CN116829162A

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

Info

Publication number: CN116829162A
Application number: CN202180087732.7A
Authority: CN
Inventors: S·伊茨库; K·伯劳; J·海耶斯
Original assignee: Mesoblast International SARL
Current assignee: Mesoblast International SARL
Priority date: 2020-12-15
Filing date: 2021-12-15
Publication date: 2023-09-29

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

Methods of treating progressive heart failure in a subject suffering from grade II heart failure

Cross Reference to Related Applications

The present application claims priority from australian application No. 2020904675 filed 12, 15, 2021, 1, 12, 2021900059, 2021902941, 2021903365, 2021, 10, 20, and U.S. application No. 63/289,868, 2021, 12, 15, each of which are incorporated herein by reference in their entirety.

Technical Field

Background

Myocardial Infarction (MI) remains one of the leading causes of death and morbidity in developed countries. Updates to the U.S. medical insurance records are published that evaluate data relating to acute MI hospitalization of 350,509 >65 year old patients who survived discharge after the occurrence of the event (Schuster et al (2004) physiology-cardiac and circulatory physiology (Physiol Heart Circa physiol.), 287 (2): 525-32). In the first year after the index event, 25.9% of MI patients die and 50.5% of patients are hospitalized again. Within one month after MI, the probability of death is 21 times that of the general medical insurance age population, and the probability of hospitalization is 12 times that of the general medical insurance age population.

In the last 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 adverse clinical courses characterized by frequent hospitalization and premature death.

Clearly, there is a need in the art for the treatment or prevention of progressive heart failure.

Disclosure of Invention

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

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

In another example, the disclosure relates to a method of reducing cardiac death in a subject suffering from 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.

In another example, the present disclosure relates to 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. 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 the myocardium at risk. Thus, in one example, the disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering to the subject a composition comprising 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 a target tissue and/or secrete factors that protect the myocardium at risk.

In one example, the cells are mesenchymal lineage precursors or stem cells (MLPSCs). 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 expanded by culture. In this example, the cells may be tnap+ prior to culture expansion. In one example, the cells have been cryopreserved.

In another example, a method of the present disclosure includes 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.

In another example, administration of the composition inhibits progression to NYHA class III progressive heart failure in the subject.

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

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

In another example, the subject has a heart failure hospitalization 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 has persistent left ventricular dysfunction.

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

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

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

In one example, the risk of ischemic MACE (non-lethal MI or non-lethal stroke) is reduced in a heart failure class III subject. In another example, the risk of cardiac death in a heart failure class III subject following treatment is reduced. In another example, the grade III heart failure subject has a reduced risk of ischemic MACE and cardiac death following treatment. In one example, the level of CRP in the class III heart failure subject is greater than or equal to 2mg/L.

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

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

In one example, the disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering to the subject a composition comprising mesenchymal 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. In another example, the disclosure relates to a method of reducing progression of heart failure in a subject, the method comprising administering to the subject a composition comprising a 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. In another example, the disclosure relates to a method of 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) classification scale, the method comprising administering to the subject a composition comprising mesenchymal precursor lineages or stem cells. In another example, the present disclosure relates to 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. In one example, active inflammation is determined based on CRP levels in the subject. In one example, the subject has heart failure grade II 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 this disclosure. 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 the myocardium at risk. In one example, the cell is an 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 expanded by culture. In this example, the cells may be tnap+ prior to 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 1000pg/ml and 2000pg/ml prior to administration of the cells. In another example, the subject has an elevated level of C-reactive protein (CRP). In another example, the subject has a CRP level of greater than or equal to 1mg/L. In another example, the subject has a CRP level of greater than or equal to 2mg/L. In another example, the subject has a CRP level between 2mg/L and 5 mg/L. In another example, the subject has a CRP level between 3mg/L and 5 mg/L.

In the embodiments of the above examples, the methods of the present disclosure include administering 1×10 ⁷ Up to 2X 10 ⁸ Individual cells.

In another exampleThe composition administered further comprises Plasma-Lyte a, dimethylsulfoxide (DMSO), human Serum Albumin (HSA). In one example, the composition administered further comprises a Plasma-Lyte a (70%), DMSO (10%), HSA (25%) solution comprising 5% HSA and 15% buffer. In one example, the composition comprises greater than 6.68X10 ⁶ Each living cell/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 an example, the ischemic event is one or more of a myocardial infarction, a 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 increased risk of cardiac death, myocardial infarction or stroke. Thus, in one example, the present disclosure relates to a method for determining an elevated risk of one or more of cardiac death, myocardial infarction, or stroke in a subject, the method comprising measuring CRP levels in a sample obtained from the subject, wherein an elevated CRP indicates an elevated risk of cardiac death, myocardial infarction, or stroke. In one example, the subject has progressive heart failure. In one example, the heart failure of the subject 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 CRP level of > 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.

Drawings

Fig. 1: the incidence of ischemic MACE (MI, stroke) was reduced.

Fig. 2: the incidence of ischemic MACEs (MI, stroke) is reduced; NYHA stages II and III.

Fig. 3: the incidence of ischemic MACEs (MI, stroke) is reduced; ischemic and non-ischemic.

Fig. 4: all treated patients (n=537); class II patients (n=206); cardiac death in class III patients (n=331).

Fig. 5: cardiac death in NYHA class II patients; ischemic and non-ischemic.

Fig. 6: cardiac death in the test patients.

Fig. 7: a) TTFE composite IMM MACE; b) Ratio normalized composite IMM MACE; c) Curves for all treated patients with baseline CRP > 2mg/L and CRP < 2 mg/ml. Curves shown for non-lethal MI or non-lethal stroke; 3-point TTFE complex IMM MACE against CV death or non-lethal MI or non-lethal stroke.

Fig. 8: a) Irreversible incidence of TTFE; b) Ratio normalized irreversible morbidity; c) All treated patients (n=537); class II patients (n=206); a curve of irreversible incidence TTFE MACE (non-lethal MI or non-lethal stroke) for patients of grade III (n=331).

