EP4264275A2 - Méthode de traitement d'une insuffisance cardiaque d'évolution progressive chez des sujets atteints d'insuffisance cardiaque de classe ii - Google Patents

Méthode de traitement d'une insuffisance cardiaque d'évolution progressive chez des sujets atteints d'insuffisance cardiaque de classe ii

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Publication number
EP4264275A2
EP4264275A2 EP21844465.1A EP21844465A EP4264275A2 EP 4264275 A2 EP4264275 A2 EP 4264275A2 EP 21844465 A EP21844465 A EP 21844465A EP 4264275 A2 EP4264275 A2 EP 4264275A2
Authority
EP
European Patent Office
Prior art keywords
subject
cells
heart failure
level
class
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21844465.1A
Other languages
German (de)
English (en)
Inventor
Silviu Itescu
Kenneth Borow
Jack Hayes
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mesoblast International SARL
Original Assignee
Mesoblast International SARL
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2020904675A external-priority patent/AU2020904675A0/en
Application filed by Mesoblast International SARL filed Critical Mesoblast International SARL
Priority claimed from PCT/US2021/063645 external-priority patent/WO2022132986A2/fr
Publication of EP4264275A2 publication Critical patent/EP4264275A2/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present disclosure relates to methods for treating and/or preventing progressive heart failure in subjects with earlier stages of heart failure. Such methods may be used for treating or preventing progressive heart failure in subjects with Class II heart failure according to the New York Heart Association (NYHA) classification scale.
  • NYHA New York Heart Association
  • MI Myocardial infarction
  • the present disclosure relates to a method for treating or preventing progressive heart failure in a subject, the method comprising administering to the subject a composition comprising cells.
  • the subject has Class II or Class III heart failure according to the New York Heart Association (NYHA) classification scale.
  • the subject may have less than Class III heart failure according to the New York Heart Association (NYHA) classification scale.
  • the subject has Class II heart failure according to the New York Heart Association (NYHA) classification scale.
  • 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 Class II heart failure according to the New York Heart Association (NYHA) classification scale.
  • NYHA New York Heart Association
  • the present disclosure relates to a method of reducing progression of heart failure in a subject, the method comprising administering to the subject a composition comprising cells, wherein the subject has Class II heart failure according to the New York Heart Association (NYHA) classification scale.
  • NYHA New York Heart Association
  • the present disclosure relates to a method of reducing cardiac death in a subject with Class II heart failure according to the New York Heart Association (NYHA) classification scale, the method comprising administering to the subject a composition comprising cells.
  • NYHA New York Heart Association
  • the present disclosure relates to a method of selecting heart failure patients for treatment with cell therapy, the method comprising i) assessing heart failure according to the New York Heart Association (NYHA) classification scale, and ii) selecting a subject having Class II heart failure according to NYHA.
  • the method further comprises administering a composition comprising cells.
  • the cells induce new blood vessel formation in target tissue.
  • the cells promote arteriogenesis.
  • the cells secrete factors that
  • 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 Class II heart failure according to the New York Heart Association (NYHA) classification scale, and wherein the cells induce new blood vessel formation in target tissue and/or secrete factors that protect at risk myocardium.
  • NYHA New York Heart Association
  • the cells are mesenchymal lineage precursor or stem cells (MLPSCs).
  • MLPSCs are STRO-1+.
  • MLPSCs mesenchymal stem cells (MSCs).
  • MLPSCs mesenchymal stem cells
  • the MLPSCs are allogeneic.
  • the cells are culture expanded.
  • the cells may be TNAP+ before they are culture expanded.
  • the cells have been cryopreserved.
  • the methods of the disclosure comprise the steps of: i) selecting a subject having Class II heart failure according to the New York Heart Association (NYHA) classification scale, and ii) administering to the subject a composition comprising cells which induce new blood vessel formation in target tissue.
  • NYHA New York Heart Association
  • administering the composition inhibits the subject’s progression to NYHA class III progressive heart failure.
  • the subject’s level of N-terminal pro-B-type natriuretic peptide is less than 2200 pg/ ml. In another example, the subject’s NT -proBNP is less than 2000 pg/ml, prior to administering the cells. In another example, the subject’s level of NT-proBNP is between 1000 pg/ml and 2000 pg/ ml prior to administering the cells.
  • the present inventors have also surprisingly found that cell therapy is particularly effective in subjects with earlier stages of progressive heart failure that have elevated C- reactive protein (CRP) levels.
  • CRP C- reactive protein
  • the subject’s CRP level is elevated.
  • the subject’s CRP level is >1 mg/L.
  • the subject’s CRP level is >1.5 mg/L.
  • the subject’s CRP level is >2 mg/L.
  • the subject’s CRP level is >2 mg/L.
  • the subject’s CRP level is between 1.5 and 5 mg/L.
  • the subject’s C-reactive protein (CRP) level is ⁇ 5 mg/L, preferably ⁇ 4 mg/L.
  • the subject’s CRP level is between 1 and 5 mg/L.
  • the subject’s CRP level is between 1.5 and 5 mg/L.
  • the subject has a LVEF of less than about 45%, preferably less than 40%. In another example, the subject has persistent left ventricular dysfunction.
  • the subject s heart failure results from an ischemic event.
  • the subject s heart failure results from a non-ischemic event.
  • the subject has a reduced risk of cardiac death after treatment.
  • the reduced risk is relative to risk of cardiac death in a subject with NYHA class III progressive heart failure.
  • the subject has a reduced risk of ischemic MACE (MI or stroke) after treatment.
  • a class III heart failure subject has a reduced risk of ischemic MACE (non-fatal MI or non-fatal stroke).
  • a class III heart failure subject has a reduced risk of cardiac death after treatment.
  • a class III heart failure subject has a reduced risk of ischemic MACE and cardiac death after treatment.
  • the class III heart failure subjects CRP level is >2 mg/L.
  • the composition is administered transendocardially and/or intravenously. In an example, the composition is administered transendocardially.
  • the present inventors also surprisingly identified that cell therapy reduced the risk of ischemic events in subjects with cardiomyopathy. Accordingly, in an example, the present disclosure also encompasses a method of reducing risk of an ischemic event in a subject, the method comprising administering to the subject a composition comprising cells.
  • the subject has a cardiomyopathy.
  • the ischemic event is formation of a cerebrovascular or cardiac occlusion.
  • the ischemic event is a stroke or myocardial infarction.
  • the subject has non-ischemic cardiomyopathy.
  • the cells are administered transendocardially.
  • the subject has Class II or Class III heart failure according to the New York Heart Association (NYHA) classification scale.
  • the subject has active inflammation.
  • the present inventors identified that administering a composition of the disclosure can treat a wider range of patients, such as patients with Class II or Class III heart failure.
  • 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 mesenchymal precursor lineage or stem cells, wherein the
  • SUBSTITUTE SHEET (RULE 26) subject has Class II or Class III heart failure according to the New York Heart Association (NYHA) classification scale and active inflammation.
  • the present disclosure relates to a method of reducing progression of heart failure in a subject, the method comprising administering to the subject a composition comprising mesenchymal precursor lineage or stem cells, wherein the subject has Class II or Class III heart failure according to the New York Heart Association (NYHA) classification scale and active inflammation.
  • the present disclosure relates to a method of reducing cardiac death in a subject with Class II or Class III heart failure according to the New York Heart Association (NYHA) classification scale active inflammation, the method comprising administering to the subject a composition comprising mesenchymal precursor lineage or stem cells.
  • the present disclosure relates to a method of selecting heart failure patients for treatment with cell therapy, the method comprising i) assessing CRP levels and heart failure according to the New York Heart Association (NYHA) classification scale, and ii) selecting a subject having Class II or Class III heart failure according to NYHA and active inflammation, preferably, wherein the method comprises administering a composition comprising mesenchymal precursor lineage or stem cells.
  • active inflammation is determined based on the subject’s CRP level.
  • the subject has Class II heart failure and “active inflammation”.
