CN116829162A - Methods of treating progressive heart failure in a subject suffering from grade II heart failure - Google Patents

Abstract

The present disclosure relates to methods for treating and/or preventing progressive heart failure in a subject suffering from early stage heart failure. Such methods may be used to treat or prevent progressive heart failure in a subject suffering from grade II heart failure according to the New York Heart Association (NYHA) classification scale.

Description

Method of treating progressive heart failure in a subject with stage II heart failure

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to Australian Application No. 2020904675 filed on December 15, 2020, Australian Application No. 2021900059 filed on January 12, 2021, Australian Application No. 2021902941 filed on September 10, 2021, Australian Application No. 2021903365 filed on October 20, 2021, and U.S. Application No. 63/289,868 filed on December 15, 2021, each of which is incorporated herein by reference in its entirety.

Technical Field

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

Background Art

Myocardial infarction (MI) remains one of the leading causes of death and morbidity in developed countries. An update of U.S. Medicare records was published, which evaluated data on acute MI hospitalization involving 350,509 patients aged >65 years who survived the event and were discharged (Schuster et al. (2004) Physiology-Heart and Circulatory Physiology, 287 (2): 525-32). Within the first year after the index event, 25.9% of MI patients died and 50.5% of patients were re-hospitalized. Within one month after MI, the likelihood of death was 21 times that of the general Medicare age group, and the likelihood of hospitalization was 12 times that of the general Medicare age group.

Over the past decade, many clinical trials evaluating novel drug therapies have been conducted in patients with advanced heart failure (HF). Despite progress in reducing morbidity and mortality in patients with HF, patients with advanced disease continue to experience an adverse clinical course characterized by frequent hospitalizations and premature death.

Clearly, there is a need in the art for treating or preventing progressive heart failure.

Summary of the invention

The inventors surprisingly found that cell therapy is particularly effective for subjects suffering from early progressive heart failure. Therefore, in one example, the present disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering a composition comprising cells to the subject. In one example, the subject suffers from grade II or grade III heart failure according to the New York Heart Association (NYHA) grading scale. In one example, the subject may suffer from heart failure less than grade III according to the New York Heart Association (NYHA) grading scale. In one example, the subject suffers from grade II heart failure according to the New York Heart Association (NYHA) grading scale. Therefore, in one example, the present disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering a composition comprising cells to the subject, wherein the subject suffers from grade II heart failure according to the New York Heart Association (NYHA) grading scale.

In another example, the present disclosure relates to a method of reducing progression of heart failure in a subject, the method comprising administering to the subject a composition comprising cells, wherein the subject has class II heart failure according to the New York Heart Association (NYHA) classification scale.

In another example, the present 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 cells.

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

In one example, the cells induce neovascularization in the target tissue. In one example, the cells promote arteriogenesis. In one example, the cells secrete factors that protect myocardium at risk. Therefore, in one example, 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, wherein the subject suffers from grade II heart failure according to the New York Heart Association (NYHA) classification scale, and wherein the cells induce neovascularization in the target tissue and/or secrete factors that protect myocardium at risk.

In one example, the cell is a mesenchymal lineage precursor or stem cell (MLPSC). In one example, the MLPSC is STRO-1+. In one example, the MLPSC is a mesenchymal stem cell (MSC). In one example, the MLPSC is allogeneic. In one example, the cell is cultured and expanded. In this example, the cell can be TNAP+ before culture expansion. In one example, the cell has been cryopreserved.

In another example, the method of the present disclosure includes 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 including cells that induce neovascularization in a target tissue.

In another example, administering the composition inhibits progression to NYHA Class III progressive heart failure in the subject.

In one example, the subject's N-terminal pro-B-type natriuretic peptide (NT-proBNP) level is less than 2200 pg/ml. In another example, the subject's NT-proBNP level is less than 2000 pg/ml prior to administration of the cells. In another example, the subject's NT-proBNP level is between 1000 pg/ml and 2000 pg/ml prior to administration of the cells.

The inventors have also surprisingly found that cell therapy is particularly effective for subjects with early progressive heart failure with elevated C-reactive protein (CRP) levels. Therefore, in one example, the CRP level of the subject is elevated. In one example, the CRP level of the subject is>1mg/L. In one example, the CRP level of the subject is>1.5mg/L. In one example, the CRP level of the subject is ≥2mg/L. In one example, the CRP level of the subject is>2mg/L. In another example, the CRP level of the subject is between 1.5mg/L and 5mg/L. In another example, the CRP level of the subject is <5mg/L, preferably <4mg/L. In another example, the CRP level of the subject is between 1mg/L and 5mg/L. In another example, the CRP level of the subject is between 1.5mg/L and 5mg/L.

In another example, the subject has had an event of hospitalization for heart failure within the previous 9 months.

In another example, the subject has a LVEF of less than about 45%, preferably less than 40%. In another example, the subject suffers from persistent left ventricular dysfunction.

In another example, the subject's heart failure is caused by an ischemic event.

In another example, the subject's heart failure is caused by a non-ischemic event.

In one example, the risk of cardiac death of the subject after treatment is reduced. In one example, the risk reduction is relative to the risk of cardiac death of a subject with NYHA III progressive heart failure. In another example, the risk of ischemic MACE (MI or stroke) of the subject after treatment is reduced.

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

In one example, the composition is administered intraendocardially and/or intravenously. In one example, the composition is administered intraendocardially.

The inventors have also surprisingly found that cell therapy reduces the risk of ischemic events in subjects with cardiomyopathy. Therefore, in one example, the present disclosure also encompasses a method for reducing the risk of ischemic events in a subject, the method comprising administering a composition comprising cells to the subject. In one example, the subject suffers from cardiomyopathy. In one example, the ischemic event is the formation of cerebrovascular or cardiac occlusion. In one example, the ischemic event is a stroke or myocardial infarction. In one example, the subject suffers from non-ischemic cardiomyopathy. In one example, the cells are administered via the endocardium. In one example, the subject suffers from grade II or grade III heart failure according to the New York Heart Association (NYHA) grading scale. In one example, the subject has active inflammation. Surprisingly, with respect to this example, the inventors determine that the composition of the present disclosure can treat a wider range of patients, such as patients suffering from grade II or grade III heart failure.