Fig. 9: the incidence of complex cardiac death or ischemic MACE (MI, stroke) is reduced; all treated patients (n=537); class II patients (n=206); class III patients (n=331).

Fig. 10: cardiac death in NYHA class II patients-years of follow-up.

Fig. 11: the risk of progression to cardiac death for NYHA class II patients with baseline hsCRP > 2mg/L is significantly increased.

Fig. 12: the risk of developing 3-point MACE (cardiac death/MI/stroke) in NYHA class II patients with baseline hsCRP > 2mg/L is significantly increased.

Detailed Description

General techniques and definitions

Unless specifically stated otherwise, all technical and scientific terms used herein should be taken 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, 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, molecular cloning Utility Specification (A practical Guide To Molecular Cloning), john Wili's father-son publishing company (John Wiley and Sons) (1984); sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), cold spring harbor laboratory Press (Cold Spring Harbour Laboratory Press) (1989); brown (edit), "basic molecular biology: practical methods (Essential Molecular Biology: A Practical Approach), volumes 1 and 2, IRL Press (1991); glover and B.D.Hames (editions), and F.M.Ausubel et al (editions), guidelines for contemporary molecular biology experiments (Current Protocols in Molecular Biology), greene Pub.associates, and Wiley International science publication (Wiley-Interscience) (1988, including all updates to date); ed Harlow and David Lane (edit) antibody: laboratory manuals (Antibodies: A Laboratory Manual), cold spring harbor laboratory, (1988); and J.E.Coligan et al (editions), "contemporary immunology guidelines (Current Protocols in Immunology)," John Wili father-son publishing company (including all updates so far).

The term "and/or", e.g. "X and/or Y", is understood to mean "X and Y" or "X or Y", and is to be taken as providing explicit support for both meanings or for either meaning.

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

The terms "level" and "amount" are used to define the amount of a particular substance in a sample from a subject or in a cell culture medium (or sample therefrom). For example, a particular concentration, weight, percentage (e.g., v/v%) or ratio may be used to define the level of a particular substance in a sample. In one example, the level is expressed in terms of how much of a particular marker is expressed by a cell of the present disclosure under culture conditions. In one example, expression refers to cell surface expression. In another example, the level is expressed in terms of how much of a particular marker is released from the cells described herein 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 may be expressed in pg/ml. In one example, the level is expressed in mg/L. For example, CRP levels may be expressed in mg/L. In another example, the level is at every 10 ⁶ Pg of individual cells.

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

The expansion of cells in culture from cryopreserved intermediates means that cells subjected to low temperature freezing 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 cells are cryopreserved and then re-inoculated into culture. For example, the level is determined after the first cryopreservation of the cells. In another example, the level is determined after a second cryopreservation of the cells. For example, cells may be culture expanded from a cryopreserved intermediate, and a second cryopreservation performed prior to re-seeding in culture, so that the level of a particular marker may be determined under culture conditions.

As used herein, the terms "treatment", "reducing progression" comprise administration of mesenchymal lineage stem cells or precursor cell populations 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 reduced progression, slowing the progression of progressive heart failure.

As used herein, the term "subject" refers to a human subject. For example, the subject may be an adult. In another example, the subject may be a child. In another example, the subject may be an adolescent. Terms such as "subject," "patient," or "individual" are terms that may be used interchangeably in the context of the present disclosure. Subjects in need of treatment include those already with progressive heart failure, as well as those in need of prevention, delay or cessation of 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 "non-genetically modified" refers to cells that have not been modified by transfection with a nucleic acid. For the avoidance of doubt, in the context of the present disclosure, mesenchymal lineage precursors or stem cells transfected with a nucleic acid encoding a protein will be considered genetically modified.

As used herein, the term "sample" refers to an extract from a subject in which CRP levels 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 sample as described above, so long as it can be collected from a subject and assayed to determine CRP levels in the subject. In one example, the sample is a blood sample. In one example, the blood sample is obtained from a subject having NYHA class II heart failure.

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

Throughout this specification, unless the context clearly indicates otherwise, reference to a single step, composition of matter, group of steps, or group of compositions of matter should be taken to encompass one or more (i.e., one or more) of those steps, compositions of matter, group of steps, or group of compositions of matter.

It will be appreciated by those skilled in the art that variations and modifications other than those specifically described may be made to the disclosure described herein. It is to be understood that the present disclosure encompasses all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, 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 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 disclosure.

Any examples disclosed herein should be considered applicable to any other examples, mutatis mutandis, unless explicitly stated otherwise.

Progressive heart failure

Cardiomyopathy is a cardiomyopathy that makes it difficult for the heart to pump blood to other parts of the body. Heart failure occurs when the heart is unable to pump blood sufficiently to maintain blood flow to meet the needs of the body. Cardiomyopathy can occur after ischemic or non-ischemic events. One cause of ischemic heart failure is contractile dysfunction following Myocardial Infarction (MI), such as a heart attack. MI occurs when blood ceases to flow normally to a certain part of the heart. Insufficient blood supply results in myocardial necrosis of the local area, known as infarction or infarct formation. The infarcted heart is unable to pump blood sufficiently to maintain blood flow to meet the needs of the body, leading to a variety of pathophysiological responses and ultimately heart failure. Non-ischemic cardiomyopathy is not associated with known coronary artery disease. One example is Dilated Cardiomyopathy (DCM), in which the pumping capacity of the heart is reduced, as the main pumping chamber left ventricle of the heart becomes dilated, dilated and weakened.

Once the heart is unable to pump blood sufficiently to maintain blood flow to meet the needs of the body, a series of compensatory mechanisms are initiated to buffer the drop in cardiac output and assist in maintaining adequate blood pressure to perfuse vital organs. Thus, patients with heart failure may not progress for a long period of time. However, the compensatory mechanisms eventually fail 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, systolic dysfunction and advanced heart failure may be used interchangeably with "progressive heart failure".