  • the active inflammation is characterised by a level of CRP >1.5 mg/L.
  • active inflammation is characterised by a level of CRP >2 mg/L.
  • the cells induce new blood vessel formation in target tissue.
  • the cells promote arteriogenesis.
  • the cells secrete factors that protect at risk myocardium.
  • the cells are MLPSCs.
  • the MLPSCs are STRO-1+.
  • the MLPSCs are mesenchymal stem cells (MSCs).
  • the MLPSCs are allogeneic.
  • the cells are culture expanded. In this example, the cells may be TNAP+ before they are culture expanded. In an example, the cells have been cryopreserved.
  • the subject’s level of N-terminal pro-B-type natriuretic peptide is between 1000 pg/ml and 2000 pg/ ml prior to administering the cells.
  • the subject’s C-reactive protein (CRP) level is elevated.
  • the subject’s CRP level is >1 mg/L.
  • SUBSTITUTE SHEET (RULE 26) level is >2 mg/L.
  • the subject’s CRP level is between 2 and 5 mg/L. In another example, the subject’s CRP level is between 3 and 5 mg/L.
  • the methods of the disclosure comprise administering between 1 x 10 7 and 2 x 10 8 cells.
  • the administered composition further comprises Plasma-Lyte A, dimethyl sulfoxide (DMSO), human serum albumin (HSA).
  • the administered composition further comprises Plasma-Lyte A (70%), DMSO (10%), HSA (25%) solution, the HSA solution comprising 5% HSA and 15% buffer.
  • the composition comprises greater than 6.68xl0 6 viable cells/mL.
  • the composition comprises human bone marrow-derived allogeneic mesenchymal precursor cells (MPCs) isolated from bone mononuclear cells with anti-STRO-3 antibodies, expanded ex vivo, and cryopreserved.
  • MPCs human bone marrow-derived allogeneic mesenchymal precursor cells
  • the ischemic events are one or more of myocardial infarction, stroke or cardiac death.
  • the method reduces risk of 3-point MACE.
  • the present inventors have also surprisingly identified that elevated CRP levels is associated with increased cardiac death risk, myocardial infarction or stroke. Accordingly, in an example, the present disclosure relates to a method for determining elevated risk of one or more of cardiac death, myocardial infarction or stroke in a subject, the method comprising measuring the level of CRP in a sample obtained from a subject, wherein elevated CRP indicates elevated risk of cardiac death, myocardial infarction or stroke.
  • the subject has progressive heart failure.
  • the subject’s heart failure is NYHA Class II heart failure.
  • a level of CRP >1 mg/L indicates elevated risk of cardiac death, myocardial infarction or stroke.
  • a level of CRP >1.5 mg/L indicates elevated risk of cardiac death, myocardial infarction or stroke.
  • a level of CRP >2 mg/L indicates elevated risk of cardiac death, myocardial infarction or stroke.
  • the method determines elevated risk of cardiac death.
  • Figure 1 Reduced Incidence of Ischemic MACE (MI, Stroke).
  • Figure 2 Reduced Incidence of Ischemic MACE (MI, Stroke); NYHA Class II and Class III.
  • Figure 5 Cardiac death in NYHA Class II patients; ischemic and non-ischemic.
  • Figure 6 Cardiac death in trial patients.
  • Figure 7 A) TTFE composite IMM MACE; B) Rate normalised composite IMM MACE; C) Curves for all treated patients with baseline CRP >2mg/L vs CRP ⁇ 2 mg/ml. Curves shown for Non-fatal MI or Non-fatal stoke; 3 -point TTFE composite IMM MACE for CV death or non-fatal MI or non-fatal stroke.
  • Figure 10 Cardiac Death in NYHA Class II patients - Multiple Years of followup.
  • cell culture techniques and assays utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J.
  • level and “amount” are used to define the amount of a particular substance in a sample from a subject or in a cell culture media (or sample therefrom). For example, a particular concentration, weight, percentage (e.g. v/v%) or ratio can be used to define the level of a particular substance in a sample.
  • the level is expressed in terms of how much of a particular marker is expressed by cells of the disclosure under culture conditions.
  • expression represents cell surface expression.
  • the level is expressed in terms of how much of a particular marker is release from cells described herein under culture conditions.
  • the sample is obtained from a patient or subject (e.g. a blood sample) and the level of a substance is measured in the sample to determine the level of the substance in the sample.
  • the level is expressed in pg/ml.
  • the level of NT- proBNP can be expressed in pg/ml.
  • the level is expressed in mg/L.
  • the level of CRP can be expressed in mg/L.
  • the level is expressed in pg per 10 6 cells.
  • the level of a particular marker in a cell culture medium is determined under culture conditions.
  • culture conditions is used to refer to cells growing in culture.
  • culture conditions refers to an actively dividing population of cells. Such cells may, in an example, in exponential growth phase.
  • the level of a particular marker can be determined by taking a sample of cell
  • SUBSTITUTE SHEET (RULE 26) culture media and measuring the level of marker in the sample.
  • the level of a particular marker can be determined by taking a sample of cells and measuring the level of the marker in the cell lysate.
  • secreted markers will be measured by sampling the culture media while markers expressed on the surface of the cell may be measured by assessing a sample of cell lysate.
  • the sample is taken when the cells are in exponential growth phase.
  • the sample is taken after at least two days in culture.
  • Culture expanding cells from a cryopreserved intermediate means thawing cells subject to cryogenic freezing and in vitro culturing under conditions suitable for growth of the cells.
  • the “level” or “amount” of a particular marker is determined after cells have been cryopreserved and then seeded back into culture.
  • the level is determined after a first cryopreservation of cells.
  • the level is determined after a second cryopreservation of cells.
  • cells may be culture expanded from a cryopreserved intermediate, cryopreserved a second time before being re-seeded in culture so that the level of a particular marker can be determined under culture conditions.
  • the terms “treating”, “treat”, “treatment”, “reducing progression” include administering a population of mesenchymal lineage stem or precursor cells and/or progeny thereof and/or soluble factors derived therefrom and/or extracellular vesicles derived therefrom to thereby reduce or eliminate at least one symptom of progressive heart failure or, in the context of reducing progression, delay development of the same.
  • subject refers to a human subject.
  • the subject can be an adult.
  • the subject can be a child.
  • the subject can be an adolescent.
  • Terms such as “subject”, “patient” or “individual” are terms that can, in context, be used interchangeably in the present disclosure.
  • Subjects in need of treatment include those already having progressive heart failure as well as those in which progressive heart failure is to be prevented, delayed or halted.
  • compositions of the disclosure comprise genetically unmodified mesenchymal precursor lineage or stem cells.
  • mesenchymal precursor lineage or stem cells As used herein, the term “genetically unmodified” 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 a mesenchymal
  • SUBSTITUTE SHEET (RULE 26) lineage precursor or stem cell transfected with a nucleic acid encoding a protein would be considered genetically modified.
  • sample refers to an extract from a subject in which CRP levels can be measured.
  • sample includes extracts and/or derivatives and/or fractions of the sample.
  • any biological material can be used as the above-mentioned sample so long as it can be collected from the subject and assayed to determine the level of CRP in the subject.
  • the sample is a blood sample.
  • the blood sample is obtained from a subject with NYHA Class II heart failure.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • Cardiomyopathy is a disease of the heart muscle that makes it harder for the heart to pump blood to the rest of the body. When the heart is unable to pump sufficiently to maintain blood flow to meet the needs of the body heart failure can occur. Cardiomyopathy can occur after an ischemic or non-ischemic event.
  • One cause of ischemic heart failure is systolic dysfunction following a myocardial infarction (MI) (e.g. heart attack).
  • MI myocardial infarction
  • a MI occurs when blood stops flowing properly to a part of the heart.
  • the lack of blood supply results in a localized area of myocardial necrosis referred to as an infarct or infarction.
  • Non-ischemic cardiomyopathy is not related to known coronary artery disease.
