent Document 4] Australian Application No. 2021 / 903365 [Patent Document 5] U.S. Application No. 63 / 289,868 [Non-patent literature]

[0006] [Non-Patent Document 1] Schuster et al.(2004)Physiol Heart Circa Physiol.,287(2):525-32 Summary of the Invention [Problem to be solved by the invention]

[0007] Clearly, there exists a need in the art for treating or preventing progressive heart failure. [Means for solving the problem]

[0008] The inventors have surprisingly found that cell therapy is particularly effective in subjects with early stages of progressive heart failure. Thus, in an embodiment, the present disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering to the subject a composition comprising cells. In an embodiment, the subject has class II or class III heart failure according to the New York Heart Association (NYHA) classification scale. In an embodiment, the subject may have heart failure below class III according to the New York Heart Association (NYHA) classification scale. In an embodiment, the subject has class II heart failure according to the New York Heart Association (NYHA) classification scale. Thus, in an embodiment, the present disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering to the subject a composition comprising cells, the subject has class II heart failure according to the New York Heart Association (NYHA) classification scale.

[0009] In another example, the disclosure relates to a method of reducing progression of heart failure in a subject, the method comprising administering to the subject a composition comprising a cell, the subject having class II heart failure according to the New York Heart Association (NYHA) classification scale.

[0010] 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, the method comprising administering to the subject a composition comprising a cell.

[0011] In another embodiment, the present 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 having class II heart failure according to NYHA. In an embodiment, the method further comprises administering a composition comprising the cells.

[0012] In examples, the cells induce new blood vessel formation in a target tissue. In examples, the cells promote arteriogenesis. In examples, the cells secrete protective factors in at-risk myocardium. Thus, in examples, the present disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering to the subject a composition comprising cells, the subject having class II heart failure according to the New York Heart Association (NYHA) classification scale, and wherein the cells induce new blood vessel formation in a target tissue and / or secrete a secreted factor in at-risk myocardium.

[0013] In an embodiment, the cells are mesenchymal lineage precursors or stem cells (MLPSCs). In an embodiment, the MLPSCs are STRO-1+. In an embodiment, the MLPSCs are mesenchymal stem cells (MSCs). In an embodiment, the MLPSCs are allogeneic. In an embodiment, the cells are culture expanded. In this embodiment, the cells may be TNAP+ before they are culture expanded. In an embodiment, the cells are cryopreserved.

[0014] In another embodiment, the method of the present disclosure comprises the steps of: 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.

[0015] In another embodiment, administering the composition inhibits progression of the subject to NYHA Class III advanced heart failure.

[0016] In an embodiment, the subject has an N-terminal pro-B-type natriuretic peptide (NT-proBNP) level of less than 2200 pg / ml. In another embodiment, the subject's NT-proBNP is less than 2000 pg / ml prior to administering the cells. In another embodiment, the subject's NT-proBNP level is between 1000 pg / ml and 2000 pg / ml prior to administering the cells.

[0017] The inventors have also surprisingly found that cell therapy is particularly effective in subjects with early stages of advanced heart failure who have elevated C-reactive protein (CRP) levels. Thus, in an embodiment, the subject's CRP level is elevated. In an embodiment, the subject's CRP level is greater than 1 mg / L. In an embodiment, the subject's CRP level is greater than 1.5 mg / L. In an embodiment, the subject's CRP level is equal to or greater than 2 mg / L. In an embodiment, the subject's CRP level is greater than 2 mg / L. In another embodiment, the subject's CRP level is between 1.5 and 5 mg / L. In another embodiment, the subject's CRP level is less than 5 mg / L, preferably less than 4 mg / L. In another embodiment, the subject's CRP level is between 1 and 5 mg / L. In another embodiment, the subject's CRP level is between 1.5 and 5 mg / L.

[0018] In another example, the subject has had a hospitalization event for heart failure over the past 9 months.

[0019] In another embodiment, the subject has an LVEF of less than about 45%, preferably less than 40%.In another embodiment, the subject has persistent left ventricular dysfunction.

[0020] In another embodiment, the subject's heart failure results from an ischemic event.

[0021] In another embodiment, the subject's heart failure results from a non-ischemic event.

[0022] In an embodiment, the subject has a reduced risk of cardiac death after treatment. In an embodiment, the reduced risk is relative to the risk of cardiac death in subjects with NYHA class III advanced heart failure. In another embodiment, the subject has a reduced risk of ischemic MACE (MI or stroke) after treatment.

[0023] In an embodiment, a subject with class III heart failure has a reduced risk of ischemic MACE (non-fatal MI or non-fatal stroke). In another embodiment, a subject with class III heart failure has a reduced risk of cardiac death after treatment. In another embodiment, a subject with class III heart failure has a reduced risk of ischemic MACE and cardiac death after treatment. In an embodiment, a subject with class III heart failure has a CRP level of 2 mg / L or greater.

[0024] In embodiments, the composition is administered transendocardially and / or intravenously. In embodiments, the composition is administered transendocardially.

[0025] The inventors have also surprisingly identified that cell therapy reduces the risk of ischemic events in subjects with cardiomyopathy. Thus, in an embodiment, the present disclosure also encompasses a method of reducing the risk of ischemic events in a subject, the method comprising administering to the subject a composition comprising cells. In an embodiment, the subject has cardiomyopathy. In an embodiment, the ischemic event is the formation of a cerebrovascular or cardiac occlusion. In an embodiment, the ischemic event is a stroke or a myocardial infarction. In an embodiment, the subject has non-ischemic cardiomyopathy. In an embodiment, the cells are administered transendocardially. In an embodiment, the subject has class II or class III heart failure according to the New York Heart Association (NYHA) classification scale. In an embodiment, the subject has active inflammation. Surprisingly, in connection with this embodiment, the inventors have identified that administering a composition of the present disclosure can treat a wider range of patients, such as patients with class II or class III heart failure.

[0026] In an embodiment, the disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering to the subject a composition comprising mesenchymal progenitor lineage or stem cells, the subject having class II or class III heart failure according to the New York Heart Association (NYHA) classification scale and active inflammation. In another embodiment, the disclosure relates to a method for reducing progression of heart failure in a subject, the method comprising administering to the subject a composition comprising mesenchymal progenitor lineage or stem cells, the subject having class II or class III heart failure according to the New York Heart Association (NYHA) classification scale and active inflammation. In another embodiment, the disclosure relates to a method for reducing cardiac death in a subject having class II or class III heart failure according to the New York Heart Association (NYHA) classification scale active inflammation, the method comprising administering to the subject a composition comprising mesenchymal progenitor lineage or stem cells. In another embodiment, the present disclosure relates to a method of selecting a heart failure patient for treatment with cell therapy, the method comprising: i) assessing CRP levels and heart failure according to the New York Heart Association (NYHA) classification scale; and ii) selecting a subject with class II or class III heart failure according to NYHA and active inflammation, preferably the method comprising administering a composition comprising a mesenchymal precursor lineage or stem cell. In an embodiment, the active inflammation is determined based on the subject's CRP level. In an embodiment, the subject has class II heart failure and "active inflammation." In an embodiment, the active inflammation is characterized by a CRP level of more than 1.5 mg / L. In another embodiment, the active inflammation is characterized by a CRP level of more than 2 mg / L. Exemplary cells are discussed above and throughout the disclosure. In an embodiment, the cells induce new blood vessel formation in the target tissue. In an embodiment, the cells promote arteriogenesis. In an embodiment, the cells secrete factors that are protective in risk myocardium. In an embodiment, the cells are MLPSCs. In an embodiment, the MLPSCs are STRO-1+. In an embodiment, the MLPSCs are mesenchymal stem cells (MSCs). In an embodiment, the MLPSCs are allogeneic. In an embodiment, the cells are expanded in culture.In this example, the cells may be TNAP+ before they are expanded in culture. In an example, the cells are cryopreserved.

[0027] In an embodiment, the subject has an N-terminal pro-B-type natriuretic peptide (NT-proBNP) level between 1000 pg / ml and 2000 pg / ml prior to administering the cells. In another embodiment, the subject has an elevated C-reactive protein (CRP) level. In another embodiment, the subject has a CRP level of 1 mg / L or greater. In another embodiment, the subject has a CRP level of 2 mg / L or greater. In another embodiment, the subject has a CRP level of 2-5 mg / L. In another embodiment, the subject has a CRP level of 3-5 mg / L.