The methods of the present disclosure are effective on a subset of patients with progressive heart failure. In one example, the subjects are defined based on the New York Heart Association (NYHA) rating scale. In one example, the subject has progressive heart failure less than grade III. In one example, the subject has heart failure grade II. In one example, NYHA classification is assigned based on symptoms of the subject. For example, the NYHA classification may be assigned based on the following table:

in one example, heart failure in a subject is caused by an ischemic event. In one example, heart failure in a subject is caused by Myocardial Infarction (MI). For example, the subject may be an MI subject. The term "Myocardial Infarction (MI) subject" is used to define a subject suffering from myocardial infarction. In one example, heart failure in a subject is caused by non-ischemic cardiomyopathy.

The methods of the present disclosure can be used to treat progressive heart failure in a particular population of MI subjects. Subjects in need of treatment include those already with progressive heart failure, as well as those in need of prevention, delay or cessation of progressive heart failure. In these examples, the subject suffers from NYHA-based grade II or grade III progressive heart failure. For example, the subject may have grade 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, the active inflammation is characterized by CRP levels of > 2mg/L.

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

In one example, the subject treated according to the present disclosure has an initial CRP level of ≡2mg/L. For example, the subject may have heart failure grade II or III, and the initial CRP level is ≡2mg/L. In another example, the subject may have grade II heart failure and the initial CRP level is greater than or equal to 2mg/L. In one example, the subject treated according to the present disclosure has an initial CRP level <5mg/L. In another example, the subject has an initial CRP level <4mg/L. In another example, the subject's initial CRP level is between 2mg/L and 6 mg/L. In another example, the subject's initial CRP level is between 3mg/L and 6 mg/L. In another example, the subject's initial CRP level is between 4mg/L and 5mg/L.

There are various assays available for measuring CRP levels, such as antibody-based immunoassays. For example, CRP levels in blood samples can be determined using enzyme-linked immunosorbent (ELISA). In one example, a blood sample is obtained from a patient and then purified prior to contact with an anti-CRP antibody. The extent of antibody binding is used to quantify the level of CRP (e.g., mg/L) in the blood sample. In one example, CRP is measured by a plasma-sensitive CRP (hsCRP) ELISA assay.

Type B Natriuretic Peptide (BNP) is a hormone produced by the heart. The N-terminal (NT) pro-hormone BNP (NT-proBNP) is an inactive pro-hormone released from the same molecule from which BNP is produced. BNP and NT-proBNP are released in response to a change in the intra-cardiac pressure. These changes may be associated with heart failure and other heart problems. The level may rise when heart failure develops or worsens, and the level may fall when heart failure stabilizes. BNP is therefore an effective marker of heart failure progression. In one example, the subject's NT-proBNP level is less than 2200pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is less than 2100pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is less than 2000pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is less than 1900pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is between 2200pg/ml and 1000pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is between 2200pg/ml and 1100pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is between 2100pg/ml and 1200pg/ml prior to administration of the composition of the present disclosure. In another example, the subject's NT-proBNP level is between 2000pg/ml and 1500pg/ml prior to administration of the composition of the present disclosure. There are various assays available for measuring the level of NT-proBNP, such as antibody-based immunoassays, e.g.ELISA assays. In one example, a blood sample is obtained from a patient and then purified prior to contact with an anti-NT-proBNP antibody. The extent of antibody binding is used to quantify the level (e.g., pg/L) of NT-proBNP in the blood sample.

In another example, the subject has a heart failure hospitalization event within the previous 12 months prior to administration of the composition disclosed herein. In another example, the subject has a heart failure hospitalization event within the previous 9 months prior to administration of the composition disclosed herein. In another example, the subject has a heart failure hospitalization event within 6 to 12 months prior to 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, heart failure hospitalization is a non-ischemic event.

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

In one example, the subject may have persistent left ventricular dysfunction. Left ventricular dysfunction is characterized by a decrease in myocardial contractility. As the contractile force of the heart muscle in the left ventricle decreases, the Left Ventricular Ejection Fraction (LVEF) decreases. Accordingly, LVEF provides a method of 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, less than 45% of subjects have left ventricular dysfunction. In other examples, less than about 44%, 43%, 42%, 41% of subjects have left ventricular dysfunction with LVEF. In another example, less than about 40% of subjects have left ventricular dysfunction with LVEF. In other examples, less than about 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30% of subjects have 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 lasts from about 1 to about 14 days or more.

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

The methods of the present disclosure relate to the treatment of gradual decline in cardiac output characterized by progressive heart failure. Thus, in the context of the present disclosure, "treatment" and "treatment" refer to therapeutic treatments and prevention or prophylaxis measures.

In one example, the treatment comprises administration of a composition of the present disclosure. In one example, the methods of the present disclosure reduce or inhibit progression of progressive heart failure. In one example, the treatment inhibits progression of progressive heart failure to NYHA class III in the subject. In another example, the treatment reduces the risk of cardiac death. In one example, the reduced risk of cardiac death is relative to the risk of cardiac death in a subject with NYHA class III 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 a complex defined as cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke (cardiac death/MI/stroke). In one example, the risk of 3-point MACE is reduced by at least 30% 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 patient survival. In one example, the treatment increases the probability that the subject survives at least 1000 days after initiation of the treatment. In another example, the treatment increases the probability that the subject survives at least 2000 days after initiation of the treatment. In one example, the increased probability is determined relative to a subject not treated with a composition of the present disclosure. In one example, the increased probability is determined relative to a subject with grade III heart failure.

In one example, treatment reduces the chance or risk of a major adverse cardiac event associated with heart failure (HF-MACE), defined as the compounding of heart-related death or resuscitating cardiac death or non-lethal 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 the compositions disclosed herein. In one example, the treatment reduces the chance or risk of total mortality.