In one example, 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 a mesenchymal precursor lineage or stem cells, wherein the subject suffers from grade II or grade III heart failure and has active inflammation according to the New York Heart Association (NYHA) grading scale. In another example, the present disclosure relates to a method for reducing the progression of heart failure in a subject, the method comprising administering to the subject a composition comprising a mesenchymal precursor lineage or stem cells, wherein the subject suffers from grade II or grade III heart failure and has active inflammation according to the New York Heart Association (NYHA) grading scale. In another example, the present disclosure relates to a method for reducing cardiac death in a subject suffering from grade II or grade III heart failure with active inflammation according to the New York Heart Association (NYHA) grading scale, the method comprising administering to the subject a composition comprising a mesenchymal precursor lineage or stem cells. In another example, the present disclosure relates to a method for selecting a patient with heart failure to be treated with cell therapy, the method comprising: i) assessing CRP levels and assessing heart failure according to the New York Heart Association (NYHA) grading scale; and ii) selecting a subject with grade II or grade III heart failure and active inflammation according to NYHA, preferably, wherein the method comprises administering a composition comprising a mesenchymal precursor lineage or stem cells. In one example, active inflammation is determined based on the CRP level of the subject. In one example, the subject suffers from grade II heart failure and has "active inflammation". In one example, the active inflammation is characterized by a level of CRP>1.5mg/L. In another example, active inflammation is characterized by a level of CRP>2mg/L. Exemplary cells are discussed above and throughout the present disclosure. In one example, the cell induces new blood vessel formation in the target tissue. In one example, the cell promotes arterial generation. In one example, the cell secretes a factor that protects the myocardium at risk. In one example, the cell is MLPSC. In one example, the MLPSC is STRO-1+. In one example, the MLPSC is a mesenchymal stem cell (MSC). In one example, the MLPSC is allogeneic. In one example, the cells are cultured and expanded. In this example, the cells may be TNAP+ before culture expansion. In one example, the cells have been cryopreserved.

In one example, the subject's N-terminal pro-B-type natriuretic peptide (NT-proBNP) level is between 1000 pg/ml and 2000 pg/ml before administering the cells. In another example, the subject's C-reactive protein (CRP) level is elevated. In another example, the subject's CRP level is ≥1 mg/L. In another example, the subject's CRP level is ≥2 mg/L. In another example, the subject's CRP level is between 2 mg/L and 5 mg/L. In another example, the subject's CRP level is between 3 mg/L and 5 mg/L.

In an embodiment of the above examples, the method of the present disclosure comprises administering 1×10 ⁷ to 2×10 ⁸ cells.

In another example, the composition administered further includes Plasma-Lyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA). In one example, the composition administered further includes Plasma-Lyte A (70%), DMSO (10%), and HSA (25%) solution, wherein the HSA solution includes 5% HSA and 15% buffer. In one example, the composition includes greater than 6.68×10 ⁶ viable cells/mL.

In another example, the composition includes human bone marrow-derived allogeneic mesenchymal precursor cells (MPCs) isolated from bone mononuclear cells using an anti-STRO-3 antibody, expanded ex vivo, and cryopreserved.

In one example, the ischemic event is one or more of myocardial infarction, stroke, or cardiac death. In one example, the method reduces the risk of 3-point MACE.

The inventors have also surprisingly determined that an increase in CRP levels is associated with an increase in the risk of cardiac death, myocardial infarction or stroke. Therefore, in one example, the present disclosure relates to a method for determining an increased risk of one or more of cardiac death, myocardial infarction or stroke in a subject, the method comprising measuring the CRP level in a sample obtained from the subject, wherein an increase in CRP indicates an increased risk of cardiac death, myocardial infarction or stroke. In one example, the subject suffers from progressive heart failure. In one example, the subject's heart failure is NYHA class II heart failure. In another example, a level of CRP>1mg/L indicates an increased risk of cardiac death, myocardial infarction or stroke. In another example, a level of CRP>1.5mg/L indicates an increased risk of cardiac death, myocardial infarction or stroke. In another example, a level of CRP≥2mg/L indicates an increased risk of cardiac death, myocardial infarction or stroke. In one example, the method determines an increased risk of cardiac death.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Reduced incidence of ischemic MACE (MI, stroke).

Figure 2: Reduced incidence of ischemic MACE (MI, stroke); NYHA class II and III.

Figure 3: Reduced incidence of ischemic MACE (MI, stroke); ischemic and non-ischemic.

Figure 4: Cardiac death in all treated patients (n=537); Class II patients (n=206); Class III patients (n=331).

Figure 5: Cardiac death in patients with NYHA class II; ischemic and non-ischemic.

Figure 6: Cardiac death in trial patients.

Figure 7: A) TTFE composite IMM MACE; B) Ratio normalized composite IMM MACE; C) Curves for all treated patients with baseline CRP ≥ 2 mg/L and CRP ≤ 2 mg/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 8: A) TTFE irreversible incidence; B) rate-normalized irreversible incidence; C) curves of irreversible incidence TTFE MACE (non-fatal MI or non-fatal stroke) for all treated patients (n=537); class II patients (n=206); class III patients (n=331).

Figure 9: Reduction in the incidence of the composite of cardiac death or ischemic MACE (MI, stroke); all treated patients (n=537); class II patients (n=206); class III patients (n=331).

Figure 10: Cardiac death in NYHA class II patients - multi-year follow-up.

Figure 11: NYHA class II patients with baseline hsCRP ≥ 2 mg/L had a significantly increased risk of progression to cardiac death.

Figure 12: NYHA class II patients with baseline hsCRP ≥ 2 mg/L had a significantly increased risk of 3-point MACE (cardiac death/MI/stroke).

DETAILED DESCRIPTION

General techniques and definitions

Unless specifically defined otherwise, all technical and scientific terms used herein should be construed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular biology, stem cell culture, immunology, clinical trials, medicine, and biochemistry).

Unless otherwise indicated, the cell culture techniques and assays used in the present disclosure are standard procedures well known to those skilled in the art. Such techniques are described and explained in the literature of the following sources: such as J. Perbal, A practical Guide To Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T. A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Vol. 1 and Vol. 2, IRL Press (IRL Press) (1991); D. M. Glover and B. D. Hames (eds.), and F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, Greene Publishing Association (Greene Publishing Association) Pub. Associates) and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988); and J. E. Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

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

As used herein, unless stated to the contrary, the term "about" means +/- 10%, more preferably +/- 5%, of the specified value.

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

In one example, the level is expressed in pg/ml. For example, the level of NT-proBNP can be expressed in pg/ml. In one example, the level is expressed in mg/L. For example, the level of CRP can be expressed in mg/L. In another example, the level is expressed in pg per 10 ⁶ cells.

In one example, the level of a specific marker in a cell culture medium is determined under culture conditions. The term "culture conditions" is used to refer to cells grown in culture. In one example, culture conditions refer to a cell population that is actively dividing. In one example, such cells may be in an exponential growth phase. For example, the level of a specific marker can be determined by obtaining a sample of a cell culture medium and measuring the level of the marker in the sample. In another example, the level of a specific marker can be determined by obtaining a cell sample and measuring the level of the marker in a cell lysate. Those skilled in the art will measure secreted markers by sampling the culture medium, while markers expressed on the cell surface can be measured by evaluating a sample of a cell lysate. In one example, a sample is obtained when the cell is in an exponential growth phase. In one example, a sample is obtained after at least two days of culture.

Expansion of cells in culture from a cryopreserved intermediate means that the cells that have been subjected to cryopreservation are thawed and cultured in vitro under conditions suitable for cell growth.