[0028] In the above example embodiment, the method of the present disclosure 7 ~2×10 8 The method includes administering the cells.

[0029] In another embodiment, the composition administered further comprises Plasma-LyteA, Dimethylsulfoxide (DMSO), and Human Serum Albumin (HSA). In an embodiment, the composition administered further comprises Plasma-LyteA (70%), DMSO (10%), and HSA (25%) solution, the HSA solution comprising 5% HSA and 15% buffer. In an embodiment, the composition comprises 6.68×10 6 Contains > viable cells / mL.

[0030] In another embodiment, the composition comprises 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.

[0031] In an embodiment, the ischemic event is one or more of myocardial infarction, stroke, or cardiac death. In an embodiment, the method reduces the risk of 3-point MACE.

[0032] The inventors have also surprisingly identified that elevated CRP levels are associated with an increased risk of cardiac death, myocardial infarction or stroke. Thus, in an embodiment, the present disclosure relates to a method for determining an 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 an elevated risk of cardiac death, myocardial infarction or stroke. In an embodiment, the subject has advanced heart failure. In an embodiment, the subject's heart failure is NYHA class II heart failure. In another embodiment, a level of CRP greater than 1 mg / L indicates an elevated risk of cardiac death, myocardial infarction or stroke. In another embodiment, a level of CRP greater than 1.5 mg / L indicates an elevated risk of cardiac death, myocardial infarction or stroke. In another embodiment, a level of CRP equal to or greater than 2 mg / L indicates an elevated risk of cardiac death, myocardial infarction or stroke. In an embodiment, the method determines an elevated risk of cardiac death. [Brief description of the drawings]

[0033] [Figure 1] Reduction in the incidence of ischemic MACE (MI, stroke). [Figure 2-1] Figure 2. Reduction in incidence of ischemic MACE (MI, stroke); NYHA class II and class III. [Figure 2-2] Continued from Figure 2. [Figure 3-1] Figure 3. Reduction in incidence of ischemic MACE (MI, stroke); ischemic and non-ischemic. [Figure 3-2] Continued from Figure 3. [Figure 4-1] Figure 4. Cardiac death in all treated (n=537); class II (n=206); class III patients (n=331). [Figure 4-2] Continued from Figure 4. [Figure 4-3] Continued from Figure 4. [Figure 5-1] Figure 5. Cardiac death in NYHA class II patients; ischemic and non-ischemic. [Figure 5-2] Continued from Figure 5. [Figure 6]Cardiac death in study patients. [Figure 7-1] Figure 7. A) TTFE composite IMM MACE; B) normalized IMM MACE rate; C) Curves for all treated patients with baseline CRP >= 2mg / L vs. CRP <= 2mg / ml. Curves shown for non-fatal MI or non-fatal stroke; 3-point TTFE composite IMM MACE for CV death or non-fatal MI or non-fatal stroke. [Figure 7-2] Continued from Figure 7. [Figure 7-3] Continued from Figure 7. [Figure 7-4] Continued from Figure 7. [Figure 8-1] Figure 8. A) TTFE irreversible morbidity; B) Normalized irreversible morbidity; C) Curves of irreversible morbidity TTFE MACE (non-fatal MI or non-fatal stroke) for all treated (n=537); class II (n=206); class III patients (n=331). [Figure 8-2] Continued from Figure 8. [Figure 8-3] Continued from Figure 8. [Figure 8-4] Continued from Figure 8. [Figure 8-5] Continued from Figure 8. [Figure 9-1] FIG. 9. Reduction in the incidence of composite cardiac death or ischemic MACE (MI, stroke); All treated (n=537); Class II (n=206); Class III patients (n=331). [Figure 9-2] Continued from Figure 9. [Figure 9-3] Continued from Figure 9. [Figure 10] Cardiac death in NYHA class II patients: A multi-year follow-up study [Figure 11] NYHA class II patients with baseline hsCRP ≥ 2 mg / L are at significantly increased risk of progression to cardiac death. [Figure 12] NYHA class II patients with baseline hsCRP ≥ 2mg / L are at significantly increased risk of 3-point MACE (cardiac death / MI / stroke). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] General Techniques and Definitions Unless expressly defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular biology, stem cell culture, immunology, clinical trials, medicine, and biochemistry).

[0035] 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), TABrown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), DMGlover and BDHames (editors), and FMAsubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and JEColigan et al. (editors) Current Protocols in Immunology,John Wiley & Sons(including all updates until These are described and explained throughout the literature in sources such as (present).

[0036] The term "and / or," e.g., "X and / or Y," shall be understood to mean either "X and Y" or "X or Y," and shall be interpreted as providing explicit support for both meanings or either meaning.

[0037] As used herein, the term "about" refers to + / - 10%, more preferably + / - 5% of the specified value, unless otherwise stated.

[0038] 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 an embodiment, the level is expressed in terms of how much of a particular marker is expressed by the cells of the present disclosure under culture conditions. In an embodiment, expression refers to cell surface expression. In another embodiment, the level is expressed in terms of how much of a particular marker is released from the cells described herein under culture conditions. In an embodiment, a sample is obtained from a patient or subject (e.g., a blood sample) and the level of the substance is measured in the sample to determine the level of the substance in the sample.

[0039] In an embodiment, the levels are expressed in pg / ml. For example, the levels of NT-proBNP may be expressed in pg / ml. In an embodiment, the levels are expressed in mg / L. For example, the levels of CRP may be expressed in mg / L. In another embodiment, the levels are expressed in mg / L. 6 Expressed in pg per cell.

[0040] In an embodiment, 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 an embodiment, culture conditions refer to a population of actively dividing cells. Such cells may be in an exponential growth phase, in an embodiment. 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 embodiment, 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. Those skilled in the art will appreciate that secreted markers may be measured by sampling the medium, while markers expressed on the surface of the cells may be measured by evaluating a sample of the cell lysate. In an embodiment, the sample is taken when the cells are in an exponential growth phase. In an embodiment, the sample is taken after at least two days in culture.

[0041] Culturing to expand cells derived from a cryopreserved intermediate refers to thawing the cells subjected to low temperature freezing and in vivo culture under conditions suitable for the expansion of the cells.

[0042] In an embodiment, the "level" or "amount" of a particular marker is determined after the cells are cryopreserved and then seeded back into culture. For example, the level is determined after the first cryopreservation of the cells. In another example, the level is determined after a second cryopreservation of the cells. For example, cells from a cryopreserved intermediate can be expanded in culture and cryopreserved a second time before being reseeded in culture so that the level of a particular marker can be determined under culture conditions.

[0043] 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 to thereby reduce or eliminate at least one symptom of progressive heart failure, or, in the context of reducing progression, slow the progression of the same.

[0044] 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 may be used interchangeably in this disclosure, in context. Subjects in need of treatment include those already with progressive heart failure and those in whom progressive heart failure is to be prevented, slowed, or halted.

[0045] In an embodiment, the compositions of the present disclosure include mesenchymal precursor lineage or stem cells that are not genetically modified. As used herein, the term "not genetically modified" refers to cells that have not been modified by transfection with a nucleic acid. For the avoidance of doubt, in the context of the present disclosure, mesenchymal precursor lineage or stem cells that have been transfected with a nucleic acid encoding a protein would be considered genetically modified.

[0046] 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 above sample as long as it can be collected from a subject and assayed to determine the level of CRP in the subject. In an embodiment, the sample is a blood sample. In an embodiment, the blood sample is obtained from a subject with NYHA class II heart failure.

[0047] Throughout this specification, the use of "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0048] Throughout this specification, unless specifically stated otherwise or unless the context requires otherwise, references to a single step, composition of matter, group of steps, or group of compositions of matter shall be taken to encompass one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0049] Those skilled in the art will understand that the disclosure described herein is susceptible to variations and modifications other than those specifically described. The present disclosure should be understood to include all such variations and modifications. The present disclosure also includes all steps, features, compositions and compounds referred to or shown herein, individually or collectively, and any and all combinations of said steps or features, or any two or more thereof.

[0050] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure as described herein.

