Biomarker for stem cell activity

Abstract

The disclosure relates to methods for evaluating the level of therapeutic stem cells comprising measuring or determining the level of a biomarker in a sample from a subject. The disclosure also relates to associated methods of prognosis and treatment.

Description

Biomarker for stem cell activityField of the invention

[0001] The disclosure relates to a biomarker for monitoring stem cell activity, determining the efficacy of stem cell treatment, and related methods of treatment.Cross reference to earlier application

[0002] This application claims priority to Australian provisional application no. 2024903213, the entire contents of which are hereby incorporated by reference in their entirety.Background of the invention

[0003] Chronic diseases are a leading cause of morbidity and mortality globally, with significant associated healthcare costs and impacts on quality of life. Despite advances in medical treatments, many chronic conditions remain challenging to manage effectively.

[0004] Stem cell therapies have emerged as a promising novel approach for the treatment of various chronic diseases. Stem cells possess the ability to migrate, modulate the immune system, secrete trophic factors to promote tissue repair and regeneration, differentiate into mature functioning cells, and potentially reverse or ameliorate disease progression.

[0005] There exists a need for improved or alternative methods for monitoring the levels and persistence of therapeutic stem cells after administration to assess the efficacy of stem cell-based treatments and determine whether additional doses may be required to maintain therapeutic effects.

[0006] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.1006117846Summary of the invention

[0007] In one aspect, there is provided a method for evaluating the level of therapeutic stem cells in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof,- determining that the subject has a high level of therapeutic stem cells when the level of the biomarker in the sample from the subject is the same or greater than a reference level of the biomarker; or- determining that the subject has a low level of therapeutic stem cells when the level of the biomarker in the sample from the subject is less than a reference level of the biomarker.

[0008] In another aspect, there is provided a method for monitoring a response to stem cell therapy in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining a level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;- comparing the level of the biomarker to a reference level of the biomarker;- determining the subject has a low probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is less than the reference level;1006117846- determining the subject has a high probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is the same or greater than the reference level; thereby monitoring the response to the stem cell therapy in the subject.

[0009] In another aspect, there is provided a method of determining the efficacy of stem cell treatment in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and- determining that the treatment has likely not been successful when the level of the biomarker in the subject is less than the reference level; or- determining that the treatment has likely been successful when the level of the biomarker in the subject is the same or greater than the reference level.

[0010] In another aspect, there is provided a method of determining the suitability of a subject to receive a further dose of stem cell therapy, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and1006117846- determining that the subject is suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is less than the reference level; or- determining that the subject is not suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is the same or greater than the reference level.

[0011] In another aspect, there is provided a method of treating a disease or condition responsive to stem cell therapy in a subject, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker;- selecting the subject for treatment only when the level of the biomarker in the subject is less than the reference level; and- administering stem cell therapy to the subject selected for treatment; thereby treating the subject having the disease or condition responsive to stem cell therapy.

[0012] In some embodiments wherein the biomarker is a polypeptide comprising a fragment of SEQ ID NO: 1 or 2, the polypeptide or fragment thereof comprises or consists of SEQ ID NO: 3.

[0013] In some embodiments wherein the biomarker is a polypeptide comprising a fragment of SEQ ID NO: 1 or 2, the polypeptide or fragment thereof does not comprise or consist of SEQ ID NO: 3.

[0014] In one aspect, there is provided a method for evaluating the level of therapeutic stem cells in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:1006117846- providing a subject who has received or is receiving stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3DKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLWD ET (SEQ ID NO: 3),- determining that the subject has a high level of therapeutic stem cells when the level of the biomarker in the sample from the subject is the same or greater than a reference level of the biomarker; or- determining that the subject has a low level of therapeutic stem cells when the level of the biomarker in the sample from the subject is less than a reference level of the biomarker.

[0015] In another aspect, there is provided a method for monitoring a response to stem cell therapy in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining a level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;- comparing the level of the biomarker to a reference level of the biomarker;- determining the subject has a low probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is less than the reference level;- determining the subject has a high probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is the same or greater than the reference level; thereby monitoring the response to the stem cell therapy in the subject.1006117846

[0016] In another aspect, there is provided a method of determining the efficacy of stem cell treatment in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and- determining that the treatment has likely not been successful when the level of the biomarker in the subject is less than the reference level; or- determining that the treatment has likely been successful when the level of the biomarker in the subject is the same or greater than the reference level.

[0017] In another aspect, there is provided a method of determining the suitability of a subject to receive a further dose of stem cell therapy, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and- determining that the subject is suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is less than the reference level; or- determining that the subject is not suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is the same or greater than the reference level.1006117846

[0018] In another aspect, there is provided a method of treating a disease or condition responsive to stem cell therapy in a subject, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker;- selecting the subject for treatment only when the level of the biomarker in the subject is less than the reference level; and- administering stem cell therapy to the subject selected for treatment; thereby treating the subject having the disease or condition responsive to stem cell therapy.

[0019] In preferred embodiments, the sample is blood, plasma, serum, saliva, or urine.

[0020] In preferred embodiments, the reference level of the biomarker is representative of one or more subjects who have not received stem cell therapy. Preferably, the reference level of the biomarker is from a sample of the subject prior to treatment.

[0021] In preferred embodiments, the reference level of the biomarker is about 3.3 lU / mL, 3.4 lU / mL, 3.5 lU / mL, 3.6 lU / mL, 3.7 lU / mL, 3.8 lU / mL, 3.9 lU / mL or about 4 ILI / mL, more preferably about 3.3 ILI / mL.

[0022] In preferred embodiments, the stem cell treatment comprises administering stem cells derived from amniotic membrane, amniotic fluid, placenta, umbilical cord, umbilical cord blood, embryo, and / or pluripotent stem cells. Most preferably the stem cell treatment comprises administering stem cells derived from umbilical cord or umbilical cord blood.

[0023] In preferred embodiments, the stem cell treatment does not comprise administering peripheral blood stem cells, adipose stem cells or bone marrow stem cells.1006117846

[0024] The disease or condition may include any disease or condition that is responsive to stem cell treatment including but not limited to neurodegenerative diseases, spinal cord injury, autoimmune diseases, inflammatory diseases, cardiovascular diseases, musculoskeletal degenerative conditions, pulmonary degenerative diseases, and endocrine disorders. Preferably the disease or condition responsive to stem cell treatment is cardiovascular disease, more preferably acute myocardial infarction (AMI).

[0025] Further aspects of the present disclosure and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0026] Fig 1. Levels of a biomarker A508in subjects receiving stem cell therapy.

[0027] Fig 2. Left ventricular ejection fraction (LVEF) of subjects receiving stem cell therapy at base line and at one year after treatment. Left bar in”Baseline” or “1-year after treatment”: Treat; right bar in “Baseline” or “1 -year after treatment”: Control.Sequence information

[0028] Table 1 - biomarker sequences1006117846Detailed description of the embodiments

[0029] Reference will now be made in detail to certain embodiments of the disclosure. While the disclosure will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the disclosure to those embodiments. On the contrary, the disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present disclosure as defined by the claims.

[0030] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. The present disclosure is in no way limited to the methods and materials described. It will be understood that the disclosure disclosed and defined in this specification extends to all alternative combinations of two or more of the individual1006117846features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the disclosure.

[0031] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0032] Current diagnostic methods for monitoring the levels and persistence of therapeutic stem cells after administration include: (i) imaging techniques such as magnetic imaging resonance (MRI), positron emission tomography (PET) and singlephoton emission computed tomography (SPECT); (ii) molecular and cellular assays such as flow cytometry, polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and measuring the level of extracellular vesicles; (iii) histological analysis; and (iv) functional assessments measured by clinical outcomes. The methods for monitoring the levels and persistence of therapeutic stem cells after administration also include other functional measurements, including cardiac echo, laboratory blood tests, physiological improvements (e.g., skin turnover time that may be measured with disappearing time of ink stains on the skin, fine wrinkle amount, skin tone), neurological performance (e.g., recall speed and response time), and mobility (ability to rise or walk, and gait).