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

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

As used herein, the term "subject" refers to a human subject. For example, the subject can be an adult. In another example, the subject can be a child. In another example, the subject can be a teenager. Terms such as "subject," "patient," or "individual" are terms that can be used interchangeably in the context of this disclosure. Subjects in need of treatment include those who already have progressive heart failure, as well as those who need to prevent, delay, or stop progressive heart failure.

In one example, the compositions of the present disclosure include mesenchymal precursor lineages or stem cells that have not been genetically modified. As used herein, the term "unmodified" refers to cells that have not been modified by transfection with nucleic acids. For the avoidance of doubt, in the context of the present disclosure, mesenchymal precursors or stem cells transfected with nucleic acids encoding proteins will be considered to be genetically modified.

As used herein, the term "sample" refers to an extract from a subject in which the CRP level can be measured. A "sample" comprises an extract and/or derivative and/or fraction of a sample. In the present disclosure, any biological material can be used as the above-mentioned sample, as long as it can be collected from a subject and measured to determine the CRP level in the subject. In an example, the sample is a blood sample. In an example, the blood sample is obtained from a subject suffering from NYHA class II heart failure.

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

Throughout this specification, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter shall be considered to cover both one and more (i.e., one or more) of such steps, compositions of matter, groups of steps, or groups of compositions of matter, unless expressly stated otherwise or the context requires otherwise.

Those skilled in the art will appreciate that variations and modifications other than those specifically described may be made to the disclosure described herein. It should be understood that the disclosure encompasses all such variations and modifications. The disclosure also encompasses all steps, features, compositions and compounds referred to or indicated in this specification, either individually or collectively, and any and all combinations of any two or more of said steps or features.

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

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

Progressive heart failure

Cardiomyopathy is a disease of the heart muscle that makes it difficult for the heart to pump blood to the rest of the body. Heart failure occurs when the heart cannot pump blood adequately to maintain blood flow to meet the body's needs. Cardiomyopathy can occur after an ischemic or non-ischemic event. One cause of ischemic heart failure is contractile dysfunction after a myocardial infarction (MI), such as a heart attack. MI occurs when blood stops flowing normally to a part of the heart. The lack of blood supply leads to localized areas of myocardial necrosis, called infarction or infarction. The infarcted heart cannot pump blood adequately to maintain blood flow to meet the body's needs, resulting in a variety of pathophysiological reactions and ultimately heart failure. Non-ischemic cardiomyopathy is not associated with known coronary artery disease. An example is dilated cardiomyopathy (DCM), in which the heart's ability to pump blood decreases because the left ventricle, the heart's main pumping chamber, becomes enlarged, dilated, and weak.

Once the heart is unable to adequately pump blood to maintain blood flow to meet the body's needs, a series of compensatory mechanisms are initiated to buffer the decline in cardiac output and assist in maintaining adequate blood pressure to perfuse vital organs. As a result, patients with heart failure may not progress for a long time. However, the compensatory mechanisms are ultimately unable to compensate for the damaged heart, resulting in a gradual decline in cardiac output, known as "progressive heart failure." In the context of the present 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."

The method of the present disclosure is effective for a subset of patients suffering from progressive heart failure. In one example, these subjects are defined based on the New York Heart Association (NYHA) classification scale. In one example, the subject's progressive heart failure is less than grade III. In one example, the subject suffers from grade II heart failure. In one example, the NYHA classification is assigned based on the subject's symptoms. For example, the NYHA classification can be assigned based on the following table:

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

The methods disclosed herein can be used to treat progressive heart failure in a specific population of MI subjects. Subjects in need of treatment include those who already have progressive heart failure, and those who need to prevent, delay or stop progressive heart failure. In these examples, the subject suffers from progressive heart failure of class II or class III based on NYHA. For example, the subject may suffer from class II progressive heart failure.

In one example, a subject treated according to the present disclosure has "active inflammation" defined by elevated C-reactive protein levels. In one example, active inflammation is characterized by CRP levels ≥ 2 mg/L.

"C-reactive protein" or "CRP" is an inflammatory mediator whose levels increase under conditions of acute inflammatory recurrence and whose levels rapidly normalize once the inflammation subsides. In one example, the risk of cardiac death is increased in subjects treated in accordance with the present disclosure. Cardiac death is death due to loss of heart function. In one example, a subject treated in accordance with the present disclosure has elevated CRP. The term "elevated CRP" is used in the context of the present disclosure to refer to CRP levels that are elevated relative to baseline CRP levels. In one example, CRP levels ≥1 mg/L are elevated. In another example, CRP levels ≥1.5 mg/L are elevated. In another example, CRP levels ≥2 mg/L are elevated.

In one example, the initial CRP level of a subject treated according to the present disclosure is ≥2 mg/L. For example, the subject may suffer from grade II or grade III heart failure, and the initial CRP level is ≥2 mg/L. In another example, the subject may suffer from grade II heart failure, and the initial CRP level is ≥2 mg/L. In one example, the initial CRP level of a subject treated according to the present disclosure is <5 mg/L. In another example, the initial CRP level of the subject is <4 mg/L. In another example, the initial CRP level of the subject is between 2 mg/L and 6 mg/L. In another example, the initial CRP level of the subject is between 3 mg/L and 6 mg/L. In another example, the initial CRP level of the subject is between 4 mg/L and 5 mg/L.

There are various assays that can be used to measure CRP levels, such as antibody-based immunoassays. For example, enzyme-linked immunosorbent assay (ELISA) can be used to measure the CRP level in a blood sample. In an example, a blood sample is obtained from a patient, and then the blood sample is purified before contacting with an anti-CRP antibody. The degree of antibody binding is used to quantify the level (e.g., mg/L) of CRP in a blood sample. In an example, CRP is measured by plasma high-sensitivity CRP (hsCRP) ELISA assay.

B-type natriuretic peptide (BNP) is a hormone produced by the heart. N-terminal (NT) prohormone BNP (NT-proBNP) is an inactive prohormone released from the same molecule that produces BNP. Both BNP and NT-proBNP are released in response to changes in pressure within the heart. These changes may be associated with heart failure and other heart problems. Levels rise as heart failure develops or worsens, and levels fall as heart failure stabilizes. Therefore, BNP is an effective marker for the progression of heart failure. In one example, the subject's NT-proBNP level is less than 2200 pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is less than 2100 pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is less than 2000 pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is less than 1900 pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is between 2200 pg/ml and 1000 pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is between 2200 pg/ml and 1100 pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is between 2100 pg/ml and 1200 pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is between 2000 pg/ml and 1500 pg/ml prior to administration of the composition of the present disclosure. There are various assays that can be used to measure NT-proBNP levels, such as antibody-based immunoassays, such as ELISA assays. In one example, a blood sample is obtained from a patient and then purified prior to contacting with an anti-NT-proBNP antibody. The extent of antibody binding is used to quantify the level of NT-proBNP (e.g., pg/L) in the blood sample.