[0051] Any embodiment disclosed herein shall be deemed to apply mutatis mutandis to any other embodiment, unless expressly stated otherwise.

[0052] progressive heart failure Cardiomyopathy is a disease of the heart muscle that makes it harder for the heart to pump blood to the rest of the body. When the heart cannot pump adequately to maintain blood flow and meet the body's needs, heart failure can occur. Cardiomyopathy can occur after an ischemic or non-ischemic event. One cause of ischemic heart failure is contractile dysfunction after a myocardial infarction (MI) (e.g., a heart attack). MI occurs when blood stops flowing properly to a part of the heart. The lack of blood supply leads to a localized area of ​​myocardial necrosis called an infarct or infarction. An infarcted heart cannot maintain blood flow and pump adequately to meet the body's needs, 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.

[0053] When the heart is no longer able to pump adequately to maintain blood flow and meet the body's needs, a series of compensatory mechanisms are initiated to buffer 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, the compensatory mechanisms eventually fail to compensate for the damaged heart, resulting in a progressive decline in cardiac output, referred to as "advanced heart failure." In the context of this disclosure, the terms chronic heart failure, congestive heart failure, congestive heart failure, systolic dysfunction, and advanced heart failure may be used interchangeably with "progressive heart failure."

[0054] The method of the present disclosure is effective in a subset of patients with advanced heart failure. In an embodiment, these subjects are defined based on the New York Heart Association (NYHA) classification scale. In an embodiment, the advanced heart failure of the subject is less than class III. In an embodiment, the subject has class II heart failure. In an embodiment, the NYHA classification is assigned based on the symptoms of the subject. For example, the NYHA classification can be assigned based on the following table: [Table 1]

[0055] In an embodiment, the subject's heart failure results from an ischemic event. In an embodiment, the subject's heart failure results from a myocardial infarction (MI). In an embodiment, the subject may be an MI subject. The term "myocardial infarction (MI) subject" is used to define a subject who has had a myocardial infarction. In an embodiment, the subject's heart failure results from a non-ischemic cardiomyopathy.

[0056] 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 and those whose progressive heart failure should be prevented, delayed or stopped.In these embodiments, subject has class II or class III progressive heart failure based on NYHA.For example, subject can have class II progressive heart failure.

[0057] In an embodiment, a subject treated according to the present disclosure has "active inflammation" defined by elevated levels of C-reactive protein. In an embodiment, active inflammation is characterized by CRP levels of 2 mg / L or greater.

[0058] "C-reactive protein" or "CRP" is an inflammatory mediator whose levels are elevated under conditions of acute inflammatory flare-ups and rapidly normalize once inflammation subsides. In an embodiment, there is an increased risk of cardiac death in accordance with the present disclosure. Cardiac death is death due to loss of cardiac function. In an embodiment, subjects treated in accordance with the present disclosure have elevated CRP. The term "elevated CRP" is used in the context of the present disclosure to refer to increased CRP levels compared to baseline CRP levels. In an embodiment, the CRP levels are elevated to 1 mg / L or greater. In another embodiment, the CRP levels are elevated to 1.5 mg / L or greater. In another embodiment, the CRP levels are elevated to 2 mg / L or greater.

[0059] In an embodiment, a subject treated according to the present disclosure has an initial CRP level of 2 mg / L or greater. For example, a subject may have class II or class III heart failure and an initial CRP level of 2 mg / L or greater. In another example, a subject may have class II heart failure and an initial CRP level of 2 mg / L or greater. In an embodiment, a subject treated according to the present disclosure has an initial CRP level of greater than 5 mg / L. In another embodiment, a subject has an initial CRP level of less than 4 mg / L. In another embodiment, a subject has an initial CRP level of 2-6 mg / L. In another embodiment, a subject has an initial CRP level of 3-6 mg / L. In another embodiment, a subject has an initial CRP level of 4-5 mg / L.

[0060] 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 an embodiment, a blood sample is obtained from a patient and then purified before contacting with anti-CRP antibodies. The degree of antibody binding is used to quantify the level of CRP (e.g., mg / L) in the blood sample. In an embodiment, CRP is measured by plasma high-sensitivity CRP (hsCRP) ELISA assay.

[0061] 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 present inside the heart. These changes may be related to heart failure and other heart problems. Levels increase when heart failure develops or worsens, and levels decrease when heart failure stabilizes. Thus, BNP is an effective marker of the progression of heart failure. In an embodiment, the subject's level of NT-proBNP is less than 2200 pg / ml before administering the composition of the present disclosure. In another embodiment, the subject's level of NT-proBNP is less than 2100 pg / ml before administering the composition of the present disclosure. In another embodiment, the subject's level of NT-proBNP is less than 2000 pg / ml before administering the composition of the present disclosure. In another embodiment, the subject's NT-proBNP level is less than 1900 pg / ml prior to administering the composition of the present disclosure. In another embodiment, the subject's NT-proBNP level is between 2200 pg / ml and 1000 pg / ml prior to administering the composition of the present disclosure. In another embodiment, the subject's NT-proBNP level is between 2200 pg / ml and 1100 pg / ml prior to administering the composition of the present disclosure. In another embodiment, the subject's NT-proBNP level is between 2100 pg / ml and 1200 pg / ml prior to administering the composition of the present disclosure. In another embodiment, the subject's NT-proBNP level is between 2000 pg / ml and 1500 pg / ml prior to administering the composition of the present disclosure. There are various assays available for measuring NT-proBNP levels, such as antibody-based immunoassays, e.g., ELISA assays. In an embodiment, a blood sample is obtained from a patient and then purified prior to contacting with an anti-NT-proBNP antibody. The degree of antibody binding is used to quantitate the level of NT-proBNP (eg, pg / L) in the blood sample.

[0062] In another embodiment, the subject has had a heart failure hospitalization event over the past 12 months prior to administration of the compositions disclosed herein. In another embodiment, the subject has had a heart failure hospitalization event over the past 9 months prior to administration of the compositions disclosed herein. In another embodiment, the subject has had a heart failure hospitalization event over the past 6-12 months prior to administration of the compositions disclosed herein. In an embodiment, the heart failure hospitalization event is worsening signs and symptoms of heart failure. In another embodiment, the heart failure hospitalization event is an ischemic event. In another embodiment, the heart failure hospitalization is a non-ischemic event.

[0063] In another embodiment, the subject can walk at least 320 meters in 6 minutes before administering the composition of the present disclosure. In another embodiment, the subject can walk at least 330 meters in 6 minutes before administering the composition of the present disclosure. In another embodiment, the subject can walk at least 340 meters in 6 minutes before administering the composition of the present disclosure. In another embodiment, the subject can walk at least 350 meters in 6 minutes before administering the composition of the present disclosure.

[0064] In an embodiment, the subject may have persistent left ventricular dysfunction.Left ventricular dysfunction is characterized by the decrease in myocardial contractility.When myocardial contractility decreases in the left ventricle, the result is the decrease in left ventricular ejection fraction (LVEF).Therefore, LVEF provides one way of determining left ventricular dysfunction.

[0065] LVEF and LVESV can be measured by a number of methods known in the art, such as echocardiogram, single photon emission computed tomography (SPECT), or cardiac magnetic resonance imaging (cMRI).

[0066] In some embodiments, subjects with an LVEF of less than 45% have left ventricular dysfunction. In other embodiments, subjects with an LVEF of less than about 44%, 43%, 42%, 41% have left ventricular dysfunction. In other embodiments, subjects with an LVEF of less than about 40% have left ventricular dysfunction. In other embodiments, subjects with an LVEF of less than about 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30% have left ventricular dysfunction.

[0067] 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" may include left ventricular dysfunction that persists for about 1 to about 14 days or longer.

[0068] In some embodiments, the subject has an LVEF of less than 45%. In other embodiments, the subject has an LVEF of less than 40%. In other embodiments, the subject has an LVEF of less than 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%.

[0069] The methods of the present disclosure relate to the treatment of the progressive decline in cardiac output characteristic of progressive heart failure. Thus, in the context of this disclosure, "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures.