[0033] The specific biomarkers used in protein based assays to measure stem cells can vary depending on the type of stem cells and the target disease. Cell surface markers include: CD34 a marker for hematopoietic stem and progenitor cells, CD105 a marker for mesenchymal stem cells, SSEA-4 a marker for pluripotent stem cells. Secreted factors include: VEGF secreted by stem cells to promote angiogenesis, HGF secreted by stem cells to stimulate tissue regeneration, TGF-[3 secreted by stem cells to modulate the immune response, GM-CSF secreted by immune cells, MIF secreted by pituitary gland, adrenal cortex or innate immune cells. Stem cell specific proteins include: Oct-4 a transcription factor essential for maintaining stem cell pluripotency, Nanog a homeobox transcription factor involved in stem cell self-renewal. Other proteins includes: PDGF-BB, SCF, SDF1 , MMP2, ICAM1 , adiponectin NT-proBNP, and hs-cTnT. Exosomal markers include: CD63, CD81 , and CD9 tetraspanin proteins present on the surface of stem cell derived exosomes.1006117846

[0034] The present inventor has identified that (i) a stem cell biomarker comprising a polypeptide having the amino acid sequence of SEQ ID NO: 1 or 2, herein also referred to as A508; or a fragment thereof comprising, or not comprising, SEQ ID NO: 3, is correlated with the level and persistence of therapeutic stem cells after administration, (ii) the biomarker levels are correlated with clinical outcomes, and / or (iii) that the biomarker levels may be used to assess whether additional doses of stem cell treatment may be required to maintain therapeutic effects.

[0035] A508 offers one or more of the following advantages as a biomarker for therapeutic stem cells over other known stem cell biomarkers.Specificity in regenerative processes

[0036] A508 demonstrates higher specificity to therapeutic stem cells actively involved in regeneration, unlike broad markers like VEGF and PDGF-BB. While VEGF is critical for angiogenesis and PDGF-BB supports tissue repair, these markers are not exclusive to stem cells, and their levels can rise due to general tissue damage or repair processes. A508, by contrast, is directly associated with stem cell-driven tissue regeneration, making it a more targeted biomarker for evaluating therapeutic efficacy. MMP-2 is more indicative of extracellular matrix remodeling at specific stages of wound healing whereas A508 reflects ongoing regenerative activity, making it more useful for monitoring direct therapeutic effects and allowing clinicians to make more informed decisions about the need for additional doses of stem cells.Correlation with therapeutic outcomes

[0037] A508 shows a stronger and more direct correlation with therapeutic success in regenerative treatments. Traditional markers like SCF (Stem Cell Factor) are essential for stem cell recruitment and proliferation, but A508 provides a more dynamic assessment of the stem cells' actual regenerative activity in tissues. This correlation allows A508 to offer better predictive value in clinical outcomes than the other biomarkers, which tend to reflect early-stage recruitment or non-specific regenerative processes.1006117846Enhanced sensitivity to regenerative effects

[0038] Compared to markers like SDF-1 , which is involved in stem cell homing, A508 is more sensitive in capturing the extent of tissue regeneration driven by stem cells. SDF-1 is useful for tracking how stem cells localize to injury sites but does not offer a continuous assessment of regenerative capacity. A508's sensitivity ensures that even subtle changes in stem cell activity are detected, which is critical for personalized treatment plans.Minimal interference from non-specific regenerative factors

[0039] Markers such as VEGF and PDGF-BB can be elevated due to general tissue damage or vascularization, leading to non-specific results. A508, on the other hand, is not influenced by unrelated regenerative processes, providing a more accurate and specific measure of stem cell activity in promoting regeneration.Correlation with clinical outcomes

[0040] A508 shows a stronger and more direct correlation with clinical outcomes in patients receiving stem cell therapy. Its levels have been reliably associated with improvements in tissue regeneration and overall recovery, making it a superior biomarker for predicting therapeutic efficacy. Other markers, such as VEGF or HGF, are linked to general regenerative processes but do not consistently reflect specific clinical improvements related to stem cell treatments.

[0041] The inventor has exemplified the disclosure for stem cell treatment of acute myocardial infarction (AMI). However, a person skilled in the art will understand that the disclosure is applicable to any disease or condition that is responsive to stem cell therapy that utilises stem cells that express the biomarker.Definitions

[0042] As used herein, except where the context reguires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.1006117846

[0043] The transitional phrase “consisting of” excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consisting of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0044] The transitional phrase “consisting essentially of” is used to define a composition, process or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed disclosure. The term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”.

[0045] Unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0046] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0047] Also, the indefinite articles “a” and “an” preceding an element or component of the disclosure are intended to be non-restrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0048] As used herein, with reference to numbers in a range of numerals, the terms “about”, “approximately” and “substantially” are understood to refer to the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1 % to + 1 % of the referenced number, most preferably -0.1 % to +0.1 % of the referenced number. Moreover, with reference to numerical ranges, these terms should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed1006117846as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, from 8 to 10, and so forth.

[0049] As used herein, "biomarker" refers to a measurable substance, detection of which typically indicates levels of therapeutic stem cells. A "biomarker" may indicate a change in expression or state of the measurable substance that correlates with the prognosis of response to stem cell therapy. A "biomarker" may be a protein or peptide. A "biomarker" may be measured in a bodily fluid such as plasma, blood or serum, saliva or urine. As used herein, "biomarker" refers to a polypeptide comprising the sequence of SEQ ID NO: 1 or 2; or a fragment thereof comprising, or not comprising, SEQ ID NO: 3.

[0050] The terms “treat”, "treatment" or "treating" of a subject includes delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the sign or symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term "treating" refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of signs or symptoms or making the injury, pathology or condition more tolerable to the individual; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating.

[0051] In particularly preferred embodiments, the methods of the present disclosure can be to prevent or reduce the severity, or inhibit or minimise progression, of a sign or symptom of a disease or condition as described herein. As such, the methods of the present disclosure have utility as treatments as well as prophylaxes.

[0052] As used herein, the term “prevention” or “preventing” are intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a disease or disorder (i.e. , causing at least one of the clinical symptoms of the disease not to develop in a patient that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease). Biological and physiological parameters for identifying such patients are known in the art.1006117846

[0053] As used herein, “favourable response” to stem cell therapy is generally understood as meaning a “therapeutic response” including an improvement in one or more measurable or observable signs or symptoms associated with the disease or condition responsive to stem cell therapy.

[0054] Herein, the term “subject”, “patient" and “individual” can be used interchangeably with each other. The term “individual” or “patient” refers to an animal that is treatable by the compound and / or method, respectively, including but not limited to, for example, dogs, cats, horses, sheep, pigs, cows, and the like, as well as human, non-human primates. Unless otherwise specified, the “subject” or “patient” may include both male and female genders. Further, it also includes a subject or patient, preferably a human, suitable for receiving treatment and / or method of the present disclosure.

[0055] As used herein, "acute myocardial infarction" or "AMI" refers to the interruption of blood supply to a part of the heart, causing restriction in blood supply ("ischaemia"), lack of oxygen, and cell death ("necrosis"), and is a type of acute coronary syndrome ("ACS"). This may result in damage or death of heart muscle tissue (myocardium).Thus, "myocardial necrosis" refers to the death of heart cells. AMI includes ST elevation myocardial infarction (STEMI), diagnosed by elevation of the ST segment of the electrocardiogram, and non-ST elevation myocardial infarction (non-STEMI), diagnosed by absence of such electrocardiographic changes.

[0056] As used herein, "Left ventricular ejection fraction" or "LVEF" is defined as the fraction of the left ventricular end-diastolic volume (LVEDV) that is ejected with each contraction (heartbeat); that is, stroke volume divided by LVEDV. In some embodiments, LVEF is calculated as the volume of blood ejected from the left ventricle during systole (end of contraction) divided by the maximum volume of blood in the left ventricle at the end of diastole (relaxation), expressed as a percentage of the total volume at the end of diastole. LVEF may be expressed as a percentage.

[0057] As used herein, "left ventricular end-diastolic volume" or "LVEDV" is defined as the volume of blood within the left ventricle immediately before contraction.1006117846

[0058] As used herein, "stroke volume" is defined as the difference between LVEDV and left ventricular end-systolic volume (LVESV) and refers to the volume of blood ejected from the left ventricle with each contraction (heartbeat).

[0059] As used herein, "left ventricular end-systolic volume" or "LVESV" is defined as the volume of blood remaining within the left ventricle at the end of contraction.Biomarker for monitoring stem cell activity

[0060] The inventor surprisingly found that in subjects who have received or are receiving stem cell treatment, the determination of the level of a biomarker comprising the amino acid sequence of SEQ ID NO: 1 or 2 or a fragment thereof, correlates with the level and persistence of therapeutic stem cells in the subject. The amino acid sequence of the biomarker may comprise or consist of, or may not comprise or consist of, the amino acid sequence of SEQ ID NO: 3.