  • DCM dilated cardiomyopathy
  • the methods of the present disclosure are effective in a subset of patients with progressive heart failure.
  • these subjects are defined based on the New York Heart Association (NYHA) classification scale.
  • the subject’s progressive heart failure is less than Class III.
  • the subject has Class II heart failure.
  • the NYHA classification is assigned based on the subject’s symptoms.
  • the NYHA classification can be assigned based on the following Table:
  • the subject’s heart failure results from an ischemic event.
  • the subject’s heart failure results from a myocardial infarction (MI).
  • MI myocardial infarction
  • the subject can be a MI subject.
  • MI myocardial infarction
  • the term “myocardial infarction (MI) subject” is used to define subjects who have had a myocardial infarction.
  • the subject’s heart failure results from a non-ischemic cardiomyopathy.
  • the methods of the present disclosure can be used to treat progressive heart failure in a specific population of MI subjects.
  • Subjects in need of treatment include those already having progressive heart failure as well as those in which progressive heart failure is to be prevented, delayed or halted.
  • the subject has Class II or Class III progressive heart failure based on the NYHA.
  • the subject can have Class II progressive heart failure.
  • subjects treating according to the present disclosure have “active inflammation” as defined by elevated C-reactive protein levels.
  • active inflammation is characterised by CRP levels >2 mg/L.
  • C-reactive protein or "CRP” is an inflammatory mediator whose levels are raised under conditions of acute inflammatory recurrence and rapidly normalize once the inflammation subsides.
  • subjects treated according to the present disclosure have elevated risk of cardiac death. Cardiac death is death due to loss of cardiac function.
  • subjects treated according to the present disclosure have elevated CRP.
  • elevated CRP is used in the context of the present disclosure to refer to CRP levels that are increased relative to baseline CRP levels. In an example, CRP levels >1 mg/L are elevated. In another example, CRP levels >1.5 mg/L are elevated. In another example, CRP levels >2 mg/L are elevated.
  • subjects treated according to the present disclosure have an initial CRP level >2 mg/L.
  • the subject can have Class II or Class III heart failure and an initial CRP level >2 mg/L.
  • the subject can have Class II heart failure and an initial CRP level >2 mg/L.
  • subjects treated according to the present disclosure have an initial CRP level ⁇ 5 mg/L.
  • subjects have an initial CRP level ⁇ 4 mg/L.
  • subjects have an initial CRP level between 2 and 6 mg/L.
  • subjects have an initial CRP level between 3 and 6 mg/L.
  • subjects have an initial CRP level between 4 and 5 mg/L.
  • CRP levels can be measured in blood samples using an Enzyme-Linked Immunosorbent (ELISA) assay.
  • ELISA Enzyme-Linked Immunosorbent
  • a blood sample is obtained from a patient and then purified before being contacted with anti-CRP antibody. Extent of antibody binding is used to quantify the level of CRP in the blood sample (e.g. mg/L).
  • CRP is measured by a plasma high sensitivity CRP (hsCRP) ELISA assay.
  • B-type natriuretic peptide is a hormone produced by the heart.
  • N-terminal (NT)-pro hormone BNP (NT-proBNP) is a non-active prohormone that is released from the same molecule that produces BNP. Both BNP and NT-proBNP are released in response to changes in pressure inside the heart. These changes can be related to heart failure and other cardiac problems. Levels goes up when heart failure develops or gets worse, and levels goes down when heart failure is stable. Accordingly, BNP is an effective marker of heart failure progression. In an example, the subject’s level of NT- proBNP is less than 2200 pg/ ml prior to administering a composition of the disclosure.
  • the subject’s level of NT-proBNP is less than 2100 pg/ ml prior to administering a composition of the disclosure. In another example, the subject’s level of NT-proBNP is less than 2000 pg/ ml prior to administering a composition of the disclosure. In another example, the subject’s level of NT-proBNP is less than 1900 pg/ ml prior to administering a composition of the disclosure. In another example, the subject’s level of NT-proBNP is between 2200 pg/ ml and 1000 pg/ml prior to administering a composition of the disclosure.
  • the subject’s level of NT-proBNP is between 2200 pg/ ml and 1100 pg/ml prior to administering a composition of the disclosure. In another example, the subject’s level of NT-proBNP is between 2100
  • SUBSTITUTE SHEET (RULE 26) pg/ ml and 1200 pg/ml prior to administering a composition of the disclosure.
  • the subject’s level of NT -proBNP is between 2000 pg/ ml and 1500 pg/ml prior to administering a composition of the disclosure.
  • assays available for measuring NT-proBNP levels such as antibody based immunoassays, for example, an ELISA assay.
  • a blood sample is obtained from a patient and then purified before being contacted with anti- NT-proBNP antibody. Extent of antibody binding is used to quantify the level of NT-proBNP in the blood sample (e.g. pg/L).
  • the subject has had a heart failure hospitalisation event over the previous 12 months prior to administration of a composition disclosed herein.
  • the subject has had a heart failure hospitalisation event over the previous 9 months prior to administration of a composition disclosed herein.
  • the subject has had a heart failure hospitalisation event over the previous 6 to 12 months prior to administration of a composition disclosed herein.
  • the heart failure hospitalisation event is worsening signs and symptoms of heart failure.
  • the heart failure hospitalisation event is an ischemic event.
  • the heart failure hospitalisation is non-ischemic event.
  • the subject is able to walk at least 320 meters in 6 minutes prior to administering a composition of the disclosure. In another example, the subject is able to walk at least 330 meters in 6 minutes prior to administering a composition of the disclosure. In another example, the subject is able to walk at least 340 meters in 6 minutes prior to administering a composition of the disclosure. In another example, the subject is able to walk at least 350 meters in 6 minutes prior to administering a composition of the disclosure.
  • subjects can have persistent left ventricular dysfunction.
  • Left ventricular dysfunction is characterised by a decrease in myocardial contractility.
  • a reduction in the left ventricular ejection fraction (LVEF) results when myocardial contractility is decreased within the left ventricle.
  • LVEF provides one way of determining left ventricular dysfunction.
  • LVEF and LVESV can be measured by a number of methods known in the art such as echocardiogram, Single Photon Emission Computed Tomography (SPECT) or cardio magnetic resonance imaging (cMRI).
  • SPECT Single Photon Emission Computed Tomography
  • cMRI cardio magnetic resonance imaging
  • a subject having an LVEF of less than 45% has left ventricular dysfunction.
  • SUBSTITUTE SHEET (RULE 26) 42%, 41% has left ventricular dysfunction.
  • a subject with a LVEF of less than about 40% has left ventricular dysfunction.
  • a subject with a LVEF of less than about 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30% has left ventricular dysfunction.
  • the term “persistent left ventricular dysfunction” is used to define left ventricular dysfunction that persists over a period of time or series of measurements.
  • “persistent left ventricular dysfunction” can include left ventricular dysfunction that persists for between about 1 to about 14 days or longer.
  • the subject has a LVEF of less than 45%. In another example, the subject has a LVEF of less than 40%. In other examples, the subject has a LVEF of less than 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%.
  • the methods of the present disclosure relate to the treatment of the progressive decline in cardiac output characteristic of progressive heart failure. Accordingly, “treat” and “treatment”, in the context of the present disclosure refers to both therapeutic treatment and prophylactic or preventative measures.
  • treatment includes administering a composition of the disclosure.
  • methods of the present disclosure reduce or inhibit progression of progressive heart failure.
  • treatment inhibits the subject’s progression to NYHA class III progressive heart failure.
  • treatment reduces the risk of cardiac death.
  • the reduced risk of cardiac death is relative to risk of cardiac death in a subject with NYHA class III progressive heart failure.
  • the risk of ischemic MACE (MI or stroke) is reduced after treatment.
  • risk of ischemic MACE (MI or stroke) is reduced by at least 50% relative to baseline.
  • risk of ischemic MACE (MI or stroke) is reduced by at least 55% relative to baseline.