[0070] In an embodiment, the treatment comprises administering a composition of the present disclosure. In an embodiment, the method of the present disclosure reduces or inhibits the progression of advanced heart failure. In an example, the treatment inhibits the progression of the subject to NYHA class III advanced heart failure. In another embodiment, the treatment reduces the risk of cardiac death. In an embodiment, the reduction in the risk of cardiac death is relative to the risk of cardiac death in subjects with NYHA class III advanced heart failure. In another embodiment, the risk of ischemic MACE (MI or stroke) is reduced after the treatment. In an embodiment, the risk of ischemic MACE (MI or stroke) is reduced by at least 50% compared to baseline. In another embodiment, the risk of ischemic MACE (MI or stroke) is reduced by at least 55% compared to baseline. In another embodiment, the risk of ischemic MACE (MI or stroke) is reduced by at least 60% compared to baseline. In another embodiment, the risk of ischemic MACE (MI or stroke) is reduced by at least 65% compared to baseline. In another embodiment, the risk of ischemic MACE (MI or stroke) is reduced by at least 70% compared to baseline. In another embodiment, the risk of ischemic MACE (MI or stroke) is reduced by at least 50%-70% compared to baseline.

[0071] In another embodiment, the risk of 3-point MACE (cardiac death / MI / stroke) is reduced after treatment. In the context of the present disclosure, "3-point MACE" is used to refer to and is defined as a composite of cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke (cardiac death / MI / stroke). In an embodiment, the risk of 3-point MACE is reduced by at least 30% compared to baseline. In another embodiment, the risk of 3-point MACE is reduced by at least 40% compared to baseline. In another embodiment, the risk of 3-point MACE is reduced by at least 45% compared to baseline. In another embodiment, the risk of 3-point MACE is reduced by at least 50% compared to baseline. In another embodiment, the risk of 3-point MACE is reduced by at least 30%-50% compared to baseline.

[0072] In an embodiment, the treatment increases the survival of the patient. In an embodiment, the treatment increases the probability of the subject surviving at least 1000 days after the start of the treatment. In another embodiment, the treatment increases the probability of the subject surviving at least 2000 days after the start of the treatment. In an embodiment, the increased probability is determined relative to a subject not treated with a composition of the present disclosure. In an embodiment, the increased probability is determined relative to a subject with class III heart failure.

[0073] In an embodiment, the treatment reduces the likelihood or risk of heart failure-associated major adverse cardiac events (HF-MACE), defined as a composite of cardiac-related death or resuscitated cardiac death, or non-fatal decompensated heart failure events. In an embodiment, 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 an embodiment, the treatment reduces the likelihood or risk of all-cause mortality.

[0074] Ischemic events In an embodiment, the present disclosure relates to a method for reducing the risk or incidence of an ischemic event in a subject, particularly a subject with cardiomyopathy. In an embodiment, the present disclosure relates to a method for reducing the risk or incidence of an ischemic event in a subject with cardiomyopathy and elevated CRP. In an embodiment, the risk or incidence is reduced compared to a subject not receiving a composition of the present disclosure. For example, the risk or incidence can be reduced compared to an untreated subject. In an embodiment, the ischemic event is caused by the formation of an occlusion. In an embodiment, the occlusion is an arterial occlusion. In an embodiment, the ischemic event is the formation of a cerebrovascular occlusion. In another embodiment, the ischemic event is the formation of a cardiac occlusion. For example, the occlusion can form in a coronary artery.

[0075] Examples of ischemic events caused by the formation of an occlusion include myocardial infarction and stroke. Thus, in an embodiment, the present disclosure relates to a method of reducing the risk or incidence of myocardial infarction or stroke in a subject with a cardiomyopathy.

[0076] 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.

[0077] In an embodiment, the subject has a non-ischemic cardiomyopathy. For example, the subject's cardiomyopathy can be caused by an enlargement of the left ventricle (dilated cardiomyopathy). In another embodiment, the cardiomyopathy is caused by a viral infection.

[0078] In another embodiment, the subject has class II or class III heart failure according to the New York Heart Association (NYHA) classification scale.

[0079] In another embodiment, the subject has an N-terminal pro-B-type natriuretic peptide (NT-proBNP) level between 1000 pg / ml and 2000 pg / ml prior to administering the cells. In another embodiment, the subject has an elevated C-reactive protein (CRP) level. In another embodiment, the subject has a CRP level of 1.5 mg / L or greater. In another embodiment, the subject has a CRP level of 2 mg / L or greater. In another embodiment, the subject has a CRP level of 1-5 mg / L. In another embodiment, the subject has a CRP level of 3-5 mg / L.

[0080] In an embodiment, the cells are administered transendocardially.

[0081] In an embodiment, the risk reduction is a 3 year risk reduction. In another embodiment, the risk reduction is a 5 year risk reduction. In these embodiments, the risk of an ischemic event is reduced over a defined period of time.

[0082] Mesenchymal progenitor cells As used herein, the term "mesenchymal lineage precursor or stem cell (MLPSC)" refers to an undifferentiated multipotent cell that has the ability to self-renew while maintaining pluripotency and to differentiate into several 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 lineage precursor cells" refers to cells that can differentiate into mesenchymal cells such as bone, cartilage, muscle and adipocytes, as well as fibrous connective tissue.

[0083] The term "mesenchymal lineage precursor or stem cell" includes both parent 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.

[0084] Mesenchymal precursors or stem cells can be autologous, allogeneic, xenogeneic, synthetic or homogeneous. Autologous cells are isolated from the same individual into whom they will be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Synthetic or homogeneous cells are isolated from genetically identical organisms such as twins, clones, or highly inbred research animal models.

[0085] In an embodiment, the mesenchymal precursors or stem cells are allogeneic. In an embodiment, the allogeneic mesenchymal precursors or stem cells are culture-expanded and cryopreserved.

[0086] Mesenchymal lineage precursors 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, cancellous 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 germ cell lineages such as mesoderm and / or endoderm and / or ectoderm. Thus, mesenchymal lineage precursors or stem cells can differentiate into a number of cell types, including, but not limited to, fat, bone, cartilage, elastic, muscle, and fibrous connective tissue. The specific lineage commitment and differentiation lineage that these cells enter into depends on various influences from endogenous bioactive factors, such as mechanical influences and / or growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue.

[0087] The terms "enriched", "enriched", or variations thereof, are used herein to describe a population of cells in which the percentage of one particular cell type or the percentage of several particular cell types is increased compared to an untreated population of cells (e.g., cells in their natural environment). In one example, a population enriched for mesenchymal progenitor or stem cells comprises at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 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 to be construed to expressly support the term "a population of cells comprising X% mesenchymal progenitor or stem cells", where X% is a percentage recited herein. Mesenchymal precursors or stem cells may, in some embodiments, 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%) may have this activity.

[0088] In an embodiment of the present disclosure, the mesenchymal lineage progenitor or stem cells are mesenchymal stem cells (MSCs). The MSCs can be a homogenous composition or a mixed cell population enriched for MSCs. The 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 with unique monoclonal antibodies. Methods for obtaining cell populations enriched for 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 an embodiment, the MSCs are allogeneic. In an embodiment, the MSCs are cryopreserved. In an embodiment, the MSCs are culture-expanded and cryopreserved.

[0089] In another embodiment, the mesenchymal progenitor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs.

[0090] Isolated or enriched mesenchymal precursors or stem cells may be expanded in vitro by culture. Isolated or enriched mesenchymal precursors or stem cells may be cryopreserved, thawed, and then expanded in vitro by culture.

[0091] In one example, isolated or enriched mesenchymal precursors or stem cells are cultured in culture medium (serum-free or serum-supplemented), for example, alpha minimal essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, at a concentration of 50,000 viable cells / cm. 2 Cells are seeded at 37° C. and allowed to attach to the culture vessel overnight at 37° C., 20% O. The culture medium is then replaced and / or changed as necessary, and the cells are cultured for an additional 68-72 hours at 37° C., 5% O.

[0092] As one skilled in the art will understand, cultured mesenchymal precursors or stem cells are phenotypically different from in vivo cells. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal precursors or stem cells are also biologically different from in vivo cells, having a high proliferation rate compared to most non-cycling (quiescent) cells in vivo.