[0061] The inventor surprisingly found that in subjects who have received or are receiving stem cell treatment, the determination of the level of a biomarker comprising the amino acid sequence of:DKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLWD ET (SEQ ID NO: 3), correlates with the level and persistence of therapeutic stem cells in the subject.

[0062] Subjects exhibit an increased level of the biomarker following stem cell treatment compared to baseline or reference levels of the biomarker in untreated subjects. Over time, the biomarker level decreases towards baseline I reference levels. The level of the biomarker may correlate with the activity of stem cells, preferably wherein the activity of stem cells includes their ability to survive, grow, proliferate and / or differentiate. Accordingly, the level of the biomarker may correlate with the level of therapeutic stem cells in the subject and prognose the subject’s response to the stem cell treatment. Therapeutic stem cells refer to stem cells that are capable of providing a therapeutic effect.1006117846

[0063] Accordingly, in one aspect, there is provided a method for evaluating the level of therapeutic stem cells in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;- determining that the subject has a high level of therapeutic stem cells when the level of the biomarker in the sample from the subject is the same or greater than a reference level of the biomarker; or- determining that the subject has a low level of therapeutic stem cells when the level of the biomarker in the sample from the subject is less than a reference level of the biomarker.

[0064] In another aspect, there is provided a method for monitoring a response to stem cell therapy in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining a level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;- comparing the level of the biomarker to a reference level of the biomarker;- determining the subject has a low probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is less than the reference level;- determining the subject has a high probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is the same or greater than the reference level;1006117846thereby monitoring the response to the stem cell therapy in the subject.

[0065] In another aspect, there is provided a method of determining the efficacy of stem cell treatment in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and- determining that the treatment has likely not been successful when the level of the biomarker in the subject is less than the reference level; or- determining that the treatment has likely been successful when the level of the biomarker in the subject is the same or greater than the reference level.

[0066] In some embodiments wherein the biomarker is a polypeptide comprising a fragment of SEQ ID NO: 1 or 2, the polypeptide or fragment thereof comprises or consists of SEQ ID NO: 3.

[0067] In some embodiments wherein the biomarker is a polypeptide comprising a fragment of SEQ ID NO: 1 or 2, the polypeptide or fragment thereof does not comprise or consist of SEQ ID NO: 3.

[0068] In another aspect, there is provided a method for evaluating the level of therapeutic stem cells in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;1006117846- determining that the subject has a high level of therapeutic stem cells when the level of the biomarker in the sample from the subject is the same or greater than a reference level of the biomarker; or- determining that the subject has a low level of therapeutic stem cells when the level of the biomarker in the sample from the subject is less than a reference level of the biomarker.

[0069] In another aspect, there is provided a method for monitoring a response to stem cell therapy in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining a level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;- comparing the level of the biomarker to a reference level of the biomarker;- determining the subject has a low probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is less than the reference level;- determining the subject has a high probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is the same or greater than the reference level; thereby monitoring the response to the stem cell therapy in the subject.

[0070] In another aspect, there is provided a method of determining the efficacy of stem cell treatment in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;1006117846- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and- determining that the treatment has likely not been successful when the level of the biomarker in the subject is less than the reference level; or- determining that the treatment has likely been successful when the level of the biomarker in the subject is the same or greater than the reference level.Biomarker

[0071] As used herein "biomarker" refers to a polypeptide comprising the sequence of SEQ ID NO: 1 or 2 or a fragment thereof. The fragment may comprise or consist of, or may not comprise or consist of, SEQ ID NO: 3.

[0072] One skilled in the art understands that the sequence of the biomarker may differ between or within different subjects due to normal genetic diversity (variation) and / or mutations. Also, native sequences may differ between or within different subjects due to post-transcriptional or post-translational modifications. Any such variants or isoforms of the biomarker are intended herein.

[0073] The reference herein to biomarker may also encompass variants thereof. The term “variant” of the biomarker refers to proteins, polypeptides or peptides the sequence (e.g. amino acid sequence) of which is substantially identical to the sequence of said recited biomarker, e.g. at least about 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. Preferably, a variant may display such degrees of identity to a recited protein, polypeptide or peptide when the whole sequence of the recited protein, polypeptide or peptide is queried in a sequence alignment (i.e. overall sequence identity).

[0074] The reference herein to biomarker may also encompass proteins, polypeptides or peptides comprising the biomarker sequence. For example, where the biomarker sequence is a peptide fragment, the biomarker may encompass the full length protein thereof.1006117846

[0075] The reference herein to biomarker may also encompass modified forms of said biomarker or a fragment thereof, such as bearing post-expression modifications including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation or oxidation of methionine to methionine sulphoxide or methionine sulphone.

[0076] The reference herein to biomarker also encompasses fragments thereof. Hence, the reference herein to measuring (or measuring the level of) the biormarker may encompass measuring the mature and / or processed soluble / secreted form(s) thereof (e.g. plasma circulating form(s)) and / or measuring one or more fragments thereof.

[0077] In one embodiment, the biomarker is full length. In another embodiment, the biomarker is a fragment thereof. A fragment may comprise up to the entire length of the reference sequence, minus at least one amino acid residue. For example, a fragment may comprise from about 5 to about 600 contiguous amino acid residues of a reference polypeptide. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous amino acid residues of a reference polypeptide. In some embodiments, a fragment may comprise at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 250, 500, or 600 contiguous amino acid residues of a reference polypeptide; prefereably, the fragment may comprise at most 608, 450, or 53 contiguous amino acid residues of the reference polypeptide.Biomarker reference level

[0078] One skilled in the art will appreciate that the magnitude of the biomarker level may vary depending on the characteristics of the assay used to measure the biomarker. Nevertheless, one skilled in the art will also appreciate that, provided the appropriate control samples are analysed, the appropriate reference level of the biomarker can be determined.

[0079] The determination of levels of the biomarker over a reference value (e.g., reference concentration) can be utilised to monitor the activity of administered stem cells and / or prognose response to a stem cell therapy. Preferably, the reference level has been predetermined or is determined from a cohort of healthy subjects or a cohort1006117846of subjects who are not receiving a stem cell therapy. Preferably, the cohort of subjects does not have, or has not been identified as having, stem cell cancer.

[0080] A level of the biomarker is greater than a reference level when it exceeds the reference level by 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more. A biomarker level that exceeds the reference level by 50% is equivalent to a 1 .5- fold greater level of the biomarker, and biomarker level that exceeds the reference level by 100% is equivalent to a 2-fold greater level of the biomarker, and so on. Accordingly, a biomarker level is greater than a reference level when it is 2-fold, 2.5-fold, 3-fold, 3.5- fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9- fold, 9.5-fold, 10-fold or more than the reference level. In another embodiment, a biomarker level is greater than a reference level when it exceeds the reference level and the difference is statistically significant as determined by methods known to the person skilled in the art. Alternatively, subjects who have a biomarker level above about the 50thpercentile, 60thpercentile, 70thpercentile, 80thpercentile, 90thpercentile, 95thpercentile, 96thpercentile, 97thpercentile, 98thpercentile, 99thpercentile, or higher, as compared with an appropriate matched control population, may be identified as having high activity of administered stem cells and / or being likely to respond to the stem cell therapy.

[0081] In some embodiments, the reference level of the biomarker is about 3.3 ILI / mL, 3.4 lU / mL, 3.5 lU / mL, 3.6 lU / mL, 3.7 lU / mL, 3.8 lU / mL, 3.9 lU / mL, or 4 lU / mL. In a preferred embodiment, the reference level of the biomarker is about 3.3 ILI / mL. It will be understood that 1 ILI / mL of the biomarker corresponds to 1 .21 ng / mL of the biomarker. For example, the aforementioned level of the biomarker corresponds to about 3.99 ng / mL, 4.11 ng / mL, 4.24 ng / mL, 4.36 ng / mL, 4.48 ng / mL, 4.60 ng / mL, 4.72 ng / mL, or 4.84 ng / mL.

[0082] One skilled in the art will understand that a biomarker level the same or greater than about 3.3 lU / mL, about 3.5 lU / mL, about 4 lU / mL, about 4.5 lU / mL, about 5 lU / mL or about 5.5 lU / mL correlates with therapeutic levels of administered stem cells and / or high likelihood of responding to stem cell therapy. Preferably, a biomarker level the1006117846same or greater than about 4.5 lU / mL correlates with a therapeutic level of administered stem cells and / or high likelihood of responding to stem cell therapy.