In another example, the subject has a heart failure hospitalization event within the previous 12 months before the administration of the composition disclosed herein. In another example, the subject has a heart failure hospitalization event within the previous 9 months before the administration of the composition disclosed herein. In another example, the subject has a heart failure hospitalization event within 6 to 12 months before the administration of the composition disclosed herein. In one example, the heart failure hospitalization event is a worsening of the signs and symptoms of heart failure. In another example, the heart failure hospitalization event is an ischemic event. In another example, the heart failure hospitalization is a non-ischemic event.

In another example, the subject is able to walk at least 320 meters in 6 minutes prior to administering the composition of the present disclosure. In another example, the subject is able to walk at least 330 meters in 6 minutes prior to administering the composition of the present disclosure. In another example, the subject is able to walk at least 340 meters in 6 minutes prior to administering the composition of the present disclosure. In another example, the subject is able to walk at least 350 meters in 6 minutes prior to administering the composition of the present disclosure.

In one example, the subject may suffer from persistent left ventricular dysfunction. Left ventricular dysfunction is characterized by decreased myocardial contractility. When myocardial contractility decreases within the left ventricle, the left ventricular ejection fraction (LVEF) decreases. Therefore, LVEF provides a method for determining left ventricular dysfunction.

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

In one example, a subject having an LVEF of less than 45% suffers from left ventricular dysfunction. In other examples, a subject having an LVEF of less than about 44%, 43%, 42%, 41% suffers from left ventricular dysfunction. In another example, a subject having an LVEF of less than about 40% suffers from left ventricular dysfunction. In other examples, a subject having an LVEF of less than about 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30% suffers from left ventricular dysfunction.

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 a 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.

In one example, the subject's LVEF is less than 45%. In another example, the subject's LVEF is less than 40%. In other examples, the subject's LVEF is less than 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%.

The methods of the present disclosure relate to the treatment of a gradual decline in cardiac output characterized by progressive heart failure.Thus, in the context of the present disclosure, "treat" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures.

In one example, treatment comprises administering a composition of the present disclosure. In one example, the method of the present disclosure reduces or inhibits the progress of progressive heart failure. In one example, treatment inhibits the progress of a subject to NYHA III grade progressive heart failure. In another example, treatment reduces the risk of cardiac death. In one example, the risk reduction of cardiac death is relative to the risk of cardiac death of a subject with NYHA III grade progressive heart failure. In another example, the risk of ischemic MACE (MI or stroke) is reduced after treatment. In one example, the risk of ischemic MACE (MI or stroke) is reduced by at least 50% relative to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 55% relative to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 60% relative to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 65% relative to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 70% relative to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 50% to 70% relative to baseline.

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

In one example, treatment increases the survival rate of the patient. In one example, treatment increases the probability that the subject survives at least 1000 days after starting treatment. In another example, treatment increases the probability that the subject survives at least 2000 days after starting treatment. In one example, the increased probability is determined relative to a subject not treated with the composition of the present disclosure. In one example, the increased probability is determined relative to a subject suffering from grade III heart failure.

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

Ischemic events

In one example, the present disclosure relates to a method for reducing the risk or incidence of ischemic events in a subject, particularly a subject with cardiomyopathy. In one example, the present disclosure relates to a method for reducing the risk or incidence of ischemic events in a subject with cardiomyopathy and elevated CRP. In one example, the risk or incidence is reduced relative to a subject not receiving a composition of the present disclosure. For example, the risk or incidence can be reduced relative to an untreated subject. In one example, the ischemic event is caused by the formation of an occlusion. In one example, the occlusion is an arterial occlusion. In one example, the ischemic event is the formation of a cerebral vascular occlusion. In another example, the ischemic event is the formation of a cardiac occlusion. For example, the occlusion can be formed in a coronary artery.

Examples of ischemic events caused by occlusion formation include myocardial infarction and stroke. Thus, in one example, the present disclosure is directed to a method of reducing the risk or incidence of myocardial infarction or stroke in a subject suffering from cardiomyopathy.

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

In one example, the subject suffers from non-ischemic cardiomyopathy. For example, the subject's cardiomyopathy may be caused by left ventricular enlargement (dilated cardiomyopathy). In another example, the cardiomyopathy is caused by a viral infection.

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

In another example, the subject's N-terminal pro-B-type natriuretic peptide (NT-proBNP) level is between 1000 pg/ml and 2000 pg/ml before administering the cells. In another example, the subject's C-reactive protein (CRP) level is elevated. In another example, the subject's CRP level is ≥1.5 mg/L. In another example, the subject's CRP level is ≥2 mg/L. In another example, the subject's CRP level is between 1 mg/L and 5 mg/L. In another example, the subject's CRP level is between 3 mg/L and 5 mg/L.

In one example, the cells are administered intraendocardially.

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

Mesenchymal lineage precursor cells

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

The term "mesenchymal lineage precursor or stem cell" includes the parent cell and its undifferentiated progeny. The term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSC), perivascular mesenchymal precursor cells and their undifferentiated progeny.

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

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

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

The terms "enriched", "enriched" or variants thereof are used herein to describe a cell population in which the proportion of a particular cell type or the proportion of a plurality of particular cell types is increased compared to an untreated cell population (e.g., cells in their natural environment). In one example, a population enriched in mesenchymal lineage precursors or stem cells includes at least about 0.1% or 0.5% or 1% or 2% or 5% or 10% or 15% or 20% or 25% or 30% or 50% or 75% of mesenchymal lineage precursors or stem cells. In this regard, the term "cell population enriched in mesenchymal lineage precursors or stem cells" will be used to clearly support the term "cell population including X% mesenchymal lineage precursors or stem cells", where X% is a percentage as described herein. In some instances, mesenchymal lineage precursors or stem cells can form clonogenic colonies, such as CFU-F (fibroblasts) or a subset thereof (e.g., 50% or 60% or 70% or 70% or 90% or 95%) can have this activity.

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

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

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

In one example, isolated or enriched mesenchymal lineage precursors or stem cells are seeded at 50,000 viable cells/cm ² in culture medium (serum-free or serum-supplemented), such as alpha minimum essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, and allowed to adhere to a culture vessel overnight at 37° C., 20% O _2. The culture medium is then replaced and/or changed as needed, and the cells are further cultured at 37° C., 5% O ₂ for 68 to 72 hours.

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

In one example, a cell population is enriched from a cell preparation including STRO-1+ cells in a selectable form. In this regard, the term "selectable form" will be understood to mean that the cell expresses a marker (e.g., a cell surface marker) that allows the selection of STRO-1+ cells. The marker may be STRO-1, but not necessarily. For example, as described and/or exemplified herein, cells (e.g., mesenchymal precursor cells) expressing STRO-2 and/or STRO-3 (TNAP) and/or STRO-4 and/or VCAM-1 and/or CD146 and/or 3G5 also express STRO-1 (and may be STRO-1bright). Therefore, the indication that a cell is STRO-1+ does not mean that the cell is selected only by STRO-1 expression. In one example, cells are selected based at least on STRO-3 expression, for example, it is STRO-3+ (TNAP+).