[0093] In one example, the cell population is enriched from a cell preparation that contains STRO-1+ cells in a selectable morphology. In this regard, the term "selectable morphology" will be 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 precursor cells) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (and may be STRO-1 bright). Thus, indicating that cells are STRO-1+ does not mean that the cells are selected solely by STRO-1 expression. In one example, the cells are selected based on at least the expression of STRO-3, e.g., that they are STRO-3+ (TNAP+).

[0094] Reference to the selection of a cell or population thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells may be selected, isolated, or enriched from a wide variety of sources. Although, in some instances, these terms provide support for selection from any tissue that contains STRO-1+ cells (e.g., mesenchymal progenitor cells), or tissue that contains vascularized tissue or surrounding cells (e.g., STRO-1 surrounding cells), or any one or more of the tissues listed herein.

[0095] 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.

[0096] "Individually" means that the disclosure encompasses the listed markers or groups of markers separately; notwithstanding that individual markers or groups of markers are not separately recited herein, the appended claims may define such markers or groups of markers separately and divisibly from one another.

[0097] "Collectively" means that the disclosure encompasses any number or combination of the listed markers or groups of markers, and notwithstanding that such numbers or combinations of markers or groups of markers may not be specifically recited herein, the appended claims may define such combinations or subcombinations separately and divisibly from any other combinations of the markers or groups of markers.

[0098] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, the term encompasses the liver isoform (LAP), the bone isoform (BAP), and the kidney isoform (KAP). In one embodiment, TNAP is BAP. In one embodiment, TNAP as used herein refers to a molecule capable of binding to the STRO-3 antibody produced by the hybridoma cell line deposited with the ATCC under the provisions of the Budapest Treaty on December 19, 2005, deposit accession number PTA-7282.

[0099] Furthermore, in one embodiment, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0100] In one embodiment, a significant proportion of STRO-1+ cells are capable of differentiation into at least two different germ cell lineages. Non-limiting examples of lineages to which STRO-1+ cells can be committed include bone progenitor cells, hepatocyte progenitors that are multipotent for bile duct epithelial cells and hepatocytes, neural restricted cells that can generate glial cell precursors that progress to oligodendrocytes and astrocytes, neuronal precursors that progress to 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, progenitor cells: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth muscle cells and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glial cells, neuronal cells, astrocytes and oligodendrocyte cells.

[0101] In an embodiment, mesenchymal precursor or stem cells are obtained from a single donor or from multiple donors, and the donor samples or mesenchymal precursor or stem cells are subsequently pooled and then expanded in culture.

[0102] The mesenchymal precursors or stem cells encompassed by the present disclosure may also be cryopreserved prior to administration to a subject, in examples, the mesenchymal precursors or stem cells are expanded in culture and cryopreserved prior to administration to a subject.

[0103] In an embodiment, the present disclosure encompasses mesenchymal lineage precursor or stem cells and their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another embodiment, the present disclosure encompasses mesenchymal lineage precursor or stem cells and extracellular vesicles isolated therefrom. For example, the mesenchymal lineage precursor or stem cells of the present disclosure can be cultured for a period of time under conditions suitable for secretion of extracellular vesicles into cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

[0104] The term "extracellular vesicles" as used herein refers to lipid particles that are naturally released from cells and range in size from about 30 nm to 10 microns, although typically less than 200 nm in size. They may contain proteins, nucleic acids, lipids, metabolites, or organelles from the releasing cells (e.g., mesenchymal stem cells; STRO-1+ cells).

[0105] The term "exosomes" as used herein generally refers to a type of extracellular vesicles that range in size from about 30 nm to about 150 nm and originate from the endosomal compartment of mammalian cells, where they are transported to and released from the cell membrane. They may contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolites, and function in intercellular communication by being secreted from one cell and taken up by other cells to deliver their cargo.

[0106] In an embodiment, the composition of the present disclosure comprises cells that induce new blood vessel formation in a target tissue. In an embodiment, the target tissue is the heart. In another embodiment, the cells secrete factors that protect at-risk or damaged myocardium. In an embodiment, the at-risk or damaged myocardium suffers from a lack of blood flow due to an ischemic event. In an embodiment, the cells secrete factors that reduce apoptosis in cardiomyocytes.

[0107] Cell culture growth In an embodiment, the mesenchymal precursors or stem cells are culture-expanded. "Culture-expanded" mesenchymal precursors or stem cells are distinct from freshly isolated cells in that they have been cultured and passaged (i.e., subcultured) in cell culture medium. In an embodiment, the culture-expanded mesenchymal precursors or stem cells are culture-expanded for about 4-10 passages. In an embodiment, the mesenchymal precursors or stem cells are culture-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 precursors or stem cells may be culture-expanded for at least 5 passages. In an embodiment, the mesenchymal precursors or stem cells may be culture-expanded for at least 5-10 passages. In an embodiment, the mesenchymal precursors or stem cells may be culture-expanded for at least 5-8 passages. In an embodiment, the mesenchymal precursors or stem cells may be culture-expanded for at least 5-7 passages. In an embodiment, the mesenchymal precursors or stem cells may be culture-expanded for more than 10 passages. In another embodiment, the mesenchymal precursors or stem cells may be culture-expanded for 7 passages. In these examples, the stem cells may be expanded in culture prior to being cryopreserved to provide an intermediate cryopreserved MLPSC population. In examples, the compositions of the present disclosure are produced by culturing cells from the intermediate cryopreserved MLPSC population, or in other words, a cryopreserved intermediate.

[0108] In an embodiment, the composition of the present disclosure includes mesenchymal lineage precursors or stem cells cultured and expanded from a cryopreserved intermediate. In an embodiment, the cell culture cultured and expanded from a cryopreserved intermediate is a culture 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 lineage precursors or stem cells may be cultured and expanded for at least 5 passages. In an embodiment, the mesenchymal lineage precursors or stem cells may be cultured and expanded for at least 5-10 passages. In an embodiment, the mesenchymal lineage precursors or stem cells may be cultured and expanded for at least 5-8 passages. In an embodiment, the mesenchymal lineage precursors or stem cells may be cultured and expanded for at least 5-7 passages. In an embodiment, the mesenchymal lineage precursors or stem cells may be cultured and expanded for more than 10 passages. In another embodiment, the mesenchymal lineage precursors or stem cells may be cultured and expanded for 7 passages.

[0109] In an embodiment, mesenchymal lineage precursor or stem cell cultures cultured and expanded from cryopreserved intermediates may be cultured and expanded in medium that does not contain animal protein. In an embodiment, mesenchymal lineage precursor or stem cell cultures cultured and expanded from cryopreserved intermediates may be cultured and expanded in xeno-free medium. In an embodiment, mesenchymal lineage precursor or stem cell cultures cultured and expanded from cryopreserved intermediates may be cultured and expanded in medium that does not contain fetal bovine serum.

[0110] In embodiments, the mesenchymal progenitor or stem cells may be obtained from a single donor, or from multiple donors, where the donor samples or mesenchymal progenitor or stem cells are subsequently pooled and then culture expanded. In an embodiment, the culture expansion process comprises: i. expanding the number of viable cells by pathway expansion to provide a preparation of at least about 1 billion viable cells, pathway expansion comprising establishing a primary culture of isolated mesenchymal lineage progenitor or stem cells and then serially establishing a first non-primary (P1) culture of isolated mesenchymal lineage progenitor or stem cells from the previous culture; ii. expanding the P1 culture of isolated mesenchymal progenitor or stem cells into a second, non-primary (P2) culture of mesenchymal progenitor or stem cells; 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; iv. thawing the cryopreserved processed intermediate mesenchymal precursor or stem cell preparation and expanding the processed intermediate mesenchymal precursor or stem cell preparation by pathway expansion.

[0111] In the examples, the expanded mesenchymal precursor or stem cell preparations having antigenic and activity profiles include: i. less than about 0.75% CD45+ cells; ii. at least about 95% CD105+ cells; iii. at least about 95% CD166+ cells.

[0112] In examples, expanded mesenchymal precursor or stem cell preparations are capable of inhibiting IL2-Rα expression by CD3 / CD28-activated PBMCs by at least about 30% compared to controls.

[0113] In examples, the cultured expanded mesenchymal precursors or stem cells are expanded for about 4-10 passages, and the mesenchymal precursors or stem cells are cryopreserved after at least 2 or 3 passages before being further cultured. In examples, the mesenchymal precursors 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.