[0083] One skilled in the art will understand that a biomarker level of less than about 3.5 lU / mL, 3.4 lU / mL, 3.3 lU / mL, 3.2 lU / mL, 3.1 lU / mL, 3 lU / mL, about 2.5 lU / mL, about 2 ILI / mL, about 1 .5 ILI / mL, about 1 ILI / mL, about 0.5 ILI / mL correlates with non- therapeutic levels of administered stem cells and / or a low likelihood of responding to stem cell therapy. Preferably, a biomarker level of less than about 3.3 ILI / mL correlates with a non-therapeutic level of administered stem cells and / or a low likelihood of responding to stem cell therapy.

[0084] In some aspects, the reference level stratifies the subjects into one of two subgroups with the following rule: if the test or subject level is less than the reference level, then the subject is assigned to the group with a lower activity of administered stem cells and / or a lower probability of responding to a stem cell therapy; if the test or subject level is greater than or equal to the reference level, then the subject is assigned to the group with a higher activity of administered stem cells and / or a higher probability of responding to a stem cell therapy.

[0085] Alternatively, there may be multiple reference levels that stratify a subject into low, moderate, or high activity of administered stem cells, and / or a low, moderate or high likelihood of responding to a stem cell therapy.

[0086] The reference level may be selected according to any method known in the art. In particular embodiments, the reference level may be a predetermined value.Alternatively, the reference level may be determined during the course of the assay. For example, samples from healthy subjects or subjects not receiving a stem cell therapy may be run concurrently with the test samples and a reference value determined therefrom. As a further alternative, test samples from a mixed population may be analysed, and the reference value is determined based on the distribution of the results, e.g., using statistical methods as known in the art.

[0087] It will be appreciated that the reference level of the biomarker for monitoring the activity of administered stem cells and / or prognosing the response to a stem cell therapy may be derived from the same individual for whom the stem cell therapy is1006117846administered, but at a different time-point, for example, hours, days, weeks or years earlier. As such, the reference level may also include the level of the biomarker before the individual was administered a stem cell therapy. Such a reference level thereby forms a baseline or basal level of the biomarker, against which the test samples may be compared.

[0088] As used herein, the baseline or basal biomarker level may refer to the level of the biomarker before stem cell therapy is administered to a subject. The sample for baseline biomarker level may be taken at any time before the stem cell therapy is administered. For example, within about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 150 minutes, 180 minutes or 210 minutes or more, within about 0.5 days, 1 .0 day, 1 .5 days, 2.0 days, 2.5 days, 3.0 days, 3.5 days, 4.0 days, 4.5 days, 5.0 days, 5.5 days, 6.0 days, 6.5 days or more, or within about 0.5 months, 1.0 month, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.0 months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 6.5 months or more before administration of the stem cell therapy.

[0089] The reference level data set will contain sufficient representative data to enable the skilled person to determine, with an appropriate degree of certainty, whether there is a high or low activity of administered stem cells and / or a high or low likelihood that an individual is responding to a stem cell therapy.

[0090] It is contemplated that over time, additional studies will generate new and additional information about the reference level of the biomarker in a healthy subject and / or in a subject not receiving a stem cell therapy. The additional information may increase the accuracy, reliability, and confidence of the reference level, and accordingly increase the accuracy, reliability, and confidence of the determinations and recommendations realised by carrying out the methods. Thus, newly generated or revised reference levels of the biomarker may be used in accordance with the methods described herein. Thus, one skilled in the art will appreciate that, reference levels may change over time, and provided that the appropriate control samples are analysed, the appropriate reference levels can be determined.1006117846

[0091] The reference data set is not limited to data pertaining to the biomarkers identified in accordance with the disclosure. For example, the data set may also contain information pertaining to other biomarkers or information which may be used to supplement the information on the activity of administered stem cells and / or the likelihood of a subject responding to a stem cell therapy, and assist the skilled practitioner in making a determination and / or deciding on the further actions to be taken.

[0092] In certain embodiments, the methods of the present disclosure include a comparison of the level of the biomarker from the individual who is a selection candidate for a given treatment. In some embodiments, that comparison may arise from an examination of the level of the biomarker, and direct visual comparison against the same amounts listed in a reference database, such as, for example, an Excel spreadsheet.Sample

[0093] In a preferred embodiment, the method is performed in vitro on a serum (or plasma or blood), saliva and / or urine sample. In other words, any method of the disclosure may be an in vitro method. For example, a step of determining, measuring or analysing in any method of the disclosure described herein is performed in vitro.

[0094] In any aspect of the disclosure, the sample for biomarker analysis may be a blood sample, plasma sample, serum sample, a saliva sample or a urine sample. In a preferred embodiment, the sample for biomarker analysis is a blood sample.

[0095] In any aspect of the disclosure, the sample for biomarker analysis may be stored in an appropriate container, preferably a serum separator tube. In some embodiments, the sample for biomarker analysis is processed according to standard laboratory procedures before it is subject to biomarker analysis. In a preferred embodiment, the sample for biomarker analysis is a serum sample separated from a blood sample by centrifugation.

[0096] Preferably, the sample for biomarker analysis is serum. Serum may be obtained by a serum separator tube that typically contains nanoparticles and coagulation factor XII. In another embodiment, the sample for biomarker analysis is plasma. Plasma may be obtained by anti-coagulating blood with EDTA, sodium heparin, lithium heparin, 1006117846sodium citrate or sodium oxalate. In yet another embodiment, the sample for biomarker analysis is blood. In one embodiment, the sample is whole blood.Measuring or determining biomarker levels

[0097] Methods for measuring or determining biomarker levels will be known to those skilled in the art and refers to any mechanism by which the level of a biomarker can be determined (assayed or quantified) and may include any molecule or reagent that is capable or directly or indirectly binding the biomarker. For instance, serum biomarker levels may be determined in a sample using any method known to those skilled in the art for detecting polypeptides including, but not limited to, for example immunoassays such as, for example enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), Western blot, slot blot, dot blot, or immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, (SDS-PAGE), chromatography, chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA) and the like. Dendrimer-enhanced radial partition immunoassays and immunofluorescence assays, for example, are known in the art and are commercially available.

[0098] As used herein, "assay", and variants thereof, refers to measurement or quantification of the level of the biomarker herein defined.

[0099] In some embodiments, the sample for biomarker analysis may be diluted in water or a buffer that is compatible with the assays or measurements prior to analysis.

[0100] The terms “antibody” and “antibodies” refer to monoclonal antibodies, polyclonal antibodies, bi-specific antibodies, multispecific antibodies, grafted antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-ld) antibodies and antigen-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e. , molecules that contain an antigen binding site. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. The heavy-chain constant domains (Fc) that correspond to the different classes of immunoglobulins are called a, 5, E, y, and p, respectively. The1006117846subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, lgG2, IgGs, lgG4, IgAi and lgA2) or subclass. The terms “antibody” and “immunoglobulin” are used interchangeably in the broadest sense. In some embodiments an antibody is part of a larger molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0101] The terms “binding fragment,” “functional fragment,” “antibody fragment” or “antigen binding fragment” are used herein, for purposes of the specification and claims, to mean a portion or fragment of an intact antibody molecule, preferably wherein the fragment retains antigen-binding function. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd (VH and CHI domains), Fd' and Fv (the VL and VH domains of a single arm of an antibody) fragments, diabodies, linear antibodies, variable light chains (VL), variable heavy chains (VH), single-chain antibody molecules, single-chain binding polypeptides, scFv, scFv2 (a tandem linkage of two scFv molecules head to tail in a chain), bivalent scFv, tetravalent scFv, one-half antibodies, dAb fragments, variable NAR domains, and bispecific or multispecific antibodies formed from antibody fragments (e.g., a bi-specific Fab2, and a tri-specific Fabs, etc.).

[0102] Methods for generating antibodies are known in the art and / or described in Harlow and Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods an antigen or immunogenic fragment or epitope thereof or a cell expressing and displaying same (i.e. , an immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal, for example, a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig. The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known route.

[0103] The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. One or more further immunizations may be given, if required to achieve a desired antibody titre. The process of boosting and titering is repeated until a suitable titre is achieved. When a desired1006117846level of immunogenicity is obtained, the immunized animal is bled and the serum isolated and stored, and / or the animal is used to generate monoclonal antibodies (mAbs).