Ischemic event

In one example, the disclosure relates to methods of reducing the risk or incidence of an ischemic event in a subject, particularly a subject having cardiomyopathy. In one example, the disclosure relates to methods of reducing the risk or incidence of an ischemic event in a subject having 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 may be reduced relative to untreated subjects. In one example, the ischemic event is caused by the formation of an occlusion. In one example, the occlusion is an arterial occlusion. In one example, the ischemic event is the formation of a cerebrovascular occlusion. In another example, the ischemic event is the formation of a heart occlusion. For example, an occlusion may be formed in a coronary artery.

Examples of ischemic events caused by occlusion formation include myocardial infarction and stroke. Thus, in one example, the disclosure relates to methods 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 suffering from cardiomyopathy is reduced by administering a cell therapy such as a composition of the present disclosure.

In one example, the subject has a non-ischemic cardiomyopathy. For example, cardiomyopathy in a subject may be caused by an enlargement of the left ventricle (dilated cardiomyopathy). In another example, cardiomyopathy is caused by a viral infection.

In another example, the subject has heart failure grade II or III 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 1000pg/ml and 2000pg/ml prior to administration of the cells. In another example, the subject has an elevated level of C-reactive protein (CRP). In another example, the subject has a CRP level of greater than or equal to 1.5mg/L. In another example, the subject has a CRP level of greater than or equal to 2mg/L. In another example, the subject has a CRP level between 1mg/L and 5mg/L. In another example, the subject has a CRP level between 3mg/L and 5mg/L.

In one example, the cells are administered endocardially.

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 period of time.

Mesenchymal precursor cells

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

The term "mesenchymal lineage precursor or stem cell" encompasses the parental cell and its undifferentiated progeny. The term also encompasses mesenchymal precursor cells, pluripotent stromal cells, mesenchymal Stem Cells (MSCs), perivascular mesenchymal precursor cells, and undifferentiated progeny thereof.

The mesenchymal lineage precursor or stem cells can be autologous, allogeneic, xenogeneic, syngeneic or isogenic. Autologous cells are isolated from the same individual in which they are to be re-implanted. Allogeneic cells are isolated from a donor of the same species. The xenogeneic cells are isolated from a donor of another species. Homologous 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 precursor or stem cells are allogeneic. In one example, allogeneic mesenchymal lineage precursors or stem cells are expanded in culture and cryopreserved.

Mesenchymal lineage precursors or stem cells are found predominantly in bone marrow, but are also shown to be present in a variety of host tissues including, for example, umbilical cord blood and cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp. It is also present in skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicle, intestine, lung, lymph node, thymus, ligament, tendon, skeletal muscle, dermis and periosteum; and is capable of differentiating into a germ line, such as mesoderm and/or endoderm and/or ectoderm. Thus, mesenchymal lineage precursors or stem cells can differentiate into a wide variety of cell types including, but not limited to, fat, bone, cartilage, elastic tissue, muscle, and fibrous connective tissue. The particular lineage commitment and differentiation pathway that these cells enter depends on various effects from mechanical influences and/or endogenous bioactive factors such as growth factors, cytokines, and/or local microenvironment conditions established by the host tissues.

The term "enriched", "enriched" or variants thereof are used herein to describe a population of cells having an increased proportion of one particular cell type or of a plurality of particular cell types as compared to an untreated population of cells (e.g., cells in their natural environment). In one example, the population enriched for 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 for mesenchymal lineage precursors or stem cells" will be employed to expressly support the term "cell population comprising X% mesenchymal lineage precursors or stem cells," where X% is a percentage as described herein. In some examples, the mesenchymal lineage precursor or stem cells can form clonogenic colonies, e.g., CFU-F (fibroblasts) or a subset thereof (e.g., 50% or 60% or 70% or 90% or 95%) can have this activity.

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

In another example, the mesenchymal lineage precursor or stem cell is cd29+, cd54+, cd73+, cd90+, cd102+, cd105+, cd106+, cd166+, MHC1+ MSC.

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

In one example, the isolated or enriched mesenchymal lineage precursor or stem cells are at 50,000 viable cells/cm ² Inoculated in a medium (serum-free or serum-supplemented), for example, an αminimum essential medium (αMEM) supplemented with 5% Fetal Bovine Serum (FBS) and glutamine, and maintained at 37℃at 20% O ₂ Adhere to the culture vessel overnight. The medium is then replaced and/or changed as required, and the cells are incubated at 37℃with 5% O ₂ Further culturing for 68-72 hours.

As will be appreciated by those skilled in the art, cultured mesenchymal lineage precursors or stem cells are phenotypically different from in vivo cells. For example, in one embodiment, it expresses one or more of the following markers: CD44, NG2, DC146, and CD140b. The cultured mesenchymal lineage precursor or stem cells are also biologically different from in vivo cells, with higher proliferation rates than most non-circulating (quiescent) cells in vivo.

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

References to the selection of cells or populations thereof do not necessarily require selection from a particular tissue source. STRO-1+ cells may be selected from or isolated or enriched from a variety of sources, as described herein. That is, in some examples, 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, either alone or in combination, selected from the group consisting of: TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90 beta), CD45+, CD146+, 3G5+, or any combination thereof.

By "individually" is meant that the present disclosure individually encompasses the markers or sets of markers, and although individual markers or sets of markers may not be individually listed herein, the appended claims may define such markers or sets of markers individually and separately from each other.

"collectively" means that the present disclosure encompasses any number or combination of the markers or sets of markers, and that although such number or combination of markers or sets of markers may not be specifically listed herein, the appended claims may define such combination or sub-combination separately and separately from any other marker combination or set of markers.

As used herein, the term "TNAP" is intended to encompass all isoforms of tissue-non-specific alkaline phosphatase. For example, the term encompasses liver isotype (LAP), bone isotype (BAP) and kidney isotype (KAP). In one example, the TNAP is BAP. In one example, TNAP as used herein refers to a molecule that can bind to STRO-3 antibodies produced by a hybridoma cell line deposited with ATCC under the provisions of the Budapest Treaty at 12/19/2005 under deposit accession number PTA-7282.

Furthermore, in one example, STRO-1+ cells are capable of producing clonogenic CFU-F.