  • risk of ischemic MACE is reduced by at least 60% relative to baseline. In another example, risk of ischemic MACE (MI or stroke) is reduced by at least 65% relative to baseline. In another example, risk of ischemic MACE (MI or stroke) is reduced by at least 70% relative to baseline. In another example, risk of ischemic MACE (MI or stroke) is reduced by at least 50% to 70% relative to baseline.
  • 3-Point MACE Cardiac death/MI/stroke
  • SUBSTITUTE SHEET (RULE 26) to refer to is defined as a composite of cardiovascular death, nonfatal myocardial infarction and nonfatal stroke (Cardiac death/MI/stroke).
  • risk of 3 point MACE is reduced by at least 30% relative to baseline.
  • MACE is reduced by at least 40% relative to baseline.
  • MACE is reduced by at least 45% relative to baseline.
  • MACE is reduced by at least 50% relative to baseline.
  • MACE is reduced by at least 30% to 50% relative to baseline.
  • treatment increases patient survival.
  • treatment increases the probability of a subject surviving for at least 1000 days after initiation of treatment.
  • treatment increases the probability of a subject surviving for at least 2000 days after initiation of treatment.
  • the increased probability is determined relative to a subject that is not treated with a composition of the disclosure.
  • the increased probability is determined relative to a subject that has Class III heart failure.
  • treatment reduces the chance or risk of heart failure-related Major Adverse Cardiac Events (HF-MACE) defined as a composite of cardiac related death or resuscitated cardiac death, or non-fatal decompensated heart failure events.
  • HF-MACE heart failure-related Major Adverse Cardiac Events
  • the chance or risk of HF-MACE is reduced over at least 6 months, at least 12 months, at least 24 months, at least 36 months after administration of a composition disclosed herein.
  • treatment reduces the chance or risk of all-cause mortality.
  • the present disclosure relates to a method of reducing the risk or incidence of ischemic events in subjects, in particular subjects with cardiomyopathy.
  • the present disclosure relates to a method of reducing the risk or incidence of ischemic events in subjects with cardiomyopathy and elevated CRP.
  • risk or incidence is reduced relative to a subject that does not receive a composition of the disclosure.
  • risk or incidence can be reduced relative to an untreated subject.
  • the ischemic event is caused by the formation of an occlusion.
  • the occlusion is an arterial occlusion.
  • the ischemic event is formation of a cerebrovascular occlusion.
  • the ischemic event is a
  • SUBSTITUTE SHEET (RULE 26) formation of a cardiac occlusion.
  • the occlusion can form in the coronary artery.
  • Examples of ischemic events caused by formation of an occlusion include myocardial infarction and stroke. Accordingly, in an example, the present disclosure relates to methods of reducing the risk or incidence of myocardial infarction or stroke in a subject with cardiomyopathy.
  • the risk or incidence of ischemic events in subjects with cardiomyopathy is reduced by administering a cell therapy such as a composition of the disclosure.
  • the subject has non-ischemic cardiomyopathy.
  • cardiomyopathy may be caused by an enlarged left ventricle (dilated cardiomyopathy.
  • the cardiomyopathy is caused by a viral infection.
  • the subject has Class II or Class III heart failure according to the New York Heart Association (NYHA) classification scale.
  • NYHA New York Heart Association
  • the subject’s level of N-terminal pro-B-type natriuretic peptide is between 1000 pg/ml and 2000 pg/ ml prior to administering the cells.
  • the subject’s C-reactive protein (CRP) level is elevated.
  • the subject’s CRP level is >1.5 mg/L.
  • the subject’s CRP level is >2 mg/L.
  • the subject’s CRP level is between 1 and 5 mg/L.
  • the subject’s CRP level is between 3 and 5 mg/L.
  • the cells are administered transendocardially.
  • reduced risk is reduced 3 year risk. In another example, the reduced risk is reduced 5 year risk. In these examples, the risk of ischemic event is reduced over a defined period of time.
  • MPSC meenchymal lineage precursor or stem cell
  • MPSC mesenchymal lineage precursor or stem cell
  • a “mesenchymal lineage precursor cell” refers to a cell which can differentiate into a mesenchymal cell such as bone, cartilage, muscle and fat cells, and fibrous connective tissue.
  • mesenchymal lineage precursor or stem cells includes both parent cells and their undifferentiated progeny.
  • the term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal precursor cells, and their undifferentiated progeny.
  • Mesenchymal lineage precursor or stem cells can be autologous, allogeneic, xenogenic, syngenic or isogenic. Autologous cells are isolated from the same individual to which they will be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogenic cells are isolated from a donor of another species. Syngenic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.
  • the mesenchymal lineage precursor or stem cells are allogeneic.
  • the allogeneic mesenchymal lineage precursor or stem cells are culture expanded and cryopreserved.
  • Mesenchymal lineage precursor or stem cells reside primarily in the bone marrow, but have also shown to be present in diverse host tissues including, for example, cord blood and umbilical cord, adult peripheral blood, adipose tissue, trabecular bone and dental pulp. They are also found in skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph node, thymus, ligament, tendon, skeletal muscle, dermis, and periosteum; and are capable of differentiating into germ lines such as mesoderm and/or endoderm and/or ectoderm.
  • mesenchymal lineage precursor or stem cells are capable of differentiating into a large number of cell types including, but not limited to, adipose, osseous, cartilaginous, elastic, muscular, and fibrous connective tissues.
  • the specific lineage-commitment and differentiation pathway which these cells enter depends upon various influences from mechanical influences and/or endogenous bioactive factors, such as growth factors, cytokines, and/or local microenvironmental conditions established by host tissues.
  • enriched enriched or enrichment or variations thereof are used herein to describe a population of cells in which the proportion of one particular cell type or the proportion of a number of particular cell types is increased when compared with an untreated population of the cells (e.g., cells in their native environment).
  • a population enriched for mesenchymal lineage precursor or stem cells comprises at least about 0.1% or 0.5% or 1% or 2% or 5% or 10% or 15% or 20% or 25% or 30% or 50% or 75% mesenchymal lineage precursor or stem cells.
  • SUBSTITUTE SHEET (RULE 26) cells enriched for mesenchymal lineage precursor or stem cells” will be taken to provide explicit support for the term “population of cells comprising X% mesenchymal lineage precursor or stem cells”, wherein X% is a percentage as recited herein.
  • the mesenchymal lineage precursor or stem cells can, in some examples, form clonogenic colonies, e.g. CFU-F (fibroblasts) or a subset thereof (e.g., 50% or 60% or 70% or 70% or 90% or 95%) can have this activity.
  • the mesenchymal lineage precursor or stem cells are mesenchymal stem cells (MSCs).
  • the MSCs may be a homogeneous composition or may be a mixed cell population enriched in MSCs. Homogeneous MSC compositions may be obtained by culturing adherent marrow or periosteal cells, and the MSCs may be identified by specific cell surface markers which are identified with unique monoclonal antibodies. A method for obtaining a cell population enriched in MSCs is described, for example, in U.S. Patent No. 5,486,359. Alternative sources for MSCs include, but are not limited to, blood, skin, cord blood, muscle, fat, bone, and perichondrium.
  • the MSCs are allogeneic.
  • the MSCs are cryopreserved. In an example, the MSCs are culture expanded and cryopreserved.
  • the mesenchymal lineage precursor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs.
  • Isolated or enriched mesenchymal lineage precursor or stem cells can be expanded in vitro by culture.
  • Isolated or enriched mesenchymal lineage precursor or stem cells can be cryopreserved, thawed and subsequently expanded in vitro by culture.
  • isolated or enriched mesenchymal lineage precursor or stem cells are seeded at 50,000 viable cells/cm 2 in culture medium (serum free or serum- supplemented), for example, alpha minimum essential media (aMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, and allowed to adhere to the culture vessel overnight at 37°C, 20% O2.
  • culture medium serum free or serum- supplemented
  • aMEM alpha minimum essential media
  • FBS fetal bovine serum
  • glutamine fetal bovine serum
  • the culture medium is subsequently replaced and/or altered as required and the cells cultured for a further 68 to 72 hours at 37°C, 5% O2.