[0104] Monoclonal antibodies are one exemplary form of antibody contemplated by the present disclosure. The term “monoclonal antibody" or “mAb” refers to a homogeneous antibody population capable of binding to the same antigen(s), for example, to the same epitope within the antigen. This term is not intended to be limited with regard to the source of the antibody or the manner in which it is made.

[0105] For the production of mAbs any one of a number of known techniques may be used, such as, for example, the procedure exemplified in LIS4196265 or Harlow and Lane (1988), supra.

[0106] For example, a suitable animal is immunized with an immunogen under conditions sufficient to stimulate antibody producing cells. Rodents such as rabbits, mice and rats are exemplary animals. Mice genetically-engineered to express human antibodies, for example, which do not express murine antibodies, can also be used to generate an antibody of the present disclosure (e.g., as described in W02002 / 066630).

[0107] Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsies of spleens, tonsils or lymph nodes, or from a peripheral blood sample. The B cells from the immunized animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal that was immunized with the immunogen.

[0108] Hybrids are amplified by culture in a selective medium comprising an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary agents are aminopterin, methotrexate and azaserine.

[0109] The amplified hybridomas are subjected to a functional selection for antibody specificity and / or titer, such as, for example, by flow cytometry and / or immunohistochemistry and / or immunoassay (e.g. radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like).1006117846

[0110] Alternatively, ABL-MYC technology (NeoClone, Madison Wl 53713, USA) is used to produce cell lines secreting MAbs (e.g., as described in Largaespada et al, J. Immunol. Methods. 197: 85-95, 1996).

[0111] Antibodies can also be produced or isolated by screening a display library, e.g., a phage display library, e.g., as described in US6300064 and / or US5885793.

[0112] Any antibody used in accordance with the present disclosure may be a synthetic antibody. For example, the antibody is a chimeric antibody, a humanized antibody, a human antibody synhumanized antibody, primatized antibody or a deimmunized antibody.

[0113] One exemplary agent for detecting a protein of interest is an antibody, or antigen binding fragment thereof, capable of specifically binding to the biomarker. The antibody may detectably labelled, either directly or indirectly.

[0114] Immunoassays for the biomarker may comprise incubating a sample with a detectably labelled antibody, or antibody fragment, capable of specifically binding to the biomarker, and detecting the bound antibody by any of a number of techniques well- known in the art. As discussed in more detail, below, the term "labelled" can refer to direct labelling of the antibody via, e.g., coupling (i.e., physically linking) a detectable substance to the antibody, and can also refer to indirect labelling of the antibody by reactivity with another reagent that is directly labelled. An example of indirect labelling includes detection of a primary antibody using a fluorescently labelled secondary antibody.

[0115] The sample can be brought in contact with and immobilised on a solid support or carrier, or other solid support, which is capable of immobilising soluble proteins. The support can then be washed with suitable buffers followed by treatment with the detectably labelled antibody. The solid support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on solid support can then be detected by conventional methods.

[0116] By "solid support or carrier" is intended to be any support capable of binding an antigen or an antibody. Well-known supports or carriers include nitrocellulose, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and 1006117846modified celluloses, polyacrylamides and magnetite. The nature of the solid support or carrier can be either soluble to some extent or insoluble.

[0117] The solid support can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface can be flat such as a sheet, test strip, etc. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

[0118] One of the ways in which an antibody specific for the biomarker can be detectably labelled is by linking the antibody to an enzyme for use in an enzyme immunoassay. The enzyme bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes that can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection and measurement can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection and measurement can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

[0119] Detection and measurement can also be accomplished using any of a variety of other immunoassays. For example, by radioactively labelling the antibody or functional antibody fragment, it is possible to detect plasma levels of biomarkers through the use of a radioimmunoassay (RIA). The radioactive isotope (e.g.,125l,1311,35S,32P or3H) can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.1006117846

[0120] It is also possible to label the antibody with a fluorescent or luminescent compound. When the fluorescently labelled antibody is exposed to light of the appropriate wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine.

[0121] The antibody can also be detectably labelled using fluorescence emitting metals such as152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Fluorescence energy transfer compounds may also be employed.

[0122] The antibody also can be detectably labelled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labelling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound can be used to label the antibody. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labelling are luciferin, luciferase and aequorin.

[0123] In another embodiment, specific binding molecules other than antibodies, such as aptamers, may be used to bind the biomarker.

[0124] Other "means for measuring" the biomarker include chromatography or electrophoresis with dye-based detection, or proteomics approaches employing spectrometry such as mass spectrometry.

[0125] Spectrometry may be used to measure dye-based assays, including visible dyes, and fluorescent or luminescent agents.

[0126] A protein chip assay may be used to measure the biomarker.1006117846

[0127] The biomarker can also be measured or assayed using of one or more of the following methods. For example, methods may include nuclear magnetic resonance (NMR) spectroscopy, a mass spectrometry method, such as electrospray ionization mass spectrometry (ESI-MS), ESI-MS / MS, ESI-MS / (MS)n (n is an integer greater than zero), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption / ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS)3 quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS / MS, APCI-(MS), atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS). Other mass spectrometry methods may include quadrupole, Fourier transform mass spectrometry (FTMS) and ion trap. Other suitable methods may include chemical extraction partitioning, column chromatography, ion exchange chromatography, hydrophobic (reverse phase) liquid chromatography, isoelectric focusing, one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) or other chromatography, such as thin- layer, gas or liquid chromatography, or any combination thereof.

[0128] In one embodiment, LDI-TOF-MS allows the generation of large amounts of information in a relatively short period of time. A biological sample is applied to one of several varieties of a support that binds the biomarker in the sample. Samples are applied directly to these surfaces in volumes as small as 0.5 pL, with or without prior purification or fractionation. The sample can be concentrated or diluted prior to application onto the support surface. Laser desorption / ionization is then used to generate mass spectra of the sample in as little as three hours.

[0129] A bead assay may also be used to measure the biomarker.

[0130] In some embodiments, a sample for biomarker analysis is analysed by ELISA. As an example, the ELISA may comprise the steps of:- Pre-coating a microplate with an antibody or antibody fragment that binds to the biomarker;- Incubating the sample in the pre-coated microplate;1006117846- Adding to the microplate a secondary enzyme-conjugated antibody or antibody fragment that binds to the biomarker, preferably wherein the antibody or antibody fragment is conjugated to horseradish peroxidase;- Adding to the microplate a substrate that the enzyme converts to a detectable signal, preferably wherein the detectable signal is a colour change, and more preferably wherein the substrate is tetramethylbenzidine (TMB); and- Measuring the intensity of the detectable signal that correlates to the concentration of the biomarker in the sample, preferably wherein the detectable signal is measured using a spectrophotometer.

[0131] In some embodiments, the sample for biomarker analysis is analysed by CLIA.As an example, the CLIA may comprise the steps of:- Pre-coating magnetic particles with an antibody or antibody fragment that binds to the biomarker;- Incubating the sample with the magnetic particles;- Adding to the mixture comprising the magnetic particles and the sample a secondary enzyme-conjugated antibody or antibody fragment that binds to the biomarker, preferably wherein the enzyme is a chemiluminescent enzyme;- Adding to the mixture a substrate that the enzyme converts to a detectable signal, preferably wherein the detectable signal is chemiluminescence, and more preferably wherein the substrate is luminol; and- Measuring the intensity of the detectable signal that correlates to the concentration of the biomarker in the sample, preferably wherein the detectable signal is measured using a luminometer.

[0132] In some embodiments, the sample for biomarker analysis is analysed by RIA.As an example, the RIA may comprise the steps of:- Incubating the sample with a radio-labelled polypeptide and an antibody or antibody fragment that binds to the biomarker, wherein the sequence of the1006117846polypeptide is identical or substantially identical to the sequence of the biomarker;- Separating the bound and free radio-labelled polypeptide using a secondary antibody or a solid-phase separation method; and- Measuring the radioactivity of the bound fraction of the polypeptide that is inversely proportional to the concentration of the biomarker in the sample, preferably wherein the radioactivity is measured using a gamma counter.

[0133] In any aspect of the disclosure, a commercially available automated immunoassay system may be employed to enhance the accuracy, precision, and throughput of the laboratory assays or measurements. In a preferred embodiment, the automated immunoassay systems automate sample handling, reagent addition, incubation and detection.

[0134] In any aspect of the disclosure, a calibration curve for the laboratory assays or measurements may be generated using standards comprising the biomarker at known concentrations.