In one example, a significant proportion of STRO-1+ cells are capable of differentiating into at least two different lineages. Non-limiting examples of lineages into which STRO-1+ cells may committed include bone precursor cells; a hepatocyte progenitor cell having multipotency for biliary epithelial cells and hepatocytes; a neural restricted cell that can generate glial cell precursors that progress into oligodendrocytes and astrocytes; a neuron precursor that progresses to a neuron; myocardium and precursors of cardiomyocytes, glucose-responsive insulin secreting pancreatic beta cell lines. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells and chondrocytes, as well as precursor cells of: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal catheter epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelial cells, glial cells, neurons, astrocytes and oligodendrocytes.

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

Mesenchymal lineage precursors or stem cells encompassed by the present disclosure can also be cryopreserved prior to administration to a subject. In one example, the mesenchymal lineage precursor 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 disclosure encompasses mesenchymal lineage precursors or stem cells and extracellular vesicles isolated therefrom. For example, mesenchymal precursor lineages or stem cells of the present disclosure may be expanded in culture for a period of time under conditions suitable for secretion of extracellular vesicles into the cell culture medium. Secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

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

As used herein, the term "exosome" refers to a type of extracellular vesicle, typically in the range of about 30nm to about 150nm in size, and derived from a mammalian cell's endosomal compartment 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 its cargo.

In one example, the compositions of the present disclosure include 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 myocardium at risk or damaged. 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 myocardial apoptosis.

Culture expansion of cells

In one example, the mesenchymal lineage precursor or stem cells are expanded by culture. The "culture expanded" mesenchymal lineage precursor or stem cell culture medium differs from freshly isolated cells in that it has been cultured and passaged (i.e., subcultured) in the cell culture medium. In one example, culture-expanded mesenchymal lineage precursors or stem cells are culture-expanded from about 4 passages to 10 passages. In one example, the mesenchymal lineage precursor or stem cells are expanded by culture 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 expanded by culture for at least 5 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 10 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 8 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 7 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded for more than 10 passages. In another example, the mesenchymal lineage precursor or stem cells can be culture expanded for more than 7 passages. In these examples, stem cells can be culture expanded prior to 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 a cryopreserved intermediate.

In one example, the compositions of the present disclosure include mesenchymal lineage precursors or stem cells expanded from cryopreserved intermediate cultures. In one example, a cell culture expanded from a cryopreserved intermediate culture is expanded for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 passages. For example, mesenchymal lineage precursors or stem cells can be expanded by culture for at least 5 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 10 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 8 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded at least 5 passages to 7 passages. In one example, the mesenchymal lineage precursor or stem cells can be culture expanded for more than 10 passages. In another example, the mesenchymal lineage precursor or stem cells can be culture expanded for more than 7 passages.

In one example, mesenchymal lineage precursors or stem cells expanded from cryopreserved intermediate cultures can be expanded in culture in a medium that does not contain animal proteins. In one example, mesenchymal lineage precursors or stem cells expanded from cryopreserved intermediate cultures can be expanded in culture in a xeno-free medium. In one example, mesenchymal lineage precursors or stem cells expanded from cryopreserved intermediate cultures can be expanded in culture in medium free of fetal bovine serum.

In one embodiment, the mesenchymal lineage precursors or stem cells can be obtained from a single donor or multiple donors, where the donor samples or mesenchymal lineage precursors or stem cells are then pooled and then culture expanded. In one example, the culture amplification process comprises:

i. expanding a plurality of living cells by passaging to provide a preparation of at least about 10 hundred million living cells, wherein passaging comprises establishing a primary culture of isolated mesenchymal lineage precursors or stem cells, and then continuously establishing a first non-primary (P1) culture of mesenchymal lineage precursors or stem cells isolated from a previous culture;

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

preparing and cryopreserving an intermediate mesenchymal lineage precursor or stem cell preparation in a process obtained from P2 culture of mesenchymal lineage precursors or stem cells; and

thawing the intermediate mesenchymal lineage precursor or stem cell preparation in the cryopreserved treatment and amplifying the intermediate mesenchymal lineage precursor or stem cell preparation in the treatment by passaging expansion.

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

i. Less than about 0.75% cd45+ cells;

at least about 95% cd105+ cells;

at least about 95% cd166+ cells.

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

In one example, the culture-expanded mesenchymal lineage precursor or stem cells are culture-expanded about 4 passages to 10 passages, with the mesenchymal lineage precursor or stem cells being cryopreserved after at least 2 passages or 3 passages before being further culture-expanded. In one example, the mesenchymal lineage precursor or stem cell is culture expanded at least 1 passage, at least 2 passages, at least 3 passages, at least 4 passages, at least 5 passages, cryopreserved, and then further culture expanded at least 1 passage, at least 2 passages, at least 3 passages, at least 4 passages, at least 5 passages prior to culturing according to the methods of the present disclosure.

The process of mesenchymal lineage precursor or stem cell isolation and ex vivo expansion can be performed using any apparatus and cell handling methods known in the art. Various culture expansion embodiments of the present disclosure employ steps that require manipulation of the cells, e.g., seeding, feeding, dissociating adherent culture, or washing. Any step of manipulating the cells may damage the cells. Although mesenchymal lineage precursors or stem cells can generally sustain a certain amount of damage during preparation, the cells are preferably manipulated by a treatment procedure and/or apparatus that adequately performs the given steps while minimizing damage to the cells.

In an example, mesenchymal lineage precursors or stem cells are washed in an apparatus comprising a cell source bag, a wash solution bag, a recycle wash bag, a rotating membrane filter with inlet and outlet ports, a filtrate bag, a mixing zone, a final product bag for washed cells, and appropriate tubing, for example, as described in US 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 CD105 positive and CD166 positive and CD45 negative. In one example, this homogeneity persists through ex vivo amplification; i.e. although multiple population doublings.