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

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

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

"Collectively" means that the present disclosure encompasses any number or combination of the markers or marker groups, and that even though such number or combination of markers or marker groups may not be specifically listed herein, the appended claims may define such combinations or subcombinations individually and separately from any other marker combination or marker group.

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

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

In one example, a significant proportion of STRO-1+ cells are able to differentiate into at least two different lineages. Non-limiting examples of lineages to which STRO-1+ cells can be committed include bone progenitor cells; hepatocyte progenitor cells that are multipotent to 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; precursors of myocardium and cardiomyocytes, glucose-responsive insulin-secreting pancreatic beta cell lineages. Other lineages include, but are not limited to, odontoblasts, cells that produce tooth texture, and chondrocytes, as well as precursors of retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, myocardium, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelial cells, glial cells, neurons, astrocytes, and oligodendrocytes.

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

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

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

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

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

In one example, the composition of the present disclosure includes cells that induce neovascularization in a target tissue. In one example, the target tissue is the heart. In another example, the cells secrete factors that protect the at-risk or damaged myocardium. In one example, the at-risk or damaged myocardium is affected by a lack of blood flow caused by an ischemic event. In one example, the cells secrete factors that reduce cardiomyocyte apoptosis.

Cell culture and expansion

In one example, mesenchymal lineage precursors or stem cells are cultured and expanded. The difference between "culture-expanded" mesenchymal lineage precursors or stem cell culture medium and freshly separated cells is that they have been cultured and passed down (i.e., passage culture) in cell culture medium. In one example, the mesenchymal lineage precursors or stem cells of culture expansion are cultured and expanded for about 4 passages to 10 passages. In one example, mesenchymal lineage precursors or stem cells are cultured and expanded for at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages. For example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5 passages to 10 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5 passages to 8 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5 passages to 7 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for more than 10 passages. In another example, mesenchymal lineage precursors or stem cells can be cultured and expanded for more than 7 passages. In these examples, stem cells can be cultured and expanded before being cryopreserved to provide an intermediate cryopreserved MLPSC population. In one example, the compositions of the present disclosure are produced by culturing cells from an intermediate cryopreserved MLPSC population or in other words, cryopreserved intermediates.

In one example, the composition of the present disclosure includes mesenchymal lineage precursors or stem cells cultured and amplified from cryopreserved intermediates. In one example, the cell culture amplified from the cryopreserved intermediates is amplified for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 generations. For example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 5 generations. In one example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 5 generations to 10 generations. In one example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 5 generations to 8 generations. In one example, mesenchymal lineage precursors or stem cells can be cultured and amplified for at least 5 generations to 7 generations. In one example, mesenchymal lineage precursors or stem cells can be cultured and amplified for more than 10 generations. In another example, mesenchymal lineage precursors or stem cells can be cultured and amplified for more than 7 generations.

In one example, the mesenchymal lineage precursors or stem cells cultured and expanded from the cryopreserved intermediates can be cultured and expanded in a medium without animal protein. In one example, the mesenchymal lineage precursors or stem cells cultured and expanded from the cryopreserved intermediates can be cultured and expanded in a medium without xenogeneic components. In one example, the mesenchymal lineage precursors or stem cells cultured and expanded from the cryopreserved intermediates can be cultured and expanded in a medium without fetal bovine serum.

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

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

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

iii. preparing and cryopreserving a processing intermediate mesenchymal lineage precursor or stem cell preparation obtained from a P2 culture of the mesenchymal lineage precursor or stem cells; and

iv. Thawing the cryopreserved intermediate mesenchymal lineage precursor or stem cell preparation and expanding the intermediate mesenchymal lineage precursor or stem cell preparation by passage expansion.

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

i. less than about 0.75% CD45+ cells;

ii. at least about 95% CD105+ cells;

iii. At least about 95% CD166+ cells.

In one example, the expanded mesenchymal lineage precursor or stem cell preparation is capable of inhibiting IL2-Ra expression by CD3/CD28 activated PBMCs by at least about 30% relative to a control.

In one example, the culture-expanded mesenchymal lineage precursors or stem cells are culture-expanded for about 4 to 10 passages, wherein the mesenchymal lineage precursors or stem cells are cryopreserved after at least 2 passages or 3 passages before being further cultured and expanded. In one example, the mesenchymal lineage precursors or stem cells are cultured and expanded for at least 1 passage, at least 2 passages, at least 3 passages, at least 4 passages, at least 5 passages, cryopreserved, and then further cultured and expanded for at least 1 passage, at least 2 passages, at least 3 passages, at least 4 passages, at least 5 passages before being cultured according to the methods disclosed herein.

Any equipment and cell processing methods known in the art can be used to separate and expand mesenchymal lineage precursors or stem cells in vitro. Various culture expansion embodiments disclosed herein use steps that require manipulation of cells, such as inoculation, feeding, dissociation of adherent cultures, or washing steps. Any step of manipulating cells is likely to damage the cells. Although mesenchymal lineage precursors or stem cells can generally withstand a certain amount of damage during preparation, cells are preferably manipulated by processing procedures and/or equipment that fully execute a given step while minimizing damage to the cells.

In one example, mesenchymal lineage precursors or stem cells are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculating wash bag, a rotating membrane filter with inlet and outlet ports, a filtrate bag, a mixing zone, a final product bag for the washed cells, and appropriate tubing, e.g., as described in U.S. Pat. No. 6,251,295, which is hereby incorporated by reference.

In one example, a mesenchymal lineage precursor or stem cell composition cultured according to the present disclosure is 95% homogeneous in terms of CD105 positivity and CD166 positivity and CD45 negativity. In one example, this homogeneity persists through ex vivo expansion; ie, despite multiple population doublings.

In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded in 3D culture. For example, the mesenchymal lineage precursors or stem cells of the present disclosure can be cultured and expanded in a bioreactor. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are first cultured and expanded in 2D culture, and then further expanded in 3D culture. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded from a master cell bank. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded from a master cell bank in 2D culture before being inoculated in 3D culture. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded from a master cell bank in 2D culture before being inoculated in 3D culture in a bioreactor for at least 3 days. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are cultured and expanded from a master cell bank in 2D culture before being inoculated in 3D culture in a bioreactor for at least 4 days. In one example, the mesenchymal lineage precursors or stem cells disclosed herein are cultured and expanded from a master cell bank in 2D culture in a bioreactor for 3 to 5 days before being inoculated in 3D culture. In these examples, 2D culture can be performed in a cell factory. Various cell factory products are commercially available (e.g., Thermofisher, Sigma).

Cell culture medium

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

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

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

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

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

In one example, the cell culture medium contains only additives of human origin. Thus, in one example, the cell culture medium is xeno-free. For the avoidance of doubt, in these examples, the culture medium is free of animal protein. In one example, the cell culture medium used in the methods of the present disclosure is free of animal components.

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

Basal culture medium;

Platelet-derived growth factor (PDGF);

Fibroblast growth factor 2 (FGF2).