[0135] In any aspect of the disclosure, quality control samples comprising the biomarker at known concentrations may be analysed alongside the sample for biomarker analysis to ensure the reliability and consistency of the laboratory assays or measurements.

[0136] In any aspect of the disclosure, a data management system may be integrated with the immunoassay platforms for standardised result reporting.

[0137] Disease or condition responsive to stem cell treatment

[0138] As used herein, “disease or condition responsive to stem cell treatment” refers to a disease or condition where the use of stem cells, which have the ability to develop into different cell types, alleviate or cure the disease or condition. Stem cell therapy involves the introduction of stem cells, or cells derived from stem cells, into the body of a subject to replace or repair damaged or diseased cells. Examples of diseases or conditions that may be responsive to stem cell treatment include but are not limited to:1006117846neurodegenerative diseases, spinal cord injury, autoimmune diseases, inflammatory diseases, cardiovascular diseases, musculoskeletal degenerative conditions, pulmonary degenerative diseases, and endocrine disorders.

[0139] In a particularly preferred embodiment, the disease or condition responsive to stem cell treatment is cardiovascular disease, more preferably acute myocardial infarction (AMI).Stem cell treatment

[0140] In any aspect of the disclosure, the biomarker may be enriched in stem cells, preferably in stem cells derived from young subjects. In a preferred embodiment, the biomarker is expressed in stem cells, including those derived from amniotic membrane, amniotic fluid, placenta, umbilical cord, umbilical cord blood, embryo, and pluripotent stem cells.

[0141] Accordingly, preferably the subject has received or is receiving stem cell treatment comprising stem cells derived from amniotic membrane, amniotic fluid, placenta, umbilical cord, umbilical cord blood, embryo, and / or pluripotent stem cells. In a particularly preferred embodiment, the stem cells are derived from umbilical cord. In another particularly preferred embodiment, the stem cells are derived from umbilical cord blood.

[0142] In a preferred embodiment, the stem cell treatment does not comprise administering peripheral blood stem cells, adipose stem cells or bone marrow stem cells.

[0143] Preferably, the stem cell treatment comprises administering stem cells at a dose between 4 x 108and 2 x 109cells per subject. Optionally, the dose corresponds to about 5.7 x 106cells / kg to about 2.9 x 107cells / kg. Alternatively, the stem cell treatment comprises administering stem cells at a dose between 3.5 x 108and 3.5 x 109cells per subject. Optionally, the dose corresponds to about 5 x 106cells / kg to about 5 x 107cells / kg.Therapeutic response1006117846

[0144] As used herein, therapeutic stem cells refer to therapeutically active stem cells that are capable of providing a therapeutic effect. A high level of therapeutic stem cells corresponds to a level of stem cells sufficient to elicit a therapeutic response. A low level of therapeutic stem cells corresponds to a level of stem cells insufficient to elicit a therapeutic response.

[0145] A therapeutic response may include one or more of:- clinical outcomes, including but not limited to improvements in symptoms or functional status of the subject, reduction in disease progression or stabilisation of the condition;- imaging and laboratory tests including, changes in the size, structure, or function of the affected tissues or organs, as observed through imaging techniques like MRI, CT scans, or ultrasound; normalisation of specific biomarkers or laboratory values related to the disease;- histological analysis including examination of biopsy samples to assess the integration, differentiation, and functionality of the transplanted stem cells; evaluation of tissue regeneration, reduced inflammation, or other structural improvements;- survival and safety including monitoring of adverse events or side effects associated with the stem cell therapy; evaluation of long-term survival and maintenance of the therapeutic benefits; and / or- functional assessment including for neurological conditions, testing of cognitive, motor, or sensory functions; for cardiovascular diseases, assessment of cardiac output, exercise capacity, or vascular function; for musculoskeletal disorders, evaluation of range of motion, strength, or physical performance.

[0146] In a particularly preferred embodiment, the disease or condition responsive to stem cell treatment is cardiovascular disease, more preferably acute myocardial infarction (AMI). The therapeutic or favourable response to stem cell treatment is survival, preferably long term survival, or non-fatal cardiac events. Survival may be selected from major adverse cardiac events (MACE)-Free survival, all-cause mortality free survival, cardiac death free survival or heart failure (HF) rehospitalisation free1006117846survival, or any other survival described herein. Non-fatal cardiac events may include MACE and adverse improvement of LVEF.

[0147] The favourable outcome may be indicative of survival or non-fatal cardiac events 1 , 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 28, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80 or more, months following stem cell therapy.

[0148] The therapeutic response may be partial or complete. A partial therapeutic response may increase the time to disease progression and / or disease recurrence. A complete therapeutic response may include wherein blood markers associated with the disease return to normal levels, or where histological or imaging scans show no detectable signs of the disease.

[0149] A low probability of a therapeutic response to stem cell therapy may include a subject that has about a 50%, 40%, 30%, 20%, 10% or less probability of a therapeutic response.

[0150] A high probability of a therapeutic response to stem cell therapy may include a subject that has about a 60%, 70%, 80%, 90% or greater probability of a therapeutic response.

[0151] As will be understood by those skilled in the art, the prediction may need not be correct for 100% of the subjects evaluated. The term, however, requires that a statistically significant portion of subjects can be identified as having an increased (or decreased) probability of having a given outcome.

[0152] In subjects who have received or are receiving stem cell treatment, the determination of the level of the biomarker, may be used to assess whether or not a given subject should be administered a further dose of stem cell treatment. Moreover, the above determination or level has prognostic impact, and accordingly early accurate prediction of response to stem cell treatment is advantageous, particularly in complex subjects, or where local health-care resources are limited.1006117846

[0153] Accordingly, in another aspect, there is provided a method of determining the suitability of a subject to receive a further dose of stem cell therapy, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and- determining that the subject is suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is lower than the reference level; or- determining that the subject is not suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is the same or higher than the reference level.

[0154] In some embodiments wherein the biomarker is a polypeptide comprising a fragment of SEQ ID NO: 1 or 2, the polypeptide or fragment thereof comprises or consists of SEQ ID NO: 3.

[0155] In some embodiments wherein the biomarker is a polypeptide comprising a fragment of SEQ ID NO: 1 or 2, the polypeptide or fragment thereof does not comprise or consist of SEQ ID NO: 3.

[0156] In another aspect, there is provided a method of determining the suitability of a subject to receive a further dose of stem cell therapy, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;1006117846- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and- determining that the subject is suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is lower than the reference level; or- determining that the subject is not suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is the same or higher than the reference level.

[0157] In subjects who have received or are receiving stem cell treatment, an increased level of the biomarker correlates with therapeutic levels of stem cells and / or is prognostic for a therapeutic response to the stem cell treatment. The measurement of the biomarker provides an accurate approach to aid monitoring of stem cell levels and activity as well as prognosing response to stem cell therapy. The disclosure provides at least one or more of the following advantages:(1 ) Increased levels of the biomarker in a subject who has received or is receiving stem cell treatment correlate with therapeutic levels of administered stem cells in the subject;(2) Increased levels of the biomarker in a subject who has received or is receiving stem cell treatment correlate with a therapeutic response to the stem cell therapy; and(3) Increased levels of the biomarker in a subject who has received or is receiving stem cell therapy to treat AMI correlate with improved left ventricular ejection fraction and / or enhanced Canadian Cardiovascular Society (CCS) class grades and / or New York Heart Association (NYHA) class grades.

[0158] In other words, increased levels of the biomarker in a subject who has received or is receiving stem cell treatment can act as an independent indicator of favourable outcomes of stem cell therapy. This approach may facilitate the identification of a subject group that are likely to require additional doses of stem cells. Those with low1006117846levels of the biomarker can be identified as having a poor prognosis following the stem cell therapy. Levels of the biomarker in a subject receiving a stem cell therapy therefore have implications for subject management and prognosis.

[0159] As used herein, Canadian Cardiovascular Society (CCS) class grades or Canadian Cardiovascular Society grading of angina pectoris is a classification system used to grade the severity of exertional angina. As an example, the CCS grading may comprise five classes. Class 0 corresponds to mild myocardial ischemia with no symptoms, or asymptomatic angina. Class I corresponds to presence of angina during strenuous, rapid, or prolonged ordinary activity, or angina only with strenuous exertion. Class II corresponds to slight limitation of ordinary activities when they are performed rapidly, after meals, in cold, in wind, under emotional stress, during the first few hours after waking up, but also walking uphill, climbing more than one flight of ordinary stairs at a normal pace and in normal conditions, or angina with moderate exertion. Class III corresponds to having difficulties walking one or two blocks or climbing one flight of stairs at normal pace and conditions, or angina with mild exertion. Class IV corresponds to no exertion needed to trigger angina, or angina at rest.