  • cultured mesenchymal lineage precursor or stem cells are phenotypically different to cells in vivo. For example, in one embodiment they express one or more of the following markers, CD44, NG2, DC 146 and CD140b. Cultured mesenchymal lineage precursor or stem cells are also biologically different to cells in vivo, having a higher rate of proliferation compared to the largely noncycling (quiescent) cells in vivo.
  • the population of cells is enriched from a cell preparation comprising STRO-1+ cells in a selectable form.
  • the term “selectable form” will be understood to mean that the cells express a marker (e.g., a cell surface marker) permitting selection of the STRO-1+ cells.
  • the marker can be STRO-1, but need not be.
  • cells e.g., mesenchymal precursor cells
  • TNAP STRO-2 and/or STRO-3
  • STRO-4 and/or VCAM-1 and/or CD146 and/or 3G5 also express STRO-1 (and can be STRO-lbright).
  • an indication that cells are STRO-1+ does not mean that the cells are selected solely by STRO-1 expression.
  • the cells are selected based on at least STRO-3 expression, e.g., they are STRO-3+ (TNAP+).
  • STRO-1+ cells can be selected from or isolated from or enriched from a large variety of sources. That said, in some examples, these terms provide support for selection from any tissue comprising STRO-1+ cells (e.g., mesenchymal precursor cells) or vascularized tissue or tissue comprising pericytes (e.g., STRO-1+ pericytes) or any one or more of the tissues recited herein.
  • tissue comprising STRO-1+ cells e.g., mesenchymal precursor cells
  • pericytes e.g., STRO-1+ pericytes
  • the cells used in the present disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1 +, THY-1+, STRO-2+, STRO-4+ (HSP-90p), CD45+, CD146+, 3G5+ or any combination thereof.
  • TNAP tissue non-specific alkaline phosphatase
  • TNAP SUBSTITUTE SHEET (RULE 26) (LAP), the bone isoform (BAP) and the kidney isoform (KAP).
  • LAP bone isoform
  • BAP bone isoform
  • KAP kidney isoform
  • the TNAP is BAP.
  • TNAP as used herein refers to a molecule which can bind the STRO-3 antibody produced by the hybridoma cell line deposited with ATCC on 19 December 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.
  • the STRO-1+ cells are capable of giving rise to clonogenic CFU-F.
  • a significant proportion of the STRO-1+ cells are capable of differentiation into at least two different germ lines.
  • the lineages to which the STRO-1+ cells may be committed include bone precursor cells; hepatocyte progenitors, which are multipotent for bile duct epithelial cells and hepatocytes; neural restricted cells, which can generate glial cell precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; precursors for cardiac muscle and cardiomyocytes, glucose-responsive insulin secreting pancreatic beta cell lines.
  • lineages include, but are not limited to, odontoblasts, dentin-producing cells and chondrocytes, and precursor cells of the following: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal duct epithelial cells, smooth and skeletal muscle cells, testicular progenitors, vascular endothelial cells, tendon, ligament, cartilage, adipocyte, fibroblast, marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericyte, vascular, epithelial, glial, neuronal, astrocyte and oligodendrocyte cells.
  • mesenchymal lineage precursor or stem cells are obtained from a single donor, or multiple donors where the donor samples or mesenchymal lineage precursor or stem cells are subsequently pooled and then culture expanded.
  • Mesenchymal lineage precursor or stem cells encompassed by the present disclosure may also be cryopreserved prior to administration to a subject.
  • mesenchymal lineage precursor or stem cells are culture expanded and cryopreserved prior to administration to a subject.
  • the present disclosure encompasses mesenchymal lineage precursor or stem cells as well as progeny thereof, soluble factors derived therefrom, and/or extracellular vesicles isolated therefrom.
  • the present disclosure encompasses mesenchymal lineage precursor or stem cells as well as extracellular vesicles isolated therefrom. For example, it is possible to culture expand
  • SUBSTITUTE SHEET (RULE 26) mesenchymal precursor lineage or stem cells of the disclosure for a period of time and under conditions suitable for secretion of extracellular vesicles into the cell culture medium. Secreted extracellular vesicles can subsequently be obtained from the culture medium for use in therapy.
  • extracellular vesicles refers to lipid particles naturally released from cells and ranging in size from about 30 nm to as a large as 10 microns, although typically they are less than 200 nm in size. They can contain proteins, nucleic acids, lipids, metabolites, or organelles from the releasing cells (e.g., mesenchymal stem cells; STRO-1 + cells).
  • exosomes refers to a type of extracellular vesicle generally ranging in size from about 30 nm to about 150 nm and originating in the endosomal compartment of mammalian cells from which they are trafficked to the cell membrane and released. They may contain nucleic acids (e.g., RNA; microRNAs), proteins, lipids, and metabolites and function in intercellular communication by being secreted from one cell and taken up by other cells to deliver their cargo.
  • nucleic acids e.g., RNA; microRNAs
  • proteins proteins
  • lipids and metabolites and function in intercellular communication by being secreted from one cell and taken up by other cells to deliver their cargo.
  • compositions of the disclosure comprise cells that induce new blood vessel formation in target tissue.
  • the target tissue is the heart.
  • the cells secrete factors that protect at risk or damaged myocardium.
  • at risk or damaged myocardium has been subject to a lack of blood flow resulting from an ischemic event.
  • the cells secrete factors that reduce apoptosis in cardiomyocytes.
  • mesenchymal lineage precursor or stem cells are culture expanded. “Culture expanded” mesenchymal lineage precursor or stem cells media are distinguished from freshly isolated cells in that they have been cultured in cell culture medium and passaged (i.e. sub -cultured). In an example, culture expanded mesenchymal lineage precursor or stem cells are culture expanded for about 4 - 10 passages. In an example, mesenchymal lineage precursor or stem cells are culture expanded for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 passages. For example, mesenchymal lineage precursor or stem cells can be culture expanded for at least 5 passages. In an example, mesenchymal lineage precursor or stem cells can be culture expanded for at least 5 - 10 passages. In an example, mesenchymal lineage precursor or stem cells can be culture expanded.
  • SUBSTITUTE SHEET expanded for at least 5 - 8 passages.
  • mesenchymal lineage precursor or stem cells can be culture expanded for at least 5 - 7 passages.
  • mesenchymal lineage precursor or stem cells can be culture expanded for more than 10 passages.
  • mesenchymal lineage precursor or stem cells can be culture expanded for more than 7 passages.
  • stem cells may be culture expanded before being cryopreserved to provide an intermediate cryopreserved MLPSC population.
  • compositions of the present disclosure are produced by culturing cells from an intermediate cryopreserved MLPSC population or, put another way, a cryopreserved intermediate.
  • compositions of the disclosure comprise mesenchymal lineage precursor or stem cells that are culture expanded from a cryopreserved intermediate.
  • the cells culture expanded from a cryopreserved intermediate are culture expanded for at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 passages.
  • mesenchymal lineage precursor or stem cells can be culture expanded for at least 5 passages.
  • mesenchymal lineage precursor or stem cells can be culture expanded for at least 5 - 10 passages.
  • mesenchymal lineage precursor or stem cells can be culture expanded for at least 5 - 8 passages.
  • mesenchymal lineage precursor or stem cells can be culture expanded for at least 5 - 7 passages. In an example, mesenchymal lineage precursor or stem cells can be culture expanded for more than 10 passages. In another example, mesenchymal lineage precursor or stem cells can be culture expanded for more than 7 passages.
  • mesenchymal lineage precursor or stem cells culture expanded from a cryopreserved intermediate can be culture expanded in medium free of animal proteins.
  • mesenchymal lineage precursor or stem cells culture expanded from a cryopreserved intermediate can be culture expanded in xeno-free medium.
  • mesenchymal lineage precursor or stem cells culture expanded from a cryopreserved intermediate can be culture expanded in medium that is fetal bovine serum free.