In one example, the culture medium includes platelet-derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2), wherein the level of FGF2 is less than about 6 ng/ml. For example, the level of FGF2 can be less than about 5 ng/ml, less than about 4 ng/ml, less than about 3 ng/ml, less than about 2 ng/ml, less than about 1 ng/ml. In other examples, the level of FGF2 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, less than about 0.2 ng/ml.

In another example, the level of FGF2 is between about 1 pg/ml and 100 pg/ml. In another example, the level of FGF2 is between about 5 pg/ml and 80 pg/ml.

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

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

In other examples, additional factors can be added to the cell culture medium. In one example, the culture medium further includes EGF. EGF is a growth factor that stimulates cell proliferation by binding to its receptor EGFR. In one example, the method of the present disclosure includes culturing a stem cell population in a cell culture medium that further includes EGF and does not contain fetal bovine serum. In one example, the EGF level is between about 0.1 ng/ml and 7 ng/ml. For example, the EGF level can be at least about 5 ng/ml.

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

In the above examples, the basal culture medium such as αMEM or StemSpan ^™ can be supplemented with reference amounts of growth factors. In one example, the culture medium includes αMEM or StemSpan ^™ supplemented with 32 ng/ml PDGF-BB, 0.8 ng/ml EGF, and 0.02 ng/ml FGF.

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

In another example, the cell culture medium of the present disclosure promotes stem cell proliferation while maintaining the stem cells in an undifferentiated state. When a stem cell has not yet become a specific differentiation lineage, it is considered to be undifferentiated. As discussed above, stem cells show morphological characteristics that are different from differentiated cells. In addition, undifferentiated stem cells express genes that can be used as markers for detecting differentiation states. Polypeptide products can also be used as markers for detecting differentiation states. Therefore, those skilled in the art can easily determine whether the method of the present disclosure maintains stem cells in an undifferentiated state using conventional morphological, genetic and/or proteomic analyses.

Cell modification

Mesenchymal lineage precursors or stem cells disclosed herein can be altered in such a way that upon administration, cell lysis is inhibited. Alteration of antigens can induce immune anergy or tolerance, thereby preventing the induction of the effector phase of the immune response (e.g., cytotoxic T cell production, antibody production, etc.), which ultimately leads to the rejection of foreign cells in a normal immune response. Antigens that can be altered to achieve this goal include, for example, MHC class I antigens, MHC class II antigens, LFA-3, and ICAM-1.

Mesenchymal lineage precursors or stem cells can also be genetically modified to express proteins that are important for differentiation and/or maintenance of striated skeletal muscle cells. Exemplary proteins include growth factors (TGF-β, insulin-like growth factor 1 (IGF-1), FGF), myogenic factors (e.g., myoD, myogenin, myogenic factor 5 (Myf5), myogenic regulatory factors (MRFs)), transcription factors (e.g., GATA-4), cytokines (e.g., cardiotropin-1), neuregulin family members (e.g., neuregulin 1, 2 and 3) and homeobox genes (e.g., Csx, tinman and NKx families).

Composition

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

In one example, the composition of the present disclosure includes 10×10 ⁶ cells to 35×10 ⁶ cells. In another example, the composition includes 20×10 ⁶ cells to 30×10 ^{6 cells. In other examples, the composition includes at least 100×10 6 cells} . In another example, the composition includes 50×10 ⁶ cells to 500×10 ⁶ cells. In other examples, the composition of the present disclosure includes 150 million cells.

In one example, the compositions of the present disclosure include pharmaceutically acceptable carriers and/or excipients. The terms "carrier" and "excipient" refer to compositions of matter that are routinely used in the art to promote storage, administration and/or biological activity of active compounds (see, for example, "Remington's Pharmaceutical Sciences", 16th edition, Mac Publishing Company (1980)). The carrier can also reduce any undesirable side effects of the active compound. Suitable carriers are, for example, stable, such as being unable to react with other components in the carrier. In one example, the carrier will not produce significant local or systemic side effects in the recipient at the dosage and concentration used for treatment.

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

In another example, the carrier is a culture medium composition, for example, in which the cells are grown or suspended. Such culture medium compositions do not cause any side effects in the subject to which they are 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 disease.

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

The compositions of the present disclosure may be cryopreserved. Cryopreservation of mesenchymal lineage 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 phenotypes, cell surface markers, and growth rates of cryopreserved cells compared to unfrozen cells.

The cryopreserved composition may include a cryopreservation solution. The pH of the cryopreservation solution is generally 6.5 to 8, preferably 7.4.

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

The cryopreservation solution may include Profreeze ^™ . The cryopreservation solution may additionally or alternatively include a culture medium, such as αMEM.

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

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

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

The cryopreserved composition can be thawed and administered directly to a subject or added to another solution, for example, including hyaluronic acid. Alternatively, the cryopreserved composition can be thawed and the mesenchymal lineage precursors or stem cells resuspended in an alternative carrier prior to administration.

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

In one example, the composition includes an effective amount or a therapeutic or preventive effective amount of a mesenchymal lineage precursor or stem cell and/or its progeny and/or a soluble factor derived therefrom. For example, the composition includes about 1×10 ⁵ stem cells to about 1×10 ⁹ stem cells or about 1.25×10 ³ stem cells to about 1.25×10 ⁷ stem cells/kg (80 kg subject). The exact amount of cells to be administered depends on a variety of factors, including the age, weight and sex of the subject and the extent and severity of the disorder to be treated.

Although the number of cells is provided in the composition, in one example, 50×10 ⁶ to 200×10 ⁷ cells are administered. In other examples, 60×10 ⁶ to 200×10 ⁶ cells or 75×10 ⁶ to 150×10 ⁶ cells are administered. In one example, 75×10 ⁶ cells are administered. In another example, 150×10 ⁶ cells are administered.

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

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

In one example, the composition can be optionally packaged in a suitable container with written instructions for its intended purpose.

The compositions of the present disclosure can be administered systemically, for example, by intravenous administration. In one example, the composition is administered transendocardially.

Risk of cardiac death, myocardial infarction, or stroke

In one example, 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 CRP level of the subject. For example, the method of the present disclosure may relate to a method for assessing the risk of cardiac death based on the CRP level of the subject. In one example, the present disclosure encompasses a method for determining the increased risk of one or more of cardiac death, myocardial infarction or stroke in a subject, the method comprising measuring the CRP level in a sample obtained from the subject, wherein an increase in CRP indicates an increased risk of cardiac death, myocardial infarction or stroke. In one example, the subject suffers from progressive heart failure. For example, the subject may suffer from NYHA II grade progressive heart failure. Therefore, in one example, the sample is obtained from a subject suffering from NYHA II grade progressive heart failure. In one example, the sample is a blood sample.

In one example, the level of CRP>1mg/L indicates an increased risk of cardiac death, myocardial infarction or stroke. In one example, the level of CRP>1.5mg/L indicates an increased risk of cardiac death, myocardial infarction or stroke. In one example, the level of CRP>2mg/L indicates an increased risk of cardiac death, myocardial infarction or stroke. In one example, the level of CRP between>2mg/L and 5mg/L indicates an increased risk of cardiac death, myocardial infarction or stroke. In one example, the level of CRP is measured after an ischemic event. In one example, the ischemic event is a myocardial infarction.