[0160] As used herein, New York Heart Association (NYHA) class grades or New York Heart Association functional classification is a classification system used to grade the extent of heart failure. As an example, the NYHA grading may comprise four classes. Class I corresponds to presence of cardiac disease with no limitation of physical activity, where ordinary physical activity does not cause undue fatigue, palpitation and / or dyspnea (shortness of breath). Class II corresponds to slight limitation of physical activity with comfort at rest, where ordinary physical activity results in fatigue, palpitation and / or dyspnea. Class III corresponds to marked limitation of physical activity with comfort at rest, where less than ordinary activity causes fatigue, palpitation and / or dyspnea. Class IV corresponds to inability to carry on any physical activity without discomfort with symptoms of heart failure at rest, where any physical activity causes an increase of discomfort.

[0161] The current disclosure provides the clinician or physician caring for a subject receiving a stem cell therapy with information about the activity of the administered stem cells and / or the likelihood of response to the stem cell therapy. On the basis of the1006117846results of the method of the disclosure, the clinician or physician can do, amongst other things:(i) administer a further dose of the stem cell treatment;(ii) enrol the subject in clinical trials for new therapies for the disease or condition responsive to stem cell treatment;(iii) treat the subject with alternative therapies such as another type of stem cell therapy, and / or medications;(iv) discuss the likely treatment and outcome scenarios with the subject; and / or(v) provide more regular or extensive post-treatment surveillance for a subject identified as having low levels of therapeutic stem cells and / or a low likelihood of response to the stem cell therapy.Method of treatment

[0162] In any embodiment of the disclosure, the method may comprise a treatment step such as administering a further dose of stem cells, administering another type of stem cell therapy, and / or administering medications. Early knowledge of prognosis during the decision-making process about subject management provides numerous advantages. Firstly, clinicians assessing subjects in whom the activity of administered stem cells is low and / or the response to stem cell therapy is not obvious or stuttering may benefit from the knowledge that an elevated biomarker is predictive of stem cell activity, which would facilitate the decision-making process about the timeliness of treatment.

[0163] In another aspect, there is provided a method of treating a disease or condition responsive to stem cell therapy in a subject, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;1006117846- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker;- selecting the subject for treatment only when the level of the biomarker in the subject is lower than the reference level; and- administering stem cell therapy to the subject selected for treatment; thereby treating the subject having the disease or condition responsive to stem cell therapy.

[0164] In some embodiments wherein the biomarker is a polypeptide comprising a fragment of SEQ ID NO: 1 or 2, the polypeptide or fragment thereof comprises or consists of SEQ ID NO: 3.

[0165] In some embodiments wherein the biomarker is a polypeptide comprising a fragment of SEQ ID NO: 1 or 2, the polypeptide or fragment thereof does not comprise or consist of SEQ ID NO: 3.

[0166] In another aspect, there is provided a method of treating a disease or condition responsive to stem cell therapy in a subject, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker;- selecting the subject for treatment only when the level of the biomarker in the subject is lower than the reference level; and- administering stem cell therapy to the subject selected for treatment; thereby treating the subject having the disease or condition responsive to stem cell therapy.1006117846

[0167] In one embodiment, wherein the disease or condition responsive to stem cell therapy is AMI, the disclosure provides for prognosis of improvement of LVEF and enhanced CCS class grades and / or NYHA class grades. In particular, in a subject who has received or is receiving stem cell therapy, increased levels of the biomarker could prognose improvement of LVEF and enhanced CCS class grades and / or NYHA class grades. In individuals who are identified as having low levels of the biomarker, additional or more intensive intervention or monitoring can be undertaken. Further, in individuals where there is a risk of not being responsive to the stem cell therapy, heart failure medication (e.g. ACE inhibitors, beta blockers etc) can be administered and heart failure preventative therapies can be applied.

[0168] The measurement of the biomarker in a subject who has received or is receiving stem cell therapy will therefore be highly valuable in the ongoing management, including the dosing of stem cells and / or use of an adjunctive or alternative therapy, as it correlates to the activity of administered stem cells and provides further prognostic information on the response to the stem cell therapy, in addition to the advantages outlined above.

[0169] As used herein, a "method" for monitoring the activity of administered stem cells and / or prognosing the response to a stem cell therapy may be presented in an alternative form. In one example, the method may be in the form of "use" of a biomarker level for monitoring the activity of administered stem cells and / or diagnosing, prognosing or treating a disease by a stem cell therapy in a subject. In a second example, the method may be in the form of a biomarker level "for use" in monitoring the activity of administered stem cells and / or prognosing or treating a disease by a stem cell therapy in a subject. In another form, the method may be in the Swiss form "use of a biomarker level in the manufacture" of a prognostic agent or a medicament. In yet another form, the method may be in the Swiss form “use of a stem cell in the manufacture” of a medicament for treating a subject, preferably for stem cell therapy, wherein the subject has been selected in accordance with any methods of the disclosure.1006117846Device

[0170] As used herein, "device" refers to a physical arrangement of components for performing an assay for measuring the biomarker. The device may be a point-of-care device used by a medical practitioner to measure the biomarker without the need for laboratory measurement. Alternatively, a point-of-care device may be used domestically, for example in a subject who has received or is receiving stem cell treatment. Alternatively, the device may be in a laboratory located separately to the subject in whom the biomarker is to be measured.

[0171] The device may employ an electrochemical cell. Electrochemical cells may use electrodes positioned within the cell in a side-by-side or "coplanar" layout to minimize the electrical interference between the electrodes. Alternatively, electrochemical cells may use non coplanar electrodes that exploit the electrical interference between the electrodes to yield additional information about the sample including information that can correct for patient to patient variations in hematocrit and interfering chemical substances that may be present in a sample.

[0172] The device may provide a qualitative output (e.g. yes / no, presence / absence / , high / low), a numerical or quantified output (e.g. concentration), or an output for visual inspection (e.g. a colour for comparison with a reference scale).Kit

[0173] As used herein, "kit" refers to a physical arrangement of components, one of which may be the device for measuring the biomarker. The kit may include a reagent such as an immunogenic moiety that binds the biomarker, a secondary detection agent for detecting the immunogenic moiety, or a reagent for sample preparation and / or processing, for example a buffer. The kit may include means, such as reagents, to perform a highly sensitive assay, such as for the detection of the biomarker.

[0174] The device or kit may be accompanied by instructions or directions for use of the device or kit in any method described herein.

[0175] As used herein, a device or kit may be in alternative forms. One form designates either suitability for or restriction to a specific use and is indicated by the1006117846word "for". Another form is restricted to a specific use only and is indicated by the words "when used for" or similar. In one embodiment of the method for treating a disease by a stem cell therapy in a subject, the biomarker level is measured using the device disclosed herein.

[0176] It will be understood that these examples are intended to demonstrate these and other aspects of the disclosure and although the examples describe certain embodiments of the disclosure, it will be understood that the examples do not limit these embodiments to these things. Various changes can be made and equivalents can be substituted and modifications made without departing from the aspects and / or principles of the disclosure mentioned above. All such changes, equivalents and modifications are intended to be within the scope of the claims set forth herein.ExamplesExample 1

[0177] Patients diagnosed with acute myocardial infarction (AMI) were pre-treated with antihistamine medications. Allogeneic umbilical cord blood stem cells (USCs) were administered to the patients intravenously at doses between 4 x 108and 2 x 109nucleated cells, corresponding to doses between approximately 5.7 x 106cells / kg and approximately 2.9 x 107cells / kg.

[0178] Patients underwent study examinations, including cardiac echo and magnetic resonance imaging (MRI). Blood laboratory studies were conducted at baseline and monthly post-treatment. Serum levels of the biomarker A508 were measured (Fig 1).