[0114] The process of mesenchymal precursor or stem cell isolation and ex vivo expansion can be carried out using any apparatus and cell handling methods known in the art. Various culture expansion embodiments of the present disclosure employ steps that require manipulation of the cells, such as seeding, feeding, dissociation of adherent cultures, or washing steps. The steps of manipulating the cells may be invasive to the cells. Although mesenchymal precursor or stem cells can generally tolerate a certain amount of invasiveness during preparation, it is preferable to manipulate the cells by handling procedures and / or apparatus that adequately perform a given step while minimizing invasiveness to the cells.

[0115] In an embodiment, the mesenchymal precursors or stem cells are washed in an apparatus comprising 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 washed cells, and appropriate tubing, for example as described in US 6,251,295, which is incorporated herein by reference.

[0116] In the examples, mesenchymal lineage precursor or stem cell compositions cultured according to the present disclosure are 95% homogeneous for being CD105 positive, CD166 positive, and CD45 negative, and in the examples, this homogeneity persists through ex vivo expansion, i.e., multiple population doublings.

[0117] In an embodiment, the mesenchymal precursors or stem cells of the present disclosure are cultured and expanded in 3D culture. For example, the mesenchymal precursors or stem cells of the present disclosure may be cultured and expanded in a bioreactor. In an embodiment, the mesenchymal precursors or stem cells of the present disclosure are cultured and expanded initially in 2D culture before being further expanded in 3D culture. In an embodiment, the mesenchymal precursors or stem cells of the present disclosure are cultured and expanded from a master cell bank. In an embodiment, the mesenchymal precursors or stem cells of the present disclosure are cultured and expanded from a master cell bank in 2D culture before being seeded in a 3D culture. In an embodiment, the mesenchymal precursors 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 a 3D culture in a bioreactor. In an embodiment, the mesenchymal precursors 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 a 3D culture in a bioreactor. In examples, mesenchymal lineage precursors or stem cells of the present disclosure are expanded in culture from a master cell bank in 2D culture for 3-5 days before seeding into 3D cultures in bioreactors. In these examples, the 2D cultures may be performed in cell factories. Various cell factory products are commercially available (e.g., Thermofisher, Sigma).

[0118] Cell culture media The mesenchymal precursors or stem cells disclosed herein may be cultured and expanded in a variety of suitable growth media.

[0119] The term "medium" or "media" as used in the context of this disclosure includes components of the environment surrounding cells. A medium provides suitable conditions to allow for cell growth. A medium can be a solid, liquid, gas, or a mixture of phases and materials. A medium can include liquid growth media that do not support cell growth, as well as liquid media. Media also include gelatinous media such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous media also include the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed.

[0120] All cell culture media used for culture growth contain essential and possibly 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).

[0121] Those skilled in the art will understand that for optimal results, the basal medium should be suitable 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 course of the culture if this energy source proves to be depleted and thus growth-limiting. In an embodiment, the dissolved oxygen (DO) level may also be controlled.

[0122] In an embodiment, the cell culture medium contains additives of human origin, for example, human serum and human platelet cell lysate can be added to the cell culture medium.

[0123] In the examples, the cell culture medium contains only additives of human origin. Thus, in the examples, the cell culture medium is xeno-free. For the avoidance of doubt, in these examples, the culture medium does not contain animal protein. In the examples, the cell culture medium used in the methods of the present disclosure does not contain animal components.

[0124] In an embodiment, the culture medium comprises serum. In another embodiment, the culture medium is a fetal bovine serum-free medium that comprises growth factors that promote mesenchymal lineage precursor or stem cell proliferation. In an embodiment, the culture medium is a serum-free stem cell culture medium. In an embodiment, the cell culture medium comprises: A basal medium; Platelet-derived growth factor (PDGF) and and fibroblast growth factor 2 (FGF2).

[0125] In an embodiment, the culture medium comprises 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 may 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.

[0126] In another embodiment, the level of FGF2 is between about 1 pg / ml and 100 pg / ml, hi another embodiment, the level of FGF2 is between about 5 pg / ml and 80 pg / ml.

[0127] In an embodiment, the PDGF is PDGF-BB. In an embodiment, the level of PDGF-BB is about 1 ng / ml to 150 ng / ml. In another embodiment, the level of PDGF-BB is about 7.5 ng / ml to 120 ng / ml. In another embodiment, the level of PDGF-BB is about 15 ng / ml to 60 ng / ml. In another embodiment, the level of PDGF-BB is at least about 10 ng / ml. In another embodiment, the level of PDGF-BB is at least about 15 ng / ml. In another embodiment, the level of PDGF-BB is at least about 20 ng / ml. In another embodiment, the level of PDGF-BB is at least about 21 ng / ml. In another embodiment, the level of PDGF-BB is at least about 22 ng / ml. In another embodiment, the level of PDGF-BB is at least about 23 ng / ml. In another embodiment, the level of PDGF-BB is at least about 24 ng / ml. In another embodiment, the level of PDGF-BB is at least about 25 ng / ml.

[0128] In another embodiment, the PDGF is PDGF-AB. In another embodiment, the level of PDGF-AB is about 1 ng / ml to 150 ng / ml. In another embodiment, the level of PDGF-AB is about 7.5 ng / ml to 120 ng / ml. In another embodiment, the level of PDGF-AB is about 15 ng / ml to 60 ng / ml. In another embodiment, the level of PDGF-AB is at least about 10 ng / ml. In another embodiment, the level of PDGF-AB is at least about 15 ng / ml. In another embodiment, the level of PDGF-AB is at least about 20 ng / ml. In another embodiment, the level of PDGF-AB is at least about 21 ng / ml. In another embodiment, the level of PDGF-AB is at least about 22 ng / ml. In another embodiment, the level of PDGF-AB is at least about 23 ng / ml. In another embodiment, the level of PDGF-AB is at least about 24 ng / ml. In another embodiment, the level of PDGF-AB is at least about 25 ng / ml.

[0129] In other examples, additional factors may be added to the cell culture medium. In examples, the medium further comprises EGF. EGF is a growth factor that promotes cell proliferation by binding to its receptor EGFR. In examples, the methods of the disclosure include culturing a population of stem cells in a fetal bovine serum-free cell culture medium further comprising EGF. In examples, the value of EGF is about 0.1-7 ng / ml. For example, the level of EGF may be at least about 5 ng / ml.

[0130] In another embodiment, the level of EGF is about 0.2 ng / ml to 3.2 ng / ml. In another embodiment, the level of EGF is about 0.4 ng / ml to 1.6 ng / ml. The level of EGF is about 0.2 ng / ml. In another embodiment, the level of EGF is at least about 0.3 ng / ml. In another embodiment, the level of EGF is at least about 0.4 ng / ml. In another embodiment, the level of EGF is at least about 0.5 ng / ml. In another embodiment, the level of EGF is at least about 0.6 ng / ml. In another embodiment, the level of EGF is at least about 0.7 ng / ml. In another embodiment, the level of EGF is at least about 0.8 ng / ml. In another embodiment, the level of EGF is at least about 0.9 ng / ml. In another embodiment, the level of EGF is at least about 1.0 ng / ml.

[0131] In the above examples, a basal medium such as alphaMEM or StemSpan™ may be supplemented with the growth factors in the amounts referenced. In the examples, the culture medium comprises alphaMEM or StemSpan™ supplemented with 32 ng / ml PDGF-BB, 0.8 ng / ml EGF, and 0.02 ng / ml FGF.

[0132] In other examples, additional factors may be added to the cell culture medium. For example, the cell culture medium may be supplemented with one or more 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 may also be cultured in the presence of at least one cytokine in an amount sufficient to support the expansion of the cells. In another embodiment, the cells may be cultured in the presence of heparin or a derivative thereof. For example, the cell culture medium may contain about 50 ng / ml of heparin. In other examples, the cell culture medium contains 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 an embodiment, the heparin derivative is 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 male and / or female mammals. Thus, exemplary heparin sulfates include male liver heparin sulfate (MML HS) and female liver heparin sulfate (FML HS).