  • mesenchymal lineage precursor or stem cells can be obtained from a single donor, or multiple donors where the donor samples or mesenchymal lineage precursor or stem cells are subsequently pooled and then culture expanded.
  • the culture expansion process comprises:
  • SUBSTITUTE SHEET (RULE 26) i. expanding by passage expansion the number of viable cells to provide a preparation of at least about 1 billion of the viable cells, wherein the passage expansion comprises establishing a primary culture of isolated mesenchymal lineage precursor or stem cells and then serially establishing a first non-primary (Pl) culture of isolated mesenchymal lineage precursor or stem cells from the previous culture; ii. expanding by passage expansion the Pl culture of isolated mesenchymal lineage precursor or stem cells to a second non-primary (P2) culture of mesenchymal lineage precursor or stem cells; and, iii.
  • the expanded mesenchymal lineage precursor or stem cell preparation has an antigen profile and an activity profile comprising: i. less than about 0.75% CD45+ cells; ii. at least about 95% CD105+ cells; iii. at least about 95% CD166+ cells.
  • the expanded mesenchymal lineage precursor or stem cell preparation is capable of inhibiting IL2-Ra expression by CD3/CD28-activated PBMCs by at least about 30% relative to a control.
  • culture expanded mesenchymal lineage precursor or stem cells are culture expanded for about 4 - 10 passages, wherein the mesenchymal lineage precursor or stem cells have been cryopreserved after at least 2 or 3 passages before being further culture expanded.
  • mesenchymal lineage precursor or stem cells are culture expanded for at least 1, at least 2, at least 3, at least 4, at least 5 passages, cryopreserved and then further culture expanded for at least 1, at least 2, at least 3, at least 4, at least 5 passages before being cultured according to the methods of the disclosure.
  • the process of mesenchymal lineage precursor or stem cell isolation and ex vivo expansion can be performed using any equipment and cell handing methods known in the art.
  • Various culture expansion embodiments of the present disclosure employ steps that require manipulation of cells, for example, steps of seeding, feeding, dissociating an
  • SUBSTITUTE SHEET adherent culture, or washing. Any step of manipulating cells has the potential to insult the cells. Although mesenchymal lineage precursor or stem cells can generally withstand a certain amount of insult during preparation, cells are preferably manipulated by handling procedures and/or equipment that adequately performs the given step(s) while minimizing insult to the cells.
  • mesenchymal lineage precursor or stem cells are washed in an apparatus that includes a cell source bag, a wash solution bag, a recirculation wash bag, a spinning membrane filter having inlet and outlet ports, a filtrate bag, a mixing zone, an end product bag for the washed cells, and appropriate tubing, for example, as described in US 6,251,295, which is hereby incorporated by reference.
  • a mesenchymal lineage precursor or stem cell composition cultured according to the present disclosure is 95% homogeneous with respect to being CD 105 positive and CD 166 positive and being CD45 negative. In an example, this homogeneity persists through ex vivo expansion; i.e. though multiple population doublings.
  • mesenchymal lineage precursor or stem cells of the disclosure are culture expanded in 3D culture.
  • mesenchymal lineage precursor or stem cells of the disclosure can be culture expanded in a bioreactor.
  • mesenchymal lineage precursor or stem cells of the disclosure are initially culture expanded in 2D culture prior to being further expanded in 3D culture.
  • mesenchymal lineage precursor or stem cells of the disclosure are culture expanded from a master cell bank.
  • mesenchymal lineage precursor or stem cells of the disclosure are culture expanded from a master cell bank in 2D culture before seeding in 3D culture.
  • mesenchymal lineage precursor or stem cells of the disclosure are culture expanded from a master cell bank in 2D culture for at least 3 days before seeding in 3D culture in a bioreactor.
  • mesenchymal lineage precursor or stem cells of the disclosure are culture expanded from a master cell bank in 2D culture for at least 4 days before seeding in 3D culture in a bioreactor.
  • mesenchymal lineage precursor or stem cells of the disclosure are culture expanded from a master cell bank in 2D culture for between 3 and 5 days before seeding in 3D culture in a bioreactor.
  • 2D culture can be performed in a cell factory.
  • Various cell factory products are available commercially (e.g. Thermofisher, Sigma).
  • Mesenchymal lineage precursor or stem cells disclosed herein can be culture expanded in various suitable growth mediums.
  • the term “medium” or “media” as used in the context of the present disclosure includes the components of the environment surrounding the cells.
  • the media contributes to and/or provides the conditions suitable to allow cells to grow.
  • Media may be solid, liquid, gaseous or a mixture of phases and materials.
  • Media can include liquid growth media as well as liquid media that do not sustain cell growth.
  • Media also include gelatinous media such as agar, agarose, gelatin and collagen matrices.
  • Exemplary gaseous media include the gaseous phase that cells growing on a petri dish or other solid or semisolid support are exposed to.
  • the cell culture media used for culture expansion contains all essential amino acids and may also contain non-essential amino acids.
  • amino acids are classified into essential amino acids (Thr, Met, Vai, Leu, He, Phe, Trp, Lys, His) and non- essential amino acids (Gly, Ala, Ser, Cys, Gin, Asn, Asp, Tyr, Arg, Pro).
  • the basal medium must be appropriate for the cell line of interest. For example, it may be necessary to increase the level of glucose (or other energy source) in the basal medium, or to add glucose (or other energy source) during the course of culture, if this energy source is found to be depleted and to thus limit growth. In an example, dissolved oxygen (DO) levels can also be controlled.
  • glucose or other energy source
  • DO dissolved oxygen
  • the cell culture medium contains human derived additives.
  • human serum and human platelet cell lysate can be added to the cell culture media.
  • the cell culture medium contains only human derived additives.
  • the cell culture media is xeno-free.
  • the culture medium is free of animal proteins.
  • cell culture medium used in the methods of the disclosure is free of animal components.
  • the culture medium comprises serum.
  • the culture medium is fetal bovine serum free culture medium comprising growth factors that promote mesenchymal lineage precursor or stem cell proliferation.
  • the culture medium is serum free stem cell culture medium.
  • the cell culture medium comprises: a basal medium; platelet derived growth factor (PDGF); fibroblast growth factor 2 (FGF2).
  • PDGF platelet derived growth factor
  • FGF2 fibroblast growth factor 2
  • the culture medium comprises platelet derived growth factor (PDGF) and fibroblast growth factor 2 (FGF2), wherein the level of FGF2 is less than about 6 ng/ml.
  • the FGF2 level may be less than about 5 ng/ml, less than about 4 ng/ml, less than about 3 ng/ml, less than about 2 ng/ml, less than about 1 ng/ml.
  • the FGF2 level is less than about 0.9 ng/ml, less than about 0.8 ng/ml, less than about 0.7 ng/ml, less than about 0.6 ng/ml, less than about 0.5 ng/ml, less than about 0.4 ng/ml, less than about 0.3 ng/ml, less than about 0.2 ng/ml.
  • the level of FGF2 is between about 1 pg/ml and 100 pg/ml. In another example, the level of FGF2 is between about 5 pg/ml and 80 pg/ml.
  • the PDGF is PDGF-BB.
  • the level of PDGF-BB is between about 1 ng/ml and 150 ng/ml. In another example, the level of PDGF-BB is between about 7.5 ng/ml and 120 ng/ml. In another example, the level of PDGF-BB is between about 15 ng/ml and 60 ng/ml. In another example, the level of PDGF-BB is at least about 10 ng/ml. In another example, the level of PDGF-BB is at least about 15 ng/ml. In another example, the level of PDGF-BB is at least about 20 ng/ml.
  • the level of PDGF-BB is at least about 21 ng/ml. In another example, the level of PDGF-BB is at least about 22 ng/ml. In another example, the level of PDGF-BB is at least about 23 ng/ml. In another example, the level of PDGF-BB is at least about 24 ng/ml. In another example, the level of PDGF-BB is at least about 25 ng/ml.