[0179] It was observed that serum levels of the biomarker following USC administration peaked at one month after USC administration, and returned to baseline levels by the second month after USC administration. Levels of the biomarker further decreased and normalised by the third month following USC administration. The increase in biomarker level was associated with improved LVEF of the patients (Fig 2 and Table 1).1006117846Table 1. Outcome of patients receiving a stem cell therapy

[0180] The increase in biomarker level was also associated with improved New York Heart Association (NYHA) and Canadian Cardiovascular Society (CSS) scores of the patients (Table 2).Table 2. New York Heart Association (NYHA) and Canadian CardiovascularSociety (CSS) scores of patients receiving a stem cell therapy

[0181] It was found that serial measurements of the biomarker over time was useful for monitoring treatment response in patients receiving USCs.Example 2

[0182] The association between high A508 levels and stem cell cellular activity was investigated. As shown in Table 3, increased cellular activity of umbilical cord blood stem cells is associated with high A508 levels (> 3.3 lll / ml). In contrast, increased cellular activity of peripheral blood, adipose and bone marrow deri ved stem cells was not associated with high A508 levels (< 3.3 lU / mL as negative).1006117846Table 3. Association between A508 levels and cellular activity of stem cells.Positive, > 3.3 lU / mL; negative, < 3.3 lU / mL1006117846

Claims

CLAIMS1 . A method for evaluating the level of therapeutic stem cells in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;- determining that the subject has a high level of therapeutic stem cells when the level of the biomarker in the sample from the subject is the same or greater than a reference level of the biomarker; or- determining that the subject has a low level of therapeutic stem cells when the level of the biomarker in the sample from the subject is less than a reference level of the biomarker.

2. A method for monitoring a response to stem cell therapy in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining a level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;- comparing the level of the biomarker to a reference level of the biomarker;- determining the subject has a low probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is less than the reference level;1006117846- determining the subject has a high probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is the same or greater than the reference level; thereby monitoring the response to the stem cell therapy in the subject.

3. A method of determining the efficacy of stem cell treatment in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and- determining that the treatment has likely not been successful when the level of the biomarker in the subject is less than the reference level; or- determining that the treatment has likely been successful when the level of the biomarker in the subject is the same or greater than the reference level.

4. A method of determining the suitability of a subject to receive a further dose of stem cell therapy, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence SEQ ID NO: 1 or 2, or a fragment thereof;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and1006117846- determining that the subject is suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is less than the reference level; or- determining that the subject is not suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is the same or greater than the reference level.

5. A method of treating a disease or condition responsive to stem cell therapy in a subject, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence SEQ ID NO: 1 or 2, or a fragment thereof;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker;- selecting the subject for treatment only when the level of the biomarker in the subject is less than the reference level; and- administering stem cell therapy to the subject selected for treatment; thereby treating the subject having the disease or condition responsive to stem cell therapy.

6. The method of any one of claims 1 to 5, wherein the biomarker is a polypeptide that comprises or consists of the sequence of SEQ ID NO: 3.

7. The method of any one of claims 1 to 5, wherein the biomarker is a polypeptide that does not comprise or consist of the sequence of SEQ ID NO: 3.

8. A method for evaluating the level of therapeutic stem cells in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;1006117846- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO:3:DKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDET (SEQ ID NO: 3);- determining that the subject has a high level of therapeutic stem cells when the level of the biomarker in the sample from the subject is the same or greater than a reference level of the biomarker; or- determining that the subject has a low level of therapeutic stem cells when the level of the biomarker in the sample from the subject is less than a reference level of the biomarker.

9. A method for monitoring a response to stem cell therapy in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received or is receiving stem cell treatment;- measuring or determining a level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;- comparing the level of the biomarker to a reference level of the biomarker;- determining the subject has a low probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is less than the reference level;- determining the subject has a high probability of favourable response to the stem cell therapy when the level of the biomarker in the subject is the same or greater than the reference level; thereby monitoring the response to the stem cell therapy in the subject.

10. A method of determining the efficacy of stem cell treatment in a subject, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:1006117846- providing a subject who has received or is receiving stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and- determining that the treatment has likely not been successful when the level of the biomarker in the subject is less than the reference level; or- determining that the treatment has likely been successful when the level of the biomarker in the subject is the same or greater than the reference level.

11. A method of determining the suitability of a subject to receive a further dose of stem cell therapy, wherein the subject has a disease or condition responsive to stem cell treatment, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker; and- determining that the subject is suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is less than the reference level; or- determining that the subject is not suitable to receive a further dose of stem cell therapy when the level of the biomarker in the subject is the same or greater than the reference level.

12. A method of treating a disease or condition responsive to stem cell therapy in a subject, the method comprising:- providing a subject who has received at least a first dose of stem cell treatment;1006117846- measuring or determining the level of a biomarker in a sample from the subject, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 3;- comparing the level of the biomarker in the sample from the subject to a reference level of the biomarker;- selecting the subject for treatment only when the level of the biomarker in the subject is less than the reference level; and- administering stem cell therapy to the subject selected for treatment; thereby treating the subject having the disease or condition responsive to stem cell therapy.

13. The method according to any one of claims 1 to 12, wherein the reference level of the biomarker is representative of one or more subjects who have not received stem cell therapy.

14. The method according to any one of claims 1 to 13, wherein the reference level of the biomarker is from a sample of the subject prior to treatment.

15. The method of any one of claims 1 to 14, wherein the sample is blood, plasma, serum, saliva, or urine.

16. The method of any one of claims 1 to 15, wherein the reference level of the biomarker is about 3.5 ILI / mL, or about 4 ILI / mL.

17. The method of claim 16, wherein the reference level of the biomarker is about 3.3 lU / mL.

18. The method of claim 14 or 15, wherein the reference level of the biomarker is measured from a sample of the subject within about 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 150 minutes, 180 minutes or 210 minutes or more, within about 0.5 days, 1 .0 day, 1 .5 days, 2.0 days, 2.5 days, 3.0 days, 3.5 days, 4.0 days, 4.5 days, 5.0 days, 5.5 days, 6.0 days, 6.5 days or more, or within about 0.5 months, 1.0 month, 1.5 months, 2.0 months, 2.5 months, 3.0 months, 3.5 months, 4.01006117846months, 4.5 months, 5.0 months, 5.5 months, 6.0 months, 6.5 months or more before the subject received the stem cell treatment.

19. The method of any one of claims 1 to 18, wherein the stem cell treatment comprises administering stem cells at a dose between 4 x 108and 2 x 109cells per subject, optionally wherein the dose corresponds to between about 5.7 x 106cells / kg and about 2.9 x 107cells / kg; or at a dose between 3.5 x 108and 3.5 x 109cells per subject, optionally wherein the dose corresponds to between about 5 x 106cells / kg to about 5 x 107cells / kg.

20. The method of claim 19, wherein the stem cell treatment comprises administering stem cells derived from amniotic membrane, amniotic fluid, placenta, umbilical cord, umbilical cord blood, embryo, and / or pluripotent stem cells.21 . The method of claim 19 or 20, wherein the stem cell treatment does not comprise administering peripheral blood stem cells, adipose stem cells or bone marrow stem cells.

22. The method of claim 20 or 21 , wherein the stem cell treatment comprises administering stem cells derived from umbilical cord or umbilical cord blood.

23. The method of any one of claims 1 to 22, wherein the disease or condition is acute myocardial infarction (AMI).

24. The method of claim 23, wherein the response to the stem cell treatment is increased left ventricular ejection fraction (LVEF), increased Canadian Cardiovascular Society (CCS) class grades, increased New York Heart Association (NYHA) class grades, decreased risk of major adverse cardiac events (MACE), increased MACE-free survival, increased heart failure-rehospitalisation-free survival, increased all-mortality free survival, and / or increased cardiac death free-survival.

25. A device comprising means for determining a level of a biomarker in a sample from a subject having a disease or condition responsive to stem cell treatment, when used in a method according to any one of claims 1 to 24, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof.100611784626. The device of claim 25, wherein the biomarker is a polypeptide that comprises or consists of the sequence of SEQ ID NO: 3.

27. The device of claim 25, wherein the biomarker is a polypeptide that does not comprise or consist of the sequence of SEQ ID NO: 3.

28. The device of any one of claims 25 to 27, comprising means for performing an immunoassay for determining the concentration of the biomarker.

29. The device of any one of claims 25 to 28, wherein the device is a point of care device.

30. A kit comprising a reagent for measuring a level of a biomarker in a sample from a subject having a disease or condition responsive to stem cell treatment, wherein the biomarker is a polypeptide comprising the sequence of SEQ ID NO: 1 or 2, or a fragment thereof, when used in a method a method according to any one of claims 1 to 24.31 . The kit of claim 30, wherein the biomarker is a polypeptide that comprises or consists of the sequence of SEQ ID NO: 3.

32. The kit of claim 30, wherein the biomarker is a polypeptide that does not comprise or consist of the sequence of SEQ ID NO: 3.1006117846

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