In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are culture expanded in 3D culture. For example, the mesenchymal lineage precursors or stem cells of the present disclosure can be culture expanded in a bioreactor. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are first culture expanded in 2D culture, then further expanded in 3D culture. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are culture expanded from a master cell bank. In one example, mesenchymal lineage precursors or stem cells of the present disclosure are culture expanded from a master cell bank in 2D culture prior to seeding in 3D culture. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are culture expanded in a bioreactor from a master cell bank in 2D culture for at least 3 days prior to seeding in 3D culture. In one example, the mesenchymal lineage precursors or stem cells of the present disclosure are culture expanded in a bioreactor from a master cell bank in 2D culture for at least 4 days prior to seeding in 3D culture. In one example, the mesenchymal lineage precursor or stem cells of the present disclosure are culture expanded in a bioreactor in 2D culture from a master cell bank for 3 days to 5 days prior to seeding in 3D culture. In these examples, the 2D culture may be performed in a cell factory. Various cell factory products are commercially available (e.g., siemens, sigma, thermofiser).

Cell culture medium

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

The term "medium" as used in the context of the present disclosure comprises a component of the surrounding environment of a cell. The culture medium facilitates and/or provides conditions suitable to allow cell growth. The medium may be solid, liquid, gaseous or a mixture of phases and materials. The medium may comprise a liquid growth medium and a liquid medium that does not sustain cell growth. The medium also comprises a gelatinous medium such as agar, agarose, gelatin and collagen matrix. Exemplary gaseous media comprise a gaseous phase to which cells grown on a petri dish or other solid or semi-solid support are exposed.

The cell culture medium used for culture expansion contains all essential amino acids and may also contain nonessential amino acids. Generally, amino acids are classified as 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 suitable for the cell line of interest. For example, if glucose (or other energy source) in the basal medium is found to be depleted and thus growth is restricted, it may be desirable to increase the level of this energy source, or to add glucose (or other energy source) during the culture process. In one example, the Dissolved Oxygen (DO) level 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 may 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 medium is animal protein free. In one example, the cell culture medium used in the methods of the present disclosure is free of animal components.

In one example, the medium includes serum. In other examples, the medium is a fetal bovine serum free medium comprising growth factors that promote proliferation of mesenchymal lineage precursors or stem cells. In one embodiment, the medium is a serum-free stem cell medium. In one example, the cell culture medium comprises:

a basal medium;

platelet Derived Growth Factor (PDGF);

fibroblast growth factor 2 (FGF 2).

In one example, the medium includes Platelet Derived Growth Factor (PDGF) and fibroblast growth factor 2 (FGF 2), wherein the level of FGF2 is less than about 6ng/ml. For example, FGF2 levels may be less than about 5ng/ml, less than about 4ng/ml, less than about 3ng/ml, less than about 2ng/ml, less than about 1ng/ml. In other examples, the FGF2 level is less than about 0.9ng/ml, less than about 0.8ng/ml, less than about 0.7ng/ml, less than about 0.6ng/ml, less than about 0.5ng/ml, less than about 0.4ng/ml, less than about 0.3ng/ml, less than about 0.2ng/ml.

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

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

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

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

In another example, the EGF level is between about 0.2ng/ml and 3.2 ng/ml. In another example, the EGF level is between about 0.4ng/ml and 1.6 ng/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, such as αMEM or StemSpan ^TM The basal medium may be supplemented with a reference amount of growth factors. In one example, the medium comprises alpha MEM or StemSpan supplemented with 32ng/ml PDGF-BB, 0.8ng/ml EGF and 0.02ng/ml FGF ^TM 。

In other examples, additional factors may be added to the cell culture medium. For example, the cell culture medium may be supplemented with one or more stimulating factors selected from the group consisting of: epidermal Growth Factor (EGF), 1 alpha, 25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor alpha (TNF-alpha), interleukin-lbeta (IL-lbeta), and stroma-derived factor lalpha (SDF-lalpha). In another embodiment, the cells may also be cultured in the presence of at least one cytokine in an amount sufficient to support the growth of the cells. In another embodiment, the cells may be cultured in the presence of heparin or a derivative thereof. For example, the cell culture medium may contain about 50ng/ml heparin. In other examples, the cell culture medium contains about 60ng/ml heparin, about 70ng/ml heparin, about 80ng/ml heparin, about 90ng/ml heparin, about 100ng/ml heparin, about 110ng/ml heparin, about 120ng/ml heparin, about 130ng/ml heparin, about 140ng/ml heparin, about 150ng/ml heparin, or derivatives thereof. In one example, the heparin derivative is sulfate. Various forms of heparin sulfate are known in the art and comprise heparin sulfate 2 (HS 2). HS2 may be from a variety of sources including, for example, the liver of male and/or female mammals. Exemplary heparan sulfates therefore include male heparin sulfate (MML HS) and female heparin sulfate (FML HS).

In another example, the cell culture media of the present disclosure promote stem cell proliferation while maintaining stem cells in an undifferentiated state. Stem cells are considered undifferentiated when they have not yet become a specific differentiation lineage. As discussed above, stem cells exhibit morphological features that are distinct from differentiated cells. In addition, undifferentiated stem cells express genes that can be used as markers for detecting the differentiation status. The polypeptide product may also be used as a marker for detecting the differentiation status. Thus, one of skill in the art can readily determine whether the methods of the present disclosure maintain stem cells in an undifferentiated state using conventional morphological, genetic, and/or proteomic analysis.

Modification of cells

The mesenchymal lineage precursors or stem cells disclosed herein can be altered in such a way that upon administration, lysis of the cells is inhibited. The alteration of the antigen may induce immune non-responses or tolerance, thereby preventing effector phases (e.g., cytotoxic T cell production, antibody production, etc.) that induce immune responses that ultimately lead to rejection of the 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 of importance for differentiation and/or maintenance of striated skeletal muscle cells. Exemplary proteins include growth factors (TGF-beta, insulin-like growth factor 1 (IGF-1), FGF), myogenic factors (e.g., myoD, myogenic factor 5 (Myf 5), myogenic Regulatory Factor (MRF)), transcription factors (e.g., GATA-4), cytokines (e.g., cardiophilin-1), neuregulin family members (e.g., neuregulin 1, 2, and 3), and homeobox genes (e.g., csx, tinman, and NKx families).