[0133] In another embodiment, the cell culture medium of the present disclosure promotes stem cell proliferation while maintaining stem cells in an undifferentiated state. Stem cells are considered undifferentiated when they are not committed to a specific differentiation lineage. As described above, stem cells exhibit morphological characteristics that distinguish them from differentiated cells. Furthermore, 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, one 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.

[0134] Cellular modification The mesenchymal precursor or stem cell disclosed herein can be modified in such a manner that cell lysis is inhibited upon administration. Modification of antigens can induce immunological unresponsiveness or tolerance, thereby preventing induction of the effector phase of immune response (e.g., cytotoxic T cell generation, antibody production, etc.) that is ultimately responsible for rejection of foreign cells in normal immune responses. Antigens that can be modified to achieve this purpose include, for example, MHC class I antigens, MHC class II antigens, LFA-3, and ICAM-1.

[0135] Mesenchymal lineage precursors or stem cells can also be genetically engineered to express proteins important for the differentiation and / or maintenance of linear skeletal muscle cells. Exemplary proteins include growth factors (TGF-β, insulin-like enhancing factor 1 (IGF-1), FGF), myogenic factors (e.g., myoD, myogenin, myogenic factor 5 (Myf5), myogenic regulatory factors (MRF)), transcription factors (e.g., GATA-4), cytokines (e.g., cardiac tropin-1), the neuregulin family (e.g., neuregulins 1, 2, and 3), and homeobox genes (e.g., Csx, tinman, and NKx families).

[0136] composition The mesenchymal lineage or stem cells disclosed herein may be expanded in culture from cryopreserved intermediates to produce a preparation containing at least one therapeutic dose.

[0137] In the examples, the compositions of the present disclosure are 6 cells ~ 35 × 10 6 In another embodiment, the composition comprises 20×10 6 cells ~ 30 × 10 6 In other embodiments, the composition comprises at least 100×10 cells. 6 In another embodiment, the composition comprises 50×10 6 cells ~ 500 x 10 6In other examples, the compositions of the disclosure comprise 150 million cells.

[0138] In one embodiment, the compositions of the present disclosure 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 storage, 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 any 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 embodiment, a carrier does not cause significant local or systemic adverse effects in a recipient at the dosages and concentrations used in therapy.

[0139] Carriers suitable for the present disclosure include those conventionally used, such as water, saline, aqueous dextrose, lactose, Ringer's solution, buffer solutions, hyaluronan, and glycols, which are exemplary liquid carriers, especially 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.

[0140] In another example, the carrier is, for example, a media composition in which the cells are grown or suspended. Such a 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 the cells and / or the ability of the cells to treat or prevent a disease.

[0141] In one example, the carrier or excipient provides a buffering activity to maintain the cells and / or soluble factors at an appropriate pH, thereby exerting biological activity, for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient as it interacts minimally with the cells and factors, allowing for rapid release of the cells and factors, and in such cases, the compositions of the present disclosure can be produced as a liquid for application, for example, by injection, into or directly into the bloodstream, or directly to a tissue or an area surrounding or adjacent to a tissue.

[0142] The compositions of the present disclosure may be cryopreserved. Cryopreservation of mesenchymal precursors or stem cells may be performed using slow cooling methods or "fast" freezing protocols known in the art. Preferably, the cryopreservation method maintains similar phenotype, cell surface markers and growth rate of cryopreserved cells compared to non-frozen cells.

[0143] The cryopreservation composition may contain a cryopreservation solution, the pH of which is usually 6.5 to 8, preferably 7.4.

[0144] Cryopreservation solutions may include sterile, non-pyrogenic, isotonic solutions (e.g., PlasmaLyte ATM). 100 mL of PlasmaLyte ATM contains 526 mg of sodium chloride USP (NaCl), 502 mg of sodium gluconate (C6H11NaO7), 368 mg of sodium acetate trihydrate USP (C2H3NaO2·3H2O), 37 mg of potassium chloride USP (KCl), and 30 mg of magnesium chloride USP (MgCl2·6H2O). No antimicrobial agents are included. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5-8.0).

[0145] The cryopreservation solution may include Profreeze™. The cryopreservation solution may additionally or alternatively include culture medium, such as αMEM.

[0146] To facilitate freezing, cryoprotectants such as dimethyl sulfoxide (DMSO) are usually added to the cryopreservation solution. Ideally, the cryoprotectant should be non-toxic to cells and patients, non-antigenic, chemically inert, provide high survival rates after thawing, and allow transplantation without washing. However, DMSO, the most commonly used cryoprotectant, exhibits some degree of cytotoxicity. Hydroxyethyl starch (HES) can be used as an alternative or in combination with DMSO to reduce the cytotoxicity of the cryopreservation solution.

[0147] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulking agents. In one embodiment, the cryopreservation solution includes Plasma Solution A (70%), DMSO (10%), HSA (25%) solution, where the HSA solution includes 5% HSA and 15% buffer.

[0148] For example, the cryopreservation solution may further comprise one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0149] 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 precursors or stem cells resuspended in an alternative carrier prior to administration.

[0150] The compositions described herein may be administered alone or as a mixture with other cells. Different types of cells may be mixed with the compositions of the present disclosure immediately or shortly before administration, or may be co-cultured for a period of time before administration.

[0151] In one embodiment, 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 may comprise about 1×10 5 Stem cells ~ approx. 1×10 9stem cells, or approximately 1.25 x 10 3 Stem cells ~ 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.

[0152] Regardless of the number of cells provided in the composition, in the examples, 50×10 6 ~200×10 7 In another embodiment, 60×10 cells are administered. 6 ~200×10 6 cells or 75 x 10 6 ~150×10 6 In the embodiment, 75×10 cells are administered. 6 In another embodiment, 150×10 cells are administered. 6 cells are administered.

[0153] In the examples, the composition is 5.00×10 6 In another embodiment, the composition comprises more than 5.50×10 viable cells / mL. 6 In another embodiment, the composition comprises more than 6.00×10 viable cells / mL. 6 In another embodiment, the composition comprises 6.50×10 viable cells / mL or more. 6 In another embodiment, the composition comprises 6.68×10 viable cells / mL or more. 6 Contains more than 10 viable cells / mL.

[0154] In embodiments, the mesenchymal precursors or stem cells comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of the cell population of the composition.

[0155] In embodiments, the compositions may be optionally packaged in a suitable container accompanied by written instructions for a desired purpose.

[0156] Compositions of the present disclosure may be administered systemically, such as, for example, by intravenous administration. In examples, the compositions are administered transendocardially.

[0157] Risk of cardiac death, myocardial infarction or stroke In an embodiment, the method of the present disclosure relates to a method for assessing the risk of one or more of cardiac death, myocardial infarction, or stroke based on the level of CRP in a subject. For example, the method of the present disclosure may relate to a method for assessing the risk of cardiac death based on the level of CRP in a subject. In an embodiment, the present disclosure encompasses a method for determining an 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, and elevated CRP indicates an elevated risk of cardiac death, myocardial infarction, or stroke. In an embodiment, the subject has progressive heart failure. For example, the subject may have NYHA class II progressive heart failure. Thus, in an embodiment, the sample is obtained from a subject with NYHA class II progressive heart failure. In an embodiment, the sample is a blood sample.

[0158] In an embodiment, a CRP level above 1 mg / L indicates an increased risk of cardiac death, myocardial infarction or stroke. In an embodiment, a CRP level above 1.5 mg / L indicates an increased risk of cardiac death, myocardial infarction or stroke. In an embodiment, a CRP level of 2 mg / L or more indicates an increased risk of cardiac death, myocardial infarction or stroke. In an embodiment, a CRP level of 2-5 mg / L or more indicates an increased risk of cardiac death, myocardial infarction or stroke. In an embodiment, the level of CRP is measured after an ischemic event. In an embodiment, the ischemic event is a myocardial infarction.

[0159] In an embodiment, a composition comprising cells that induce neovascularization in a target tissue is administered to a subject that is assessed to have an elevated risk of cardiac death, myocardial infarction, or stroke.Accordingly, in an embodiment, the present disclosure relates to a method for treating advanced heart failure, the method comprising: i) selecting a subject with class II heart failure according to the New York Heart Association (NYHA) classification scale and elevated CRP level; and ii) administering a composition according to the present disclosure to the subject.In an embodiment, the subject's CRP level is greater than 2mg / ml.