  • the PDGF is PDGF-AB.
  • the level of PDGF - AB is between about 1 ng/ml and 150 ng/ml. In another example, the level of PDGF-AB is between about 7.5 ng/ml and 120 ng/ml. In another example, the level of PDGF-AB is between about 15 ng/ml and 60 ng/ml. In another example, the level of PDGF-AB is at least about 10 ng/ml. In another example, the level of PDGF-AB is at least about 15 ng/ml. In another example, the level of PDGF-AB is at least about 20 ng/ml. In another example, the level of PDGF-AB is at least about 21 ng/ml. In another example, the level of PDGF-AB is at least about 22 ng/ml. In another example, the level of PDGF-AB is at
  • SUBSTITUTE SHEET (RULE 26) least about 23 ng/ml.
  • the level of PDGF-AB is at least about 24 ng/ml.
  • the level of PDGF-AB is at least about 25 ng/ml.
  • the culture medium further comprising EGF.
  • EGF is a growth factor that stimulates cell proliferation by binding to its receptor EGFR.
  • the method of the present disclosure comprises culturing a population of stem cells in a fetal bovine serum free cell culture medium further comprising EGF.
  • the level of EGF is between about 0.1 and 7 ng/ml.
  • the level of EGF can be at least about 5 ng/ml.
  • the level of EGF is between about 0.2 ng/ml and 3.2 ng/ml. In another example, the level of EGF is between about 0.4 ng/ml and 1.6 ng/ml. In another example, the level of EGF is between about 0.2 ng/ml. In another example, the level of EGF is at least about 0.3 ng/ml. In another example, the level of EGF is at least about 0.4 ng/ml. In another example, the level of EGF is at least about 0.5 ng/ml. In another example, the level of EGF is at least about 0.6 ng/ml. In another example, the level of EGF is at least about 0.7 ng/ml. In another example, the level of EGF is at least about 0.8 ng/ml. In another example, the level of EGF is at least about 0.9 ng/ml. In another example, the level of EGF is at least about 1.0 ng/ml.
  • basal medium such as Alpha MEM or StemSpanTM can be supplemented with the referenced quantity of growth factor.
  • the culture medium comprises Alpha MEM or StemSpanTM supplemented with 32 ng/ml PDGF-BB, 0.8 ng/ml EGF and 0.02 ng/ml FGF.
  • the cell culture media can be supplemented with one or more stimulatory factors selected from the group consisting of epidermal growth factor (EGF), la, 25- dihydroxyvitamin D3 (1,25D), tumor necrosis factor a (TNF- a), interleukin -ip (IL-ip) and stromal derived factor la (SDF-la).
  • EGF epidermal growth factor
  • TNF- a tumor necrosis factor a
  • IL-ip interleukin -ip
  • SDF-la stromal derived factor la
  • cells may also be cultured in the presence of at least one cytokine in an amount adequate to support growth of the cells.
  • cells can be cultured in the presence of heparin or a derivative thereof.
  • the cell culture medium may contain about 50ng/ml of heparin.
  • the cell culture medium contains about 60ng/ml of heparin, about 70ng/ml of heparin, about 80ng/ml of heparin, about 90ng/ml of heparin, about lOOng/ml of heparin, about HOng/ml of heparin, about HOng/ml of heparin, about
  • SUBSTITUTE SHEET 120ng/ml of heparin, about 130ng/ml of heparin, about 140ng/ml of heparin, about 150ng/ml of heparin or a derivative thereof.
  • the heparin derivative is a sulphate).
  • Various forms of heparin sulphate are known in the art and include heparin sulphate 2 (HS2).
  • HS2 can be derived from various sources including for example, the liver of male and/or female mammals.
  • an exemplary heparin sulphate includes male liver heparin sulphate (MML HS) and female liver heparin sulphate (FML HS).
  • the cell culture medium of the present disclosure promotes stem cell proliferation while maintaining stem cells in an undifferentiated state.
  • Stem cells are considered to be undifferentiated when they have not committed to a specific differentiation lineage.
  • stem cells display morphological characteristics that distinguish them from differentiated cells.
  • undifferentiated stem cells express genes that may be used as markers to detect differentiation status.
  • the polypeptide products may also be used as markers to detect differentiation status. Accordingly, one of skill in the art could readily determine whether the methods of the present disclosure maintain stem cells in an undifferentiated state using routine morphological, genetic and/or proteomic analysis.
  • the mesenchymal lineage precursor or stem cells disclosed herein may be altered in such a way that upon administration, lysis of the cell is inhibited.
  • Alteration of an antigen can induce immunological non-responsiveness or tolerance, thereby preventing the induction of the effector phases of an immune response (e.g., cytotoxic T cell generation, antibody production etc.) which are ultimately responsible for rejection of foreign cells in a normal immune response.
  • Antigens that can be altered to achieve this goal include, for example, MHC class I antigens, MHC class II antigens, LFA-3 and ICAM-1.
  • the mesenchymal lineage precursor or stem cells may also be genetically modified to express proteins of importance for the differentiation and/or maintenance of striated skeletal muscle cells.
  • Exemplary proteins include growth factors (TGF-P, insulin-like growth factor 1 (IGF-1), FGF), myogenic factors (e.g. myoD, myogenin, myogenic factor 5 (Myf5), myogenic regulatory factor (MRF)), transcription factors (e.g. GATA-4), cytokines (e.g. cardiotropin- 1), members of the neuregulin family (e.g. neuregulin 1, 2 and 3) and homeobox genes (e.g. Csx, tinman and NKx family).
  • TGF-P insulin-like growth factor 1
  • FGF insulin-like growth factor 1
  • myogenic factors e.g. myoD, myogenin, myogenic factor 5 (Myf5), myogenic regulatory factor (MRF)
  • transcription factors e.g. GATA-4
  • cytokines e.g. cardio
  • Mesenchymal lineage or stem cells disclosed herein can be culture expanded from a cryopreserved intermediate to produce a preparation containing at least one therapeutic dose.
  • compositions of the disclosure comprise between 10 x 10 6 cells and 35 x 10 6 cells. In another example, the composition comprises between 20 x 10 6 cells and 30 x 10 6 cells. In other examples, the composition comprises at least 100 x 10 6 cells. In another example, the composition comprises between 50 x 10 6 cells and 500 x 10 6 cells. In other examples, compositions of the disclosure comprise 150 million cells.
  • compositions of the disclose comprise a pharmaceutically acceptable carrier and/or excipient.
  • carrier and/or excipient refer to compositions of matter that are conventionally used in the art to facilitate the storage, administration, and/or the biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980).
  • a carrier may also reduce any undesirable side effects of the active compound.
  • a suitable carrier is, for example, stable, e.g., incapable of reacting with other ingredients in the carrier. In one example, the carrier does not produce significant local or systemic adverse effect in recipients at the dosages and concentrations employed for treatment.
  • Suitable carriers for the present disclosure include those conventionally used, e.g., water, saline, aqueous dextrose, lactose, Ringer's solution, a buffered solution, hyaluronan and glycols are exemplary liquid carriers, particularly (when isotonic) for solutions.
  • Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.
  • a carrier is a media composition, e.g., in which a cell is grown or suspended. Such a media composition does not induce any adverse effects in a subject to whom it is administered. Exemplary carriers and excipients do not adversely affect the viability of a cell and/or the ability of a cell to treat or prevent disease.
  • the carrier or excipient provides a buffering activity to maintain the cells and/or soluble factors at a suitable pH to thereby exert a biological activity
  • the carrier or excipient is phosphate buffered saline (PBS).
  • PBS represents an attractive
  • composition of the disclosure may be produced as a liquid for direct application to the blood stream or into a tissue or a region surrounding or adjacent to a tissue, e.g., by injection.
  • compositions of the disclosure may be cryopreserved.
  • Cryopreservation of mesenchymal lineage precursor or stem cells can be carried out using slow-rate cooling methods or 'fast' freezing protocols known in the art.