In one example, a composition comprising cells inducing new blood vessel formation in a target tissue is administered to a subject assessed to have an elevated risk of cardiac death, myocardial infarction, or stroke. Therefore, in one example, the present disclosure relates to a method for treating progressive heart failure, comprising: i) selecting a subject with grade II heart failure and elevated CRP levels according to the New York Heart Association (NYHA) grading scale; and ii) administering a composition according to the present disclosure to the subject. In one example, the subject's CRP level is ≥2 mg/ml.

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

This application claims priority to AU2020904675 filed on December 15, 2020, 2021900059 filed on January 12, 2021, AU2021902941 filed on September 10, 2021, and AU2021903365 filed on October 20, 2021, the disclosures of which are incorporated herein in their entirety.

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

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

Examples

Composition

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

patient

Baseline data

The primary and secondary outcome measures were as follows:

1) Non-fatal major adverse cardiac events (MACE):

heart failure MACE (recurrent decompensated heart failure events or high-grade arrhythmias);

Ischemic MACE (heart attack or stroke)

2) Death due to cardiac causes

Relative to the control, cell therapy unexpectedly reduced the incidence of ischemic MACE (MI, stroke) by 60% (N = 537 patients; p = 0.002; Figure 1). Figure 2 shows that the incidence of ischemic MACE (MI, stroke) observed in NYHA class II and III was significantly reduced relative to the control. These data indicate that cell therapy can reduce the risk of ischemic events in patients with cardiomyopathy. Figure 3 shows that the incidence of ischemic MACE (MI, stroke) was significantly reduced in both ischemic and non-ischemic patients. Cardiomyopathy patients are at risk of occlusive plaque production due to the persistent inflammatory process in these patients. In view of the general reduction in ischemic MACE observed in patients with ischemic and non-ischemic cardiomyopathy, cell therapy appears to inhibit these problem processes. Therefore, the current data seem to support a general concept by which cell therapy can be administered to reduce the risk of ischemic events in patients with cardiomyopathy.

Surprisingly, cell therapy reduced cardiac death in NYHA class II patients, but did not reduce cardiac death in NYHA class III patients (Figures 4 and 6). This result is surprising because it shows that a certain threshold level of viable myocardium is required to reduce cardiac death using cell therapy. In other words, patients with class III heart failure may progress too fast in the disease continuum and cannot use cell therapy to improve their survival rate. These findings show that cell therapy will be particularly effective for NYHA class II patients. In view of the ability of the administered cells to induce new blood vessel formation in target tissues, the inventor's discovery proposes a general concept of reducing cardiac death in patients with grades below NYHA class III by administering cell therapy. As further support for this concept, the inventor noted that regardless of the cause of cardiomyopathy, a reduction in cardiac death in NYHA class II patients was observed, wherein a reduction in cardiac death was observed in ischemic and non-ischemic NYHA class II patients (Figure 5).

3) Improved results

3-point MACE

Subsequent analysis revealed that, compared with the control, in all 537 treated patients, a single injection of cell therapy significantly reduced the risk of the composite outcome of 3-point irreversible morbidity or mortality MACE. In subjects with CRP ≥ 2 mg/ml, the risk reduction was even more obvious. The risk of 3-point MACE was reduced by 33% [HR 0.667; 95% CI (0.472, 0.941); P = 0.021; Fig. 7A] using the time analysis to the first event, and reduced by 35% [HR 0.646; 95% CI (0.466, 0.895); P = 0.009; Fig. 7B] in the analysis of the recurrence event rate normalized for follow-up time (i.e., the number of events per 100 patient-years). The Kaplan-Meier curve (Kaplan-Meier curve) of this composite outcome for patients with plasma hsCRP levels > 2 mg/L or <2 mg/L is depicted in Fig. 7C. As shown in Figure 7C, among all treated patients with CRP > 2 mg/L, cell therapy significantly reduced the risk of:

Nonfatal MI and nonfatal stroke ( Figure 7C1 ); and

- Composite of cardiac death or ischemic MACE (MI or stroke) (Figure 7C2).

Ischemic MACE

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

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

Cell therapy significantly reduced the composite of cardiac death or ischemic MACE (MI or stroke) of all patients by 33% (Fig. 9).Subsequently, further analysis of the patient group revealed that, compared with the control, cell therapy significantly reduced the composite of cardiac death or ischemic MACE (MI or stroke) of NYHA II grade patients by 60%, which further supported the rationale (Fig. 9) of selecting and treating the patient with NYHA II grade heart failure.Also noted that, after many years of follow-up, cell therapy prevented the progression (Fig. 10) of cardiac death of NYHA II grade patients.

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, especially CRP levels ≥ 2 mg/L. These beneficial effects were not evident in patients with baseline CRP < 2 mg/L, suggesting that cell therapy may be particularly beneficial in the presence of active inflammation.

Further data analysis revealed that CRP is an important marker for cardiac death. As shown in Figure 11, patients with elevated CRP levels (≥2 mg/L) have a significantly increased risk of cardiac death. These data further support the use of cell therapy to treat NYHA class II patients, especially when these patients have elevated CRP levels, such as CRP ≥ 2 mg/L. These data also indicate the utility of CRP levels as an indicator or in patients at risk of cardiac death.

Claims (60)

1. A method for treating or preventing progressive heart failure in a subject, the method comprising administering to the subject a composition comprising cells, wherein the subject has grade II heart failure according to the New York Heart Association (NYHA) classification scale.

2. A method of reducing progression of heart failure in a subject, the method comprising administering to the subject a composition comprising cells, wherein the subject has grade II heart failure according to the New York Heart Association (NYHA) classification scale.

3. A method of reducing cardiac death in a subject having grade II heart failure according to the New York Heart Association (NYHA) classification scale, the method comprising administering to the subject a composition comprising cells.

4. A method of selecting a heart failure patient to be treated with cell therapy, the method comprising: i) Heart failure was assessed according to the New York Heart Association (NYHA) rating scale; and II) selecting a subject having grade II heart failure according to NYHA, preferably wherein the method comprises administering a composition comprising cells.

5. The method of any one of claims 1-4, wherein the CRP level of the subject is elevated prior to administration of the cells.

6. The method of claim 5, wherein the subject has a CRP level of ≡2mg/L.

7. The method of any one of claims 1 to 6, wherein the cell:

inducing neovascularization in the target tissue, preferably wherein said cells promote arteriogenesis; and/or

Factors are secreted that protect the myocardium at risk.

8. The method according to claim 1 or claim 2, comprising the steps of: i) Selecting a subject having grade 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 neovascularization in the target tissue.

9. The method of any one of claims 1-8, wherein administration of the composition inhibits progression to nyhail grade progressive heart failure in the subject.