Composition and method for producing the same

The mesenchymal lineage or stem cells disclosed herein can be expanded from cryopreserved intermediate cultures to produce a formulation containing at least one therapeutic dose.

In one example, the compositions of the present disclosure comprise 10 x 10 ⁶ Individual cells to 35X 10 ⁶ Individual cells. In another example, the composition includes 20X 10 ⁶ Individual cells to 30X 10 ⁶ Individual cells. In other examples, the composition comprises at least 100 x 10 ⁶ Individual cells. In another example, the composition includes 50X 10 ⁶ Individual cells up to 500X 10 ⁶ Individual cells. In other examples, the compositions of the present disclosure include 1.5 hundred million cells.

In one example, the compositions of the present disclosure include a pharmaceutically acceptable carrier and/or excipient. The terms "carrier" and "excipient" refer to compositions of matter conventionally used in the art to facilitate storage, administration and/or bioactivity of an active compound (see, e.g., remington's Pharmaceutical Sciences, 16 th edition, mark publication (Mac Publishing Company) (1980)). The carrier may also reduce any undesirable side effects of the active compound. Suitable carriers are, for example, stable, e.g., not reactive with other components of the carrier. In one example, the carrier does 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, buffer solutions, hyaluronic acid, and glycols are exemplary liquid carriers, particularly (when isotonic) for solutions. Suitable pharmaceutical carriers and excipients include starch, cellulose, dextrose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol and the like.

In another example, the carrier is a culture medium composition, e.g., in which cells are grown or suspended. Such a culture medium composition does not cause any side effects in the subject to which it is administered. Exemplary carriers and excipients do not adversely affect cell viability and/or the ability of the cells to treat or prevent disease.

In one example, the carrier or excipient provides buffer activity to maintain the cells and/or soluble factors at a suitable pH to exert biological activity, e.g., the carrier or excipient is Phosphate Buffered Saline (PBS). PBS represents an attractive carrier or excipient because of minimal interaction with and allows for rapid release of cells and factors, in which case the compositions of the present disclosure may be produced as a liquid for direct application into the blood stream or into tissue or areas surrounding or adjacent to tissue, for example by injection.

The compositions of the present disclosure may be cryopreserved. Cryopreservation of mesenchymal lineage precursors or stem cells can 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 comprise a cryopreservation solution. The pH of the cryopreservation solution is typically 6.5 to 8, preferably 7.4.

The cryopreservation solution may comprise a sterile, pyrogen-free isotonic solution, such as PlasmaLyte ATM.100mL of PlasmaLyte ATM contained 526mg sodium chloride, USP (NaCl); 502mg sodium gluconate (C6H 11NaO 7); 368mg sodium acetate trihydrate, USP (C2H 3 NaO2.3H2O); 37mg potassium chloride, USP (KCl); and 30mg magnesium chloride, USP (MgCl2.6H2O). It is free of antimicrobial agents. The pH was adjusted with sodium hydroxide. The pH was 7.4 (6.5 to 8.0).

The cryopreservation solution may include Profreize ^TM . The cryopreservation solution may additionally or alternatively comprise a culture medium, such as αmem.

To facilitate freezing, cryoprotectants, such as Dimethylsulfoxide (DMSO), are typically added to the cryopreservation solution. Ideally, cryoprotectants should be non-toxic, 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) may be used as a surrogate or in combination with DMSO to reduce 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 cryopreserved solution comprises a Plasma-Lyte a (70%), DMSO (10%), HSA (25%) solution comprising 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 may be thawed and administered directly to a subject or added to another solution comprising, for example, hyaluronic acid. Alternatively, the cryopreserved composition may be thawed and the mesenchymal lineage precursor or stem cells resuspended in an alternative carrier prior to administration.

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

In one example, the composition comprises an effective amount or a therapeutically or prophylactically effective amount of mesenchymal lineage precursors or stem cells and/or progeny thereof and/or soluble factors derived therefrom. For example, the composition comprises about 1X 10 ⁵ Stem cells to about 1X 10 ⁹ Individual stem cells or about 1.25X10 ³ Stem cells to about 1.25X10 ⁷ Individual 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 condition to be treated.

Although the number of cells is provided in the composition, in one example, 50X 10 is administered ⁶ From 200X 10 ⁷ Individual cells. In other examples, 60X 10 is applied ⁶ From 200X 10 ⁶ Individual cells or 75X 10 ⁶ From 150X 10 ⁶ Individual cells. In one example, 75X 10 is applied ⁶ Individual cells. In the other of the examples of the present invention,application of 150X 10 ⁶ Individual cells.

In one example, the composition comprises greater than 5.00×10 ⁶ Each living cell/mL. In another example, the composition comprises greater than 5.50X10 ⁶ Each living cell/mL. In another example, the composition comprises greater than 6.00×10 ⁶ Each living cell/mL. In another example, the composition comprises greater than 6.50X10 ⁶ Each living cell/mL. In another example, the composition comprises greater than 6.68X10 ⁶ Each living cell/mL.

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

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

The compositions of the present disclosure may be administered systemically, e.g., by intravenous administration. In one example, the composition is administered endocardially.

Risk of cardiac death, myocardial infarction or stroke

In an example, the methods of the present disclosure relate to methods of assessing risk of one or more of cardiac death, myocardial infarction, or stroke based on CRP levels in a subject. For example, the methods of the present disclosure may relate to methods of assessing risk of cardiac death based on CRP levels in a subject. In an example, the present disclosure encompasses a method for determining an elevated risk of one or more of cardiac death, myocardial infarction, or stroke in a subject, the method comprising measuring CRP levels in a sample obtained from the subject, wherein an elevated CRP indicates an elevated risk of cardiac death, myocardial infarction, or stroke. In one example, the subject has progressive heart failure. For example, the subject may have NYHA class II progressive heart failure. Thus, in one example, the sample is obtained from a subject having NYHA class II progressive heart failure. In one example, the sample is a blood sample.