[0160] It will be appreciated by those skilled in the art that numerous variations and / or modifications may be made to the invention as illustrated in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0161] This application claims priority from AU2020904675, filed December 15, 2020, 2021900059, filed January 12, 2021, AU2021902941, filed September 10, 2021, and AU2021903365, filed October 20, 2021, the disclosures of which are incorporated herein in their entireties.

[0162] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0163] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention, and is not to be construed as an admission that any or all of such matters formed part of the prior art or were general general knowledge in the art relevant to the present invention as they existed prior to the priority date of each claim of this application. EXAMPLES

[0164] composition The composition consists of human bone marrow-derived allogeneic MPCs isolated from bone mononuclear cells using anti-STRO-3 antibodies, expanded ex vivo, and cryopreserved.

[0165] patient Baseline data [Table 2] [Table 3]

[0166] The primary and secondary outcome measures were: 1) Non-fatal major adverse cardiovascular events (MACE): heart failure MACE (recurrent decompensated heart failure events or severe arrhythmias); Ischemic MACE (heart attack or stroke) 2) Death due to cardiac causes

[0167] Cell therapy unexpectedly reduced the non-significant rate of ischemic MACE (MI, Stroke) by 60% compared to controls (N=537 patients; p=0.002; Figure 1). Figure 2 shows that a significant reduction in the incidence of ischemic MACE (MI, Stroke) was observed compared to controls in NYHA classes II & III. 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 ischemic MACE (MI, Stroke) was observed in both ischemic and non-ischemic patients. Cardiomyopathy patients 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, considering the general reduction in ischemic MACE observed in both ischemic and non-ischemic cardiomyopathy patients. Thus, the present data seem to support the general concept that cell therapy can be administered to reduce the risk of ischemic events in patients suffering from cardiomyopathy.

[0168] Surprisingly, cell therapy reduced cardiac death in NYHA class II patients but not in NYHA class III patients (Figures 4 and 6). This result was surprising because it suggested that a threshold level of viable myocardium was required to reduce cardiac death with cell therapy. In other words, patients with class III heart failure may have progressed too far along the disease continuum for cell therapy to improve survival. These findings suggest that cell therapy is particularly effective in NYHA class 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 below NYHA class III by administering cell therapy. As further support for this concept, we noted that the observed reduction in cardiac death for NYHA class II patients was maintained despite the cause of the cardiomyopathy, with reduction in cardiac death observed in ischemic and non-ischemic NYHA class II patients (Figure 5).

[0169] 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 death MACE compared with controls in all 537 treated patients. 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 the time to first event analysis [HR 0.667; 95% CI (0.472, 0.941); P = 0.021; Figure 7A] and by 35% in the recurrent event rate analysis normalized by 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 illustrated in Figure 7C. As shown in FIG. 7C, in all treated patients with CRP ≥ 2 mg / L, cell therapy significantly reduced the risk of: Non-fatal MI and non-fatal stroke (Figure 7C1), and · Composite of cardiac death or ischemic MACE (MI or stroke) (Figure 7C2).

[0170] Ischemic MACE Across all 537 patients treated, a single injection of cell therapy reduced the risk of occurrence of irreversible morbidity (non-fatal MI or non-fatal stroke) by 65% ​​compared to control using time to first event (TTFE) [HR 0.346; 95% CI (0.180, 0.664); P = 0.001; Figure 8A], and by 69% using normalized recurrent event rate [HR 0.306; 95% CI (0.162, 0.579); P < 0.001; Figure 8B].

[0171] A prespecified subgroup analysis was performed on 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]. Combined with the above 3-point MACE analysis, these data support the rationale for selecting and treating patients with heart failure, preferably with active inflammation, as defined by CRP > 2 mg / L.

[0172] 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 a significant 60% reduction in the composite of cardiac death or ischemic MACE (MI or stroke) in NYHA class II patients compared to controls, further supporting the rationale for selecting and treating patients with NYHA class II heart failure (Figure 9). It was also noted that cell therapy prevented the progression of cardiac death in NYHA class II patients over multiple years of follow-up (Figure 10).

[0173] Cell therapy also unexpectedly reduced the risk of cardiac death (Figure 11) and 3-point MACE (cardiac death / MI / stroke; Figure 12) in NYHA class II patients with elevated CRP levels, particularly CRP levels above 2 mg / L. These beneficial effects were not evident in patients with baseline CRP below 2 mg / L, suggesting that cell therapy may be particularly beneficial in the presence of active inflammation.

[0174] Further data analysis revealed that CRP is an important marker of cardiac death. As shown in FIG. 11, patients with elevated CRP levels (greater than 2 mg / L) had a significantly increased risk of cardiac death. These data further support the treatment of NYHA class II patients with cell therapy, especially when these patients have elevated CRP levels, e.g., CRP greater than 2 mg / L. These data also show the utility of CRP levels as an indicator or patients at risk of cardiac death.

Claims

1. A composition for treating or preventing progressive heart failure in a subject, comprising mesenchymal progenitor or stem cells (MLPSCs), wherein the subject has Class II heart failure according to the New York Heart Association (NYHA) classification scale, and the subject's CRP level is 2 mg / L or greater.

2. The composition described in claim 1, wherein treatment reduces the subject's progression to NYHA Class III advanced heart failure.

3. A composition comprising mesenchymal progenitor or stem cells (MLPSCs) for use in a method for reducing the risk of an ischemic event or cardiac death in a subject having Class II heart failure according to the New York Heart Association (NYHA) classification scale, wherein the subject has a CRP level of 2 mg / L or greater.

4. 1. A method for selecting heart failure patients 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 subjects with class II heart failure according to NYHA and a CRP level of 2 mg / L or greater.

5. A composition described in any one of claims 1 to 3, wherein the treatment comprises the steps of 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 MLPSCs that induce new blood vessel formation in a target tissue.

6. the subject's level of N-terminal pro-B-type natriuretic peptide (NT-proBNP); less than 2200 pg / ml, preferably less than 2000 pg / ml, prior to administration of the cells; or The composition of any one of claims 1 to 3 or 5, wherein the composition is at 1000 pg / ml to 2000 pg / ml prior to administration of the cells.

7. The composition of any one of claims 1 to 3, 5 or 6, wherein the subject has a C-reactive protein (CRP) level of less than 5 mg / L, preferably less than 4 mg / L.

8. The subject is - Heart failure hospitalization events over the past 9 months; - LVEF less than 45%, or less than 40%; - persistent left ventricular dysfunction; - Heart failure resulting from ischemic or non-ischemic events The composition of any one of claims 1 to 3 or 5 to 7, comprising one or more of:

9. The composition of any one of claims 1-3 or 5-8, wherein the subject has a reduced risk of ischemic MACE (MI or stroke) or cardiac death following treatment.

10. 10. The composition of claim 9, wherein the risk reduction is relative to the risk of cardiac death in subjects with NYHA Class III advanced heart failure.

11. The composition of any one of claims 1 to 3 or 5 to 10, wherein the MLPSCs are mesenchymal stem cells (MSCs).

12. 1x10 ７ ~2×10 ８ The composition of any one of claims 1 to 3 or 5 to 11, comprising cells.

13. The composition of any one of claims 1-3 or 5-12, wherein the composition further comprises a Plasma-Lyte A (70%), DMSO (10%), HSA (25%) solution, wherein the HSA solution comprises 5% HSA and 15% buffer, or wherein the composition comprises greater than 6.68 x 10 6 viable cells / mL.

14. The composition of any one of claims 1 to 3, 5 to 10, 12, or 13, wherein the MLPSCs are mesenchymal progenitor cells.

15. The composition of claim 14, wherein said composition comprises human bone marrow-derived allogeneic MPCs isolated from bone mononuclear cells by anti-STRO-3 antibodies, expanded ex vivo, and cryopreserved.

16. the ischemic event: - formation of arterial blockages; - formation of cerebrovascular occlusion; - formation of cardiac blockages; - stroke; or - Myocardial infarction The composition according to any one of claims 3 or 5 to 15,

17. The composition of any one of claims 1 to 3 or 5 to 16, wherein the subject has non-ischemic cardiomyopathy.

18. A composition described in any one of claims 1 to 3 or 5 to 17 for transendocardial administration.