10. The method of any one of claims 1-9, wherein the subject's N-terminal pro-B-type natriuretic peptide (NT-proBNP) level is:

less than 2200pg/ml, preferably less than 2000pg/ml, prior to administration of the cells; or,

between 1000pg/ml and 2000pg/ml prior to administration of the cells.

11. The method according to any one of claims 1 to 10, wherein the subject has a C-reactive protein (CRP) level <5mg/L, preferably <4mg/L.

12. The method of any one of claims 1-11, wherein the subject has a CRP level of between 1.5mg/L and 5 mg/L.

13. The method of any one of claims 1-12, wherein the subject has a heart failure hospitalization within the previous 9 months.

14. The method of any one of claims 1 to 13, wherein the subject has a LVEF of less than about 45%, preferably less than 40%.

15. The method of any one of claims 1 to 14, wherein the subject has persistent left ventricular dysfunction.

16. The method of any one of claims 1-15, wherein heart failure of the subject is caused by an ischemic event or a non-ischemic event.

17. The method of any one of claims 1, 2, or 5-16, wherein the subject has a reduced risk of cardiac death following treatment.

18. The method of claim 17, wherein the reduced risk is relative to the risk of cardiac death in a subject with NYHA class III progressive heart failure.

19. The method of any one of claims 1 to 18, wherein the subject has a reduced risk of ischemic MACE (MI or stroke) following treatment.

20. The method of any one of claims 1 to 19, wherein the composition is administered endocardially and/or intravenously.

21. The method of any one of claims 1 to 20, wherein the cells are mesenchymal lineage precursors or stem cells (MLPSCs).

22. The method of claim 21, wherein the MLPSC is STRO-1+.

23. The method of claim 21, wherein the MLPSC is a Mesenchymal Stem Cell (MSC).

24. The method of claim 21 or claim 22, wherein the MLPSC is allogeneic.

25. The method of any one of claims 21 to 24, wherein the cells are expanded in culture.

26. The method of claim 25, wherein the cells are tnap+ prior to culture expansion.

27. The method of any one of claims 21 to 26, wherein the cells have been cryopreserved.

28. The method of any one of claims 1 to 27, comprising administering 1 x 10 ⁷ Up to 2X 10 ⁸ Individual cells.

29. The method of any one of claims 1 to 28, wherein the composition further comprises Plasma-Lyte a, dimethylsulfoxide (DMSO), human Serum Albumin (HSA).

30. The method of any one of claims 1-29, wherein the composition further comprises a Plasma-Lyte a (70%), DMSO (10%), HSA (25%) solution comprising 5% HSA and 15% buffer.

31. The method of any one of claims 1 to 30, wherein the composition comprises greater than 6.68 x 10 ⁶ Each living cell/mL.

32. The method of any one of claims 1-25 or 27-31, wherein the composition comprises human bone marrow-derived allogeneic Mesenchymal Precursor Cells (MPCs) isolated from bone mononuclear cells using anti-STRO-3 antibodies, expanded ex vivo, and cryopreserved.

33. A method of reducing the risk of an ischemic event in a subject, the method comprising administering to the subject a composition comprising cells.

34. The method of claim 33, wherein the subject has a CRP level of ≡2mg/L.

35. The method of claim 33 or 34, wherein the ischemic event is the formation of arterial occlusion.

36. The method of claim 33 or 34, wherein the ischemic event is the formation of a cerebrovascular or cardiac occlusion.

37. The method of claim 33 or 34, wherein the ischemic event is a stroke or myocardial infarction.

38. The method of any one of claims 33-37, wherein the subject has a non-ischemic cardiomyopathy.

39. The method of any one of claims 33 to 38, wherein the cells are administered endocardially.

40. The method of any one of claims 33-39, wherein the subject has grade II or III heart failure according to the New York Heart Association (NYHA) classification scale.

41. The method of any one of claims 33 to 40, wherein the cell:

inducing neovascularization in the target tissue, preferably wherein said cells promote arteriogenesis; and/or

Factors are secreted that protect the myocardium at risk.

42. The method of any one of claims 33 to 41, wherein the cells are mesenchymal lineage precursors or stem cells (MLPSCs).

43. The method of any one of claims 33-42, wherein the subject's N-terminal pro-B-type natriuretic peptide (NT-proBNP) level is between 1000pg/ml and 2000pg/ml prior to administration of the cells.

44. The method of any one of claims 33 or 35-44, wherein the subject has a C-reactive protein (CRP) level of between 1.5mg/L and 5 mg/L.

45. The method of claim 42, wherein the MLPSC is one or more of: STRO-1+, allogeneic, culture expanded, subject to cryopreservation.

46. The method of claim 45, wherein the cells are culture expanded and express tnap+ prior to culture expansion.

47. The method of any one of claims 33 to 46, comprising administering 1 x 10 ⁷ Up to 2X 10 ⁸ Individual cells.

48. The method of any one of claims 33-47, wherein the composition comprises a solution of Plasma-Lyte a (70%), DMSO (10%), HSA (25%), the HSA solution comprising 5% HSA and 15% buffer.

49. The method of any one of claims 33 to 48, wherein the composition comprises human bone marrow derived allogeneic Mesenchymal Precursor Cells (MPCs) isolated from bone mononuclear cells using anti-STRO-3 antibodies, expanded ex vivo, and cryopreserved.

50. 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 CRP levels in a sample obtained from the subject, wherein an elevated CRP indicates an elevated risk of cardiac death, myocardial infarction, or stroke.

51. The method of claim 50, wherein the subject has progressive heart failure.

52. The method of claim 51, wherein the subject has grade II progressive heart failure.

53. The method of any one of claims 50 to 52, wherein a CRP level ≡2mg/L indicates an increased risk of cardiac death, myocardial infarction or stroke.

54. The method of any one of claims 50 to 53, wherein the method determines an increased risk of cardiac death.

55. A method for treating or preventing progressive heart failure in a subject, the method comprising administering to the subject a composition comprising a mesenchymal precursor lineage or stem cells, wherein the subject has grade II or III heart failure and has active inflammation according to the New York Heart Association (NYHA) classification scale.

56. A method of reducing progression of heart failure in a subject, the method comprising administering to the subject a composition comprising mesenchymal precursor lineages or stem cells, wherein the subject suffers from grade II or III heart failure and has active inflammation according to the New York Heart Association (NYHA) classification scale.

57. A method of reducing cardiac death in a subject suffering from grade II or III heart failure with active inflammation according to the New York Heart Association (NYHA) classification scale, the method comprising administering to the subject a composition comprising mesenchymal precursor lineages or stem cells.

58. A method of selecting a heart failure patient to be treated with cell therapy, the method comprising: i) CRP levels were assessed and heart failure was assessed according to the New York Heart Association (NYHA) rating scale; and II) selecting a subject having grade II or III heart failure and having active inflammation according to NYHA, preferably wherein the method comprises administering a composition comprising mesenchymal precursor lineages or stem cells.

59. The method of any one of claims 55-58, wherein active inflammation is determined based on CRP levels in the subject.

60. The method of claim 59, wherein the active inflammation is characterized by CRP levels of > 2mg/L.