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

In one example, a composition comprising cells that induce neovascularization in a target tissue is administered to a subject assessed as having an elevated risk of cardiac death, myocardial infarction, or stroke. Accordingly, in one example, the present disclosure relates to a method of treating progressive heart failure, the method comprising: i) Selecting a subject having grade II heart failure and having elevated CRP levels according to the New York Heart Association (NYHA) classification scale; and ii) administering to the subject a composition according to the present disclosure. In one example, the CRP level of the subject is greater than or equal to 2mg/ml.

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

The disclosure of this document is incorporated herein in its entirety by claims of priority from AU2020904675 submitted at 15 of 12 in 2020, 2021900059 submitted at 12 of 1 in 2021, AU2021902941 submitted at 10 of 9 in 2021 and AU2021903365 submitted at 20 of 10 in 2021.

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

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

Examples

Composition and method for producing the same

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

Patient(s)

Baseline data

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The primary and secondary results were measured as follows:

1) Non-lethal Major Adverse Cardiac Events (MACEs):

heart failure MACE (recurrent decompensated heart failure event or high level arrhythmia);

ischemic MACE (heart attack or stroke)

2) Death due to heart disease

Cell therapy unexpectedly reduced the incidence of ischemic MACEs (MI, stroke) by 60% relative to control (n=537 patients; p=0.002; fig. 1). Figure 2 shows that the incidence of ischemic MACEs (MI, stroke) observed in NYHA class II and III is 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 a significant reduction in the incidence of ischemic MACEs (MI, stroke) was observed in both ischemic and non-ischemic patients. Cardiomyopathy patients are at risk of occlusive plaque production due to a sustained inflammatory process in these patients. Given the general reduction in ischemic MACEs observed in ischemic and non-ischemic cardiomyopathy patients, cell therapy appears to inhibit these problematic processes. Thus, the current data appears to support a general concept by which cell therapies 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 not in NYHA class III patients (figures 4 and 6). This result is surprising because it suggests that a certain threshold level of viable myocardium is required for reducing cardiac death using cell therapy. In other words, patients with grade III heart failure may progress too fast in the disease continuum to use cell therapy to increase their survival. These findings indicate that cell therapy will be particularly effective in NYHA class II patients. In view of the ability of administered cells to induce neovascularization in target tissue, the inventors' findings suggest the general concept of reducing cardiac death in patients rated below NYHA class III by administration of cell therapies. As a further support for this concept, the inventors noted that a reduction in cardiac death was observed in NYHA class II patients, regardless of the cause of cardiomyopathy, with a reduction in cardiac death observed in ischemic and non-ischemic NYHA class II patients (fig. 5).

3) Improved results

3-Point MACE

Subsequent analysis revealed that single injection cell therapy significantly reduced the risk of compounding the 3-point irreversible morbidity or mortality MACE in all 537 treated patients compared to the control. In subjects with CRP > 2mg/ml, the risk reduction is even more pronounced. The risk of 3-point MACE was reduced by 33% using time analysis to first event [ HR 0.667 ] The method comprises the steps of carrying out a first treatment on the surface of the 95% CI (0.472,0.941); p=0.021; FIG. 7A]And a 35% [ HR 0.646 ] decrease in recurrence event rate analysis normalized to follow-up time (i.e., number of events per 100 patient-years); 95% CI (0.466,0.895); p=0.009; FIG. 7B]. Plasma hsCRP levels>2mg/L or<The Kaplan-Meier curve (Kaplan-Meier) of this composite result for a 2mg/L patient is depicted in FIG. 7C. As shown in FIG. 7C, at CRP>In all treated patients at 2mg/L, cell therapy significantly reduced the risk of:

● Non-lethal MI and non-lethal stroke (fig. 7C 1); and

● Compounding of cardiac death or ischemic MACE (MI or stroke) (fig. 7C 2).

Ischemic MACE

In all 537 treated patients, single injection cell therapy reduced the risk of occurrence of irreversible morbidity (non-lethal MI or non-lethal stroke) by 65% [ HR 0.346 ] using time to first event analysis (TTFE) compared to control; 95% CI (0.180,0.664); p=0.001; fig. 8A ], and reduces the risk by 69% [ HR 0.306 with normalization of recurrent event rate; 95% CI (0.162,0.579); p <0.001; FIG. 8B ].

All treated patients were subjected to a pre-specified subgroup analysis based on the presence or absence of inflammation at the time of treatment. 301 patients with inflammation (CRP) >2 mg/L) reduced the risk of non-lethal MI or non-lethal stroke by 79% [ HR 0.206 with TTFE; 95% CI (0.070,0.611); p=0.004; FIG. 8A]And reducing the risk by 83% [ HR 0.170 with normalization of recurrence event rate; 95% CI (0.059,0.492); p=0.001; FIG. 8B]. In combination with the 3-point MACE analysis mentioned above, these data support the selection and treatment of a heart failure with active inflammation (preferably as by CRP>2 mg/L).

Cell therapy significantly reduced the compounding of cardiac death or ischemic MACE (MI or stroke) in all patients by 33% (fig. 9). Subsequent further analysis of the patient group revealed that cell therapy significantly reduced the combination of cardiac death or ischemic MACE (MI or stroke) in NYHA class II patients by 60% compared to the control, further supporting the rationale for selecting and treating patients with NYHA class II heart failure (fig. 9). It was also noted that over years of follow-up, cell therapy prevented progression of cardiac death in NYHA class II patients (fig. 10).

Cell therapy also unexpectedly reduced the risk of elevated CRP levels, particularly for NYHA class II patients with CRP levels > 2mg/L for cardiac death (FIG. 11) and for 3-point MACE (cardiac death/MI/stroke; FIG. 12). These benefits were not apparent in patients with baseline CRP <2 mg/indicating that cell therapy may be particularly beneficial in the presence of active inflammation.

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

Claims

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 alternatively, the process may be performed,

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:

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.