  • the method of cry opreservation maintains similar phenotypes, cell surface markers and growth rates of cryopreserved cells in comparison with unfrozen cells.
  • the cryopreserved composition may comprise a cryopreservation solution.
  • the pH of the cry opreservation solution is typically 6.5 to 8, preferably 7.4.
  • the cyropreservation solution may comprise a sterile, non-pyrogenic isotonic solution such as, for example, PlasmaLyte ATM.
  • PlasmaLyte ATM contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H1 lNaO7); 368 mg of sodium acetate trihydrate, USP (C2H3NaO2»3H2O); 37 mg of potassium chloride, USP (KC1); and 30 mg of magnesium chloride, USP (MgC12»6H2O). It contains no antimicrobial agents.
  • the pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).
  • the cryopreservation solution may comprise ProfreezeTM.
  • the cryopreservation solution may additionally or alternatively comprise culture medium, for example, aMEM.
  • a cryoprotectant such as, for example, dimethylsulfoxide (DMSO)
  • DMSO dimethylsulfoxide
  • the cryoprotectant should be nontoxic for cells and patients, nonantigenic, chemically inert, provide high survival rate after thawing and allow transplantation without washing.
  • the most commonly used cryoprotector, DMSO shows some cytotoxicity .
  • Hydroxylethyl starch (HES) may be used as a substitute or in combination with DMSO to reduce cytotoxicity of the cry opreservation solution.
  • the cry opreservation solution may comprise one or more of DMSO, hydroxy ethyl starch, human serum components and other protein bulking agents.
  • the cryopreserved solution comprises Plasma-Lyte A (70%), DMSO (10%), HSA (25%) solution, the HSA solution comprising 5% HSA and 15% buffer.
  • the cry opreservation solution may further comprise one or more of methycellulose, polyvinyl pyrrolidone (PVP) and trehalose.
  • PVP polyvinyl pyrrolidone
  • cryopreserved composition may be thawed and administered directly to the subject or added to another solution, for example, comprising hyaluronic acid.
  • the cryopreserved composition may be thawed and the mesenchymal lineage precursor or stem cells resuspended in an alternate carrier prior to administration.
  • compositions described herein may be administered alone or as admixtures with other cells.
  • the cells of different types may be admixed with a composition of the disclosure immediately or shortly prior to administration, or they may be co-cultured together for a period of time prior to administration.
  • the composition comprises an effective amount or a therapeutically or prophylactically effective amount of mesenchymal lineage precursor or stem cells and/or progeny thereof and/or soluble factor derived therefrom.
  • the composition comprises about IxlO 5 stem cells to about IxlO 9 stem cells or about 1.25xl0 3 stem cells to about 1.25xl0 7 stem cells/kg (80 kg subject). The exact amount of cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the subject, and the extent and severity of the disorder being treated.
  • x 10 6 to 200 x 10 7 cells are administered.
  • 60 x 10 6 to 200 x 10 6 cells or 75 x 10 6 to 150 x 10 6 cells are administered.
  • 75 x 10 6 cells are administered.
  • 150 x 10 6 cells are administered.
  • the composition comprises greater than 5.00xl0 6 viable cells/mL. In another example, the composition comprises greater than 5.50xl0 6 viable cells/mL. In another example, the composition comprises greater than 6.00xl0 6 viable cells/mL. In another example, the composition comprises greater than 6.50xl0 6 viable cells/mL. In another example, the composition comprises greater than 6.68xl0 6 viable cells/mL.
  • 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.
  • composition may optionally be packaged in a suitable container with written instructions for a desired purpose.
  • compositions of the disclosure may be administered systemically, such as, for example, by intravenous administration.
  • compositions are administered transendocardially.
  • 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 the level of CRP in a subject.
  • the methods of the present disclosure can relate to methods of assessing risk of cardiac death based on the level of CRP in a subject.
  • the present disclosure encompasses a method for determining elevated risk of one or more of cardiac death, myocardial infarction or stroke in a subject, the method comprising measuring the level of CRP in a sample obtained from a subject, wherein elevated CRP indicates elevated risk of cardiac death, myocardial infarction or stroke.
  • the subject has progressive heart failure.
  • the subject can have NYHA Class II progressive heart failure.
  • the sample is obtained from a subject with NYHA Class II progressive heart failure.
  • the sample is a blood sample.
  • a level of CRP >1 mg/L indicates elevated risk of cardiac death, myocardial infarction or stroke. In an example, a level of CRP >1.5 mg/L indicates elevated risk of cardiac death, myocardial infarction or stroke. In an example, a level of CRP >2 mg/L indicates elevated risk of cardiac death, myocardial infarction or stroke. In an example, a level of CRP between >2 and 5 mg/L indicates elevated risk of cardiac death, myocardial infarction or stroke. In an example, the level of CRP is measured after an ischemic event. In an example, the ischemic event is a myocardial infarction.
  • a composition comprising cells which induce new blood vessel formation in target tissue is administered to subjects assessed as having elevated risk of cardiac death, myocardial infarction or stroke.
  • the present disclosure relates to a method of treating progressive heart failure, the method comprising i) selecting a subject having Class II heart failure according to the New York Heart Association (NYHA) classification scale and elevated CRP level, and ii) administering to the subject a composition according to the present disclosure.
  • the subject s CRP level is >2 mg/ml.
  • the composition consists of human bone marrow-derived allogeneic MPCs isolated from bone mononuclear cells with anti-STRO-3 antibodies, expanded ex vivo, and cryopreserved.
  • MACE Non-Fatal Major Adverse Cardiac Events
  • Heart Failure MACE recurrent decompensated heart failure events or high-grade arrhythmias
  • Figure 2 shows the significantly reduced incidence of ischemic MACE (MI, Stroke) observed relative to controls in NYHA Class II & III. These data suggest that cell therapy can reduce risk of ischemic events in patients with cardiomyopathy.
  • Figure 3 shows that the significantly reduced incidence of ischemic MACE (MI, Stroke) was observed in both ischemic and non-ischemic patients. Cardiomyopathy patients are at risk of occlusive plaque development due to ongoing inflammatory processes in these patients.

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Abstract

La présente invention concerne des méthodes de traitement et/ou de prévention d'une insuffisance cardiaque d'évolution progressive chez des sujets présentant des stades précoces d'insuffisance cardiaque. Une telle méthode peut être utilisée pour traiter ou prévenir une insuffisance cardiaque d'évolution progressive chez des sujets atteints d'une insuffisance cardiaque de classe II selon l'échelle de classification de la New York Heart Association (NYHA).
EP21844465.1A 2020-12-15 2021-12-15 Méthode de traitement d'une insuffisance cardiaque d'évolution progressive chez des sujets atteints d'insuffisance cardiaque de classe ii Pending EP4264275A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
AU2020904675A AU2020904675A0 (en) 2020-12-15 Method of treating progressive heart failure in subjects with Class II heart failure
AU2021900059A AU2021900059A0 (en) 2021-01-12 Method of treating progressive heart failure in subjects with Class II heart failure
AU2021902941A AU2021902941A0 (en) 2021-09-10 Method of treating progressive heart failure in subjects with Class II heart failure
AU2021903365A AU2021903365A0 (en) 2021-10-20 Method of treating progressive heart failure in subjects with Class II heart failure
PCT/US2021/063645 WO2022132986A2 (fr) 2020-12-15 2021-12-15 Méthode de traitement d'une insuffisance cardiaque d'évolution progressive chez des sujets atteints d'insuffisance cardiaque de classe ii

Publications (1)

Publication Number Publication Date
EP4264275A2 true EP4264275A2 (fr) 2023-10-25

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EP21844465.1A Pending EP4264275A2 (fr) 2020-12-15 2021-12-15 Méthode de traitement d'une insuffisance cardiaque d'évolution progressive chez des sujets atteints d'insuffisance cardiaque de classe ii

Country Status (1)

Country Link
EP (1) EP4264275A2 (fr)

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